Final Report Summary - AMBULUNG (Ambulatory Bio-Artificial Lung)
Executive Summary:
Executive summary
The idea to initiate the Ambulung project was based on the unmet medical needs to adequately treat patients, who suffer from severe COPD, a chronic pulmonary disease, which is the 4th leading cause of death worldwide. Following this idea, we formed a team of clinicians, scientists, engineers and entrepreneurs from four different institutions, who are convinced that the combined effort of this European team will create a door opening pioneer product that will trigger consecutive research and development efforts to relieve suffering from end stage lung failure. Consequently, we defined it as our common goal to create the world’s first wearable bioartificial lung for longterm application on COPD patients in an outpatient setting.The main objectives of this three years project were to develop a miniaturized and wearable extracorporeal lung support system, to cellularize the diffusion membrane with endothelial cells and to evaluate the developed system in pre-clinical and clinical studies.
After completion of the three year project period we can summarize:
• We have been successful to develop appropriate procedures and processes for the differentiation of endothelial cells from Human Empryonic Stem Cells.
• We were able to expand these endothelial cells by suitable, partly newly developed methods, up to a number that is necessary for complete cellularization the diffusion membrane.
• The gas-exchange surface of the diffusion membrane was functionalized with various biochemical and physical methods in order to create an ideal base for cellularization.
• We have successfully seeded these surfaces with endothelial cells and have tested the stability of the cell monolayers unter static and dynamic conditions using a newly developed dynamic bioreactor. As result of these seeding tests, we have defined the optimal surface for a durable and stable cell coating.
• We have developed a gas exchanger with a diffusion membrane, which is suitable for the cellularization with endothelial cells. The gas exchanger is designed small and lightweight while maintaining the required performance parameters. The innovative shape of the gas exchanger with circular cavity and tangential flow onto the membrane allows the required wearabilty.
• The miniaturized hardware was developed from scratch with state-of-the-art components, which weigh less than 3 kg including the disposable components, so that we have surpassed one of our main objectives. The entire system is designed for autonomous and mobile use and for attachment to the patient body.
• We have manufactured prototypes of the entire system that have been successfully tested in the laboratory and in the course of an animal study.
• The clinical protocol for the intended first-in-man was approved by the competent ethics committee, but the final approval by the Ministry of Health has not yet been issued.
• We have started our dissemination activities at an early stage of the project and have already gained great interest of potential users worlwide.
• In summary, all our objectives except the implementation of the first-in-man trial have been achieved within the given timeframe.
This project is not finished after the three-year term. The AmbuLung team has mutually committed to stay together as a product group even after the end of this project phase. We will continue to work intensively on the further development, verification and validation of this product family with the aim to obtain the CE certification in the next year and to start commercializing afterwards.
Project Context and Objectives:
Project context and main objectives
Chronic obstructive pulmonary disease (COPD) is actually the 4th leading cause of death worldwide and is going to become the 3rd. Acute exacerbations of COPD dramatically change the course of the disease, since they are associated with a rapid decline in lung function and worsening quality of life. AECOPD accounts for 750,000 hospitalizations, for 120,000 deaths and 1.5 million emergency department visits annually in the US alone. Patients with AECOPD and hypercapnia have mortality rates of 33% at six months and 49% at two years. In patients suffering from severe acute exacerbations of COPD, when drug treatment is not sufficient, non- invasive ventilation has been established as the treatment of choice. However, when NIV fails, which occurs in about 10-15% of patients treated with NIV, invasive mechanical ventilation will be employed, resulting in high mortality and lasting decline in lung function.
As opposed to the treatment of end stage heart failure with ventricular assist devices (VAD), long term respiratory support with any kind of mechanical ventilation currently relies on the gas exchange function of the diseased lung, rather than providing gas exchange independent of the human lung. Long-term use of invasive mechanical ventilation has drawbacks, such as limited mobility, airway infection (VAP), ventilator associated lung injury (VILI, VALI), and ventilator induced diaphragmatic dysfunction (VIDD), tracheotomy and others that affect both outcomes and quality of life in advanced respiratory failure.
More importantly, an extracorporeal artificial lung will reduce over distension of the diseased lung. This “functional volume unloading” performed with an extracorporeal artificial lung might even affect the dismal course of the disease.
Therefore, our concept relies on a wearable, ambulatory, least invasive, long-term bioartificial lung that “breathes” outside of the human lung. This will unload the lung i.e. remove over distension, support mobile, spontaneously breathing patients in advanced chronic lung failure (subgroup of GOLD IV pts.) and bridge patients across severe exacerbations, when NIV fails. Our concept and development will allow avoiding invasive mechanical ventilation, which is associated with poor outcomes in this group of patients.
Long-term respiratory support increases the life expectancy and the quality of life of COPD patients and decreases the cost of care. Currently available artificial lungs, such as Novalung’s interventional Lung Assist (iLA) system, fail after about one month, mainly due to thrombus formation at the blood/machine interface. In addition, the size of current systems limits their use to the intensive care unit, restricts patient mobility and avoids outpatent use.
The AmbuLung consortium aims to create a wearable bioartificial lung (AmbuLung) for long-term application in an outpatient setting.
The main objectives are:
• The miniaturisation of the current available iLA activve system, including the supporting technology and patient monitoring system.
• The cellularisation of the diffusion membrane with endothelial cells to decrease thrombogenicity, to increase the gas-exchange rate and to increase operation time of the membrane ventilator.
• The evaluation of the developed system in pre-clinical and clinical studies.
For cellularisation, endothelial cells derived from FDA approved clinical grade human embryonic stem cells will be used. Cell differentiation, scale-up, seeding, and maintenance will be performed using established automatable and scalable dynamic bioreactor technology. A mathematical model will be developed to predict and refine the function of this complex system in vitro and in vivo. AmbuLung will be evaluated in a pig model, assessing functionality, and non-thrombogenicity. The data will provide information required for potential clinical transfer. If successful, a clinical trial will be carried out on five COPD patients after acquisition of the required regulatory approvals.
We will develop a prototype of a miniaturized disposable device that provides an optimal physiological environment to function effectively as a dynamic cell culture chamber and extracorporeal gas exchanger to be used as an ambulatory lung support. We will address the optimization of fibre configuration with respect to hollow fibre spacing, orientation and distance, taking into account the future task to surface-functionalise the hollow fibres with adhesion and crosslinking molecules and cellularise the outer, blood-contacting fibre surfaces with endothelial cells. We will modify the gas exchange surface and housing/inlet geometry of the current iLA gasexchanger to allow pulsatile mode and optimized device inflow characteristics. We will also carriy out initial conceptual studies to create an optimized vascular access and to optimize the tubing set. In addition, we will also focus on the “system concept” of the project, as evidenced by the emphasis put on the development of a mobile unit, i.e. the carrying system for the integrated pump/gas exchanger.
In the general context of the AmbuLung project, it is the goal to miniaturize the hardware of the artificial lung to a size and a weight, which allows the patient to wear the complete system on his body during operation. The mobile system should operate independently of a central processing unit and a main power supply. It will be designed to be powerful enough to control and operate the bioartificial lung, yet small enough to allow for wearability by COPD GOLD IV patients, which are generally unable to carry more than about 3 kg.
We will design and manufacture prototypes, which will be extensively tested and verified in vitro and in vivo by an animal trial and will be suitable for human use in a first-in-man study.
In order to cellularize the diffusion membrane with endothelial cells it is the overall objective to produce high quality human endothelial cells for preclinical testing and for clinical application of the AmbuLung device. For this purpose, we will analyze the whole range of molecules (from amino acids to nutrients/metabolites to growth factors) and identify the optimal operating conditions when correlated with cellular phenotype & functionality for pluripotent cell expansion and differentiation. This will lead to the optimization/standardisation of the bioprocess for the expansion & differentiation of human pluripotent stem cells using model-based design of experiments. In order to generate the endothelial (and epithelial) cells required for the cellularisation of the AmbuLung device, we will acquire, characterize and expand clinical grade hESCs prior to differentiation. By using the expanded hESCs, we will define a standardized differentiation protocol for the production of endothelial cells. This task will be successful if we can managed to validate the generation of endothelial cells. The protocol should provide a sufficient number of differentiated endothelial cells, which maintain their endothelial characteristics until their third passage, at least, and their number is expanded 5 times. This amount of cells will cover the requirements for the number of the cells for the AmbuLung device of ~3-5 x 108 cells. Finally, we will take undifferentiated human pluripotent stem cells, expand them and differentiate them into pulmonary epithelial cells in a defined medium under hypoxic condition and cultured on specific basement membrane molecules (collagen IV) using established protocols.
One of the main objective is to optimize functionalization and cell seeding of the gas exchange membranes using chemical and physicochemical approaches. We will develop and use dynamic bioreactors to bench test the shear-resistance of the endothelial cell-seeded AmbuLung gas exchange units under simulated physiological conditions of pulsatile flow. We will further define and refine computational models, analyzing and predicting fluid dynamics and cell-biomaterials interactions in the AmbuLung gas exchanger.
The outer surface of the poly(methylpenten) (PMP) hollow fibre membrane will be equipped with a surface coating that promotes adhesion and formation of a shear-resistant confluent endothelial cell layer. We will develop and evaluate a method to seed endothelial cells on the hollow-fibrous membranes in the AmbuLung gas exchanger for the use in animal experiments and for human use. For this pupose, we will design and manufacture a specific bioreactor system to assess seeding and “maturation” of the endothelial cell monolayers under physiologic, dynamic conditions. This dynamic bioreactor will be used to test optimum seeding and culture conditions by gradually adapting mechanical loading/shear stress, rather then suddenly exposing the system to detrimental mechanical forces,(e.g. high shear stress).
After the completion of the first functional samples of the AmbuLung device, we will test their safety and functionality in preclinical animal studies in vivo. For that, we will merge the specific ethical approval needs for animal trials and for a first human use and will develop a suitable study protocol for animal experiments. We will deploy the AmbuLung device in preclinical in vivo studies using the anesthetized and ventilated pig as animal model and will analyse critical physiological and hematological parameters required to evaluate the performance and efficacy of the AmbuLung device under physiological conditions.
The final objective of the AmbuLung project is to test the clinical feasability, wearability and safety of the newly designed device in a first-in-man trial. Within the 36 months tenure of this project the consortium does not plan to develop the AmbuLung device to the stage of serial production. Specifically, we do not expect that the device would require/receive CE approval prior to the first-in-man trial. Hence, approval for the trial on humans will be requested and obtained from the Institutional Review Board (IRB) of the performing Hospital. After ethical approval, we will carry out a small-scale first-in-man clinical trial as a prelude to obtaining a Clinical Trial Authorization and as proof-of-concept for the wearability, safety, durability, and efficacy of the new medical device.
It is in the interest of the entire project team to publish R&D results in an appropriate manner. Is not only just about publishing project outcomes but also about establishing a bi-directional communication process with the targeted audience, i.e. potential end-users (patients/physicians), key opinion leaders (KOLs), and policy makers.
Given the importance of targeted dissemination of the AmbuLung concept, we will pondering early on how to inform the public without jeopardizing the success of the project. We will bring into line the partially divergent interests of the scientific partners with those of the industrial partner. For that, we will coordinate all planned dissemination activities closely and implement only those that we agreed upon unanimously. The main objectives for exploitation and dissemination are:
• Create broad awareness of the project objectives, benefits and value among all the stakeholders
• Create the conditions for a sustainable exploitation of the AmbuLung results at European level
• Prepare AmbuLung exploitation during the post-project phase
• Publish peer-reviewed papers and communications at relevant conferences, meetings and congresses.
• Organize workshops with experts and stakeholders
• Maximise collaboration with the European community
• Transfer knowledge and dissemination through contacts with industry, health centres and academia
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Project Results:
General remarks
The tasks within the AmbuLung project were divided into 3 groups:
1.) Basic research and scientific activities
2.) Engineering research and development
3.) Application testing, verification, validation
Basic research and scientific activities were performed in the fields of research on stem cells and in particular in differentiation, expansion and production of endothelial cells from human embryonic stem cells. This work has been executed by the beneficiary Imperial College in London.
In the field of tissue engineering the benficiary Fraunhofer IGB in Stuttgart examined the functionalization of artificial gas exchange surfaces with biochemical and physical methods and developed procedures to seed such functionalized surfaces durable with endothelial cells.
It was the task of the industrial partner Novalung GmbH in Heilbronn to develop the medical device and produce first prototypes. This task was in turn divided into two areas: the development of the device disposables and the device hardware. Novalung was also the coordinator of the project.
The prototype of the newly developed medical device were investigated in vivo in animals, using a pig model. This task was under the supervision of NovaLung and was conducted by a renowned animal laboratory.
At the end of the project, it was planned to conduct a first use of the medical device on humans in form of a first-in-man trial. For this task, the beneficiary University Hospital in Florence (UNIFI) was responsible, prepared and available.
S & T results / Foregrounds
Basic research and scientific activities on cells:
1.) Information-driven Bioprocessing
We installed and commissioned the bioprocess monitoring and cell culture equipment. This equipment provided us with the capability to monitor the metabolic state of the cells (both undifferentiated and differentiated) and to link it with functional characterisation at the genetic and protein levels.
Furthermore, we installed the environmental culture chamber, which facilitates the high-specification and regulated production of the cells for the requirements of the project. The environmental chamber was used to evaluate the role of oxygen in the differentiation of hESCs towards the endothelial cell lineage.
Consequently, the technical means for the generation of high quality, regulated production of hESC-derived endothelial and epithelial cells was achieved. The “metabolic signature” of the cells was employed to characterise both undifferentiated hESCs and the differentiated cells.
We developed and experimentally validated a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.
This model-based framework has been experimentally validated for the expansion of ESCs.
Specifically, our system demonstrated how batch cultures were susceptible to stress from metabolite accumulation, which exceeded toxic levels leading to the loss of pluripotency over time. In contrast, perfusion cultures maintained relatively low levels of metabolic stress, which facilitates the maintenance of high pluripotency levels.
A method for the metabolomics analysis of the cell culture metabolic physiology was developed. The metabolomics analysis of stem cells and other cell cultures has been established and the results have been validated as accurate. Metabolomics data have helped on the understanding of the metabolic physiology of stem cell cultures towards differentiation.
Results
• The combined experimental-modeling platform for the bioprocessing of ESCs facilitates efficient in silico identification of optimal culture protocols that are implemented in a novel perfusion scalable bioreactor that provides a robust and controlled metabolic environment that is conducive to high cell growth and quality ESC bioprocessing.
2.) Expansion of undifferentiated stem cells
The expansion of hPSCs, both hESCs and hiPSCs, has been successful in a feeder-independent culture system and in sufficient numbers.
The expansion of the cells has been applied in 3D cultures and the results showed successful expansion. The pluripotency of the hPSCs was maintained at 70%.
