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High yield and performance stem cell lab

Final Report Summary - HYPERLAB (High yield and performance stem cell lab)

Stem cell research is one of the most promising areas of biomedical research: besides its usefulness for basic science to study developmental biology, it offers a hope for treatment of many unconquered degenerative diseases including Parkinson's, diabetes or heart disease as well as cancer.

But the success of stem cell therapy is highly dependent on a safe and reliable supply of human stem cells and stem cell-derived differentiated cells, which must be assured by efficient and robust culture methods. Current cultivation of human embryonic stem cell (hESC)s and adult stem cell (ASC)s is not optimised (with regards to media, growth factors, supporting biomaterials, and differentiation techniques) and is far from fulfilling the demands in terms of reproducibility and preciseness. Although much effort and costs have been invested, growth and expansion of hESCs and proliferation of ASCs, as well as efficient generation of differentiated cell types has not been completely mastered. The unavailability of standard protocols and tools and the resulting lack of reproducibility between laboratories cause a significant duplication of effort and cost. Additionally, current methods do not allow fast screening of culture conditions for their systematic optimisation. These limitations currently slow down the development of stem cell applications and are addressed and will be overcome by the HYPERLAB project. To reach the overall aim of developing new and improved culture methods, media and protocols for stem cell cultivation and differentiation, HYPERLAB adapts novel microfluidic-based cell cultivation technologies to the specific needs of stem cell culture. The developed culture systems have two major advantages over existing approaches: on the one hand, they improve stem cell culture in terms of microenvironment control, reproducibility, robustness and efficiency. On the other hand, these microscale technologies, together with developed transgenic readout systems, allow medium to high-throughput screening of culture conditions, enabling determination of optimal protocols in shorter time. Since these new tools are highly amenable to parallelisation, conditions can be investigated in a rapid, cost-efficient and highly precise way: these tools hence enable not only much easier cell culturing with a given protocol, but also automated investigation of thousands of different conditions, thus allowing determination of optimal culture and differentiation protocols.

Project context and objectives:

To reach the overall aim of developing new and improved culture methods, media and protocols for stem cell cultivation and differentiation, HYPERLAB adapted novel microfluidics-based cell cultivation technologies to the specific needs of stem cell culture. The developed culture systems have two major advantages over existing approaches: on the one hand, they improve stem cell culture in terms of microenvironment control, reproducibility, robustness and efficiency. On the other hand, these microscale technologies, together with developed transgenic readout systems, allow medium to high throughput screening of culture conditions, enabling determination of optimal protocols in shorter time. Since these new tools are highly amenable to parallelisation, conditions can be investigated in a rapid, cost-efficient and highly precise way: these tools hence enable not only much easier cell culturing with a given protocol, but also automated investigation of thousands of different conditions, thus allowing determination of optimal culture and differentiation protocols. Once established, technologies, protocols and conditions have been evaluated for their upscalability and good manufacturing practice (GMP) compliance to form a solid basis for progress in human stem cell therapy. To implement this innovative strategy, HYPERLAB followed an integrated approach, bringing together renowned experts from stem cell biology, microsystem technologies, biomaterial design and relevant regulatory bodies.

Objectives

Technologies, protocols and conditions are evaluated for their upscalability and made GMP-compliant to form a solid basis for progress in human stem cell therapy. To implement this innovative strategy, HYPERLAB follows an integrated approach, bringing together renowned experts from stem cell biology, microsystem technologies, biomaterial design and relevant regulatory bodies.

Project results:

Work package (WP) 1: Knowledge management

UKK constantly maintained and updated the HYPERLAB knowledge base. Protocols for the knowledge base were obtained from HYPERLAB project partners and by screening of literature. The protocols had been evaluated by HYPERLAB partners, further improved via identification of optimal media, screening of small molecules, growth factors and genetic engineering to permit lineage selection and concluded in a form of standard operating procedures (SOPs) used during the project duration. The compiled protocols describe optimal conditions for erythroid progenitor isolation, cultivation and differentiation into erythrocytes; for maintenance and differentiation of hESC and induced pluripotent stem cell (iPSC)s into either cardiac or hepatic lineages and mesenchymal stem cell (MSC)s into osteoblasts. Priority was given to protocols which support serum-free and xenobiotic free stem cell cultivation and differentiation, as well as the cell expansion and differentiation compatible with bioreactors, HYPERLAB microfluidics devices, hESC or EB encapsulation and cultivation on microcarriers.

