Final Report Summary - MEHTRICS (Micropattern-Enhanced High Throughput RNA Interference for Cell Screening)
Executive Summary:
The MEHTRICS project challenged the current limits of HT cell-based screening by combining HT-RNAi with an emerging new technology for normalizing cultured cell behaviors, namely the growth of cells on adhesive micropatterns. Initial applications of this technology had already demonstrated its potential for enhancing the quality of existing high content analyses by radically reducing the cell population’s phenotypic variability, resulting in much lower cell sampling requirements. This approach also promised to open up major new “assay development space” by broadening the range of analysis strategies that drive novel cell-based assay designs, whose evolution has otherwise stagnated in recent years. The consortium carried out several parallel and complementary lines of development to diversify the applicability of the MEHTRICS platform for both academic and industrial uses, ultimately validating the new capabilities through proof of principle screens.
Our key objectives were:
• to optimize micropattern geometries and compositions to accommodate extended timelines typical of siRNA assays,
• to integrate the promising Transfected Cell Array (TCA) technique to decrease cost and increase throughput,
• to develop novel cell models of key diseases based on micropatterned adult stem cells and polarized epithelia/endothelial architectures and
• to validate each of these implementations in key industry-relevant siRNA screening applications.
Thanks to the outstanding synergy stemming from the complementary expertise of the consortium partners, the MEHTRICS platform has achieved major accomplishments showing i) that the tight control of the microenvironment provided by the micropattern technology can not only improve assay performance but also enable the development of innovative and relevant assays for both industry and academic applications, ii) that the improvements of TCA technique will reduce the screening cost, iii) that long term siRNA knock-down should be achievable and iv) that image analysis and models will increase overall data quality and significance.
Since experimental designs requiring siRNA screens are among the most demanding of all HT/HC studies and encompass virtually all technical challenges also encountered in compound screens, we expect the envisioned scope of activities to deliver the maximal potential for impactful innovation, widespread adoption and clear relevance for all major applications of HT/HC cell screening. The resulting new tools and methodologies were incorporated into CYTOO commercial offerings. As the availability of new classes of approaches as described here usually fosters new applications, an important source of value of the accomplished research is the potential usefulness to develop new approaches for the drug development and fundamental research fields.
Project Context and Objectives:
Mammalian cell-based assays are powerful for basic studies of eukaryotic cell physiology and represent crucial models for the development of novel pharmacological compounds. The vast majority of HT/HC screens carried out today make use of adherent immortalized cancer cell lines growing on plastic or glass surfaces used either as such or instead evenly coated with a variety of possible extracellular matrix components. Since those cells adhere, spread and divide in a largely uncontrolled manner, the resulting high variability in the behavior of individual cells within each sample population is an inherent weakness of this experimental approach, as it requires the experimentalist to sample large enough cell numbers such that this variability can get normalized down to background “noise”. This is a basic obstacle in the optimization of nearly all HT assays using this standard paradigm, namely, the need to minimize sample variability to maximize assay windows. This difficulty is further compounded by the need to maximize analysis throughputs and overall cost-effectiveness in these studies, both of which push users towards higher-density plate formats (384-well, 1536-well…). These, in turn, allow fewer cells in each sample well, thereby potentially increasing assay variability by reducing sampling sizes for each treated cell population. This problem represents a basic impasse that plagues all large-scale HT cell-based studies, whether they be siRNA screens, pharmacological studies or combinations of both.
The development and application of precisely-controlled adhesive micropatterns as new growth substrates for large scale HT cell-based studies is now emerging as a breakthrough for addressing this problem. By yielding highly homogeneous “standardized” cell behaviors within each sample population, this approach has already been shown in initial feasibility studies to vastly reduce assay noise levels and sampling sizes. The direct implications are clear in promising higher quality datasets through a broader screening window and higher statistical relevance, all with higher throughputs, and therefore better cost-efficiency overall. These observations provide a very encouraging basis on which this project will build further by first determining and characterizing the degree to which this new technology can similarly enhance a hopefully-broad range of other HT/HC applications. In fact, the main limitation of this technology today resides in micropattern sizes and geometries that do not maintain cell normalization after the first division. Thus, as a key element of the proposed MEHTRICS development, combining a network of micropatterns standardizing cells over several divisions with unlimited design possibilities will drastically reduce cell variability in a much broader range of HT/HC assays.
Beyond such enhancements of existing assays, the MEHTRICS platform is also expected to open up brand new “assay development space”. Thanks to the high level of control afforded by the ability to finely modulate all functional aspects of the micropatterns, combined with the inherent ability to characterize the phenotypic fate of individual cells in both live and endpoint assays, this will enable a new range of HT/HC assay strategies that have not been possible until now. Of particular interest are more advanced disease models using primary cells, including adult stem cells, which can maximize patho-physiological relevance but are often not available in adequate supply to enable large-scale screens. The smaller sampling sizes required on adhesive micropatterns promises to overcome the limited cellular scale-up that has hampered these models. In addition, the tight control of adhesion areas introduces a spatial reference frame against which cells not only organize but also differentiate. Incorporating CYTOO’s technology in cell-based assays promises therefore a tighter control over cell physiology in culture methods. Such technological development would represent a veritable quantum leap for the field and a broad applicability in HT cell-based screening in general.
In addition, the synergistic combination of micropattern technology with TCA is a promising niche to develop siRNA cell based assays with lower costs, faster data collection and improved comparability between assays or platforms. Indeed, by bringing down the cell number to analyze, the siRNA spot size can be reduced, resulting in an increased sample number per cell array. The complete genome could then be located on 1-3 single cell arrays, which is up to 150 times less than what is currently needed for full genome screening today. In addition, high sample densities per field of view of a microscope objective will allow a fast, parallel microscopic readout after stimuli of many samples using different approaches such as fluorescence live-time imaging (FLIM) or fluorescence correlation spectroscopy (FCS). Another expected benefit is a higher concordance of siRNA experiments done on different platforms, due to the combination of exact siRNA array replicates provided by TCA and standardization of cell behavior achieved on micropatterns. Finally, the implementation of a progressive siRNA release system will avoid dilution of active molecules after each cell cycle, which is a completely new approach that promises longer durations of siRNA silencing. Such improvements of the TCA technique will open exciting new classes of parallel and combinatorial assays, and in addition, will make siRNA screens accessible to a wider range of academic users.
Key objectives
Our key objectives were:
• To optimize micropattern geometries and compositions to accommodate extended timelines typical of siRNA assays,
• To explore novel cell models of key diseases based on micropatterned adult stem cells and polarized epithelia/endothelial architectures and
• To develop modeling approach as well as automated tools for enhancing image processing and data analysis
• To combine micropattern technology with the Transfected Cell Array (TCA) technique to decrease cost and increase throughput,
• To develop novel methods for delayed or triggered siRNA release
• To optimize MEHTRICS platform for both academic and industry-relevant siRNA screening applications
• To validate each of these implementations thanks to Proof of principle screen.
Project Results:
The main results achieved during the MEHTRICs project are the following:
- Successful development of micropattern networks for long term siRNA
- Successful development of micropatterns for new cellular models including epithelial cells, endothelial cells, stem cell, cardiomyocytes, hepatocytes and spindle positioning assay
- Easy integration of micropattern technology into partner’s workflow
- Development of assays relevant to industry and academia
- Efficient combination of micropattern technology with the Transfected Cell array approach
- Improvement of TCA including spot size reduction, increased spot density and absence of cross-contamination
- Proof of concept for triggered siRNA release at will
- Development of nanofoil approach for delayed siRNA delivery
- Establishment of mathematical models predicting cell behavior on micropatterns networks and integrin trafficking
- Implementation of automated image and data analysis tools
- Proof of concept screens: oncology, inflammation, stem cell differentiation, spindle pole positioning, integrin trafficking
- Demonstration of micropattern benefits for different applications (academic and industry)
- Dissemination of project results through posters, banners, press releases, talks, publications and marketing materials
- Integration of micropattern networks into CYTOO product offer with associated revenues
- Protection of foreground: The work performed in WP02 on micropattern networks has been protected through patent filing (Patent #: EP 13305119.3 filed by CYTOO on 01/02/2013). IP protection on a newly developed “Migration assay” has been abounded after several rounds of Review. The newly developed nanofoil approach for delayed siRNA should be protected in the coming months after MEHTRICS end (planned for May 2015).
Note that three PoP screens as well as further developments of image analysis tools, modeling approach and delayed siRNA were performed during the last four months of the project. The extension was therefore critical to achieve major results for the project.
A brief description of the main achievements is given below.
Micropatterns Development
Partner1/CYTOO designed and produced different types of micropattern adapted to assays and readouts in each workpackage:
- Standard geometries were used for the development and evaluation of integrin trafficking (HeLa, WP06) and spindle positioning (HeLa, WP05) assays
- Interconnected micropatterns networks were achieved and used to develop ciliogenesis assay (WP02), oncology assay (WP05), LDL uptake (HepaRG, WP05), stem cell differentiation (ADSCs, WP05)
- Structuring and differentiating micropatterns were created for hypertrophy (rat primary cardiomyocytes, WP05), inflammation assay (NHBE, WP05), endothelial tube formation (HUVEC, WP02), centriolar inheritance (HeLa, WP05), migration (MCF7 and MDA-MB-231, WP02) and stem cell cell differentiation (ADSCs, WP05).