To achieve the reproducible and controlled 3D cultures, single cell suspensions of hPSCs were obtained through the use of ROCK inhibition.
Results:
• Pluripotency levels dropped following treatment with ROCK inhibitor, especially following prolonged exposure.
• The effects of ROCK inhibitor use on the pluripotency and metabolic state of the hPSCs were studied. Results indicated specific switches on the metabolic physiology of the cells in both hESCs and hiPSCs cultures. Consequently, prolonged exposure (over 1 day) of hPSCs to ROCK inhibitor is not recommended.
• A publication has been submitted. (see dissemination activities)
3.) Production of endothelial cells
The objective was to have a standardized differentiation protocol for the production of endothelial cells by using the expanded hESCs. The task was successful, as we have managed to validate the generation of endothelial cells. The protocol provides a sufficient number of differentiated endothelial cells, which maintain their endothelial characteristics until their third passage, at least, and their number is possible to be expanded 5 times. This amount of cells covers the requirements for the number of the cells for the AmbuLung device of ~3-5 x 108 cells.
The protocol that was used was adapted from Orlova et al. Our results confirm that the protocol works properly in terms of endothelial differentiation. In this step we verified three important components of the methodology used:
a) An efficient differentiation protocol of hESCs towards an endothelial phenotype,
b) The ability to sort and purify the hESC derived endothelial- cells, and
c) The generation of sufficient cell number per 6 well plate (2 x 106 cells) that renders scale-up feasible for the production of the required 2-3 x 108 endothelial cells for cellularization of an AmbuLung device.
In order to optimise the differentiation protocol, we investigated the role of oxygen in endothelial cell differentiation. Specifically, the protocol was carried out at both ambient oxygen levels (21% oxygen, “normoxia”) and at reduced oxygen levels (5% oxygen hypoxia). The number of differentiated ECs on day 10 was essentially similar, suggesting that, at least with this protocol there is no effect of reduced oxygen on the directed differentiation of ECS from hESCs.
Aside from phenotypic characterisation, we also performed functional analysis of the produced endothelial cells. Populations of endothelial cells differentiated and purified were expanded until passage 5 (9-12 days). The cells were treated with the inflammatory cytokine TNF-α (0, 10, and 100 ng/mL) and checked for the expression of VCAM1 (CD106+) by flow cytometry. VCAM1 expression was obvious following stimulation with TNF-α. Similar levels of expression (approximately 67%) was observed at both 10 ng/mL and 100 ng/mL.
Cells were also checked for the cytokine-inducible expression of ICAM1 (CD54+). A fraction (33%) of the expanded endothelial cell population constitutively expressed low levels of ICAM1 even in the absence TNF-α (0 ng/mL). As with VCAM1 expression, treatment with different concentrations of TNF-α (10 and 100 ng/mL) yielded a significant increase in both the level of ICAM expression and in the fraction of the cells (approximately 98%), expressing CD54, although the increase of the concentration above 10u/mL did not changed the results.
Tissue Factor (CD142) expression was similar to ICAM1 expression. Specifically, approximately 1/3 of the untreated endothelial cells constitutively expressed TF. Upon treatment with different concentrations of TNF-α, the number of cells expressing TF increased significantly, albeit not the level of TF in each cell. Interestingly, eNOS expression was observed, which has not been reported before in the literature for hESC-derived ECs. Following treatment with TNF-α in an apparent TNF-α dose response 10 (70%) and 100 (92%) u/mL.
The homogeneity and functionality of the cultures was also confirmed by the uniform staining of the entire cell populations with an endothelial cell specific marker vWF and the histiotypic uptake of Ac-LDL.
There has also been applied a metabolomics analysis on hESCs, the human derived endothelial cells, both CD31+ and CD31+CD144+, we acquired at passage 1 and HUVEC line which could be comparable to endothelial cells. The metabolic profiles showed a bigger similarity of CD31+ endothelial cells with HUVECs rather than hESCs. CD31+CD144+ cells seem to have a more distinct metabolic profile compare to HUVECs. As expected, hESCs have the most distinct metabolic profile compared to the other cells. These results could be explained by the phenotypical heterogeneity of endothelial cells, which is high and dependent on their functional role and developmental stage. The CD31+ cell population may not be as homogeneous, which is also the case for the HUVECs, in contrast to the more homogeneous CD31+CD144+ cell population.
Results:
• An efficient differentiation protocol has been established
• The endothelial derived had the phenotypic, functional and metabolomics of endothelial cells
• CD31+ sorted were obtained with high purity; alas, when these cells were expanded they would progressively lose their endothelial cell characteristics.
It was postulated that we should investigate the effect of differentiation time and obtain an earlier differentiated cell population of CD31+CD144+ cells, which produced a homogeneous cell population that has the desired phenotypic and functional characteristics of endothelial cells and that can be expanded.
We were successful at acquiring CD31+CD144+ cells after 7 days of differentiation. The results showed a significant extent of differentiation (24%) at day 7 of double positive cells. These cells were then sorted and purified at greater than 95% to be used in further analysis.
The functional analysis of the double-sorted cells (CD31+CD144+) showed the expression of VCAM1, tissue factor and eNOS after the treatment of the cells with 100 ng/mL of TNF-α. The unstained cells did not express any TF. The treated cells showed a high level of functional expression. The differentiated endothelial cells maintained the positive expression for Ac-LDL uptake at 10 μg/mL of concentration. The cells also maintained the expression of CD31 marker and VCAM1 as shown by immunostaining.
The double positive cells were expended up to passage 5 which showed >95% cells were positive for endothelial markers, i.e. CD144+ and CD31+, yielded up to 15x106 cells. Double positive cells were frozen directly after sorting and were thawed after 1 month kept in liquid nitrogen. The expanded cells (passage 5) retained the double positive phenotype. Furthermore, expansion produced approximately 1x107 cells, which proved that the required cell number for the cellularisation of the AmbuLung device can be achieved. The expanded double positive endothelial cells (passage 1) retained their functional properties since they remained positive for all functional endothelial markers, with >90% of the cells expressing VCAM1, ICAM1, Tissue factor (CD142) and eNOS.
Results:
• We established an efficient protocol for the directed differentiation of endothelial cells form hESCs (hESC-ECs)
• The endothelial derived had the phenotypic, functional and metabolomics of established neonatal endothelial cells (HUVEC),
• CD31+ sorted hESC-ECs were obtained with high purity;
• The initial differentiation process seems to be independent of the ambient oxygen concentrations, while hypoxia impairs the expansion of hESC-ECs
• As a caveat when these cells were expanded they progressively lost their endothelial cell characteristics.
In future studies we will investigate the effects of the duration of hESC-EC differentiation and analyse an earlier differentiating cell populations of CD31+CD144+ cells at earlier time points.
4.) Production of alveolar epithelial cells
We initiated the work on the directed differentiation of pluripotent stem cells (hIPSCs) towards the epithelial cell lineage. The first task was to develop an efficient differentiation protocol. To achieve this, we evaluated the role of several established growth/differentiation factors, alone and in various combinations, such as Activin A, Wtn3a, BMP4, and compared them with the efficacy of A549 conditioned medium to induce AT2 cell differentiation. The efficiency of differentiation was evaluated by analyzing the expression of specific marker genes (at day 6, which was normalized with day 0). Down-regulation of the pluripotency / stemness markers OCT3/4 and Sox2 was observed in the A549 + Activin A + Wnt3a + BMP4 group (AAWB). Furthermore, there was concomitant up-regulation of the epithelial differentiation genes (SOX17 and FOXA2).
To further enhance alveolar epithelial differentiation and to move away from the use of a cancer-line (A549) derived conditioned medium for clinical applications, we proceeded to develop a new (Imperial College) medium, based in parts on a 1995 publication by McCormick et al. entitled “Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells”. Based on our preliminary results we conclude that the Imperial medium (IC) in most of its variants significantly down-regulated the pluripotency genes by day 6 as compared to the A549 conditioned medium. Furthermore, at day 6 of differentiation marker genes for definitive endoderm induction were upregulated by some of the IC formulations in comparison to the level of induction by A549 conditioned medium.
Results:
• Supplementation of a new differentiation medium (IC) with Activin A, BMP4 and Wnt3a results in a significant down regulation of some pluripotency marker gene expression in particulary SOX2, helping the transition from the pluripotency state.
• Different combination of Activin A, BMP4 and Wnt3a produce an increased expression of endodermal linage markers.
• Use of different combination of Activin A, BMP4 and Wnt3a enhances the differentiating properties of A549 condition media.
• Interferon alfa, interleukin 6, and insulin antagonize the expression of pluripotency genes and produce a synergism in the endodermal formation.
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Basic research and scientific activities on functionalization and cell seeding of gas-exchange surfaces:
1.) Optimisation of Cell / Material interactions – in vitro testing -
In the first step we investigated potential surface coatings based on considerations that
a) The coating procedure for the AmbuLung had to be compatible with the established workflow at Novalung GmbH with view to the planned AmbuLung preparation for in vivo investigations.
b) The potential risk for failure should be minimized.
Therefore, our first priority was to utilize the approved ILA® multilayer coatings as basis for the coupling of cell adhesion peptides.
Peptide conjugation was done by 1-Ethyl-3-(3-imethylaminopropyl) carbodiimide (EDC) mediated amide-formation of peptide-bound amino-functions with the carboxy-functions present within the ILA® albumin heparin multilayer coating.
Secondly, benzophenone-modified heparin chemically coupled to the “bare” PMP surfaces and cell adhesion peptides were coupled to the modified heparin triggered by UV irradiation of benzophenone-functions. For this, we selected two peptide sequences found in fibronectin. REDV found in the IIICS region of plasma fibronectin is reportedly specifically recognized by endothelial cells. EILDVPST or “CS1-fragment” of fibronectin is reportedly selective for endothelial cells and is not recognised by thrombocytes. All peptide sequences used had glycine-lysine (GK) extensions in order to provide an additional amino group and an additional aliphatic side chain for EDC mediated coupling to [alb/hep]3, and for reaction with benzophenone-residues, respectively.
In addition to X-ray photoelectron spectroscopy (XPS) analysis of surface coatings, the presence of heparin within [alb/hep]3 multilayers as well as benzophenone-modified heparin was visualized using critical electrolyte staining with alcian blue. Peptide coupling was visualized using fluorescently labelled peptides.
Our data indicate that both, [alb/hep]3 multilayer coatings, which attach to the PMP surfaces via physical adsorption, and [alb/hep]3 coatings attached via benzophenone-heparin, which chemically reacts with the PMP surfaces upon UV irradiation, covered the entire fibre surfaces. Moreover, all layers were stable during flushing with PBS buffer in bioreactors at 11.5 dyn cm-2 shear stress. Microscopic analysis of the coatings revealed that the benzophenone-modified heparin coatings exhibited some degree of inhomogeneity due to molecular aggregations.
Very good hemocompatibility was confirmed for [alb/hep]3 based coatings in terms of white and red blood cell counts, hemolysis, platelet counts, platelet activation, activation of coagulation cascade and thrombogenicity using a Chandler loop model. All parameters were in the range of the negative controls, except for a moderate increase in fibrin formation and blood cell clotting on the functionalized fibre surfaces. Peptide coupling did not affect any of these parameters significantly and no differences were observed between surfaces with the REDVGK sequence or the EILDVPST sequence. For surface coatings based on benzophenone-modified heparin we found an increased potential for platelet activation and coagulation in equivalent to that seen on uncoated PMP mats, while in terms of the other parameters the performance was equal to the [alb/hep]3 based coatings. Again, no significant differences were observed between the peptides and whether the peptides had been immobilized to the heparin coatings or not.
An AmbuLung prototyp was coated with [alb/hep]3 using the automated coating procedure at NovaLung GmbH. Subsequently, a solution of 5(6)-carboxyfluorescein-labeled REDVGK peptide was flushed through the device, followed by flushing with PBS buffer. The analysis of random hollow fiber surfaces revealed essentially homogeneous coating with the fluorescently labeled peptide.
Gamma sterilization of PMP hollow fibre mats coated with benzophenone-modified heparin and RGD peptides did not result in changes in surface composition according to XPS analysis. Subsequently, the effect of sterilization of biofunctionalized membranes on cell adhesion and proliferation was also investigated. Membranes coated with benzophenone-modified heparin alone and in combination with RGD peptides were immersed in 70% ethanol or gamma sterilized and then seeded with endothelial cells. Cell proliferation was impaired on fibres immersed in alcohol when compared with gamma-irradiation treated fibres, presumably because of residual alcohol within the hollow fibres. Gamma-sterilized surfaces were well/uniformly seeded with endothelial monolayers.
At this stage we did not explicitly test ethylene oxide (EtO) sterilization for peptide functionalized surfaces. This method is approved for the sterilization of albumin-heparin coated surfaces. Yet, in view of the unexpected high thrombogenicity of the peptide-coated surfaces after EtO treatment the effect of EtO on the interaction of the peptide coated surfaces with cells will have to be further investigated.
PMP surfaces coated with [alb/hep]3 or benzophenone-modified heparin, as well as both coatings additionally functionalized with the peptide sequences have been evaluated in terms of hemocompatibility under static and dynamic testing conditions. The inflammatory potential of peptide-functionalized surfaces ([based on alb/hep]3) was evaluated in static testing by ELISA based quantification of the PNM-elastase release in fresh samples of heparin blood from four healthy volunteers. The contact time with the samples was 1h contact with the samples. The inflammatory potential of all coated surfaces was in the range of the negative controls.
In preliminary tests the thrombogenicity of peptide-functionalized surfaces was evaluated in a static set-up by incubating the mats with samples of fresh heparinized whole blood. [Alb/hep]3 coated PMP surfaces and surfaces additionally functionalized with one of the peptide-sequences EDVGK, RGDGK, or EILDVPSTGK were incubated with blood for 1 h at 37°C, carefully rinsed with PBS- buffer, fixed (Histofix) and frozen at -20°C until analysed by SEM. SEM analysis of the surfaces displayed no or minimally increased fibrin and blood cell adhesion to the fibre surfaces on peptide-functionalized surfaces in comparison with [alb/hep]3 coated surfaces for two out of four donors. For two out of four donors blood cell adhesion was very little augmented on peptide-functionalized surfaces but no difference was observed between the peptide sequences used. Compared to collagen coated surfaces which served as a positive control, the thrombogenicity of all peptide-functionalized surfaces was on a low level.