UKK compiled a list of conditions potentiating or inhibiting hESC and iPSC cells into cardiac or hepatic lineages, MSC cells into osteoblasts and erythroid progenitors into erythrocytes. Scientific publications and reports on international conferences relevant to miniaturised cell cultivation technologies, devices and technologies accelerating specified cell generation yield and quality, cell growth interacting surfaces, extracellular matrixes and biomaterials, small molecules and bioactive macromolecules, novel medium compositions as well as cardiac, hepatic, erythrocytes and bone differentiation were continuously screened and evaluated.

Links to other on-going projects working on complementary topics were defined and the patent landscape for miniaturised cell cultivation devices was monitored.

WP2: Development of microfluidic-based technologies for stem cell cultivation

Investigation of stem cells is a field of interest for diagnostic and therapeutic applications in medical research. Considering cost efficiency and high reproducibility scientists focus more and more besides established platforms and protocols on microfluidic techniques as middle- and high-throughput systems for stem cell investigation.

In the HYPERLAB project three microfluidics based technologies had been developed and applied for stem cell cultivation:

i) pipette robot-based technology;
ii) modified hanging droplet technology; and
iii) pipe based bioreactors technology.

Whereas both, the pipette robot technology and the modified hanging droplet technology could run on commercial pipette robots the pipe-based bioreactors technology serves as new approach as a closed system for stem cell cultivation.

For the modified hanging droplet technology, a special plate for hanging droplets (droplets = bioreactors) has been developed.

The pipe based bioreactors technology bases on the principle of the 'segmented flow'. As well as in the hanging droplets, embryoid bodies form spontaneously. By means of functional modules e.g. the culture medium could be exchanged and an optical readout realised.

WP3: Development of miniaturised systems for stem cell culture and differentiation

Aim of WP3 was to develop and test the fundamental principles and setups of the new microfluidics-based, technological approaches in their effect on stem cell culture and differentiation; those were then evaluated and benchmarked against selected state-of-the-art technologies and tools identified in WP1.

Therefore, it was necessary to transfer established protocols to miniaturised cultivation units and to investigate the effects of miniaturised and closed systems on different stem cell types in terms of viability, differentiation, adhesion and proliferation. First of all, physicochemical changes in open and closed miniaturised systems, as osmolarity and evaporation, have been investigated to define and determine the parameters for stem cell cultivation and differentiation. After prototypes of all three technologies have been successfully installed at IBMT, experiments with all partners, cell lines (hESCs and ASCs) and relevant protocols have been conducted to evaluate the HYPERLAB technologies and to show the proof-of-principle of these innovative approaches. In the final project period, the pipe-based bioreactors were further optimised with focus on cytotoxicity tests, whereas the pipette robot and the modified hanging droplet technology were used for upscaling. For this, both approaches were implemented on a fully automated cell cultivation platform and joint experiments with different partners, cell lines (EGFP reporter cell lines) and protocols were conducted. To guarantee optimal conditions for differentiation and cultivation in the microfluidics based HYPERLAB technologies, the potential of scaffolds and biomaterials like ultrahigh-viscosity alginate was evaluated.

WP4: Establishment of stem cell expansion conditions

Optimal protocols for culturing hESCs and ASCs do not exist at present. hESCs are not adapted to bulk culture, requiring the use of either feeder layers, extracellular matrices, complex media or media containing serum (or serum fractions) and close visual inspection. Routine subculturing using enzymes to dissociate cells have been associated with chromosomal abnormalities and excessive cell death. Long term culture of ASCs (with the exception of haematopoietic stem cells) is hampered by the strictly adherent growth requirements of these cells and a tendency to lose their plasticity with increasing passage numbers.

WP4 addressed these issues both for ASCs as well as ESCs applying the cultivation systems developed in WP2 and adapted for use with stem cells in WP3. In close collaboration with WP5 (covering the differentiation conditions), WP4 developed culture conditions and protocols capable of producing stem cells for therapeutic application in vitro. In addition, the work on isolation and expansion of HSC from cord blood was continued.

We could establish protocols for the reproducible expansion / outgrowth of MSCs from umbilical cord samples. Working stocks of low-passage MSCs from different donors were banked for further use during the project. MSCs were thoroughly characterised by flow cytometry, and stemness was confirmed by performing multi-lineage differentiation experiments for every sample. As reference, we used commercially available UC-MSCs, BM-MSCs and primary osteoblasts from Promocell. Self-isolated MSCs performed well in all assays, with the known differences to BM-MSCs (which give better adipogenic differentiation). Self-isolated UC-MSCs were then used to fulfil all tasks for MUW in WP5. For HSC, the successful quantitative CD34+ cultivation system was used to identify new candidate factors with positive effects on HSC expansion. This in-vitro cultivation assay was combined with multi-parameter immunophenotyping to characterise the early progenitor populations, which emerge during in-vitro cultivation. Addition of single factors led to strong shifts in e.g. CD90+/CD38- populations. The expansion protocol was finally transferred into a closed bag cultivation system with GMP-grade medium and factors. Successful expansion of CD34+ cells in this system was demonstrated.