Collaboration with the different partners went well with loops of design improvement that allowed major progresses on the different applications. As initially planned, a modeling approach was used to create new designs. Micropatterns networks were successfully developed and enabled single cell approaches for longer time period and combination with siRNA screening. In addition, new micropatterns were obtained and allowed accessing more relevant multicellular models and approaches.
CYTOO produced and delivered all the required materials for the different partners to perform the work planned. A problem with residual glue toxicity in CYTOOplates was observed but only delayed some applications (inflammation and endothelial tube formation) for which solutions were found too late in the project to plan new developments.
Assay Development
Results from the different workpackages showed that all the assays, protocol, cell lines and type of readouts could be developed on micropatterns. Micropattern performance was evaluated for each applications and showed different level of success:
- Not achievable as planned: centriole inheritance and integrin trafficking could not be developed on micropattern networks but alternative solutions on standard micropatterns were identified
- Similar level of performance: LDL uptake and oncology assays
- Improved performance: inflammation assay with H292, osteogenesis differentiation and hypertrophy
- Enabling: spindle positioning assay, which could not be performed on standard culture condition, was successfully developed on micropatterns
Overall, the different assays demonstrated that micropatterns technology is highly versatile and can be adapted to the development of very different applications, cell lines and readouts.
Image processing, data analysis and modeling Development
Image processing and analysis tools were an important part of the developments made during the project. Indeed, because micropatterned cells possess specific features, dedicated tools (image processing and analysis algorithms geared towards the organization of the cytoskeleton and the adhesions) were required to fully exploit the new capabilities of the MEHTRICS platform. In addition, we aimed to rationalize or guide assay design and developments by mathematical modeling approaches. Different tools and models have been successfully developed during the project, including:
- Image processing tool (used in WP05 and WP06)
- Single cell shape model (used in WP05 and WP06, published)
- Multi-scale model for integrin trafficking on micropatterns (used in WP06)
- Cell community model predicting the effect of micropatterning design for multicellular ensembles (used in WP02)
TCA development
For combining micropattern with TCA, Partner4/UHEI-Erfle developed improved high throughput printing technologies and fully automated workarounds for high density cell array applications on micropatterns. The work performed included i) tests on the composition of the transfection mixture, ii) optimization of the printing techniques for siRNA delivery into spots of TCA, iii) reduction of spot size and iv) increased spot density. Subsequently, a protocol for the solid phase reverse transfection of siRNAs on interconnected micropattern networks and in spots of TCAs has been established. Solid phase transfection mixture contact printing on CYTOO micropatterns did not interfere with their adhesive properties. Cross contamination effects were eliminated by improving the printing protocols. To confirm and validate these developments, siRNAs were used to demonstrate efficient delivery of the siRNA into cells on micropatterns as well as loss of function phenotype without cross contamination problems. To carefully evaluate these parameters, a dedicated automated image analysis in the Konstanz Information Miner (KNIME) was developed. In conclusion, high throughput printing technologies and fully automated workflows for high density cell array applications on micropatterns were successfully achieved.
Time-resolved siRNA release
Downregulation of gene products with low turnover have been difficult to address with siRNA screens up to now due to the progressive dilution of active molecules over several cell cycles. Therefore, we planned to develop new methods allowing either a delayed or a triggered siRNA delivery for longer durations of siRNA silencing.
Triggered siRNA delivery
To assemble molecular machineries for triggered siRNA delivery, we explored siRNA immobilization via photocleavable groups. Preliminary protocols for fabrication of a molecular film that contains a photolabile linker have been developed by Partner3/CPST. Results confirmed the feasibility of the “release-on-demand” concept. However, we could not find conditions where the cleavage was compatible with cell culture conditions. As modifications of such chemistry would have been too long for the project duration, our efforts had focused on the second approach (delayed siRNA release).
Delayed siRNA release
For this approach, we have developed a method for stabilization of cell transfection reagents printed by standard TCA fabrication technologies, e.g. pin arraying. It is based on application of a nanometer-thick layer of PEG MA hydrogel-fibronectin composite onto the transfection mixture spots. Our results demonstrated that:
1. The nanofoil itself could form cell-adhesive micropatterns, defined by the surface topography of the PDMS stamp.
2. The nanofoil is not toxic for HeLa cells.
3. The removal of the nanofoil-covered transfection mixture spots from the surface started in 3 hours of HeLa cell culture, with material release occurring after 11 hours from the cell seeding for approximately 30hours.
4. siRNA was efficiently internalized into the HeLa cells.
These results clearly showed that the method opens perspectives for true surface-supported, prolonged siRNA transfection technologies in TCA format, over periods longer than 48 h. Also, it possibly could be applied for controlled drug release and encapsulation. Additional developments are needed for ultimate integration of the method with the CYTOO platform. This method will be patented soon.
POP screen
Our overall strategy was to carry out several parallel and complementary lines of development, each geared towards enabling a subset of high-value HT/HC-RNAi applications. As initially planned, the project was staged, including exploratory phases with decision points narrowing down on successful applications, for which resulting new capabilities were demonstrated through Proof of Principle (PoP) screens. Our key objectives and results are the following :
1) Optimization of MEHTRICS platform to enhance existing industry-relevant siRNA screening applications
The potential benefits of micropattern technology have been evaluated in two established industrial assay setups by Partner2/CENIX:
• A microscopy-based oncology related assay, representing a multiplex high content assay, and
• An ELISA-based inflammation assay, representing a single-readout plate reader assay.
For each assay, critical parameters, including performance of controls, screening window, variability, performance (Z’-Factor) and hit selection were assessed.
Results for Oncology assay
Two cell lines, the colon cancer cell line HCT116 and the breast cancer line MDA-MB-231, were chosen for the PoP screen comparing tiremark network micropattern and non-patterned plates. 29 genes shown to have influence on cell health and/or viability were chosen for this assay based. Although the optimization had hinted that tiremark micropattern might yield an improvement of the oncology assay, statistical analysis of the screening scenario did not show significant improvement over non-patterned plates.
Results for Inflammation assay
In the inflammation assay, the response of the lung epithelial cell line H292 to TNFalpha (tumor necrosis factor alpha) and treatment with steroid and interferon gamma was studied. 84 genes were selected and screened in parallel on homogeneously coated and diamond micropattern CYTOOplates. In summary, the inflammation assay with single parameter plate reader readout shows impressive improvement on micropatterned plates. Assay variability is significantly lower resulting in good Z’-factors for the data set (0.3 - 0.5) and eventually in higher confidence in hit selection.
2) Application of MEHTRICS platform to create relevant cellular models for industrial application
Beyond the enhancement of existing assays, the increased control of microenvironment provided by adhesive micropatterns opened up the potential for developing new models and assays of disease-relevant processes. The MEHTRICS platform was therefore used to drive the development of new patho-physiologically predictive models from epithelial cells, endothelial cells stem cells derived from adipose tissue, hepatocytes and cardiomyocytes. Our strategy was to carried out parallel development of these models and to select the most promising to conduct a PoP screen. 5 models were developed by Partner1/CYTOO and Partner2/CENIX:
• Stem cell differentiation assay: We established adipogenisis, osteogenesis as well as "mixed cue" high throughput RNAi assays in 96well format on micropattern plates using adipose tissue derived adult human stem cells (ADSCs). Adipogenesis alone was not improved on micropattern compared to non-patterned plates. Osteogenesis, in contrast, was boosted up to 10fold on micropatterns compared to standard culture condition and showed superior detection of siRNA knockdown. A micropattern design allowing mixed a differentiation approach and resulting in higher percentage of differentiated cells was also identified.
• Inflammation assay in primary human bronchoepithelial cells could not be established on time as the cells showed unforeseen sensitivity towards the micropattern plates (residual glue toxicity) and this problem was only solved at the end of the project.
• LDL-uptake assay was developed with HepaRG cells. Condition of culture, polarization and LDL-uptake were set up on micropatterns, however RNAi screening with few genes did not show significant advantage over cell growth on non-patterned plates.
• Hypertrophy assay was developed with primary rat neonatal cardiomyocytes. Rectangular micropatterns showed promising performance for a microscopy based hypertrophy assay in primary neonatal rat cardiomyocytes.
• Tube formation assay was initiated with primary endothelial cells (HUVEC). A preliminary protocol has been established but further developments were delayed due to residual glue toxicity problem, which was only solved at the end of the project.
As the osteogenesis assay was the most advanced and the most promising in terms of benefits, we decided to perform a Proof-of-Principle screen for osteogenesis using L-stairs micropattern networks.