Dynamic hemocompatibility testing was performed at the Clinical Research Laboratory, Department for Thoracigal and Cardiovascular Surgery at the University of Tübingen by using a Chandler loop model with fresh heparinized human whole blood. Four parameters were assessed in accordance with DIN ISO 10993-4: Thrombogenicity, coagulation, platelet activation and hemolysis using six different surface modifications: [alb/hep]3, [alb/hep]3 + REDVGK, [alb/hep]3 + EILDVPSTGK, benzophenone-modified heparin, benzophenonemodified heparin + REDVGK, benzophenone-modified heparin + EILDVPSTGK. These have been compared to uncoated PMP. Blood contacted only to the Chandler loop was used for the negative controls. Four samples of each surface were tested. The results from the dynamic in vitro tests indicate that [alb/hep]3, [alb/hep]3 + REDVGK, [alb/hep]3 + EILDVPSTGK each reflected a very good hemocompatibility, while surface coatings based on benzophenone-modified heparin showed no improvement in comparison with the uncoated surface. No abnormality was detected in terms of hematology (red and white blood cell counts, hemolysis) and platelet counts for any surface including negative and positive controls.
The sensitive biochemical marker analysis (ELISA) of ß-thromboglobulin (ß-TG) and thrombin-antithrombin-III complex (TAT) indicated significant improvement of the surface properties in terms of platelet activation and coagulation for all [alb/hep]3 based coatings which performed in the range of the negative controls. In contrast, with relation to non-coated PMP no improvement was detected on benzophenone-modified heparin based surface coatings. SEM images show that fibrin formation and blood cell aggregation was observed at some fibre surfaces, while others seemed to be coated by a proteinaceous layer. Within the tested timeframe of 1 h no three-dimensional thrombus formation occurred on any surface. While differences in the degree of clotting were obvious between the four donors, systematic influences of the fibre surfaces could not be deduced from SEM images.
Results:
• The ILA® multilayer coating ([alb/hep]3) was selected as base for the coupling of cell recognition peptides to PMP hollow fibres in order to enable endothelial cell adhesion, monolayer formation and stabilization of the endothelial layer under dynamic flow conditions.
• The coatings have been evaluated and performed very well in terms of shear stability (11.5 dyn cm-2), coupling of the peptides REDVGK and EILDVSTGK, gamma sterilization and hemocompatibility.
• Hemocompatibility of the coated surfaces was tested in terms of white and red blood cell counts, hemolysis, platelet counts, platelet activation, activation of the coagulation cascade and thrombogenicity applying a Chandler loop model:
• All parameters were in the range of the negative controls, except for moderate fibrin formation and blood cell clotting observed in SEM images for some of the surfaces. Peptide coupling to [alb/hep]3 coatings did not affect these parameters significantly. In particular, no differences were observed when using the REDVGK sequence or the EILDVPST sequence. Based on these data [alb/hep]3 coating functionalized with the REDVGK peptide was chosen for in vivo testing of the surface hemocompatibility.
• Successful functionalization of the AmbuLung prototype was demonstrated by the use of 5(6)-carboxyfluorescein-labelled REDVGK peptides. In vivo testing of [alb/hep]3 and REDVGK coated AmbuLung prototype resulted in premature clotting of the device and abortion of the test. The reason for the discrepancy between the in vitro hemocompatibility and the in vivo performance can neither be confirmed nor interpreted in the frame of the project at this stage. We hypothesize that one possible reason can be that ethylene-oxide (EtO) sterilization was used for sterilizing the AmbuLung device, whereas gamma-sterilization had been applied for the sterilization of all samples for in vitro testing.
• In addition to the [alb/hep]3 multilayer coatings, benzophenone-modified heparin was also evaluated as base layer for peptide coupling. The UV-triggered chemical coupling of the heparin derivative to PMP surfaces and peptide immobilization was successful. Yet, the surface coatings based on benzophenone-modified heparin showed no improvement in terms of hemocompatibility of the surfaces when compared with uncoated PMP. Therefore, benzophenone-mediated coupling may be relevant for chemical immobilization of coatings to the PMP polymer for subsequent multilayer assembly, but not for direct blood contact.
2.) Dynamic testing of cell seeded gas exchange units in vitro
The aim of this task was to test if it is possible to completely seed an AmbuLung gas exchanger with endothelial cells for the animal trials. In the first step we seeded individual membranes from PMP fibers with one million cells per mat in a cell culture dish and then integrated them in a dynamic bioreactor, which was developed for this purpose by Novalung GmbH and Fraunhofer IGB.
The AmbuLung gas exchanger, however, contains a stack of 66 mats. Thus, the seeding procedure had to be adjusted. Up scaling was achieved in two steps. Starting with a double membrane, a stack of eight mats was used in the bioreactor, followed by the use of the complete 66-fold stack in the gas exchanger.
Microvascular endothelial cells where isolated from human skin biopsies and expanded. For primary culture, the cells where seeded at a density of 5000 cells per cm²;in the following culture steps, a cell density of 3000 cells per cm² was used for seeding. Before reaching confluence, the cells where passaged up to the third passage at most. We performed each test with one separate batch of cells.
To prevent the membranes from floating on the media surface, the membranes were fixed to the bottom of Petri dishes with magnetic stainless steel rings as used in the bioreactors. Because of the hydrophobicity of the PMP fibres, the cells were seeded by directly placing 500 µL of endothelial cell suspension (conc. = 2 x 106 cells/mL) onto the membrane and letting the cells adhere overnight. The next day, the membranes were turned upside-down in a new dish and the seeding procedure was repeated. On the third day of the procedure, the seeded membranes were integrated in the bioreactors and the dynamic culture was started.
For seeding of stacks of more than two mats, the procedure was changed. The membrane stacks were inserted into the bioreactor chamber; the chamber was closed and filled with cell suspension (5 ml per reactor). After incubation overnight, the suspension was removed, the bioreactor was turned upside down and the procedure was repeated. The seeding results were evaluated by counting the cells that did not adhere to the fibres and were left in the seeding suspension. These results showed that 84 – 90 % of the cells used for the seeding attached to the fibres in the bioreactor and the viability of the residual non-adhered cells was about 60 – 70 %.
The cell seeding procedure for the mats in the bioreactor was also used for seeding the AmbuLung gas exchanger, which were coated with albumin/heparin. To account for the larger dimensions of the device in comparison to the bioreactor, we increased the cell number to 50 million endothelial cells per seeded device, re-suspended in a volume of 80 ml.
To analyse the efficacy of endothelial cell seeding on individual PMP membranes, 48 h after the initial seeding and before starting the dynamic culture, the cells were characterized with staining against PECAM-1, the assessment of acLDL-uptake and/or live/dead staining. On all modified coatings, fibres were almost completely covered with endothelial cells after 48 h. The cells showed typical endothelial morphology and expressed specific markers, such as PECAM-1. Additionally, the images also reveal the high purity of the seeded cells, since no contaminating cells (not expressing PECAM-1) were detected.
We performed the two stack dynamic culture of PMP membranes to establish the seeding procedure and the dynamic culture of the seeded fibre mats, and to narrow down or to identify the best membrane modification for endothelial cell attachment and culture. For the dynamic culture, two different initial shear stress levels (1.08 dyn/cm2 and 2.22 dyn/cm2) were applied to check the stability of the adherent cells under low-shear dynamic culture conditions. The preliminary experiments showed no significant differences in terms of seeding and attachment of endothelial cells on the differently coated membranes. The fibres were fully covered with cells for the most parts, showing the typical EC-specific morphology, as well as the expression of cell-cell contacts. Most of the cells took up acLDL, indicating the functionality as well as the EC-identity and purity of the cells used. Under dynamic conditions, the CS-1 coating appeared to result in a slight loss of attached cells after 7 days in areas facing the incoming flow-front in comparison to the other coatings. RGD coatings reportedly showed thrombogenic effects and in our experiments did not effectively improve cell adhesion in comparison to pure [alb/hep]3 or [alb/hep]3 + REDV coatings. On the basis of those findings, only REDV and pure albumin/heparin [alb/hep]3 coatings were selected for further studies and for up-scaling the culture conditions.
Results:
• With the established seeding and culture protocols, it is possible to obtain PMP fibres fully covered with ECs after an initial period of static culture for 48 h. The cells can be characterized as pure populations of ECs, as inferred from PECAM-1 and VE-Cadherin positive cell-cell contacts.
• For a dynamic culture of the seeded fibre mats in the bioreactor, it is beneficial to gradually increase the applied shear stress to allow cells to adapt to the altered conditions. That way, we were able to increase the shear stress up to 13.3 dyn/cm2 without causing any detectable cell loss or alteration in cell or monolayer properties.
• Like in other studies, good cell retention was found on surfaces covalently coated with heparin and albumin.
• To further improve cell adhesion, especially under dynamic conditions, the alb/hep surfaces were modified with various putative adhesion peptides.
• For our subsequent experiments we decided to narrow down the surface selection for a more detailed analysis to alb/hep and alb/hep + REDV, since REDV is expected to specifically enhance the adhesion of ECs.
• In a set of experiments with different culture conditions and various rates of shear stress, no improvement in cell adhesion was detected when coupling the additional REDV peptide to the surface. In comparison to other studies, which described profound cell loss after dynamic culture for 24 h under similar shear conditions, though, there was no excessive cell loss detectable in our system. Additionally, we were able to extend the culture period in the bioreactors up to 14 d without any sign of damage or loss of cells.
• In a last set of experiments, we were able to utilize and scale-up our dynamic seeding and culture protocol for use with the prototype of the AmbuLung gasexchanger, containing 66 PMP fibre mats. In contrast to the configuration with a stack of 8 mats we found distinct regional differences in the AmbuLung device, especially in terms of cell morphology and pattern of marker expression, as assessed form the loss of cell-cell contacts and an upregulation of EC activation markers. At this stage, we hypothesized that the difference in the properties of the seeded cells are mainly reflecting unique, distinct flow conditions in the AmbuLung prototype, which might be quite different from the conditions in the bioreactor due to the altered geometry.
3.) Computational modelling of fluid dynamics and cell-biomaterials interactions
Focusing mainly on developing mathematical models to predict the performance of the AmbuLung prototypes, this task is at the interface between research of the surface functionalisation/ cell seeding and the technical development of the gasexchanger, and the results were important for both partners. Detailed computational fluid dynamics (CFD) simulations of the blood flow through the membrane oxygenator were carried out in order to determine whether the amount of shear stress acting on the fibres is within normal physiological levels. The CFD simulation models served as the basis for the bioreactor cell seeding experiments. These models allow us to predict the density of adherent endothelial cells on the PMP substrate over time as a function of the dynamic culture conditions. The proposed model entails parameters such as the fluid-flow rate, volumetric cell density, and cell-cell and cell membrane adhesion coefficients. The models are beneficial for the optimisation of key design criteria of the AmbuLung, such as minimising the time for cell seeding, maximising the adherent cell density and minimising the extent of cell loss from the hollow fibres. More generally, the work represents a significant advance beyond current models describing the dynamics of cell seeding of biomaterials. These models helped us to predict and optimize the functionality and durability of a cell seeded gas exchanger, which is at the core of the AmbuLung concept.
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Engineering research and development
1.) Engineering - Gas Exchange Membrane + vascular access design
In a first step, we determined theoretically and validated experimentally the optimum fibre arrangement, which would maximize the performance of the minimized gas exchanger in terms of efficient gas exchange and enhanced hemocompatibility. For the verification of the selected fibre arrangement, we carried out in vitro tests in the Novalung wet lab. As performance factors we primarily investigated a) the capability to eliminate CO2, b) the pressure drop and c) the priming volume. In addition to the first CO2 exchange performance tests, which we intended for validating the performance of the prototypes as compared to the mathematical model and CFD, we carried out extensive performance tests for the detailed characterization of the gas exchanger.
Results:
• The measured CO2 values at the outlet of the gas exchanger confirmed the expectation based on the mathematical model that a nearly identical CO2 gas exchange can be achieved compared with the iLA gas exchanger, despite the more than 50% smaller gas exchange surface area of 0.6 m2 of the AmbuLung versus 1.3 m2 of the iLA exchanger
• Especially within the low-flow range (0.5-1.2 l/min) and therefore within the target range of the AmbuLung the performance of the newly designed AmbuLung gas exchanger is excellent.
• These in vitro results exceeded the calculated and simulated theoretical performance for this fibre configuration.
The original computational model for the gas exchanger predicted a pressure drop of about 20 mmHg at 1.5 l/min. In fact, we observed a pressure drop between 25-35 mmHg in vitro, therefore, an adaptation of the model was required to describe the overall behaviour of the prototypes. The revised model is based on a pressure drop of 30 mmHg at 1.5 l/min. In the subsequent animal studies, the average pressure drop at 1.2 l/min blood flow was approximately 20 mmHg. This fits well with the corrected model. According to this revised model, the maximum shear stress is at about 30 dyne/cm2, which is in the physiological range and well below the critical level of 70 dyne/cm2 that reportedly damages the integrity of endothelial monolayers.
A major factor for a homogeneous distribution of blood flow through the AmbuLung gas exchanger was the design of a cylindrical cavity for the miniaturized device, versus the rectangular cavity of the iLA device. Flow experiments with this new cavity design showed two circulating areas of recirculating eddies due to the flat, lateral inflow into the cylindrical cavity. However, these areas of recirculation were deemed safe/uncritical, since the flow velocity and thus the shear rate in these areas was assumed to remain below a critical level.
The analysis showed that at the minimum flow rate of 0.5 l/min the flow velocity exceeded 2 mm/s in certain areas of the cavity. The resulting blood compatibility, i.e. the risk of hemolysis, was assessed by evaluating the combination of shear rate and residence times. With respect to hemolysis and platelet activation, no potential “threat” was identified. Nevertheless, low-shear-stress areas of recirculation may represent a location for the deposition of already activated/aggregated blood constituents, such as platelets, even if the gas exchanger per se may not be the cause for their activation. Thus, there is room for improvement in the design of the gas exchanger geometry. However, as vortex formation cannot be eliminated completely even after opting for a lateral inflow, we decided to accept this potential risk in favor of increased patient mobility.
Minimal structural space and a low priming volume are of central importance for the AmbuLung system. For this reason we decided to use ¼ " tubings for the AmbuLung tubing set. We developed a pump head with ¼ " connectors and cannulas with corresponding dimensions. We minimized the complexity of the AmbuLung tubing set, since more complex units generally require more space. The set includes an inline pressure sensor, the blood pump head and the gas exchanger, as well as three connecting ¼ " tubing. This tubing set is by far smaller, less complex and much easier to handle as tubing sets, used on current systems.
The total length of the tubing is about 780 mm. and the priming volume, hence the total extracorporal blood volume is less than 250 ml. The gas and blood ports of the gas exchanger are guided in parallel and oriented in the same direction in order to allow a more compact tubing arrangement. The use of an inline pressure sensor provides a luer-free tubing set. By completly abandoning luer connectors, the risk of air embolism decreases significantly. For this reason, we also omitted a de-airing membrane in the gas exchanger, which in turn positively influenced the flow through the gas exchanger, because the integration of a de-airing membrane would have meant integrating a discontinuity. During operation of a mobile device, such an external de-airing membrane will only be of limited use, because the spatial position of the gas exchanger varies in a mobile application. Instead, we integrated the de-airing membrane into the priming set.