WP5: Establishment of stem cell differentiation conditions

Stable transgenic cell lines for tracking differentiation of hESC into either cardiac or hepatic cell fate, and MSC into osteoblasts had been generated and tested for screening applications.

Optimal differentiation protocols yielding high proportion of hESC into either cardiac or hepatic, and MSC into osteoblasts lineage fate had been established. Furthermore, a range of promising factors potentiating or inhibiting differentiation into cardiac, hepatic, and osteoblasts had been evaluated. Established transgenic hESC and MSC cell lines had been validated for screening for differentiation modulating factors.

WP6: Validation of robustness and stabilisation of protocols

One of the major goals of the HYPERLAB project was to develop strategies for the production and storage of pluripotent stem cells in relevant numbers for cell therapy applications. Within this scope, iBET and IBMT have established cryopreservation methods for hESCs using different approaches. For adherent cells, strategies using slow rate freezing were compared with the surface-based vitrification method, which has revealed to be the most efficient for the storage of intact hESC colonies. In alternative, three-dimensional (3D) strategies based on microencapsulation for the integration of expansion and cryopreservation of hESCs were also implemented, being the alginate immobilisation of hESC on microcarriers the approach that led to the highest expansion ratios combined with highest post-thawing viability and maintenance of pluripotency. In addition, iBET has developed tools for toxicological testing using different systems. For long term, repeated dose toxicity, spheroids of primary human hepatocytes were cultivated in stirred tank bioreactors with perfusion and could be maintained up to six weeks in culture with functional detoxification enzymes. A dual reporter hepatic cell line was also developed and was validated using the pipette robots developed at HYPERLAB, constituting a powerful tool for high throughput screening.

WP7: Dissemination and exploitation

The HYPERLAB project and updated results of scientific progress are presented on a public internet website (see http://www.HYPERLAB.eu online). For the scientific community, experimental data are presented by all partners during international conferences and published in peer-reviewed journals. The official HYPERLAB leaflet is available for scientists and general audience to present the focus of the project and recent research advances. A HYPERLAB workshop session has been held in October 2011 within the frame of the annual meeting of the European Society of Gene and Cell Therapy. In August 2012 the HYPERLAB consortium presented all the project results during the HYPERLAB public workshop at IBMT in St. Ingbert.

WP8: Project management

The overall objective of WP8 is to ensure the smooth run of the project by taking care of day-to-day operational management in view of financial, contractual and logistical aspects. In course of financial and contractual management ART and IBMT administered EC financial contribution and distributed partner shares, coordinated contractual periodic reporting, monitored timely production of deliverables and reports according to project milestones and indicators. ART established and regularly updated project management tools (project management plan, budget and effort indicators, deliverable and milestone indicators). In view of meetings and logistics management, IBMT chaired board teleconferences and six-monthly general assembly meetings with focus on progress review, strategy and decision making. ART provided logistics support and electronic tools (via private web space, conference service) and coordinated reports for internal meetings.

Potential impact:

Socio-economic impact

The socio-economic impact can be great as the technologies can in principle be adapted to all kind of cell based assays, e.g. for drug or toxicity testing. The technologies are scalable and do have the potential to replace several currently completely manual cell handling protocols, which will positively influence clinical developments in general.

The technology developers would benefit from marketing the technologies through licence fees, which could be used to invest in further technology development. Users of the technologies could potentially increase their competitiveness as they would allow significantly increasing throughput and reducing cost (as less cells, medium and chemicals have to be used) at the same time.

Wider societal implications of the project

The HYPERLAB technologies offer the potential in medium to long term to largely reduce animal testing as they will allow application of (even individualised) cell models with the highest possible biological relevance, i.e. human cell models. The application of more specific and human cell models will also improve the development of new drugs and therapies as it will reduce the dropout rates in the process of preclinical and clinical development.

Main dissemination activities

The HYPERLAB results were published in peer reviewed journals and presented at national and international conferences. In addition HYPERLAB presented its results in October 2011 within a dedicated HYPERLAB session at the European Society for Gene and Cell Therapy Meeting, as well as in a public workshop organised in August 2012 at IBMT premises in Sulzbach, Germany.

Exploitation of results

The HYPERLAB project could successfully demonstrate the proof of concept for the developed technologies. Nevertheless they need further research and development before actual product development can start. With continued efforts it can be expected that the first technologies can enter the market in less than five years from the end of the project. Depending on the results of the further technology development, the exploitation could take place through licencing of single technologies and components or even through the creation of new business entities offering cell based services or technology solutions.

Project website: http://www.hyperlab.eu

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