Unfortunately, in November 2014 Cenix BioScience was forced to file for insolvency. The PoP screen could therefore not be conducted at the anticipated level in terms of extent and depth of analysis. However, data of a mini-screen (10 genes) were generated. Differentiation was assessed by microscopy using a fluorescent substrate for Alkaline Phosphatase (induced during differentiation), ELF97. Analysis of the results showed:
- Osteogenic differentiation is boosted 5 fold on L-stairs micropattern networks compared to cell growth on uniform substrate
- The assay window is increased 2-3 fold on L-stairs micropatterns. Consequently, a graduation or dosing of the effects of individual genes is more readily assessable on micropattern
In lack of in depth analysis, the evaluation remains incomplete, however, consistent superior performance of micropatterns throughout multiple rounds of assay optimization suggests that we have developed a new, more powerful screening assay for osteogenic differentiation.
3) Optimization of MEHTRICS platform to enhance hit validation and classification in siRNA functional genomic screens
Thanks to the development of novel assays during the project, two PoP screens were planned to address wether the MEHTRICS platform can enhance functional genomic studies:
- Spindle positioning screen of approximately 1000 genes
- Centriolar inheritance screen of approximately 50 genes
Results for Spindle positionning screen
Partner5/EPFL first optimized assay conditions for the spindle positioning siRNA-based screen of fixed specimens disposed in CYTOO’s 96-well plates harboring L shapes and standard plates uniformly coated with fibronectin. Results indicated that neither method is robust enough to conduct a high throughput screen. Therefore, a live imaging approach, only possible on CYTOO’s 96-well plates, was explored. After several round of protocol optimization and improvement of micropattern design, this assay was successfully developed and allowed performing a Proof of Principle screen of a 1200-membered kinase/phosphatase siRNA library.
Until the end of the MEHTRICS project, 7 out of 16 screening plates could be analysed using the custom algorithm yielding 13 candidate genes, among which genes already implicated in spindle positioning as well as unknown genes could be identified.
In conclusion, taking advantage of CYTOO’s micropattern technology, we were able to develop and perform a challenging live imaging screen for spindle positioning in human cells, a first in the field. Since the fibronectin coating of 96-well plates is not achievable to date with sufficient quality for such a sensitive assay, we come to the conclusion that CYTOO’s micropattern technology is clearly superior for high throughput spindle positioning assays in human cells.
Results for Centriolar inheritance screen
Tools important for studies of centriole inheritance in human cells (a centriolar protein that is detectable at centrioles for multiple cell cycles in human cells) have been developed. Unfortunately, however, despite advances in development of a micropattern network suited to study centriole inheritance, we were not yet able to develop a network that would fully meet all requirements for this assay. The PoP screen was therefore not performed.
4) Integration of MEHTRICS platform with Transfected Cell Array (TCA) technology
As described above, thanks to several technical improvements integration of micropatterns into the TCA approach has been successfully achieved. To validate the benefits of this new platform, it was initially planned to run a PoP screen focusing on integrin trafficking, which requires i) the development of dedicated micropattern networks, ii) the adaptation of the integrin trafficking protocol and iii) a dedicated image analysis to determine how trafficking correlates with the geometry of the micropattern as a function of the knocked down genes. Such an approach is only possible on micropattern where adhesion can be completely controlled and modified at will.
The initial strategy was to conduct parallel development of i) the integrin assay with standard siRNA transfection and micropatterns, ii) integration of TCA with dedicated micropattern networks, and iii) image analysis/modeling then to combine all developments to conduct the PoP screen. However, due to delays in setting up the assay, difficulties in developing dedicated micropattern networks and lack of time at the end of the project, we decided to perform the integrin assay with standard transfection procedure on standard micropatterns to fully exploit all developments made on the assay (protocols and image analysis). Then, to address the PoP screen on TCA, we decided to take advantage of the development made in WP06 & WP03, selecting a micropattern network already compatible with TCA (WP06) and image analysis developed for cellular shape and actin readouts (WP03).
Integrin trafficking assay
After several rounds of protocol improvement, the integrin internalization assay has been established on micropatterns and combined with gene knock-down (6 genes). The effects of the selected genes on the efficiency of integrin internalization and its subcellular distribution were analyzed as a function of defined micropattern geometry. Preliminary results were obtained at the very end of the project and are still under investigation.
POP screen on TCA
A PoP screen was conducted at the very end of the project making use of 15 genes inducing nuclear and actin distribution phenotypes, for which image analysis has been developed in WP03 and WP06. Results should be obtained shortly after project end.
Conclusions:
Thanks to the excellent complementarity of consortium partner’s expertise, the MEHTRICS platform has achieved its major aims showing i) that the tight control of the microenvironment provided by the micropattern technology can not only improve assay performance but also enable the development of innovative and relevant assays for both industry and academic applications, ii) that the improvements of TCA technique will reduce the screening cost, iii) that delayed siRNA delivery will allow long term siRNA knock-down and iv) that image analysis and models will increase overall data quality and significance.
Potential Impact:
The foreseen impact of the project as described in the DOW is quoted below and then commented regarding MEHTRICS achievements.
« MEHTRICS focuses on the development of new approaches, cellular models and know-how for applications in academic research and pharmaceutical industry. Such innovative technologies aim at addressing common bottlenecks in the area of HT/HC cell-based screens and could therefore become a mainstream technology in a few years. To succeed in demonstrating the new capabilities of MEHTRICS platform and stimulate a wide adoption, the consortium will exploit the PoP screens and assays that support the different objectives of the project: 1) demonstrate a higher performance on standard assay, 2) develop new relevant assays and 3) streamline HT/HC cell-based screen in both academic and industrial context. The results from the PoP screens will be rapidly disseminated by the consortium to the global scientific community, to the public but also to specific target groups, such as key opinion leaders, pharmaceutical industry and biotech companies. By focusing on these aspects of dissemination, the consortium expects to increase the impact of the project and to strongly enhance a wide adoption of MEHTRICS technological advances which in turn will have both a direct and indirect effects on the global competitiveness of the Europe and the METHTRICS consortium partners. A direct effect will be obtained by the strengthening of all consortium partners acting as direct providers and developers for such technologies. Indeed, the new capabilities co-developed by CYTOO, CENIX and their academic partners will benefit to all members of the consortium through joint publications and the advancement of ongoing research programs, but also, through potential licensing of joint inventions that will be commercialized by CYTOO and CENIX. An indirect effect is also anticipated due to the fulfillment of the needs of first users of such technologies. For example, the technological development proposed here, is expected to drastically improve cell imaging processes which is up to now a considerable obstacle to a wider use of HCS in drug screening. As the availability of new class of approaches such as described here usually fosters new applications, an important source of value of the research is the potential usefulness to develop new approaches for the drug development and biotechnology field.
Indeed, since the cost of drug development increases exponentially from the discovery phase to the preclinical stage and even more significantly when entering clinical trial, the clear objectives of the pharmaceutical industry has been to improve the means of selecting the most efficient and the least toxic molecules as early as possible in the drug development process. For that purpose, pharmaceutical and biotechnology industries laboratories are progressively investing in novel technologies to advance drug discovery and development with a particular focused on cell based assays and HCS (High Content Screening) in particular. HCS that was essentially used in secondary screens only a few years ago, has now pushed both upstream into most primary screens but has also been recently integrated downstream in the drug development process in applications covering efficacy, compound profiling and predictive toxicology. However, HCS did not reach all expectations due mainly to the lack of relevant cellular models. One of the biggest steps of the project to reach the maximum impact will thus reside in the development and validation of at least one fully standardized relevant cellular model suitable for automation with ready-to-use analytical tool and instrumentation. To quote Hirschhaeuser et al. on the possible exploitation of micropattern technologies for the production of uniformly sized spheroid cultures. “Its further development (of micropatterning) into a culture platform or screening tool for cancer stem or tumor-initiating cells would be an ambitious venture requiring the expertise of various scientific disciplines. However, in light of recent literature which implies that sphere-formation is useful if not necessary to maintain and expand cells with tumor-initiating capability from various primary tumor entities, such devices could revolutionize anti-tumor screening operations and also provide a rationale for personalized therapeutic test strategies.”
In addition, the development foreseen in the MEHTRICS project will be an extraordinary basis to address other bottlenecks of the drug industry such as the establishment of pertinent cell-based assays for toxicity testing. Indeed, the reduced number of new marketed drugs and the increased number of withdrawals in the recent years are mostly due to toxicity reasons, which put pharmaceutical and biotechnology industries under high pressure to develop tools for the prediction of toxicity as early as possible in the development pipeline. Moreover, they have to adapt to new regulation, such as REACH (European Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals) entered into force on 1st June 2007, which aims to improve the protection of human health and the environment from the risks that can be posed by chemicals as well as to restrict toxicology testing on animals by promoting cell-based assays as an alternative. In this context, the innovative developments made through the MEHTRICS platform are of particular interest for Europe but also for the consortium partners, especially SMEs, which can envisage acquiring a leading advantage on the cell based assay market with unique solutions for the development of relevant models to the drug industry.
The proposed project offers a genuinely innovative opportunity to push beyond the limitations of current HT/HC genomic-driven research paradigms, offering the dual impact of significantly enhancing a wide range of existing approaches while at the same time opening up truly new “assay development space”. While our focus on such a novel paradigm inherently carries more risk than the re-hashing of established methods to merely fund a few new screens, we expect that our approach promises more meaningful, broadly-applicable and longer-lasting advances for the HT screening community. By focusing on enabling technologies, which have a potentially huge impact and where SMEs can play a fundamental role, this research project will give participating partners and Europe a leading edge and has the potential of increasing their competitiveness in the long term. Such a proposal is perfectly in line with the present SME-focused FP7-call, supporting the development of technological tools for HT and expertise by SMEs for improving the health of European citizens and increasing the competitiveness and boosting the innovative capacity of European health-related industries while addressing global health issues.