The setup of the mobile unit, i.e. the carrying system, is inextricably linked with the cannulation of the patient and the design of the tubing set. The mobile unit consists of three main components: Disposable-Case, Power&Control-Unit and the load-carrying elements (waistbelt + shirt). The Disposable-Case consists of two textile-covered shells made of expanded polypropylene (EPP) foam, into which the essential components for therapy are inserted.
We designed the Disposable-Case in a way that the tubing set can be inserted without moving previously inserted components. The dimensions of the case are approx. 220 mm x 310 mm x 105 mm.
The EPP foam acts as a thermal and acoustic insulator and allows a high mechanical protection through its energy absorbing properties. From a thermal perspective, EPP has proven to be an ideal material. The waste heat from the various components is partially stored in the case. As a result, the internal temperature adjusts itself to a constant level that is virtually independent of the outside temperature. This reduces considerably the risk of hypothermia to the patient and avoids the occurrence of condensation in the gas circuit, which could damage the vacuum pump. Specifically placed vents further reduce any remaining heat in the case.
2.) Engineering – Hardware and peripherals -
In a first step, we defined the overall concept of the AmbuLung hardware system. We defined the basic requirements in terms of performance, usability, and safety for users and patients, with special focus on compliance with the rules and regulations for the development of class 2b medical devices. We implemented the basic considerations and concepts for the design of the hardware components and developed and manufactured a small series of prototypes that were extensively tested in the laboratory and in an animal studies. The special challenge was to reduce the components to minimum weight and size without compromising their performance. For this purpose we completely redevelop major components such as the pump drive; other purchased parts had to be modify to meet our specifications.
In developing the AmbuLung hardware we decided to use as many components as possible of the iLA activve®. The iLA activve® is Novalung’s lung support system, which is CE-marked and has been released in 2011. This device consists of a bedside console and a disposable set and enables extracorporeal lung support in a veno-venous mode for awake, but immobile patients in the ICU.
Due to the complexity of the task to radically reduce size and weight of the system while concomittantly guaranteeing the specified system performance, we could use only a few components of the iLA activve® without modifications. Specifcially we used existing iLA activve® components and algorithms only for the development of the central unit and the blood pump control. All other components and control systems were developed from scratch.
Results:
As part of the AmbuLung project, we developed following hardware components:
• The Central Unit has been development based on the current iLA activve console using state of the art processors and operating system, touch screen, modern small size batteries and a user interface design, that meets the latest requirements for user-friendliness and ergonomics.
• The mobile Power & Control Unit has been developed from scratch and contains all electronic components necessary for the self-sufficient and safe long-term operation of the AmbuLung system. This unit is worn on the patient’s body.
• The blood pump drive is conceptually based on the Novalung DP3® pump, which drives i.a. the iLA activve® system, but is radically reduced in weight by 65% and its size by 50%.
• We developed an entirely new sweep-gas supply system, which is unique and has never been used in comparable systems so far. It works autonomously without external gas supplies. It is optimized for small size, weight and noise levels and is also worn on the patient's body.
• For sensors we used standard products, which we modified for use in a mobile miniaturized unit, worn directly on the patient’s body.
• All hardware components, which are connected to blood- or gas-tubes, have been integrated together with the disposable components in the disposable case. This case is lockable and protects sensitive components against mechanical stress, moisture and dirt. The disposable case contains the central components of the AmbuLung system and is worn on the patient's body.
• All hardware components have been extensively and successfully tested on reliability, performance and compliance with their specifications.
• We reached one of our main objectives and were able to develop a system that weighs less than 3 kg and can be worn by the patient in any body position without compromising comfort.
Application testing, verification, validation
1.) Preclinical in vivo study
All disposable and hardware components were validated and verified in vitro in the laboratory.
In addition, we tested their safety and functionality in a preclinical animal study in vivo, using the anesthetized and ventilated pig as animal model. We analyzed critical physiological and hematological parameters required to evaluate the performance and efficacy of the AmbuLung device under physiological conditions. In this study we used the iLA-activve® system as a benchmark and as a comparative system to the AmbuLung device. The overall goal of the study was to assess the decarboxylation efficacy of the AmbuLung prototype compared with the current iLA-activve® system over a period of 72 hours in a large animal model.
Six (6) animals were treated for 72 hours in a randomized fashion with the established iLA-activve® system and nine (9) animals with the AmbuLung® prototype.
Results:
• All except of one animal survived until the end of the study. One pig developed septic complications and had to be euthanized after 56 hours.
• For both lung assist devices, we observed no technical problems over the observation period of 72 hours.
• The AmbuLung prototype showed a comparable efficiency in CO2 elimination as the established iLA-activve® system both based on the arterial blood gases and the gas concentrations in the sweep gas under constant conditions with a blood flow of 1.2 L/min and an sweep gas flow of 8 L/min.
• The average PaCO2 value was around 40 mmHg throughout the 72h-observation time with no marked differences between both devices.
• The average drainage pressure was approximately -30 mmHg and thus within a safe range. The CO2 elimination performance of the gas exchangers was in the order of magnitude of the in vitro results. A direct comparison with the measured values obtained in vitro, however, was not feasible, as expected, because the base line blood gas values before testing the gas exchangers varied due to the different vital condition of the individual animals.
• Overall, the CO2 diffusion rates of the AmbuLung gas exchanger and the benchmark device were rather similar. The maximum rates were in the range of about 130-140 ml/l (Bloodflow: 1.2 l/min; Gasflow: 8 l/min), which led to a reduction of the CO2 partial pressure by 30-40 mmHg, depending on the input value. Such a reduction of the partial pressure level provides a significant improvement in the perceived status of the patient
• The reduction in gas exchange performance of the AmbuLung gas exchanger at reduced gas flow rates was generally lower than that of the benchmark device. However, in controlled laboratory tests, this effect was less pronounced.
• The gas exchange performance of the prototypes was largely constant during the in vivo tests that lasted for up to 72 hours. The pressure gradient across the membrane was between 20-25 mmHg and increased only marginally over the entire test period.
• With a sufficient anticoagulation in place, no clotting was observed in both extracorporeal gasexchangers.
• There were no signs of hemodynamic instability, acid-base disorders, thromboembolic events or hemolysis at both extracorporeal gasexchangers.
• In terms of inflammatory response, there were no differences between both study groups. However, towards the end of the observation time, modest increases of all cytokines were recorded in both systems.
• The number of CAE-positive cells in lung tissue showed no difference between animals which have been treated with either AmbuLung or iLA-activve®.
In summary, we could demonstrate:
• The AmbuLung system operates reliably and without any technical problems over at least 72 hours.
• The gas exchange performance of the AmbuLung system is at least as good as the one of the iLA activve, which serves as a benchmark, although the gas-exchange surface of the AmbuLung gasexchanger is less than 50% of the ILA one.
• The use of the AmbuLung system showed no adverse effects in terms of biocompatibility, hemocompatibility, hemodynamic, acid-base disorders, thromboembolic events, hemolysis or inflammatory response.
2.) Clinical Implantation: First-in-man Study
After the successful implementation of the animal study, it was the final objective of the AmbuLung project to test the clinical feasability, wearability and safety of the newly designed device in a first-in-man trial. Within the 36 months tenure of this project the consortium did not plan to develop the AmbuLung device to the stage of serial production. Specifically, we did not expect that the device would require/receive CE approval prior to the first-in-man trial. Hence, approval for the trial on humans was requested and obtained from the Institutional Review Board (IRB) of the performing Hospital. After ethical approval, it was planned to carry out a small-scale first-in-man clinical trial as a prelude to obtaining a Clinical Trial Authorization and as proof-of-concept for the wearability, safety, durability, and efficacy of the new medical device.
Results:
• We set up and trained a team of doctors and medical staff from the intensive care and respiratory subject areas.
• The team has gained extensive experience in treating COPD patients by extracorporeal CO2 removal using the Novalung iLA® activve device.
• We received approval for a specific clinical protocol for the implementation of a first-in-man trial by the local ethic committee of the Careggi Hospital in Florence.
• This locally approved request was submitted to the Italian Ministry of Health for final approval.
• We created all documents for the final approval by the Ministry of Health, for which the manufacturer was able to provide the necessary data.
• At this stage, the Ministry decided to withhold final approval until such a time that the Ambulung Device will be fully validated by the manufacturer according to the rules and regulations for the development of class 2b medical devices.
• This unforeseen demand for complete device validation prior to a first-in-man trial reversed the original plan and timeline of action (i.e. first-in man-trials followed by complete device validation), as originally approved by the EU commission.
• The demand by the Italian Ministry of Health now necessitates the delay of the first-in-man trials until the end of the current EU funding.
• The team and the infrastructure at the hospital is ready to start the use of AmbuLung on humans immediately after receipt of approval, which is tentativly scheduled for Q4/2015.
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Potential Impact:
The product generation emerged from the AmbuLung project represents an innovative and first-of-a-kind family of fully wearable bioartificial lungs for long-term use in an outpatient setting. Once CE-marked, which is scheduled for Q3/2016, these systems will be commercialized by the established Marketing & Sales organisation of Novalung. These medical devices are unique because of their optimized gas exchange membrane /blood interface for long-term use, which is characterized by a high degree of hemocompatibility.
Our new bioartificial lungs will create pulmonary volume unloading, avoid dyspnea, prevent invasive mechanical ventilation and improve overall quality of patient life. Compared with current non-invasive and invasive mechanical ventilation, the newly developed bioartificial lungs create a novel treatment modality and option for patients suffering from advanced COPD by freeing up the diseased lung from the need to perform. We fully expect that the results of the upcoming clinical studies in the next project phase will show that the extracorporeal volume unloading enabled by the bioartificial lung will positively affect the hitherto unstoppable progressive course of COPD. The outcomes of the AmbuLung project is groundbreaking and paradigm shifting, resulting in the first set of wearable bioartificial lungs with miniaturized support technology. These bioartificial lungs represent the world's first extracorporeal lung support systems that are fully mobile and are worn by the patient on the body during treatment. The outcomes of the AmbuLung project pave the way for use of bioartificial lungs outside of the hospital environment in analogy with current artificial heart protocols.
The development of commercial miniaturized artificial lung systems also catalyzes a clinical paradigm shift in the approach to long-term respiratory support. We anticipate that our technology will shift the treatment environment for lung failure towards avoiding dyspnea and exertion, improved patient mobility and quality of life. By providing functional volume unloading the bioarticial lung may improve the dismal course of COPD. For those patients currently undergoing invasive mechanical ventilation in acute exacerbations of COPD (AECOPD) the use of a bioartificial lung will avoid sedation and immobility thus creating self-managed, active patients. It will lead to a more patient driven treatment scenario for cases of advanced COPD. A wearable bioartifical lung can also be excellently used to bridge the waiting time for a donor organ (bridge-to-transplant). It may even avoid the need of lung transplantations, as was already observed in the use of ventricular assist devices (VAD) in heart transplantations. The methodology developed here for a bioartificial lung will also be applicable for and influence other bioartificial devices (liver, kidney, etc.), where biolization/cell coverage might improve both durability and function. The AmbuLung project will lead to a CE marked, first generation, wearable bioartificial lung systems for clinical use in less than one year. Once available commercially, the medical device will positively influence patient’s quality of life, reduce health care costs and overall expense to society by decreasing hospital stay length, hospitalization rate and palliative costs.
The AmbuLung project already created new positions at Novalung and the hiring of five people in R&D and Marketing, which continue to work fulltime on the project. Until CE marked in one year, the project will create up to approximatly 15 further new positions for Novalung in Manufacturing, Marketing, Sales and Clinical Support.
We were able to initiate five theses by the AmbuLung project with topics in the field of stem cell research (1), tissue engineering (1) and human medicine (3). These theses are completed and published shortly. After completion of their thesis, some of these PhD students will have the opportunity of a permanent employment at the respective institution to deepen their expertise obtained within the AmbuLung project. We expect that, initiated by the AmbuLung project, follow-up projects will be generated, which will create additional positions for young scientists, bioengineers and MDs at the scientific and medical institutes involved in the AmbuLung project.
Dissemination activities
As a Consortium we focused on the implementation of an iterative dissemination plan, which was continously discussed and agreed by all partners. This plan includes several actions to enhance the overall visibility of the main results of the project to target groups and relevant stakeholders. This group includes potential users and patients, the scientific community, policy makers as well as health authorities. The goals of dissemination extend beyond the EU funding phase of the project and will leverage future commercialization concepts.
Our dissemination plan the following activities:
• Creation of a product website based on the project homepage
• Participation on congresses, scientific and medical meetings
• Scientific publications and presentations
• Organisation of workshops with stakeholders and the medical community
• Initiation of press releases
• Production of marketing material like videos, leaflets, brochures, training & education material
• Application for patents
So far, we have carried out the following dissemination activities:
• We attended 15 congresses and meetings mainly in Europe and the US, covering the topics of stem cell research, biomaterials, biomedical engineering and medicine. In these meetings we were represented either with a lecture, of attending a workshop or as an exhibitor.
• On 10 meetings, members of the consortium presented their specific topics by invited talks (9) and posters (7).
• We generated five publications so far. One is published, two are submitted but not yet reviewed and two are in preparation.
• There were a couple of press releases, of which six very exclusively devoted to the AmbuLung topic.
• We organized a workshop with expert in the field of intersive care medicine, pulmology and biomedical engineering on an international level and gained import input for the further cause of the development process.
• We already applied for three international patents, which are at the stage of the approval process and have not yet been officially granted.
With all due caution with regard to confidentiality and secrecy of the key project results we have already achieved great interest in the professional public with our previous dissemination activities.
The market is waiting for the outcomes of the AmbuLung project.
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Exploitation
As main outcome of the project, we will further verify and validate the product, resulting from the project results, with emphasis in the coming months with the aim to obtain CE-Mark approval in Q3/2016 und in order to start market release and commercialisation.
As further exploitable foreground, we plan to commercialize the dynamic bioreactor, which we developed within the task of the cell seeding on the gasexchage membranes. Because there is not yet such a specialized laboratory instrument on the market, we expect a commercial success in marketing this bioreactor.