To achieve such transformational leadership, the consortium needs to rely on a synergy between key leaders in their respective fields. As leading institutes and companies are spread over Europe, the FP7 programme is the right framework to join these expertises and RTD resources for the establishment of outstanding innovations. In addition, the support of European RTD programmes for MEHTRICS is largely justified regarding the potential benefits for the European society and economy. In other words, this public funding will help the consortium to keep its technological advantage in terms of know-how while turning it into a competitive solution. Indeed, because the MEHTRICS consortium brings together the provider of new technologies with a service company that has access to potential end-users benefiting from the MEHTRICS platform developments, the results will directly lead to the commercialization of new products or services that will put the two European SMEs, CENIX and CYTOO, in stronger competitive situations versus existing players on the markets in Europe and world-wide. Since the technology development is strongly synergic between CYTOO and CENIX core business, the exploitation will follow a standard path of technology transfer into CENIX existing HT/HC-RNAi-based contract research offerings, creating an important added value thanks to the improvement of current assays performance and the extension of offers to more relevant cellular models. For CYTOO, micropatterned growth substrates for long term HT/HC siRNA screen or relevant cellular models as developed in the project will be directly available as a validated technology but most importantly should stimulate an increase demand on custom-designed micropatterns to adapt specificities of new assays or cell lines used by its customers. These new products and services have potential applications in a wide range of areas including large scale RNAi screens, fine study of drug effects as well as development of alternative drug testing methods. The foreseen commercialization of these new offers represents a strong potential for operational growth and new job creation at both CYTOO and CENIX. Thanks to the MEHTRICS project a mutually beneficial innovation synergy will be initiated, which should gain momentum and continue well after the end of this project. »
Comments:
Most of the foreseen impact described in the DOW is still valid. Indeed, as described in the above section “Main S&T results”, the MEHTRICS platform developed several new technologies, models and fully integrated assays by combining the expertise of each Consortium partner and demonstrated their performance thanks to robust data and PoP screens. These data and PoP screens supported our initial objectives: 1) demonstrate a higher performance on standard assays, 2) develop new relevant assays and 3) streamline HT/HC cell-based screen in both academic and industrial context. As most of the PoP screens were performed at the end of the project, dissemination is not yet fully engaged and real impact of these developments is not yet quantifiable. The consortium will therefore now focus on dissemination aspects to widely diffuse results of the MEHTRICS platform. Dissemination will include several publications, a patent protecting the development of delayed siRNA release and new products offer for CYTOO. These actions should in turn reinforce consortium members and EU competitiveness.
The MEHTRICS platforms considerably strengthen the position of consortium partners . Partner1/CYTOO developed new micropatterns, innovative assays and addressed critical points of its production process. In addition, thanks to the development in the different workpackages, not only the versatility of the technology, its compatibility with various applications and its ability to improve and/or enable innovative approach for both academia and industrial assays were clearly established but fully integrated assays are now available for its commercial offering. Thanks to the results generated during the first half of the project, CYTOO communicated on the MEHTRICS platform objectives, achievements and potential applications via conferences, email campaigns or face to face meetings with customers. This approach already generated products and service sales for CYTOO’s CRO activity. The latest development of the MEHTRICS platform will for sure reinforce its position as a leader in cell-based assay development, which should in turn generate additional incomes in the coming years. In addition to this direct impact, the technological development, but most importantly the fully integrated assays, clearly triggered a lot of interest from potential customers, which opened up new opportunities of developments for innovative assays on different cellular models.
Developments made by Partner4/UHEI_Schwarz put them at the forefront of quantitative analysis and modeling of cells on micropatterns in a high-throughput context and will result in 4-6 publications. Based on the accumulated expertise, Schwarz’s group will push the envelope of this exciting field and merge it with other state-of-the-art developments like evaluation of screens based on the CRISPR/Cas9 system or three-dimensional patterning of cells (combined with appropriate imaging methods like light-sheet microscopy). This will be conducted in collaboration with experimental groups, which puts Schwarz’s group in a unique position to achieve quantitative insight into the regulation of cells in health and disease.
Partner4/UHEI_Erfle successfully combined CYTOO’s micropatterns to Transfected Cell Arrays (TCAs) thanks to major development of the RNAi Screening Facility platform with implementation of improved high throughput printing technologies for spot restricted transfection in the spots of TCAs without crosscontamination. This valuable expertise combined to the possibility to offer experimentation and/or assay development on micropatterns is a true added value as it clearly enriches the portfolio of the RNAi Screening Facility at Heidelberg University. In addition, the very close collaboration established with Partner3/CPST during MEHTRICS will be followed up after MEHTRICS ends.
A joint UHEI/CPST publication is being prepared summarizing results of integrin internalization studies. Another paper describing the new nanfoil technology for controlled release of siRNA is planned by the end of 2015 by UHEI/CPST. Publication date will depend on the patentability search on the nanofoil technology currently performed. The CPST lab is currently leading a bi-lateral Swiss-Lithuanian project with the world-famous protein chemistry group of Prof. Kai Johnsson, EPFL. The project is dedicated to single-cell level analysis of metabolic activity of cells by combining engineered protein sensor and nanopattern technologies. However, among the possibilities the partners envision also to include in this work micro-scale control of the analyzed cells by the CYTOO pattern and network technologies, developed in MEHTRICS. The CPST has been active in enabling the access of research results to SMEs, among others by establishing dedicated open access facilities. One of these, BALTFAB (www.baltfab.com) is offering services and technologies related to unconventional fabrication, e.g. production of bio-active surfaces, patterns and micro/nanodevices. As a result of MEHTRICS, UHEI is already listed as a partner of the BALTFAB facility, thus offering the external partners a tandem platform by combining micro/nanofabrication with robotic printing, HTC and HC microscopy. As the BALTFAB platform is further improved and developed, there are also good possibilities to include other partners of MEHTRICS, based on mutual interests. In turn, BALTFAB is a member of a pan-Baltic virtual fabrication facility Technet_nano, a membership that will further ensure the visibility of MEHTRICS results in this region. Dr. Ramūnas Valiokas is a co-founder of a Vilnius-based international startup Ferentis UAB, a biomaterials company with the focus on 3D in vitro tissue models and regenerative medicine. There are good possibilities to further cooperate with UHEI, CYTOO and other partners of MEHTRICS, as micropattern technology already proved powerful in development of a new generation of implants for corneal surgery applications (see publication below).
Finally, Partner5/EPFL has been able to develop and perform a challenging live imaging screen for spindle positioning in human cells, which is a first in the field. Gönczy’s laboratory plans to utilize the 96-well plate format developed by CYTOO to complete targeted live imaging RNAi-based screens for spindle positioning genes, as well as continue the development and application of assays for monitoring centriole inheritance in human cells.
MEHTRICS has strongly shaped research agenda of all consortium partners for the last 3 years, it further reinforced partner’s leadership in their respective field but, most importantly, it allowed a true translational synergy between partner’s expertise, which enabled major progresses in HT/HC screening applications. Collaborations will be therefore prolonged far beyond the MEHTRICS project.
Conferences & Publications: All MEHTRICS partners presented results to several conferences and events. As expected, results were first presented in conferences, then published or will be published in journals, presenting a more complete description of models and results.
Conferences:
- Invited talk by U. Schwarz at World Congress of Biomechanics Boston USA July 6-12 2014 on cell shape on patterns
- Invited talk by U. Schwarz (UHEI) at Symposium Physics of Living Matter on cell shape on patterns, September 19th, 2014, Cambridge, UK
- P Gönczy (EPFL) Poster presentation at NCCR Chemical Biology Retreat; Villars, Switzerland, June 11-13, 2014
- Invited talk by R. Valiokas (CPST), Biopatterning techniques in tissue engineering, Gliwice Science Meeting, Gliwice, Poland, 21 November 2014
Published paper: Dynamics of cell shape and forces on micropatterned substrates predicted by a cellular Potts model. Albert PJ, Schwarz US. Biophys J. 2014 Jun 3;106(11):2340-52.
Planned dissemination activities beyond the project:
- Planned publication on multicellular ensembles on networks (CYTOO and UHEI - Schwarz)
- Publication on pattern design (UHEI – Schwarz)
- 2 planned publications UHEI – Schwartz group with Starkuviene on integrin trafficking (one biological, one biophysical)
- Invited talk by U. Schwarz (UHEI) at European CellMech Meeting Barcelona May 13, 2015 on cell shape on patterns
- Publication on Spindle positioning screen and follow-up analysis (P. Gönczy-EPFL)
- Publication on Identification of Cep41 as a stable centriolar protein (P. Gönczy – EPFL)
- Press release for MEHTRICS close up
List of Websites:
www.mehtrics.com
For any contact please contact: Sebastien DEGOT (sdegot@cytoo.com) - +33 4 38884746
The MEHTRICS project challenged the current limits of HT cell-based screening by combining HT-RNAi with an emerging new technology for normalizing cultured cell behaviors, namely the growth of cells on adhesive micropatterns. Initial applications of this technology had already demonstrated its potential for enhancing the quality of existing high content analyses by radically reducing the cell population’s phenotypic variability, resulting in much lower cell sampling requirements. This approach also promised to open up major new “assay development space” by broadening the range of analysis strategies that drive novel cell-based assay designs, whose evolution has otherwise stagnated in recent years. The consortium carried out several parallel and complementary lines of development to diversify the applicability of the MEHTRICS platform for both academic and industrial uses, ultimately validating the new capabilities through proof of principle screens.
Our key objectives were:
• to optimize micropattern geometries and compositions to accommodate extended timelines typical of siRNA assays,
• to integrate the promising Transfected Cell Array (TCA) technique to decrease cost and increase throughput,
• to develop novel cell models of key diseases based on micropatterned adult stem cells and polarized epithelia/endothelial architectures and
• to validate each of these implementations in key industry-relevant siRNA screening applications.
Thanks to the outstanding synergy stemming from the complementary expertise of the consortium partners, the MEHTRICS platform has achieved major accomplishments showing i) that the tight control of the microenvironment provided by the micropattern technology can not only improve assay performance but also enable the development of innovative and relevant assays for both industry and academic applications, ii) that the improvements of TCA technique will reduce the screening cost, iii) that long term siRNA knock-down should be achievable and iv) that image analysis and models will increase overall data quality and significance.
Since experimental designs requiring siRNA screens are among the most demanding of all HT/HC studies and encompass virtually all technical challenges also encountered in compound screens, we expect the envisioned scope of activities to deliver the maximal potential for impactful innovation, widespread adoption and clear relevance for all major applications of HT/HC cell screening. The resulting new tools and methodologies were incorporated into CYTOO commercial offerings. As the availability of new classes of approaches as described here usually fosters new applications, an important source of value of the accomplished research is the potential usefulness to develop new approaches for the drug development and fundamental research fields.
Project Context and Objectives:
Mammalian cell-based assays are powerful for basic studies of eukaryotic cell physiology and represent crucial models for the development of novel pharmacological compounds. The vast majority of HT/HC screens carried out today make use of adherent immortalized cancer cell lines growing on plastic or glass surfaces used either as such or instead evenly coated with a variety of possible extracellular matrix components. Since those cells adhere, spread and divide in a largely uncontrolled manner, the resulting high variability in the behavior of individual cells within each sample population is an inherent weakness of this experimental approach, as it requires the experimentalist to sample large enough cell numbers such that this variability can get normalized down to background “noise”. This is a basic obstacle in the optimization of nearly all HT assays using this standard paradigm, namely, the need to minimize sample variability to maximize assay windows. This difficulty is further compounded by the need to maximize analysis throughputs and overall cost-effectiveness in these studies, both of which push users towards higher-density plate formats (384-well, 1536-well…). These, in turn, allow fewer cells in each sample well, thereby potentially increasing assay variability by reducing sampling sizes for each treated cell population. This problem represents a basic impasse that plagues all large-scale HT cell-based studies, whether they be siRNA screens, pharmacological studies or combinations of both.
The development and application of precisely-controlled adhesive micropatterns as new growth substrates for large scale HT cell-based studies is now emerging as a breakthrough for addressing this problem. By yielding highly homogeneous “standardized” cell behaviors within each sample population, this approach has already been shown in initial feasibility studies to vastly reduce assay noise levels and sampling sizes. The direct implications are clear in promising higher quality datasets through a broader screening window and higher statistical relevance, all with higher throughputs, and therefore better cost-efficiency overall. These observations provide a very encouraging basis on which this project will build further by first determining and characterizing the degree to which this new technology can similarly enhance a hopefully-broad range of other HT/HC applications. In fact, the main limitation of this technology today resides in micropattern sizes and geometries that do not maintain cell normalization after the first division. Thus, as a key element of the proposed MEHTRICS development, combining a network of micropatterns standardizing cells over several divisions with unlimited design possibilities will drastically reduce cell variability in a much broader range of HT/HC assays.
Beyond such enhancements of existing assays, the MEHTRICS platform is also expected to open up brand new “assay development space”. Thanks to the high level of control afforded by the ability to finely modulate all functional aspects of the micropatterns, combined with the inherent ability to characterize the phenotypic fate of individual cells in both live and endpoint assays, this will enable a new range of HT/HC assay strategies that have not been possible until now. Of particular interest are more advanced disease models using primary cells, including adult stem cells, which can maximize patho-physiological relevance but are often not available in adequate supply to enable large-scale screens. The smaller sampling sizes required on adhesive micropatterns promises to overcome the limited cellular scale-up that has hampered these models. In addition, the tight control of adhesion areas introduces a spatial reference frame against which cells not only organize but also differentiate. Incorporating CYTOO’s technology in cell-based assays promises therefore a tighter control over cell physiology in culture methods. Such technological development would represent a veritable quantum leap for the field and a broad applicability in HT cell-based screening in general.
In addition, the synergistic combination of micropattern technology with TCA is a promising niche to develop siRNA cell based assays with lower costs, faster data collection and improved comparability between assays or platforms. Indeed, by bringing down the cell number to analyze, the siRNA spot size can be reduced, resulting in an increased sample number per cell array. The complete genome could then be located on 1-3 single cell arrays, which is up to 150 times less than what is currently needed for full genome screening today. In addition, high sample densities per field of view of a microscope objective will allow a fast, parallel microscopic readout after stimuli of many samples using different approaches such as fluorescence live-time imaging (FLIM) or fluorescence correlation spectroscopy (FCS). Another expected benefit is a higher concordance of siRNA experiments done on different platforms, due to the combination of exact siRNA array replicates provided by TCA and standardization of cell behavior achieved on micropatterns. Finally, the implementation of a progressive siRNA release system will avoid dilution of active molecules after each cell cycle, which is a completely new approach that promises longer durations of siRNA silencing. Such improvements of the TCA technique will open exciting new classes of parallel and combinatorial assays, and in addition, will make siRNA screens accessible to a wider range of academic users.
Key objectives
Our key objectives were:
• To optimize micropattern geometries and compositions to accommodate extended timelines typical of siRNA assays,
• To explore novel cell models of key diseases based on micropatterned adult stem cells and polarized epithelia/endothelial architectures and
• To develop modeling approach as well as automated tools for enhancing image processing and data analysis
• To combine micropattern technology with the Transfected Cell Array (TCA) technique to decrease cost and increase throughput,
• To develop novel methods for delayed or triggered siRNA release
• To optimize MEHTRICS platform for both academic and industry-relevant siRNA screening applications
• To validate each of these implementations thanks to Proof of principle screen.
Project Results:
The main results achieved during the MEHTRICs project are the following:
- Successful development of micropattern networks for long term siRNA
- Successful development of micropatterns for new cellular models including epithelial cells, endothelial cells, stem cell, cardiomyocytes, hepatocytes and spindle positioning assay
- Easy integration of micropattern technology into partner’s workflow
- Development of assays relevant to industry and academia
- Efficient combination of micropattern technology with the Transfected Cell array approach
- Improvement of TCA including spot size reduction, increased spot density and absence of cross-contamination
- Proof of concept for triggered siRNA release at will
- Development of nanofoil approach for delayed siRNA delivery
- Establishment of mathematical models predicting cell behavior on micropatterns networks and integrin trafficking
- Implementation of automated image and data analysis tools
- Proof of concept screens: oncology, inflammation, stem cell differentiation, spindle pole positioning, integrin trafficking
- Demonstration of micropattern benefits for different applications (academic and industry)
- Dissemination of project results through posters, banners, press releases, talks, publications and marketing materials
- Integration of micropattern networks into CYTOO product offer with associated revenues
- Protection of foreground: The work performed in WP02 on micropattern networks has been protected through patent filing (Patent #: EP 13305119.3 filed by CYTOO on 01/02/2013). IP protection on a newly developed “Migration assay” has been abounded after several rounds of Review. The newly developed nanofoil approach for delayed siRNA should be protected in the coming months after MEHTRICS end (planned for May 2015).
Note that three PoP screens as well as further developments of image analysis tools, modeling approach and delayed siRNA were performed during the last four months of the project. The extension was therefore critical to achieve major results for the project.
A brief description of the main achievements is given below.
Micropatterns Development
Partner1/CYTOO designed and produced different types of micropattern adapted to assays and readouts in each workpackage:
- Standard geometries were used for the development and evaluation of integrin trafficking (HeLa, WP06) and spindle positioning (HeLa, WP05) assays
- Interconnected micropatterns networks were achieved and used to develop ciliogenesis assay (WP02), oncology assay (WP05), LDL uptake (HepaRG, WP05), stem cell differentiation (ADSCs, WP05)
- Structuring and differentiating micropatterns were created for hypertrophy (rat primary cardiomyocytes, WP05), inflammation assay (NHBE, WP05), endothelial tube formation (HUVEC, WP02), centriolar inheritance (HeLa, WP05), migration (MCF7 and MDA-MB-231, WP02) and stem cell cell differentiation (ADSCs, WP05).