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List of Websites:
Public Website Address: www.ambulung.com
Contact details for the design and the regular update of the website:
PD Dr. med. Georg Matheis, General Manager, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Georg.Matheis@xenios-ag.com
Dr. Esther Novosel, Manager Business Development, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Esther.Novosel@xenios-ag.com
Dr. Timo Hammer, Productmanager, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Timo.Hammer@xenios-ag.com
Executive summary
The idea to initiate the Ambulung project was based on the unmet medical needs to adequately treat patients, who suffer from severe COPD, a chronic pulmonary disease, which is the 4th leading cause of death worldwide. Following this idea, we formed a team of clinicians, scientists, engineers and entrepreneurs from four different institutions, who are convinced that the combined effort of this European team will create a door opening pioneer product that will trigger consecutive research and development efforts to relieve suffering from end stage lung failure. Consequently, we defined it as our common goal to create the world’s first wearable bioartificial lung for longterm application on COPD patients in an outpatient setting.The main objectives of this three years project were to develop a miniaturized and wearable extracorporeal lung support system, to cellularize the diffusion membrane with endothelial cells and to evaluate the developed system in pre-clinical and clinical studies.
After completion of the three year project period we can summarize:
• We have been successful to develop appropriate procedures and processes for the differentiation of endothelial cells from Human Empryonic Stem Cells.
• We were able to expand these endothelial cells by suitable, partly newly developed methods, up to a number that is necessary for complete cellularization the diffusion membrane.
• The gas-exchange surface of the diffusion membrane was functionalized with various biochemical and physical methods in order to create an ideal base for cellularization.
• We have successfully seeded these surfaces with endothelial cells and have tested the stability of the cell monolayers unter static and dynamic conditions using a newly developed dynamic bioreactor. As result of these seeding tests, we have defined the optimal surface for a durable and stable cell coating.
• We have developed a gas exchanger with a diffusion membrane, which is suitable for the cellularization with endothelial cells. The gas exchanger is designed small and lightweight while maintaining the required performance parameters. The innovative shape of the gas exchanger with circular cavity and tangential flow onto the membrane allows the required wearabilty.
• The miniaturized hardware was developed from scratch with state-of-the-art components, which weigh less than 3 kg including the disposable components, so that we have surpassed one of our main objectives. The entire system is designed for autonomous and mobile use and for attachment to the patient body.
• We have manufactured prototypes of the entire system that have been successfully tested in the laboratory and in the course of an animal study.
• The clinical protocol for the intended first-in-man was approved by the competent ethics committee, but the final approval by the Ministry of Health has not yet been issued.
• We have started our dissemination activities at an early stage of the project and have already gained great interest of potential users worlwide.
• In summary, all our objectives except the implementation of the first-in-man trial have been achieved within the given timeframe.
This project is not finished after the three-year term. The AmbuLung team has mutually committed to stay together as a product group even after the end of this project phase. We will continue to work intensively on the further development, verification and validation of this product family with the aim to obtain the CE certification in the next year and to start commercializing afterwards.
Project Context and Objectives:
Project context and main objectives
Chronic obstructive pulmonary disease (COPD) is actually the 4th leading cause of death worldwide and is going to become the 3rd. Acute exacerbations of COPD dramatically change the course of the disease, since they are associated with a rapid decline in lung function and worsening quality of life. AECOPD accounts for 750,000 hospitalizations, for 120,000 deaths and 1.5 million emergency department visits annually in the US alone. Patients with AECOPD and hypercapnia have mortality rates of 33% at six months and 49% at two years. In patients suffering from severe acute exacerbations of COPD, when drug treatment is not sufficient, non- invasive ventilation has been established as the treatment of choice. However, when NIV fails, which occurs in about 10-15% of patients treated with NIV, invasive mechanical ventilation will be employed, resulting in high mortality and lasting decline in lung function.
As opposed to the treatment of end stage heart failure with ventricular assist devices (VAD), long term respiratory support with any kind of mechanical ventilation currently relies on the gas exchange function of the diseased lung, rather than providing gas exchange independent of the human lung. Long-term use of invasive mechanical ventilation has drawbacks, such as limited mobility, airway infection (VAP), ventilator associated lung injury (VILI, VALI), and ventilator induced diaphragmatic dysfunction (VIDD), tracheotomy and others that affect both outcomes and quality of life in advanced respiratory failure.
More importantly, an extracorporeal artificial lung will reduce over distension of the diseased lung. This “functional volume unloading” performed with an extracorporeal artificial lung might even affect the dismal course of the disease.
Therefore, our concept relies on a wearable, ambulatory, least invasive, long-term bioartificial lung that “breathes” outside of the human lung. This will unload the lung i.e. remove over distension, support mobile, spontaneously breathing patients in advanced chronic lung failure (subgroup of GOLD IV pts.) and bridge patients across severe exacerbations, when NIV fails. Our concept and development will allow avoiding invasive mechanical ventilation, which is associated with poor outcomes in this group of patients.
Long-term respiratory support increases the life expectancy and the quality of life of COPD patients and decreases the cost of care. Currently available artificial lungs, such as Novalung’s interventional Lung Assist (iLA) system, fail after about one month, mainly due to thrombus formation at the blood/machine interface. In addition, the size of current systems limits their use to the intensive care unit, restricts patient mobility and avoids outpatent use.
The AmbuLung consortium aims to create a wearable bioartificial lung (AmbuLung) for long-term application in an outpatient setting.
The main objectives are:
• The miniaturisation of the current available iLA activve system, including the supporting technology and patient monitoring system.
• The cellularisation of the diffusion membrane with endothelial cells to decrease thrombogenicity, to increase the gas-exchange rate and to increase operation time of the membrane ventilator.
• The evaluation of the developed system in pre-clinical and clinical studies.
For cellularisation, endothelial cells derived from FDA approved clinical grade human embryonic stem cells will be used. Cell differentiation, scale-up, seeding, and maintenance will be performed using established automatable and scalable dynamic bioreactor technology. A mathematical model will be developed to predict and refine the function of this complex system in vitro and in vivo. AmbuLung will be evaluated in a pig model, assessing functionality, and non-thrombogenicity. The data will provide information required for potential clinical transfer. If successful, a clinical trial will be carried out on five COPD patients after acquisition of the required regulatory approvals.
We will develop a prototype of a miniaturized disposable device that provides an optimal physiological environment to function effectively as a dynamic cell culture chamber and extracorporeal gas exchanger to be used as an ambulatory lung support. We will address the optimization of fibre configuration with respect to hollow fibre spacing, orientation and distance, taking into account the future task to surface-functionalise the hollow fibres with adhesion and crosslinking molecules and cellularise the outer, blood-contacting fibre surfaces with endothelial cells. We will modify the gas exchange surface and housing/inlet geometry of the current iLA gasexchanger to allow pulsatile mode and optimized device inflow characteristics. We will also carriy out initial conceptual studies to create an optimized vascular access and to optimize the tubing set. In addition, we will also focus on the “system concept” of the project, as evidenced by the emphasis put on the development of a mobile unit, i.e. the carrying system for the integrated pump/gas exchanger.
In the general context of the AmbuLung project, it is the goal to miniaturize the hardware of the artificial lung to a size and a weight, which allows the patient to wear the complete system on his body during operation. The mobile system should operate independently of a central processing unit and a main power supply. It will be designed to be powerful enough to control and operate the bioartificial lung, yet small enough to allow for wearability by COPD GOLD IV patients, which are generally unable to carry more than about 3 kg.
We will design and manufacture prototypes, which will be extensively tested and verified in vitro and in vivo by an animal trial and will be suitable for human use in a first-in-man study.
In order to cellularize the diffusion membrane with endothelial cells it is the overall objective to produce high quality human endothelial cells for preclinical testing and for clinical application of the AmbuLung device. For this purpose, we will analyze the whole range of molecules (from amino acids to nutrients/metabolites to growth factors) and identify the optimal operating conditions when correlated with cellular phenotype & functionality for pluripotent cell expansion and differentiation. This will lead to the optimization/standardisation of the bioprocess for the expansion & differentiation of human pluripotent stem cells using model-based design of experiments. In order to generate the endothelial (and epithelial) cells required for the cellularisation of the AmbuLung device, we will acquire, characterize and expand clinical grade hESCs prior to differentiation. By using the expanded hESCs, we will define a standardized differentiation protocol for the production of endothelial cells. This task will be successful if we can managed to validate the generation of endothelial cells. The protocol should provide a sufficient number of differentiated endothelial cells, which maintain their endothelial characteristics until their third passage, at least, and their number is expanded 5 times. This amount of cells will cover the requirements for the number of the cells for the AmbuLung device of ~3-5 x 108 cells. Finally, we will take undifferentiated human pluripotent stem cells, expand them and differentiate them into pulmonary epithelial cells in a defined medium under hypoxic condition and cultured on specific basement membrane molecules (collagen IV) using established protocols.
One of the main objective is to optimize functionalization and cell seeding of the gas exchange membranes using chemical and physicochemical approaches. We will develop and use dynamic bioreactors to bench test the shear-resistance of the endothelial cell-seeded AmbuLung gas exchange units under simulated physiological conditions of pulsatile flow. We will further define and refine computational models, analyzing and predicting fluid dynamics and cell-biomaterials interactions in the AmbuLung gas exchanger.
The outer surface of the poly(methylpenten) (PMP) hollow fibre membrane will be equipped with a surface coating that promotes adhesion and formation of a shear-resistant confluent endothelial cell layer. We will develop and evaluate a method to seed endothelial cells on the hollow-fibrous membranes in the AmbuLung gas exchanger for the use in animal experiments and for human use. For this pupose, we will design and manufacture a specific bioreactor system to assess seeding and “maturation” of the endothelial cell monolayers under physiologic, dynamic conditions. This dynamic bioreactor will be used to test optimum seeding and culture conditions by gradually adapting mechanical loading/shear stress, rather then suddenly exposing the system to detrimental mechanical forces,(e.g. high shear stress).
After the completion of the first functional samples of the AmbuLung device, we will test their safety and functionality in preclinical animal studies in vivo. For that, we will merge the specific ethical approval needs for animal trials and for a first human use and will develop a suitable study protocol for animal experiments. We will deploy the AmbuLung device in preclinical in vivo studies using the anesthetized and ventilated pig as animal model and will analyse critical physiological and hematological parameters required to evaluate the performance and efficacy of the AmbuLung device under physiological conditions.
The final objective of the AmbuLung project is to test the clinical feasability, wearability and safety of the newly designed device in a first-in-man trial. Within the 36 months tenure of this project the consortium does not plan to develop the AmbuLung device to the stage of serial production. Specifically, we do not expect that the device would require/receive CE approval prior to the first-in-man trial. Hence, approval for the trial on humans will be requested and obtained from the Institutional Review Board (IRB) of the performing Hospital. After ethical approval, we will carry out a small-scale first-in-man clinical trial as a prelude to obtaining a Clinical Trial Authorization and as proof-of-concept for the wearability, safety, durability, and efficacy of the new medical device.
It is in the interest of the entire project team to publish R&D results in an appropriate manner. Is not only just about publishing project outcomes but also about establishing a bi-directional communication process with the targeted audience, i.e. potential end-users (patients/physicians), key opinion leaders (KOLs), and policy makers.
Given the importance of targeted dissemination of the AmbuLung concept, we will pondering early on how to inform the public without jeopardizing the success of the project. We will bring into line the partially divergent interests of the scientific partners with those of the industrial partner. For that, we will coordinate all planned dissemination activities closely and implement only those that we agreed upon unanimously. The main objectives for exploitation and dissemination are:
• Create broad awareness of the project objectives, benefits and value among all the stakeholders
• Create the conditions for a sustainable exploitation of the AmbuLung results at European level
• Prepare AmbuLung exploitation during the post-project phase
• Publish peer-reviewed papers and communications at relevant conferences, meetings and congresses.
• Organize workshops with experts and stakeholders
• Maximise collaboration with the European community
• Transfer knowledge and dissemination through contacts with industry, health centres and academia
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Project Results:
General remarks
The tasks within the AmbuLung project were divided into 3 groups:
1.) Basic research and scientific activities
2.) Engineering research and development
3.) Application testing, verification, validation
Basic research and scientific activities were performed in the fields of research on stem cells and in particular in differentiation, expansion and production of endothelial cells from human embryonic stem cells. This work has been executed by the beneficiary Imperial College in London.
In the field of tissue engineering the benficiary Fraunhofer IGB in Stuttgart examined the functionalization of artificial gas exchange surfaces with biochemical and physical methods and developed procedures to seed such functionalized surfaces durable with endothelial cells.
It was the task of the industrial partner Novalung GmbH in Heilbronn to develop the medical device and produce first prototypes. This task was in turn divided into two areas: the development of the device disposables and the device hardware. Novalung was also the coordinator of the project.
The prototype of the newly developed medical device were investigated in vivo in animals, using a pig model. This task was under the supervision of NovaLung and was conducted by a renowned animal laboratory.
At the end of the project, it was planned to conduct a first use of the medical device on humans in form of a first-in-man trial. For this task, the beneficiary University Hospital in Florence (UNIFI) was responsible, prepared and available.
S & T results / Foregrounds
Basic research and scientific activities on cells:
1.) Information-driven Bioprocessing
We installed and commissioned the bioprocess monitoring and cell culture equipment. This equipment provided us with the capability to monitor the metabolic state of the cells (both undifferentiated and differentiated) and to link it with functional characterisation at the genetic and protein levels.
Furthermore, we installed the environmental culture chamber, which facilitates the high-specification and regulated production of the cells for the requirements of the project. The environmental chamber was used to evaluate the role of oxygen in the differentiation of hESCs towards the endothelial cell lineage.
Consequently, the technical means for the generation of high quality, regulated production of hESC-derived endothelial and epithelial cells was achieved. The “metabolic signature” of the cells was employed to characterise both undifferentiated hESCs and the differentiated cells.
We developed and experimentally validated a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.
This model-based framework has been experimentally validated for the expansion of ESCs.
Specifically, our system demonstrated how batch cultures were susceptible to stress from metabolite accumulation, which exceeded toxic levels leading to the loss of pluripotency over time. In contrast, perfusion cultures maintained relatively low levels of metabolic stress, which facilitates the maintenance of high pluripotency levels.
A method for the metabolomics analysis of the cell culture metabolic physiology was developed. The metabolomics analysis of stem cells and other cell cultures has been established and the results have been validated as accurate. Metabolomics data have helped on the understanding of the metabolic physiology of stem cell cultures towards differentiation.
Results
• The combined experimental-modeling platform for the bioprocessing of ESCs facilitates efficient in silico identification of optimal culture protocols that are implemented in a novel perfusion scalable bioreactor that provides a robust and controlled metabolic environment that is conducive to high cell growth and quality ESC bioprocessing.
2.) Expansion of undifferentiated stem cells
The expansion of hPSCs, both hESCs and hiPSCs, has been successful in a feeder-independent culture system and in sufficient numbers.
The expansion of the cells has been applied in 3D cultures and the results showed successful expansion. The pluripotency of the hPSCs was maintained at 70%.