Collaboration with the different partners went well with loops of design improvement that allowed major progresses on the different applications. As initially planned, a modeling approach was used to create new designs. Micropatterns networks were successfully developed and enabled single cell approaches for longer time period and combination with siRNA screening. In addition, new micropatterns were obtained and allowed accessing more relevant multicellular models and approaches.
CYTOO produced and delivered all the required materials for the different partners to perform the work planned. A problem with residual glue toxicity in CYTOOplates was observed but only delayed some applications (inflammation and endothelial tube formation) for which solutions were found too late in the project to plan new developments.
Assay Development
Results from the different workpackages showed that all the assays, protocol, cell lines and type of readouts could be developed on micropatterns. Micropattern performance was evaluated for each applications and showed different level of success:
- Not achievable as planned: centriole inheritance and integrin trafficking could not be developed on micropattern networks but alternative solutions on standard micropatterns were identified
- Similar level of performance: LDL uptake and oncology assays
- Improved performance: inflammation assay with H292, osteogenesis differentiation and hypertrophy
- Enabling: spindle positioning assay, which could not be performed on standard culture condition, was successfully developed on micropatterns
Overall, the different assays demonstrated that micropatterns technology is highly versatile and can be adapted to the development of very different applications, cell lines and readouts.
Image processing, data analysis and modeling Development
Image processing and analysis tools were an important part of the developments made during the project. Indeed, because micropatterned cells possess specific features, dedicated tools (image processing and analysis algorithms geared towards the organization of the cytoskeleton and the adhesions) were required to fully exploit the new capabilities of the MEHTRICS platform. In addition, we aimed to rationalize or guide assay design and developments by mathematical modeling approaches. Different tools and models have been successfully developed during the project, including:
- Image processing tool (used in WP05 and WP06)
- Single cell shape model (used in WP05 and WP06, published)
- Multi-scale model for integrin trafficking on micropatterns (used in WP06)
- Cell community model predicting the effect of micropatterning design for multicellular ensembles (used in WP02)
TCA development
For combining micropattern with TCA, Partner4/UHEI-Erfle developed improved high throughput printing technologies and fully automated workarounds for high density cell array applications on micropatterns. The work performed included i) tests on the composition of the transfection mixture, ii) optimization of the printing techniques for siRNA delivery into spots of TCA, iii) reduction of spot size and iv) increased spot density. Subsequently, a protocol for the solid phase reverse transfection of siRNAs on interconnected micropattern networks and in spots of TCAs has been established. Solid phase transfection mixture contact printing on CYTOO micropatterns did not interfere with their adhesive properties. Cross contamination effects were eliminated by improving the printing protocols. To confirm and validate these developments, siRNAs were used to demonstrate efficient delivery of the siRNA into cells on micropatterns as well as loss of function phenotype without cross contamination problems. To carefully evaluate these parameters, a dedicated automated image analysis in the Konstanz Information Miner (KNIME) was developed. In conclusion, high throughput printing technologies and fully automated workflows for high density cell array applications on micropatterns were successfully achieved.
Time-resolved siRNA release
Downregulation of gene products with low turnover have been difficult to address with siRNA screens up to now due to the progressive dilution of active molecules over several cell cycles. Therefore, we planned to develop new methods allowing either a delayed or a triggered siRNA delivery for longer durations of siRNA silencing.
Triggered siRNA delivery
To assemble molecular machineries for triggered siRNA delivery, we explored siRNA immobilization via photocleavable groups. Preliminary protocols for fabrication of a molecular film that contains a photolabile linker have been developed by Partner3/CPST. Results confirmed the feasibility of the “release-on-demand” concept. However, we could not find conditions where the cleavage was compatible with cell culture conditions. As modifications of such chemistry would have been too long for the project duration, our efforts had focused on the second approach (delayed siRNA release).
Delayed siRNA release
For this approach, we have developed a method for stabilization of cell transfection reagents printed by standard TCA fabrication technologies, e.g. pin arraying. It is based on application of a nanometer-thick layer of PEG MA hydrogel-fibronectin composite onto the transfection mixture spots. Our results demonstrated that:
1. The nanofoil itself could form cell-adhesive micropatterns, defined by the surface topography of the PDMS stamp.
2. The nanofoil is not toxic for HeLa cells.
3. The removal of the nanofoil-covered transfection mixture spots from the surface started in 3 hours of HeLa cell culture, with material release occurring after 11 hours from the cell seeding for approximately 30hours.
4. siRNA was efficiently internalized into the HeLa cells.
These results clearly showed that the method opens perspectives for true surface-supported, prolonged siRNA transfection technologies in TCA format, over periods longer than 48 h. Also, it possibly could be applied for controlled drug release and encapsulation. Additional developments are needed for ultimate integration of the method with the CYTOO platform. This method will be patented soon.
POP screen
Our overall strategy was to carry out several parallel and complementary lines of development, each geared towards enabling a subset of high-value HT/HC-RNAi applications. As initially planned, the project was staged, including exploratory phases with decision points narrowing down on successful applications, for which resulting new capabilities were demonstrated through Proof of Principle (PoP) screens. Our key objectives and results are the following :
1) Optimization of MEHTRICS platform to enhance existing industry-relevant siRNA screening applications
The potential benefits of micropattern technology have been evaluated in two established industrial assay setups by Partner2/CENIX:
• A microscopy-based oncology related assay, representing a multiplex high content assay, and
• An ELISA-based inflammation assay, representing a single-readout plate reader assay.
For each assay, critical parameters, including performance of controls, screening window, variability, performance (Z’-Factor) and hit selection were assessed.
Results for Oncology assay
Two cell lines, the colon cancer cell line HCT116 and the breast cancer line MDA-MB-231, were chosen for the PoP screen comparing tiremark network micropattern and non-patterned plates. 29 genes shown to have influence on cell health and/or viability were chosen for this assay based. Although the optimization had hinted that tiremark micropattern might yield an improvement of the oncology assay, statistical analysis of the screening scenario did not show significant improvement over non-patterned plates.
Results for Inflammation assay
In the inflammation assay, the response of the lung epithelial cell line H292 to TNFalpha (tumor necrosis factor alpha) and treatment with steroid and interferon gamma was studied. 84 genes were selected and screened in parallel on homogeneously coated and diamond micropattern CYTOOplates. In summary, the inflammation assay with single parameter plate reader readout shows impressive improvement on micropatterned plates. Assay variability is significantly lower resulting in good Z’-factors for the data set (0.3 - 0.5) and eventually in higher confidence in hit selection.
2) Application of MEHTRICS platform to create relevant cellular models for industrial application
Beyond the enhancement of existing assays, the increased control of microenvironment provided by adhesive micropatterns opened up the potential for developing new models and assays of disease-relevant processes. The MEHTRICS platform was therefore used to drive the development of new patho-physiologically predictive models from epithelial cells, endothelial cells stem cells derived from adipose tissue, hepatocytes and cardiomyocytes. Our strategy was to carried out parallel development of these models and to select the most promising to conduct a PoP screen. 5 models were developed by Partner1/CYTOO and Partner2/CENIX:
• Stem cell differentiation assay: We established adipogenisis, osteogenesis as well as "mixed cue" high throughput RNAi assays in 96well format on micropattern plates using adipose tissue derived adult human stem cells (ADSCs). Adipogenesis alone was not improved on micropattern compared to non-patterned plates. Osteogenesis, in contrast, was boosted up to 10fold on micropatterns compared to standard culture condition and showed superior detection of siRNA knockdown. A micropattern design allowing mixed a differentiation approach and resulting in higher percentage of differentiated cells was also identified.
• Inflammation assay in primary human bronchoepithelial cells could not be established on time as the cells showed unforeseen sensitivity towards the micropattern plates (residual glue toxicity) and this problem was only solved at the end of the project.
• LDL-uptake assay was developed with HepaRG cells. Condition of culture, polarization and LDL-uptake were set up on micropatterns, however RNAi screening with few genes did not show significant advantage over cell growth on non-patterned plates.
• Hypertrophy assay was developed with primary rat neonatal cardiomyocytes. Rectangular micropatterns showed promising performance for a microscopy based hypertrophy assay in primary neonatal rat cardiomyocytes.
• Tube formation assay was initiated with primary endothelial cells (HUVEC). A preliminary protocol has been established but further developments were delayed due to residual glue toxicity problem, which was only solved at the end of the project.
As the osteogenesis assay was the most advanced and the most promising in terms of benefits, we decided to perform a Proof-of-Principle screen for osteogenesis using L-stairs micropattern networks.
Unfortunately, in November 2014 Cenix BioScience was forced to file for insolvency. The PoP screen could therefore not be conducted at the anticipated level in terms of extent and depth of analysis. However, data of a mini-screen (10 genes) were generated. Differentiation was assessed by microscopy using a fluorescent substrate for Alkaline Phosphatase (induced during differentiation), ELF97. Analysis of the results showed:
- Osteogenic differentiation is boosted 5 fold on L-stairs micropattern networks compared to cell growth on uniform substrate
- The assay window is increased 2-3 fold on L-stairs micropatterns. Consequently, a graduation or dosing of the effects of individual genes is more readily assessable on micropattern
In lack of in depth analysis, the evaluation remains incomplete, however, consistent superior performance of micropatterns throughout multiple rounds of assay optimization suggests that we have developed a new, more powerful screening assay for osteogenic differentiation.