To achieve the reproducible and controlled 3D cultures, single cell suspensions of hPSCs were obtained through the use of ROCK inhibition.
Results:
• Pluripotency levels dropped following treatment with ROCK inhibitor, especially following prolonged exposure.
• The effects of ROCK inhibitor use on the pluripotency and metabolic state of the hPSCs were studied. Results indicated specific switches on the metabolic physiology of the cells in both hESCs and hiPSCs cultures. Consequently, prolonged exposure (over 1 day) of hPSCs to ROCK inhibitor is not recommended.
• A publication has been submitted. (see dissemination activities)
3.) Production of endothelial cells
The objective was to have a standardized differentiation protocol for the production of endothelial cells by using the expanded hESCs. The task was successful, as we have managed to validate the generation of endothelial cells. The protocol provides a sufficient number of differentiated endothelial cells, which maintain their endothelial characteristics until their third passage, at least, and their number is possible to be expanded 5 times. This amount of cells covers the requirements for the number of the cells for the AmbuLung device of ~3-5 x 108 cells.
The protocol that was used was adapted from Orlova et al. Our results confirm that the protocol works properly in terms of endothelial differentiation. In this step we verified three important components of the methodology used:
a) An efficient differentiation protocol of hESCs towards an endothelial phenotype,
b) The ability to sort and purify the hESC derived endothelial- cells, and
c) The generation of sufficient cell number per 6 well plate (2 x 106 cells) that renders scale-up feasible for the production of the required 2-3 x 108 endothelial cells for cellularization of an AmbuLung device.
In order to optimise the differentiation protocol, we investigated the role of oxygen in endothelial cell differentiation. Specifically, the protocol was carried out at both ambient oxygen levels (21% oxygen, “normoxia”) and at reduced oxygen levels (5% oxygen hypoxia). The number of differentiated ECs on day 10 was essentially similar, suggesting that, at least with this protocol there is no effect of reduced oxygen on the directed differentiation of ECS from hESCs.
Aside from phenotypic characterisation, we also performed functional analysis of the produced endothelial cells. Populations of endothelial cells differentiated and purified were expanded until passage 5 (9-12 days). The cells were treated with the inflammatory cytokine TNF-α (0, 10, and 100 ng/mL) and checked for the expression of VCAM1 (CD106+) by flow cytometry. VCAM1 expression was obvious following stimulation with TNF-α. Similar levels of expression (approximately 67%) was observed at both 10 ng/mL and 100 ng/mL.
Cells were also checked for the cytokine-inducible expression of ICAM1 (CD54+). A fraction (33%) of the expanded endothelial cell population constitutively expressed low levels of ICAM1 even in the absence TNF-α (0 ng/mL). As with VCAM1 expression, treatment with different concentrations of TNF-α (10 and 100 ng/mL) yielded a significant increase in both the level of ICAM expression and in the fraction of the cells (approximately 98%), expressing CD54, although the increase of the concentration above 10u/mL did not changed the results.
Tissue Factor (CD142) expression was similar to ICAM1 expression. Specifically, approximately 1/3 of the untreated endothelial cells constitutively expressed TF. Upon treatment with different concentrations of TNF-α, the number of cells expressing TF increased significantly, albeit not the level of TF in each cell. Interestingly, eNOS expression was observed, which has not been reported before in the literature for hESC-derived ECs. Following treatment with TNF-α in an apparent TNF-α dose response 10 (70%) and 100 (92%) u/mL.
The homogeneity and functionality of the cultures was also confirmed by the uniform staining of the entire cell populations with an endothelial cell specific marker vWF and the histiotypic uptake of Ac-LDL.
There has also been applied a metabolomics analysis on hESCs, the human derived endothelial cells, both CD31+ and CD31+CD144+, we acquired at passage 1 and HUVEC line which could be comparable to endothelial cells. The metabolic profiles showed a bigger similarity of CD31+ endothelial cells with HUVECs rather than hESCs. CD31+CD144+ cells seem to have a more distinct metabolic profile compare to HUVECs. As expected, hESCs have the most distinct metabolic profile compared to the other cells. These results could be explained by the phenotypical heterogeneity of endothelial cells, which is high and dependent on their functional role and developmental stage. The CD31+ cell population may not be as homogeneous, which is also the case for the HUVECs, in contrast to the more homogeneous CD31+CD144+ cell population.
Results:
• An efficient differentiation protocol has been established
• The endothelial derived had the phenotypic, functional and metabolomics of endothelial cells
• CD31+ sorted were obtained with high purity; alas, when these cells were expanded they would progressively lose their endothelial cell characteristics.
It was postulated that we should investigate the effect of differentiation time and obtain an earlier differentiated cell population of CD31+CD144+ cells, which produced a homogeneous cell population that has the desired phenotypic and functional characteristics of endothelial cells and that can be expanded.
We were successful at acquiring CD31+CD144+ cells after 7 days of differentiation. The results showed a significant extent of differentiation (24%) at day 7 of double positive cells. These cells were then sorted and purified at greater than 95% to be used in further analysis.
The functional analysis of the double-sorted cells (CD31+CD144+) showed the expression of VCAM1, tissue factor and eNOS after the treatment of the cells with 100 ng/mL of TNF-α. The unstained cells did not express any TF. The treated cells showed a high level of functional expression. The differentiated endothelial cells maintained the positive expression for Ac-LDL uptake at 10 μg/mL of concentration. The cells also maintained the expression of CD31 marker and VCAM1 as shown by immunostaining.
The double positive cells were expended up to passage 5 which showed >95% cells were positive for endothelial markers, i.e. CD144+ and CD31+, yielded up to 15x106 cells. Double positive cells were frozen directly after sorting and were thawed after 1 month kept in liquid nitrogen. The expanded cells (passage 5) retained the double positive phenotype. Furthermore, expansion produced approximately 1x107 cells, which proved that the required cell number for the cellularisation of the AmbuLung device can be achieved. The expanded double positive endothelial cells (passage 1) retained their functional properties since they remained positive for all functional endothelial markers, with >90% of the cells expressing VCAM1, ICAM1, Tissue factor (CD142) and eNOS.
Results:
• We established an efficient protocol for the directed differentiation of endothelial cells form hESCs (hESC-ECs)
• The endothelial derived had the phenotypic, functional and metabolomics of established neonatal endothelial cells (HUVEC),
• CD31+ sorted hESC-ECs were obtained with high purity;
• The initial differentiation process seems to be independent of the ambient oxygen concentrations, while hypoxia impairs the expansion of hESC-ECs
• As a caveat when these cells were expanded they progressively lost their endothelial cell characteristics.
In future studies we will investigate the effects of the duration of hESC-EC differentiation and analyse an earlier differentiating cell populations of CD31+CD144+ cells at earlier time points.
4.) Production of alveolar epithelial cells
We initiated the work on the directed differentiation of pluripotent stem cells (hIPSCs) towards the epithelial cell lineage. The first task was to develop an efficient differentiation protocol. To achieve this, we evaluated the role of several established growth/differentiation factors, alone and in various combinations, such as Activin A, Wtn3a, BMP4, and compared them with the efficacy of A549 conditioned medium to induce AT2 cell differentiation. The efficiency of differentiation was evaluated by analyzing the expression of specific marker genes (at day 6, which was normalized with day 0). Down-regulation of the pluripotency / stemness markers OCT3/4 and Sox2 was observed in the A549 + Activin A + Wnt3a + BMP4 group (AAWB). Furthermore, there was concomitant up-regulation of the epithelial differentiation genes (SOX17 and FOXA2).
To further enhance alveolar epithelial differentiation and to move away from the use of a cancer-line (A549) derived conditioned medium for clinical applications, we proceeded to develop a new (Imperial College) medium, based in parts on a 1995 publication by McCormick et al. entitled “Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells”. Based on our preliminary results we conclude that the Imperial medium (IC) in most of its variants significantly down-regulated the pluripotency genes by day 6 as compared to the A549 conditioned medium. Furthermore, at day 6 of differentiation marker genes for definitive endoderm induction were upregulated by some of the IC formulations in comparison to the level of induction by A549 conditioned medium.
Results:
• Supplementation of a new differentiation medium (IC) with Activin A, BMP4 and Wnt3a results in a significant down regulation of some pluripotency marker gene expression in particulary SOX2, helping the transition from the pluripotency state.
• Different combination of Activin A, BMP4 and Wnt3a produce an increased expression of endodermal linage markers.
• Use of different combination of Activin A, BMP4 and Wnt3a enhances the differentiating properties of A549 condition media.
• Interferon alfa, interleukin 6, and insulin antagonize the expression of pluripotency genes and produce a synergism in the endodermal formation.
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Basic research and scientific activities on functionalization and cell seeding of gas-exchange surfaces:
1.) Optimisation of Cell / Material interactions – in vitro testing -
In the first step we investigated potential surface coatings based on considerations that
a) The coating procedure for the AmbuLung had to be compatible with the established workflow at Novalung GmbH with view to the planned AmbuLung preparation for in vivo investigations.
b) The potential risk for failure should be minimized.
Therefore, our first priority was to utilize the approved ILA® multilayer coatings as basis for the coupling of cell adhesion peptides.
Peptide conjugation was done by 1-Ethyl-3-(3-imethylaminopropyl) carbodiimide (EDC) mediated amide-formation of peptide-bound amino-functions with the carboxy-functions present within the ILA® albumin heparin multilayer coating.
Secondly, benzophenone-modified heparin chemically coupled to the “bare” PMP surfaces and cell adhesion peptides were coupled to the modified heparin triggered by UV irradiation of benzophenone-functions. For this, we selected two peptide sequences found in fibronectin. REDV found in the IIICS region of plasma fibronectin is reportedly specifically recognized by endothelial cells. EILDVPST or “CS1-fragment” of fibronectin is reportedly selective for endothelial cells and is not recognised by thrombocytes. All peptide sequences used had glycine-lysine (GK) extensions in order to provide an additional amino group and an additional aliphatic side chain for EDC mediated coupling to [alb/hep]3, and for reaction with benzophenone-residues, respectively.
In addition to X-ray photoelectron spectroscopy (XPS) analysis of surface coatings, the presence of heparin within [alb/hep]3 multilayers as well as benzophenone-modified heparin was visualized using critical electrolyte staining with alcian blue. Peptide coupling was visualized using fluorescently labelled peptides.
Our data indicate that both, [alb/hep]3 multilayer coatings, which attach to the PMP surfaces via physical adsorption, and [alb/hep]3 coatings attached via benzophenone-heparin, which chemically reacts with the PMP surfaces upon UV irradiation, covered the entire fibre surfaces. Moreover, all layers were stable during flushing with PBS buffer in bioreactors at 11.5 dyn cm-2 shear stress. Microscopic analysis of the coatings revealed that the benzophenone-modified heparin coatings exhibited some degree of inhomogeneity due to molecular aggregations.
Very good hemocompatibility was confirmed for [alb/hep]3 based coatings in terms of white and red blood cell counts, hemolysis, platelet counts, platelet activation, activation of coagulation cascade and thrombogenicity using a Chandler loop model. All parameters were in the range of the negative controls, except for a moderate increase in fibrin formation and blood cell clotting on the functionalized fibre surfaces. Peptide coupling did not affect any of these parameters significantly and no differences were observed between surfaces with the REDVGK sequence or the EILDVPST sequence. For surface coatings based on benzophenone-modified heparin we found an increased potential for platelet activation and coagulation in equivalent to that seen on uncoated PMP mats, while in terms of the other parameters the performance was equal to the [alb/hep]3 based coatings. Again, no significant differences were observed between the peptides and whether the peptides had been immobilized to the heparin coatings or not.
An AmbuLung prototyp was coated with [alb/hep]3 using the automated coating procedure at NovaLung GmbH. Subsequently, a solution of 5(6)-carboxyfluorescein-labeled REDVGK peptide was flushed through the device, followed by flushing with PBS buffer. The analysis of random hollow fiber surfaces revealed essentially homogeneous coating with the fluorescently labeled peptide.
Gamma sterilization of PMP hollow fibre mats coated with benzophenone-modified heparin and RGD peptides did not result in changes in surface composition according to XPS analysis. Subsequently, the effect of sterilization of biofunctionalized membranes on cell adhesion and proliferation was also investigated. Membranes coated with benzophenone-modified heparin alone and in combination with RGD peptides were immersed in 70% ethanol or gamma sterilized and then seeded with endothelial cells. Cell proliferation was impaired on fibres immersed in alcohol when compared with gamma-irradiation treated fibres, presumably because of residual alcohol within the hollow fibres. Gamma-sterilized surfaces were well/uniformly seeded with endothelial monolayers.
At this stage we did not explicitly test ethylene oxide (EtO) sterilization for peptide functionalized surfaces. This method is approved for the sterilization of albumin-heparin coated surfaces. Yet, in view of the unexpected high thrombogenicity of the peptide-coated surfaces after EtO treatment the effect of EtO on the interaction of the peptide coated surfaces with cells will have to be further investigated.
PMP surfaces coated with [alb/hep]3 or benzophenone-modified heparin, as well as both coatings additionally functionalized with the peptide sequences have been evaluated in terms of hemocompatibility under static and dynamic testing conditions. The inflammatory potential of peptide-functionalized surfaces ([based on alb/hep]3) was evaluated in static testing by ELISA based quantification of the PNM-elastase release in fresh samples of heparin blood from four healthy volunteers. The contact time with the samples was 1h contact with the samples. The inflammatory potential of all coated surfaces was in the range of the negative controls.
In preliminary tests the thrombogenicity of peptide-functionalized surfaces was evaluated in a static set-up by incubating the mats with samples of fresh heparinized whole blood. [Alb/hep]3 coated PMP surfaces and surfaces additionally functionalized with one of the peptide-sequences EDVGK, RGDGK, or EILDVPSTGK were incubated with blood for 1 h at 37°C, carefully rinsed with PBS- buffer, fixed (Histofix) and frozen at -20°C until analysed by SEM. SEM analysis of the surfaces displayed no or minimally increased fibrin and blood cell adhesion to the fibre surfaces on peptide-functionalized surfaces in comparison with [alb/hep]3 coated surfaces for two out of four donors. For two out of four donors blood cell adhesion was very little augmented on peptide-functionalized surfaces but no difference was observed between the peptide sequences used. Compared to collagen coated surfaces which served as a positive control, the thrombogenicity of all peptide-functionalized surfaces was on a low level.