3) Optimization of MEHTRICS platform to enhance hit validation and classification in siRNA functional genomic screens
Thanks to the development of novel assays during the project, two PoP screens were planned to address wether the MEHTRICS platform can enhance functional genomic studies:
- Spindle positioning screen of approximately 1000 genes
- Centriolar inheritance screen of approximately 50 genes
Results for Spindle positionning screen
Partner5/EPFL first optimized assay conditions for the spindle positioning siRNA-based screen of fixed specimens disposed in CYTOO’s 96-well plates harboring L shapes and standard plates uniformly coated with fibronectin. Results indicated that neither method is robust enough to conduct a high throughput screen. Therefore, a live imaging approach, only possible on CYTOO’s 96-well plates, was explored. After several round of protocol optimization and improvement of micropattern design, this assay was successfully developed and allowed performing a Proof of Principle screen of a 1200-membered kinase/phosphatase siRNA library.
Until the end of the MEHTRICS project, 7 out of 16 screening plates could be analysed using the custom algorithm yielding 13 candidate genes, among which genes already implicated in spindle positioning as well as unknown genes could be identified.
In conclusion, taking advantage of CYTOO’s micropattern technology, we were able to develop and perform a challenging live imaging screen for spindle positioning in human cells, a first in the field. Since the fibronectin coating of 96-well plates is not achievable to date with sufficient quality for such a sensitive assay, we come to the conclusion that CYTOO’s micropattern technology is clearly superior for high throughput spindle positioning assays in human cells.
Results for Centriolar inheritance screen
Tools important for studies of centriole inheritance in human cells (a centriolar protein that is detectable at centrioles for multiple cell cycles in human cells) have been developed. Unfortunately, however, despite advances in development of a micropattern network suited to study centriole inheritance, we were not yet able to develop a network that would fully meet all requirements for this assay. The PoP screen was therefore not performed.
4) Integration of MEHTRICS platform with Transfected Cell Array (TCA) technology
As described above, thanks to several technical improvements integration of micropatterns into the TCA approach has been successfully achieved. To validate the benefits of this new platform, it was initially planned to run a PoP screen focusing on integrin trafficking, which requires i) the development of dedicated micropattern networks, ii) the adaptation of the integrin trafficking protocol and iii) a dedicated image analysis to determine how trafficking correlates with the geometry of the micropattern as a function of the knocked down genes. Such an approach is only possible on micropattern where adhesion can be completely controlled and modified at will.
The initial strategy was to conduct parallel development of i) the integrin assay with standard siRNA transfection and micropatterns, ii) integration of TCA with dedicated micropattern networks, and iii) image analysis/modeling then to combine all developments to conduct the PoP screen. However, due to delays in setting up the assay, difficulties in developing dedicated micropattern networks and lack of time at the end of the project, we decided to perform the integrin assay with standard transfection procedure on standard micropatterns to fully exploit all developments made on the assay (protocols and image analysis). Then, to address the PoP screen on TCA, we decided to take advantage of the development made in WP06 & WP03, selecting a micropattern network already compatible with TCA (WP06) and image analysis developed for cellular shape and actin readouts (WP03).
Integrin trafficking assay
After several rounds of protocol improvement, the integrin internalization assay has been established on micropatterns and combined with gene knock-down (6 genes). The effects of the selected genes on the efficiency of integrin internalization and its subcellular distribution were analyzed as a function of defined micropattern geometry. Preliminary results were obtained at the very end of the project and are still under investigation.
POP screen on TCA
A PoP screen was conducted at the very end of the project making use of 15 genes inducing nuclear and actin distribution phenotypes, for which image analysis has been developed in WP03 and WP06. Results should be obtained shortly after project end.
Conclusions:
Thanks to the excellent complementarity of consortium partner’s expertise, the MEHTRICS platform has achieved its major aims showing i) that the tight control of the microenvironment provided by the micropattern technology can not only improve assay performance but also enable the development of innovative and relevant assays for both industry and academic applications, ii) that the improvements of TCA technique will reduce the screening cost, iii) that delayed siRNA delivery will allow long term siRNA knock-down and iv) that image analysis and models will increase overall data quality and significance.
Potential Impact:
The foreseen impact of the project as described in the DOW is quoted below and then commented regarding MEHTRICS achievements.
« MEHTRICS focuses on the development of new approaches, cellular models and know-how for applications in academic research and pharmaceutical industry. Such innovative technologies aim at addressing common bottlenecks in the area of HT/HC cell-based screens and could therefore become a mainstream technology in a few years. To succeed in demonstrating the new capabilities of MEHTRICS platform and stimulate a wide adoption, the consortium will exploit the PoP screens and assays that support the different objectives of the project: 1) demonstrate a higher performance on standard assay, 2) develop new relevant assays and 3) streamline HT/HC cell-based screen in both academic and industrial context. The results from the PoP screens will be rapidly disseminated by the consortium to the global scientific community, to the public but also to specific target groups, such as key opinion leaders, pharmaceutical industry and biotech companies. By focusing on these aspects of dissemination, the consortium expects to increase the impact of the project and to strongly enhance a wide adoption of MEHTRICS technological advances which in turn will have both a direct and indirect effects on the global competitiveness of the Europe and the METHTRICS consortium partners. A direct effect will be obtained by the strengthening of all consortium partners acting as direct providers and developers for such technologies. Indeed, the new capabilities co-developed by CYTOO, CENIX and their academic partners will benefit to all members of the consortium through joint publications and the advancement of ongoing research programs, but also, through potential licensing of joint inventions that will be commercialized by CYTOO and CENIX. An indirect effect is also anticipated due to the fulfillment of the needs of first users of such technologies. For example, the technological development proposed here, is expected to drastically improve cell imaging processes which is up to now a considerable obstacle to a wider use of HCS in drug screening. As the availability of new class of approaches such as described here usually fosters new applications, an important source of value of the research is the potential usefulness to develop new approaches for the drug development and biotechnology field.
Indeed, since the cost of drug development increases exponentially from the discovery phase to the preclinical stage and even more significantly when entering clinical trial, the clear objectives of the pharmaceutical industry has been to improve the means of selecting the most efficient and the least toxic molecules as early as possible in the drug development process. For that purpose, pharmaceutical and biotechnology industries laboratories are progressively investing in novel technologies to advance drug discovery and development with a particular focused on cell based assays and HCS (High Content Screening) in particular. HCS that was essentially used in secondary screens only a few years ago, has now pushed both upstream into most primary screens but has also been recently integrated downstream in the drug development process in applications covering efficacy, compound profiling and predictive toxicology. However, HCS did not reach all expectations due mainly to the lack of relevant cellular models. One of the biggest steps of the project to reach the maximum impact will thus reside in the development and validation of at least one fully standardized relevant cellular model suitable for automation with ready-to-use analytical tool and instrumentation. To quote Hirschhaeuser et al. on the possible exploitation of micropattern technologies for the production of uniformly sized spheroid cultures. “Its further development (of micropatterning) into a culture platform or screening tool for cancer stem or tumor-initiating cells would be an ambitious venture requiring the expertise of various scientific disciplines. However, in light of recent literature which implies that sphere-formation is useful if not necessary to maintain and expand cells with tumor-initiating capability from various primary tumor entities, such devices could revolutionize anti-tumor screening operations and also provide a rationale for personalized therapeutic test strategies.”
In addition, the development foreseen in the MEHTRICS project will be an extraordinary basis to address other bottlenecks of the drug industry such as the establishment of pertinent cell-based assays for toxicity testing. Indeed, the reduced number of new marketed drugs and the increased number of withdrawals in the recent years are mostly due to toxicity reasons, which put pharmaceutical and biotechnology industries under high pressure to develop tools for the prediction of toxicity as early as possible in the development pipeline. Moreover, they have to adapt to new regulation, such as REACH (European Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals) entered into force on 1st June 2007, which aims to improve the protection of human health and the environment from the risks that can be posed by chemicals as well as to restrict toxicology testing on animals by promoting cell-based assays as an alternative. In this context, the innovative developments made through the MEHTRICS platform are of particular interest for Europe but also for the consortium partners, especially SMEs, which can envisage acquiring a leading advantage on the cell based assay market with unique solutions for the development of relevant models to the drug industry.
The proposed project offers a genuinely innovative opportunity to push beyond the limitations of current HT/HC genomic-driven research paradigms, offering the dual impact of significantly enhancing a wide range of existing approaches while at the same time opening up truly new “assay development space”. While our focus on such a novel paradigm inherently carries more risk than the re-hashing of established methods to merely fund a few new screens, we expect that our approach promises more meaningful, broadly-applicable and longer-lasting advances for the HT screening community. By focusing on enabling technologies, which have a potentially huge impact and where SMEs can play a fundamental role, this research project will give participating partners and Europe a leading edge and has the potential of increasing their competitiveness in the long term. Such a proposal is perfectly in line with the present SME-focused FP7-call, supporting the development of technological tools for HT and expertise by SMEs for improving the health of European citizens and increasing the competitiveness and boosting the innovative capacity of European health-related industries while addressing global health issues.