Dynamic hemocompatibility testing was performed at the Clinical Research Laboratory, Department for Thoracigal and Cardiovascular Surgery at the University of Tübingen by using a Chandler loop model with fresh heparinized human whole blood. Four parameters were assessed in accordance with DIN ISO 10993-4: Thrombogenicity, coagulation, platelet activation and hemolysis using six different surface modifications: [alb/hep]3, [alb/hep]3 + REDVGK, [alb/hep]3 + EILDVPSTGK, benzophenone-modified heparin, benzophenonemodified heparin + REDVGK, benzophenone-modified heparin + EILDVPSTGK. These have been compared to uncoated PMP. Blood contacted only to the Chandler loop was used for the negative controls. Four samples of each surface were tested. The results from the dynamic in vitro tests indicate that [alb/hep]3, [alb/hep]3 + REDVGK, [alb/hep]3 + EILDVPSTGK each reflected a very good hemocompatibility, while surface coatings based on benzophenone-modified heparin showed no improvement in comparison with the uncoated surface. No abnormality was detected in terms of hematology (red and white blood cell counts, hemolysis) and platelet counts for any surface including negative and positive controls.
The sensitive biochemical marker analysis (ELISA) of ß-thromboglobulin (ß-TG) and thrombin-antithrombin-III complex (TAT) indicated significant improvement of the surface properties in terms of platelet activation and coagulation for all [alb/hep]3 based coatings which performed in the range of the negative controls. In contrast, with relation to non-coated PMP no improvement was detected on benzophenone-modified heparin based surface coatings. SEM images show that fibrin formation and blood cell aggregation was observed at some fibre surfaces, while others seemed to be coated by a proteinaceous layer. Within the tested timeframe of 1 h no three-dimensional thrombus formation occurred on any surface. While differences in the degree of clotting were obvious between the four donors, systematic influences of the fibre surfaces could not be deduced from SEM images.
Results:
• The ILA® multilayer coating ([alb/hep]3) was selected as base for the coupling of cell recognition peptides to PMP hollow fibres in order to enable endothelial cell adhesion, monolayer formation and stabilization of the endothelial layer under dynamic flow conditions.
• The coatings have been evaluated and performed very well in terms of shear stability (11.5 dyn cm-2), coupling of the peptides REDVGK and EILDVSTGK, gamma sterilization and hemocompatibility.
• Hemocompatibility of the coated surfaces was tested in terms of white and red blood cell counts, hemolysis, platelet counts, platelet activation, activation of the coagulation cascade and thrombogenicity applying a Chandler loop model:
• All parameters were in the range of the negative controls, except for moderate fibrin formation and blood cell clotting observed in SEM images for some of the surfaces. Peptide coupling to [alb/hep]3 coatings did not affect these parameters significantly. In particular, no differences were observed when using the REDVGK sequence or the EILDVPST sequence. Based on these data [alb/hep]3 coating functionalized with the REDVGK peptide was chosen for in vivo testing of the surface hemocompatibility.
• Successful functionalization of the AmbuLung prototype was demonstrated by the use of 5(6)-carboxyfluorescein-labelled REDVGK peptides. In vivo testing of [alb/hep]3 and REDVGK coated AmbuLung prototype resulted in premature clotting of the device and abortion of the test. The reason for the discrepancy between the in vitro hemocompatibility and the in vivo performance can neither be confirmed nor interpreted in the frame of the project at this stage. We hypothesize that one possible reason can be that ethylene-oxide (EtO) sterilization was used for sterilizing the AmbuLung device, whereas gamma-sterilization had been applied for the sterilization of all samples for in vitro testing.
• In addition to the [alb/hep]3 multilayer coatings, benzophenone-modified heparin was also evaluated as base layer for peptide coupling. The UV-triggered chemical coupling of the heparin derivative to PMP surfaces and peptide immobilization was successful. Yet, the surface coatings based on benzophenone-modified heparin showed no improvement in terms of hemocompatibility of the surfaces when compared with uncoated PMP. Therefore, benzophenone-mediated coupling may be relevant for chemical immobilization of coatings to the PMP polymer for subsequent multilayer assembly, but not for direct blood contact.
2.) Dynamic testing of cell seeded gas exchange units in vitro
The aim of this task was to test if it is possible to completely seed an AmbuLung gas exchanger with endothelial cells for the animal trials. In the first step we seeded individual membranes from PMP fibers with one million cells per mat in a cell culture dish and then integrated them in a dynamic bioreactor, which was developed for this purpose by Novalung GmbH and Fraunhofer IGB.
The AmbuLung gas exchanger, however, contains a stack of 66 mats. Thus, the seeding procedure had to be adjusted. Up scaling was achieved in two steps. Starting with a double membrane, a stack of eight mats was used in the bioreactor, followed by the use of the complete 66-fold stack in the gas exchanger.
Microvascular endothelial cells where isolated from human skin biopsies and expanded. For primary culture, the cells where seeded at a density of 5000 cells per cm²;in the following culture steps, a cell density of 3000 cells per cm² was used for seeding. Before reaching confluence, the cells where passaged up to the third passage at most. We performed each test with one separate batch of cells.
To prevent the membranes from floating on the media surface, the membranes were fixed to the bottom of Petri dishes with magnetic stainless steel rings as used in the bioreactors. Because of the hydrophobicity of the PMP fibres, the cells were seeded by directly placing 500 µL of endothelial cell suspension (conc. = 2 x 106 cells/mL) onto the membrane and letting the cells adhere overnight. The next day, the membranes were turned upside-down in a new dish and the seeding procedure was repeated. On the third day of the procedure, the seeded membranes were integrated in the bioreactors and the dynamic culture was started.
For seeding of stacks of more than two mats, the procedure was changed. The membrane stacks were inserted into the bioreactor chamber; the chamber was closed and filled with cell suspension (5 ml per reactor). After incubation overnight, the suspension was removed, the bioreactor was turned upside down and the procedure was repeated. The seeding results were evaluated by counting the cells that did not adhere to the fibres and were left in the seeding suspension. These results showed that 84 – 90 % of the cells used for the seeding attached to the fibres in the bioreactor and the viability of the residual non-adhered cells was about 60 – 70 %.
The cell seeding procedure for the mats in the bioreactor was also used for seeding the AmbuLung gas exchanger, which were coated with albumin/heparin. To account for the larger dimensions of the device in comparison to the bioreactor, we increased the cell number to 50 million endothelial cells per seeded device, re-suspended in a volume of 80 ml.
To analyse the efficacy of endothelial cell seeding on individual PMP membranes, 48 h after the initial seeding and before starting the dynamic culture, the cells were characterized with staining against PECAM-1, the assessment of acLDL-uptake and/or live/dead staining. On all modified coatings, fibres were almost completely covered with endothelial cells after 48 h. The cells showed typical endothelial morphology and expressed specific markers, such as PECAM-1. Additionally, the images also reveal the high purity of the seeded cells, since no contaminating cells (not expressing PECAM-1) were detected.
We performed the two stack dynamic culture of PMP membranes to establish the seeding procedure and the dynamic culture of the seeded fibre mats, and to narrow down or to identify the best membrane modification for endothelial cell attachment and culture. For the dynamic culture, two different initial shear stress levels (1.08 dyn/cm2 and 2.22 dyn/cm2) were applied to check the stability of the adherent cells under low-shear dynamic culture conditions. The preliminary experiments showed no significant differences in terms of seeding and attachment of endothelial cells on the differently coated membranes. The fibres were fully covered with cells for the most parts, showing the typical EC-specific morphology, as well as the expression of cell-cell contacts. Most of the cells took up acLDL, indicating the functionality as well as the EC-identity and purity of the cells used. Under dynamic conditions, the CS-1 coating appeared to result in a slight loss of attached cells after 7 days in areas facing the incoming flow-front in comparison to the other coatings. RGD coatings reportedly showed thrombogenic effects and in our experiments did not effectively improve cell adhesion in comparison to pure [alb/hep]3 or [alb/hep]3 + REDV coatings. On the basis of those findings, only REDV and pure albumin/heparin [alb/hep]3 coatings were selected for further studies and for up-scaling the culture conditions.
Results:
• With the established seeding and culture protocols, it is possible to obtain PMP fibres fully covered with ECs after an initial period of static culture for 48 h. The cells can be characterized as pure populations of ECs, as inferred from PECAM-1 and VE-Cadherin positive cell-cell contacts.
• For a dynamic culture of the seeded fibre mats in the bioreactor, it is beneficial to gradually increase the applied shear stress to allow cells to adapt to the altered conditions. That way, we were able to increase the shear stress up to 13.3 dyn/cm2 without causing any detectable cell loss or alteration in cell or monolayer properties.
• Like in other studies, good cell retention was found on surfaces covalently coated with heparin and albumin.
• To further improve cell adhesion, especially under dynamic conditions, the alb/hep surfaces were modified with various putative adhesion peptides.
• For our subsequent experiments we decided to narrow down the surface selection for a more detailed analysis to alb/hep and alb/hep + REDV, since REDV is expected to specifically enhance the adhesion of ECs.
• In a set of experiments with different culture conditions and various rates of shear stress, no improvement in cell adhesion was detected when coupling the additional REDV peptide to the surface. In comparison to other studies, which described profound cell loss after dynamic culture for 24 h under similar shear conditions, though, there was no excessive cell loss detectable in our system. Additionally, we were able to extend the culture period in the bioreactors up to 14 d without any sign of damage or loss of cells.
• In a last set of experiments, we were able to utilize and scale-up our dynamic seeding and culture protocol for use with the prototype of the AmbuLung gasexchanger, containing 66 PMP fibre mats. In contrast to the configuration with a stack of 8 mats we found distinct regional differences in the AmbuLung device, especially in terms of cell morphology and pattern of marker expression, as assessed form the loss of cell-cell contacts and an upregulation of EC activation markers. At this stage, we hypothesized that the difference in the properties of the seeded cells are mainly reflecting unique, distinct flow conditions in the AmbuLung prototype, which might be quite different from the conditions in the bioreactor due to the altered geometry.
3.) Computational modelling of fluid dynamics and cell-biomaterials interactions
Focusing mainly on developing mathematical models to predict the performance of the AmbuLung prototypes, this task is at the interface between research of the surface functionalisation/ cell seeding and the technical development of the gasexchanger, and the results were important for both partners. Detailed computational fluid dynamics (CFD) simulations of the blood flow through the membrane oxygenator were carried out in order to determine whether the amount of shear stress acting on the fibres is within normal physiological levels. The CFD simulation models served as the basis for the bioreactor cell seeding experiments. These models allow us to predict the density of adherent endothelial cells on the PMP substrate over time as a function of the dynamic culture conditions. The proposed model entails parameters such as the fluid-flow rate, volumetric cell density, and cell-cell and cell membrane adhesion coefficients. The models are beneficial for the optimisation of key design criteria of the AmbuLung, such as minimising the time for cell seeding, maximising the adherent cell density and minimising the extent of cell loss from the hollow fibres. More generally, the work represents a significant advance beyond current models describing the dynamics of cell seeding of biomaterials. These models helped us to predict and optimize the functionality and durability of a cell seeded gas exchanger, which is at the core of the AmbuLung concept.
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Engineering research and development
1.) Engineering - Gas Exchange Membrane + vascular access design
In a first step, we determined theoretically and validated experimentally the optimum fibre arrangement, which would maximize the performance of the minimized gas exchanger in terms of efficient gas exchange and enhanced hemocompatibility. For the verification of the selected fibre arrangement, we carried out in vitro tests in the Novalung wet lab. As performance factors we primarily investigated a) the capability to eliminate CO2, b) the pressure drop and c) the priming volume. In addition to the first CO2 exchange performance tests, which we intended for validating the performance of the prototypes as compared to the mathematical model and CFD, we carried out extensive performance tests for the detailed characterization of the gas exchanger.
Results:
• The measured CO2 values at the outlet of the gas exchanger confirmed the expectation based on the mathematical model that a nearly identical CO2 gas exchange can be achieved compared with the iLA gas exchanger, despite the more than 50% smaller gas exchange surface area of 0.6 m2 of the AmbuLung versus 1.3 m2 of the iLA exchanger
• Especially within the low-flow range (0.5-1.2 l/min) and therefore within the target range of the AmbuLung the performance of the newly designed AmbuLung gas exchanger is excellent.
• These in vitro results exceeded the calculated and simulated theoretical performance for this fibre configuration.
The original computational model for the gas exchanger predicted a pressure drop of about 20 mmHg at 1.5 l/min. In fact, we observed a pressure drop between 25-35 mmHg in vitro, therefore, an adaptation of the model was required to describe the overall behaviour of the prototypes. The revised model is based on a pressure drop of 30 mmHg at 1.5 l/min. In the subsequent animal studies, the average pressure drop at 1.2 l/min blood flow was approximately 20 mmHg. This fits well with the corrected model. According to this revised model, the maximum shear stress is at about 30 dyne/cm2, which is in the physiological range and well below the critical level of 70 dyne/cm2 that reportedly damages the integrity of endothelial monolayers.
A major factor for a homogeneous distribution of blood flow through the AmbuLung gas exchanger was the design of a cylindrical cavity for the miniaturized device, versus the rectangular cavity of the iLA device. Flow experiments with this new cavity design showed two circulating areas of recirculating eddies due to the flat, lateral inflow into the cylindrical cavity. However, these areas of recirculation were deemed safe/uncritical, since the flow velocity and thus the shear rate in these areas was assumed to remain below a critical level.
The analysis showed that at the minimum flow rate of 0.5 l/min the flow velocity exceeded 2 mm/s in certain areas of the cavity. The resulting blood compatibility, i.e. the risk of hemolysis, was assessed by evaluating the combination of shear rate and residence times. With respect to hemolysis and platelet activation, no potential “threat” was identified. Nevertheless, low-shear-stress areas of recirculation may represent a location for the deposition of already activated/aggregated blood constituents, such as platelets, even if the gas exchanger per se may not be the cause for their activation. Thus, there is room for improvement in the design of the gas exchanger geometry. However, as vortex formation cannot be eliminated completely even after opting for a lateral inflow, we decided to accept this potential risk in favor of increased patient mobility.
Minimal structural space and a low priming volume are of central importance for the AmbuLung system. For this reason we decided to use ¼ " tubings for the AmbuLung tubing set. We developed a pump head with ¼ " connectors and cannulas with corresponding dimensions. We minimized the complexity of the AmbuLung tubing set, since more complex units generally require more space. The set includes an inline pressure sensor, the blood pump head and the gas exchanger, as well as three connecting ¼ " tubing. This tubing set is by far smaller, less complex and much easier to handle as tubing sets, used on current systems.
The total length of the tubing is about 780 mm. and the priming volume, hence the total extracorporal blood volume is less than 250 ml. The gas and blood ports of the gas exchanger are guided in parallel and oriented in the same direction in order to allow a more compact tubing arrangement. The use of an inline pressure sensor provides a luer-free tubing set. By completly abandoning luer connectors, the risk of air embolism decreases significantly. For this reason, we also omitted a de-airing membrane in the gas exchanger, which in turn positively influenced the flow through the gas exchanger, because the integration of a de-airing membrane would have meant integrating a discontinuity. During operation of a mobile device, such an external de-airing membrane will only be of limited use, because the spatial position of the gas exchanger varies in a mobile application. Instead, we integrated the de-airing membrane into the priming set.