To achieve such transformational leadership, the consortium needs to rely on a synergy between key leaders in their respective fields. As leading institutes and companies are spread over Europe, the FP7 programme is the right framework to join these expertises and RTD resources for the establishment of outstanding innovations. In addition, the support of European RTD programmes for MEHTRICS is largely justified regarding the potential benefits for the European society and economy. In other words, this public funding will help the consortium to keep its technological advantage in terms of know-how while turning it into a competitive solution. Indeed, because the MEHTRICS consortium brings together the provider of new technologies with a service company that has access to potential end-users benefiting from the MEHTRICS platform developments, the results will directly lead to the commercialization of new products or services that will put the two European SMEs, CENIX and CYTOO, in stronger competitive situations versus existing players on the markets in Europe and world-wide. Since the technology development is strongly synergic between CYTOO and CENIX core business, the exploitation will follow a standard path of technology transfer into CENIX existing HT/HC-RNAi-based contract research offerings, creating an important added value thanks to the improvement of current assays performance and the extension of offers to more relevant cellular models. For CYTOO, micropatterned growth substrates for long term HT/HC siRNA screen or relevant cellular models as developed in the project will be directly available as a validated technology but most importantly should stimulate an increase demand on custom-designed micropatterns to adapt specificities of new assays or cell lines used by its customers. These new products and services have potential applications in a wide range of areas including large scale RNAi screens, fine study of drug effects as well as development of alternative drug testing methods. The foreseen commercialization of these new offers represents a strong potential for operational growth and new job creation at both CYTOO and CENIX. Thanks to the MEHTRICS project a mutually beneficial innovation synergy will be initiated, which should gain momentum and continue well after the end of this project. »
Comments:
Most of the foreseen impact described in the DOW is still valid. Indeed, as described in the above section “Main S&T results”, the MEHTRICS platform developed several new technologies, models and fully integrated assays by combining the expertise of each Consortium partner and demonstrated their performance thanks to robust data and PoP screens. These data and PoP screens supported our initial objectives: 1) demonstrate a higher performance on standard assays, 2) develop new relevant assays and 3) streamline HT/HC cell-based screen in both academic and industrial context. As most of the PoP screens were performed at the end of the project, dissemination is not yet fully engaged and real impact of these developments is not yet quantifiable. The consortium will therefore now focus on dissemination aspects to widely diffuse results of the MEHTRICS platform. Dissemination will include several publications, a patent protecting the development of delayed siRNA release and new products offer for CYTOO. These actions should in turn reinforce consortium members and EU competitiveness.
The MEHTRICS platforms considerably strengthen the position of consortium partners . Partner1/CYTOO developed new micropatterns, innovative assays and addressed critical points of its production process. In addition, thanks to the development in the different workpackages, not only the versatility of the technology, its compatibility with various applications and its ability to improve and/or enable innovative approach for both academia and industrial assays were clearly established but fully integrated assays are now available for its commercial offering. Thanks to the results generated during the first half of the project, CYTOO communicated on the MEHTRICS platform objectives, achievements and potential applications via conferences, email campaigns or face to face meetings with customers. This approach already generated products and service sales for CYTOO’s CRO activity. The latest development of the MEHTRICS platform will for sure reinforce its position as a leader in cell-based assay development, which should in turn generate additional incomes in the coming years. In addition to this direct impact, the technological development, but most importantly the fully integrated assays, clearly triggered a lot of interest from potential customers, which opened up new opportunities of developments for innovative assays on different cellular models.
Developments made by Partner4/UHEI_Schwarz put them at the forefront of quantitative analysis and modeling of cells on micropatterns in a high-throughput context and will result in 4-6 publications. Based on the accumulated expertise, Schwarz’s group will push the envelope of this exciting field and merge it with other state-of-the-art developments like evaluation of screens based on the CRISPR/Cas9 system or three-dimensional patterning of cells (combined with appropriate imaging methods like light-sheet microscopy). This will be conducted in collaboration with experimental groups, which puts Schwarz’s group in a unique position to achieve quantitative insight into the regulation of cells in health and disease.
Partner4/UHEI_Erfle successfully combined CYTOO’s micropatterns to Transfected Cell Arrays (TCAs) thanks to major development of the RNAi Screening Facility platform with implementation of improved high throughput printing technologies for spot restricted transfection in the spots of TCAs without crosscontamination. This valuable expertise combined to the possibility to offer experimentation and/or assay development on micropatterns is a true added value as it clearly enriches the portfolio of the RNAi Screening Facility at Heidelberg University. In addition, the very close collaboration established with Partner3/CPST during MEHTRICS will be followed up after MEHTRICS ends.
A joint UHEI/CPST publication is being prepared summarizing results of integrin internalization studies. Another paper describing the new nanfoil technology for controlled release of siRNA is planned by the end of 2015 by UHEI/CPST. Publication date will depend on the patentability search on the nanofoil technology currently performed. The CPST lab is currently leading a bi-lateral Swiss-Lithuanian project with the world-famous protein chemistry group of Prof. Kai Johnsson, EPFL. The project is dedicated to single-cell level analysis of metabolic activity of cells by combining engineered protein sensor and nanopattern technologies. However, among the possibilities the partners envision also to include in this work micro-scale control of the analyzed cells by the CYTOO pattern and network technologies, developed in MEHTRICS. The CPST has been active in enabling the access of research results to SMEs, among others by establishing dedicated open access facilities. One of these, BALTFAB (www.baltfab.com) is offering services and technologies related to unconventional fabrication, e.g. production of bio-active surfaces, patterns and micro/nanodevices. As a result of MEHTRICS, UHEI is already listed as a partner of the BALTFAB facility, thus offering the external partners a tandem platform by combining micro/nanofabrication with robotic printing, HTC and HC microscopy. As the BALTFAB platform is further improved and developed, there are also good possibilities to include other partners of MEHTRICS, based on mutual interests. In turn, BALTFAB is a member of a pan-Baltic virtual fabrication facility Technet_nano, a membership that will further ensure the visibility of MEHTRICS results in this region. Dr. Ramūnas Valiokas is a co-founder of a Vilnius-based international startup Ferentis UAB, a biomaterials company with the focus on 3D in vitro tissue models and regenerative medicine. There are good possibilities to further cooperate with UHEI, CYTOO and other partners of MEHTRICS, as micropattern technology already proved powerful in development of a new generation of implants for corneal surgery applications (see publication below).
Finally, Partner5/EPFL has been able to develop and perform a challenging live imaging screen for spindle positioning in human cells, which is a first in the field. Gönczy’s laboratory plans to utilize the 96-well plate format developed by CYTOO to complete targeted live imaging RNAi-based screens for spindle positioning genes, as well as continue the development and application of assays for monitoring centriole inheritance in human cells.
MEHTRICS has strongly shaped research agenda of all consortium partners for the last 3 years, it further reinforced partner’s leadership in their respective field but, most importantly, it allowed a true translational synergy between partner’s expertise, which enabled major progresses in HT/HC screening applications. Collaborations will be therefore prolonged far beyond the MEHTRICS project.
Conferences & Publications: All MEHTRICS partners presented results to several conferences and events. As expected, results were first presented in conferences, then published or will be published in journals, presenting a more complete description of models and results.
Conferences:
- Invited talk by U. Schwarz at World Congress of Biomechanics Boston USA July 6-12 2014 on cell shape on patterns
- Invited talk by U. Schwarz (UHEI) at Symposium Physics of Living Matter on cell shape on patterns, September 19th, 2014, Cambridge, UK
- P Gönczy (EPFL) Poster presentation at NCCR Chemical Biology Retreat; Villars, Switzerland, June 11-13, 2014
- Invited talk by R. Valiokas (CPST), Biopatterning techniques in tissue engineering, Gliwice Science Meeting, Gliwice, Poland, 21 November 2014
Published paper: Dynamics of cell shape and forces on micropatterned substrates predicted by a cellular Potts model. Albert PJ, Schwarz US. Biophys J. 2014 Jun 3;106(11):2340-52.
Planned dissemination activities beyond the project:
- Planned publication on multicellular ensembles on networks (CYTOO and UHEI - Schwarz)
- Publication on pattern design (UHEI – Schwarz)
- 2 planned publications UHEI – Schwartz group with Starkuviene on integrin trafficking (one biological, one biophysical)
- Invited talk by U. Schwarz (UHEI) at European CellMech Meeting Barcelona May 13, 2015 on cell shape on patterns
- Publication on Spindle positioning screen and follow-up analysis (P. Gönczy-EPFL)
- Publication on Identification of Cep41 as a stable centriolar protein (P. Gönczy – EPFL)
- Press release for MEHTRICS close up
List of Websites:
www.mehtrics.com
For any contact please contact: Sebastien DEGOT (sdegot@cytoo.com) - +33 4 38884746