The setup of the mobile unit, i.e. the carrying system, is inextricably linked with the cannulation of the patient and the design of the tubing set. The mobile unit consists of three main components: Disposable-Case, Power&Control-Unit and the load-carrying elements (waistbelt + shirt). The Disposable-Case consists of two textile-covered shells made of expanded polypropylene (EPP) foam, into which the essential components for therapy are inserted.
We designed the Disposable-Case in a way that the tubing set can be inserted without moving previously inserted components. The dimensions of the case are approx. 220 mm x 310 mm x 105 mm.
The EPP foam acts as a thermal and acoustic insulator and allows a high mechanical protection through its energy absorbing properties. From a thermal perspective, EPP has proven to be an ideal material. The waste heat from the various components is partially stored in the case. As a result, the internal temperature adjusts itself to a constant level that is virtually independent of the outside temperature. This reduces considerably the risk of hypothermia to the patient and avoids the occurrence of condensation in the gas circuit, which could damage the vacuum pump. Specifically placed vents further reduce any remaining heat in the case.
2.) Engineering – Hardware and peripherals -
In a first step, we defined the overall concept of the AmbuLung hardware system. We defined the basic requirements in terms of performance, usability, and safety for users and patients, with special focus on compliance with the rules and regulations for the development of class 2b medical devices. We implemented the basic considerations and concepts for the design of the hardware components and developed and manufactured a small series of prototypes that were extensively tested in the laboratory and in an animal studies. The special challenge was to reduce the components to minimum weight and size without compromising their performance. For this purpose we completely redevelop major components such as the pump drive; other purchased parts had to be modify to meet our specifications.
In developing the AmbuLung hardware we decided to use as many components as possible of the iLA activve®. The iLA activve® is Novalung’s lung support system, which is CE-marked and has been released in 2011. This device consists of a bedside console and a disposable set and enables extracorporeal lung support in a veno-venous mode for awake, but immobile patients in the ICU.
Due to the complexity of the task to radically reduce size and weight of the system while concomittantly guaranteeing the specified system performance, we could use only a few components of the iLA activve® without modifications. Specifcially we used existing iLA activve® components and algorithms only for the development of the central unit and the blood pump control. All other components and control systems were developed from scratch.
Results:
As part of the AmbuLung project, we developed following hardware components:
• The Central Unit has been development based on the current iLA activve console using state of the art processors and operating system, touch screen, modern small size batteries and a user interface design, that meets the latest requirements for user-friendliness and ergonomics.
• The mobile Power & Control Unit has been developed from scratch and contains all electronic components necessary for the self-sufficient and safe long-term operation of the AmbuLung system. This unit is worn on the patient’s body.
• The blood pump drive is conceptually based on the Novalung DP3® pump, which drives i.a. the iLA activve® system, but is radically reduced in weight by 65% and its size by 50%.
• We developed an entirely new sweep-gas supply system, which is unique and has never been used in comparable systems so far. It works autonomously without external gas supplies. It is optimized for small size, weight and noise levels and is also worn on the patient's body.
• For sensors we used standard products, which we modified for use in a mobile miniaturized unit, worn directly on the patient’s body.
• All hardware components, which are connected to blood- or gas-tubes, have been integrated together with the disposable components in the disposable case. This case is lockable and protects sensitive components against mechanical stress, moisture and dirt. The disposable case contains the central components of the AmbuLung system and is worn on the patient's body.
• All hardware components have been extensively and successfully tested on reliability, performance and compliance with their specifications.
• We reached one of our main objectives and were able to develop a system that weighs less than 3 kg and can be worn by the patient in any body position without compromising comfort.
Application testing, verification, validation
1.) Preclinical in vivo study
All disposable and hardware components were validated and verified in vitro in the laboratory.
In addition, we tested their safety and functionality in a preclinical animal study in vivo, using the anesthetized and ventilated pig as animal model. We analyzed critical physiological and hematological parameters required to evaluate the performance and efficacy of the AmbuLung device under physiological conditions. In this study we used the iLA-activve® system as a benchmark and as a comparative system to the AmbuLung device. The overall goal of the study was to assess the decarboxylation efficacy of the AmbuLung prototype compared with the current iLA-activve® system over a period of 72 hours in a large animal model.
Six (6) animals were treated for 72 hours in a randomized fashion with the established iLA-activve® system and nine (9) animals with the AmbuLung® prototype.
Results:
• All except of one animal survived until the end of the study. One pig developed septic complications and had to be euthanized after 56 hours.
• For both lung assist devices, we observed no technical problems over the observation period of 72 hours.
• The AmbuLung prototype showed a comparable efficiency in CO2 elimination as the established iLA-activve® system both based on the arterial blood gases and the gas concentrations in the sweep gas under constant conditions with a blood flow of 1.2 L/min and an sweep gas flow of 8 L/min.
• The average PaCO2 value was around 40 mmHg throughout the 72h-observation time with no marked differences between both devices.
• The average drainage pressure was approximately -30 mmHg and thus within a safe range. The CO2 elimination performance of the gas exchangers was in the order of magnitude of the in vitro results. A direct comparison with the measured values obtained in vitro, however, was not feasible, as expected, because the base line blood gas values before testing the gas exchangers varied due to the different vital condition of the individual animals.
• Overall, the CO2 diffusion rates of the AmbuLung gas exchanger and the benchmark device were rather similar. The maximum rates were in the range of about 130-140 ml/l (Bloodflow: 1.2 l/min; Gasflow: 8 l/min), which led to a reduction of the CO2 partial pressure by 30-40 mmHg, depending on the input value. Such a reduction of the partial pressure level provides a significant improvement in the perceived status of the patient
• The reduction in gas exchange performance of the AmbuLung gas exchanger at reduced gas flow rates was generally lower than that of the benchmark device. However, in controlled laboratory tests, this effect was less pronounced.
• The gas exchange performance of the prototypes was largely constant during the in vivo tests that lasted for up to 72 hours. The pressure gradient across the membrane was between 20-25 mmHg and increased only marginally over the entire test period.
• With a sufficient anticoagulation in place, no clotting was observed in both extracorporeal gasexchangers.
• There were no signs of hemodynamic instability, acid-base disorders, thromboembolic events or hemolysis at both extracorporeal gasexchangers.
• In terms of inflammatory response, there were no differences between both study groups. However, towards the end of the observation time, modest increases of all cytokines were recorded in both systems.
• The number of CAE-positive cells in lung tissue showed no difference between animals which have been treated with either AmbuLung or iLA-activve®.
In summary, we could demonstrate:
• The AmbuLung system operates reliably and without any technical problems over at least 72 hours.
• The gas exchange performance of the AmbuLung system is at least as good as the one of the iLA activve, which serves as a benchmark, although the gas-exchange surface of the AmbuLung gasexchanger is less than 50% of the ILA one.
• The use of the AmbuLung system showed no adverse effects in terms of biocompatibility, hemocompatibility, hemodynamic, acid-base disorders, thromboembolic events, hemolysis or inflammatory response.
2.) Clinical Implantation: First-in-man Study
After the successful implementation of the animal study, it was the final objective of the AmbuLung project to test the clinical feasability, wearability and safety of the newly designed device in a first-in-man trial. Within the 36 months tenure of this project the consortium did not plan to develop the AmbuLung device to the stage of serial production. Specifically, we did not expect that the device would require/receive CE approval prior to the first-in-man trial. Hence, approval for the trial on humans was requested and obtained from the Institutional Review Board (IRB) of the performing Hospital. After ethical approval, it was planned to carry out a small-scale first-in-man clinical trial as a prelude to obtaining a Clinical Trial Authorization and as proof-of-concept for the wearability, safety, durability, and efficacy of the new medical device.
Results:
• We set up and trained a team of doctors and medical staff from the intensive care and respiratory subject areas.
• The team has gained extensive experience in treating COPD patients by extracorporeal CO2 removal using the Novalung iLA® activve device.
• We received approval for a specific clinical protocol for the implementation of a first-in-man trial by the local ethic committee of the Careggi Hospital in Florence.
• This locally approved request was submitted to the Italian Ministry of Health for final approval.
• We created all documents for the final approval by the Ministry of Health, for which the manufacturer was able to provide the necessary data.
• At this stage, the Ministry decided to withhold final approval until such a time that the Ambulung Device will be fully validated by the manufacturer according to the rules and regulations for the development of class 2b medical devices.
• This unforeseen demand for complete device validation prior to a first-in-man trial reversed the original plan and timeline of action (i.e. first-in man-trials followed by complete device validation), as originally approved by the EU commission.
• The demand by the Italian Ministry of Health now necessitates the delay of the first-in-man trials until the end of the current EU funding.
• The team and the infrastructure at the hospital is ready to start the use of AmbuLung on humans immediately after receipt of approval, which is tentativly scheduled for Q4/2015.
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Potential Impact:
The product generation emerged from the AmbuLung project represents an innovative and first-of-a-kind family of fully wearable bioartificial lungs for long-term use in an outpatient setting. Once CE-marked, which is scheduled for Q3/2016, these systems will be commercialized by the established Marketing & Sales organisation of Novalung. These medical devices are unique because of their optimized gas exchange membrane /blood interface for long-term use, which is characterized by a high degree of hemocompatibility.
Our new bioartificial lungs will create pulmonary volume unloading, avoid dyspnea, prevent invasive mechanical ventilation and improve overall quality of patient life. Compared with current non-invasive and invasive mechanical ventilation, the newly developed bioartificial lungs create a novel treatment modality and option for patients suffering from advanced COPD by freeing up the diseased lung from the need to perform. We fully expect that the results of the upcoming clinical studies in the next project phase will show that the extracorporeal volume unloading enabled by the bioartificial lung will positively affect the hitherto unstoppable progressive course of COPD. The outcomes of the AmbuLung project is groundbreaking and paradigm shifting, resulting in the first set of wearable bioartificial lungs with miniaturized support technology. These bioartificial lungs represent the world's first extracorporeal lung support systems that are fully mobile and are worn by the patient on the body during treatment. The outcomes of the AmbuLung project pave the way for use of bioartificial lungs outside of the hospital environment in analogy with current artificial heart protocols.
The development of commercial miniaturized artificial lung systems also catalyzes a clinical paradigm shift in the approach to long-term respiratory support. We anticipate that our technology will shift the treatment environment for lung failure towards avoiding dyspnea and exertion, improved patient mobility and quality of life. By providing functional volume unloading the bioarticial lung may improve the dismal course of COPD. For those patients currently undergoing invasive mechanical ventilation in acute exacerbations of COPD (AECOPD) the use of a bioartificial lung will avoid sedation and immobility thus creating self-managed, active patients. It will lead to a more patient driven treatment scenario for cases of advanced COPD. A wearable bioartifical lung can also be excellently used to bridge the waiting time for a donor organ (bridge-to-transplant). It may even avoid the need of lung transplantations, as was already observed in the use of ventricular assist devices (VAD) in heart transplantations. The methodology developed here for a bioartificial lung will also be applicable for and influence other bioartificial devices (liver, kidney, etc.), where biolization/cell coverage might improve both durability and function. The AmbuLung project will lead to a CE marked, first generation, wearable bioartificial lung systems for clinical use in less than one year. Once available commercially, the medical device will positively influence patient’s quality of life, reduce health care costs and overall expense to society by decreasing hospital stay length, hospitalization rate and palliative costs.
The AmbuLung project already created new positions at Novalung and the hiring of five people in R&D and Marketing, which continue to work fulltime on the project. Until CE marked in one year, the project will create up to approximatly 15 further new positions for Novalung in Manufacturing, Marketing, Sales and Clinical Support.
We were able to initiate five theses by the AmbuLung project with topics in the field of stem cell research (1), tissue engineering (1) and human medicine (3). These theses are completed and published shortly. After completion of their thesis, some of these PhD students will have the opportunity of a permanent employment at the respective institution to deepen their expertise obtained within the AmbuLung project. We expect that, initiated by the AmbuLung project, follow-up projects will be generated, which will create additional positions for young scientists, bioengineers and MDs at the scientific and medical institutes involved in the AmbuLung project.
Dissemination activities
As a Consortium we focused on the implementation of an iterative dissemination plan, which was continously discussed and agreed by all partners. This plan includes several actions to enhance the overall visibility of the main results of the project to target groups and relevant stakeholders. This group includes potential users and patients, the scientific community, policy makers as well as health authorities. The goals of dissemination extend beyond the EU funding phase of the project and will leverage future commercialization concepts.
Our dissemination plan the following activities:
• Creation of a product website based on the project homepage
• Participation on congresses, scientific and medical meetings
• Scientific publications and presentations
• Organisation of workshops with stakeholders and the medical community
• Initiation of press releases
• Production of marketing material like videos, leaflets, brochures, training & education material
• Application for patents
So far, we have carried out the following dissemination activities:
• We attended 15 congresses and meetings mainly in Europe and the US, covering the topics of stem cell research, biomaterials, biomedical engineering and medicine. In these meetings we were represented either with a lecture, of attending a workshop or as an exhibitor.
• On 10 meetings, members of the consortium presented their specific topics by invited talks (9) and posters (7).
• We generated five publications so far. One is published, two are submitted but not yet reviewed and two are in preparation.
• There were a couple of press releases, of which six very exclusively devoted to the AmbuLung topic.
• We organized a workshop with expert in the field of intersive care medicine, pulmology and biomedical engineering on an international level and gained import input for the further cause of the development process.
• We already applied for three international patents, which are at the stage of the approval process and have not yet been officially granted.
With all due caution with regard to confidentiality and secrecy of the key project results we have already achieved great interest in the professional public with our previous dissemination activities.
The market is waiting for the outcomes of the AmbuLung project.
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Exploitation
As main outcome of the project, we will further verify and validate the product, resulting from the project results, with emphasis in the coming months with the aim to obtain CE-Mark approval in Q3/2016 und in order to start market release and commercialisation.
As further exploitable foreground, we plan to commercialize the dynamic bioreactor, which we developed within the task of the cell seeding on the gasexchage membranes. Because there is not yet such a specialized laboratory instrument on the market, we expect a commercial success in marketing this bioreactor.
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List of Websites:
Public Website Address: www.ambulung.com
Contact details for the design and the regular update of the website:
PD Dr. med. Georg Matheis, General Manager, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Georg.Matheis@xenios-ag.com
Dr. Esther Novosel, Manager Business Development, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Esther.Novosel@xenios-ag.com
Dr. Timo Hammer, Productmanager, Novalung GmbH, Im Zukunftspark 1, 74076 Heilbronn, Germany email: Timo.Hammer@xenios-ag.com