Microfluidic systems handle and manipulate tiny amounts of fluids at volumes thousands times smaller than a tear drop. The goals of our proposal are (i) to design, fabricate and test a microfluidic technology platform (i.e., hESC-Chips) for performing human embryonic stem cell (hESC) culture and assay with significantly improved efficiency, (ii) to utilize these hESC-Chips in search of new culture conditions for hESC self renewal and (iii) to disseminate this technology for routine use in other hESC laboratories. Compared to those macroscopic setting employed for the conventional hESC research, the advantages of the proposed hESC-Chips are low sample/reagent consumption, fast processing, precise control over physical/chemical environments and automated operation. As the proposed project unfolds, we anticipate having contributions on the following three aspects: First, the successful demonstration of the proposed hESC-Chips will provide a powerful technology for contemporary hESC research. In our design, a hESC-Chip will contain 100 culture chambers for a simultaneous examination of 100 hESC experiments. Since the sizes of these hESC culture chambers are very small, the consumptions of hESC samples and the associated reagents will be significantly reduced (4 to 5 orders of magnitude lower than the conventional setting). In addition, due to the use of PC-operated interface, critical parameters for hESC experiments can be monitored and controlled in the hESC-Chips with superior precision, which is unattainable using the conventional macroscopic setting. Second, the hESC-Chips promise to accelerate the screening process in search of animal product-free culture conditions for hESC self renewal and differentiation. Generally, hESC culture requires feeders, e.g., mouse embryonic fibroblasts (mEFs) or Matrigel, and mEF-conditioned medium to maintain hESCs at the undifferentiated stage. These culture conditions lead to contamination of animal products, thus restrict the therapeutic applications of hESCs in clinic settings. Right now, it is critical to define animal product-free hESC culture conditions by screening a large number of combinations of extracellular matrices and soluble factors. Using the conventional hESC research setting this screening process is time and cost-consuming. Third, we will disseminate this microfluidic technology for routine use in other hESC laboratories. A user friendly version of hESC-Chips will be developed and then tested in the co-PI’s research group and other hESC groups at UCLA. The final versions of the chip design and control programs will be freely available for download from the PI’s research group web site (http://labs.pharmacology.ucla.edu/tsenglab/), where some existing designs and programs can be accessed.
Statement of Benefit to California:
As the proposed project unfolds, we anticipate will benefit the State of California and it citizens on five fronts: 1. The successful demonstration of the proposed microfluidic platform (i.e., hESC-Chips) will present a new technology for far-reaching application to all types of stem cell research with significantly improved operation efficiency. In contrast, the conventional stem cell research is plagued by the use of macroscopic setting, resulting in several constraints, e.g., high sample/reagent consumption, poor precision to control the environments of hESC experiments and the lack of integrated platforms for accurate measurements. 2. Using the proposed hESC-Chips the costs for hESC research will be greatly reduced. We estimate that each hESC experiment in the hESC-Chip consumes 30 nanoliter of cell culture reagents/medium, which is 4 to 5 orders of magnitude lower than a commonly used 6-well plate (requiring 2.5 mL of reagents/medium for a single study). Many hESC studies that require large-scale hESC experiments can, therefore, be implemented using the hESC-Chips in a cost-efficient manner. 3. The proposed hESC-Chip allows greater precision of measurements. Due to the use of PC-operated interface, critical parameters for hESC experiments can be monitored and controlled in the hESC-Chips with superior precision, which is unattainable using the conventional macroscopic setting. 4. The hESC-Chips promise to accelerate the discovery of new understanding of hESC growth control and to speed up the drug screening processes for hESC therapy. In our design, a hESC-Chip will contain 100 culture chambers for a simultaneous examination of 100 hESC experiments. A custom-designed interface allows 50 hESC-Chips to function in parallel. 5. User-friendly instrumentations of the proposed technology will be created for routine use in other hESC laboratories. The final versions of the chip design and control programs will be freely available for download from the PI’s research group web site (http://labs.pharmacology.ucla.edu/tsenglab/), where some existing designs and programs can be accessed.
SYNOPSIS: In this proposal, the PI aims to ultimately develop an automated, integrated microfluid platform for hESC studies that he nicknames hESC-Chips. He proposes to attack this in three aims. The first is to design, fabricate, and test new-generation hESC-Chips for fully automated hESC culture and assays. The second is to utilize these hESC-Chips as high-throughput screening platforms for defining optimal culture conditions for hESC self renewal. The third specific aim is to develop user-friendly versions of the hESC-Chips. SIGNIFICANCE AND INNOVATION: This proposal is highly innovative and significant, and addresses a longstanding issue in the field. The preparation of standardized ways of measuring hESC pluripotency as well as differentiation requires a standard approach not currently available in any laboratory. The successful resolution of the aims of this proposal will provide a manner of applying consistency across different platforms and laboratories investigating hESCs. This is a strong proposal by a great team of collaborators who wish to apply mature multilayer soft lithography (MSL) to problems in stem cell biology. High-throughput low-volume automated screening of culture conditions is sorely needed for stem cell research. STRENGTHS: One reviewers states is currently no standardized approach among different laboratories working with hESCs. Thus, the availability of a standardized hESC chip allows for not only fast, reliable, and statistically significant measurements of behavior in response to different culture mediums or different compounds, but will also allow for a more homogenous approach to synergize among different independent efforts. The webpage referenced by the PI already includes some examples of what these chips might look like. It therefore qualifies as a high-risk, high-reward project. Another reviewer states the strengths of this proposal are: --the team, which has strength in both stem cell biology and microfluidics --the preliminary results, which are convincing, and show that they have made progress with the basic issues of culturing cells on chips --the research plan is well thought out WEAKNESSES: There are a couple of issues not addressed by the PI in this version of the proposal that demand clarification for this platform to useful across different investigators. 1) It is not very clear even if the project is 100% successful what will be the cost to purchase, use and monitor the platform. If the expenses surrounding this new technology become overwhelming, it might not attract as many investigators as the PI might wish. 2) This grant could have been easily submitted to NIH and other agencies for funding, as it really does not require cell lines out of the non-registry collection for the establishment of proof of principle. 3) How user-friendly will the interface be? Despite the claim of specific aim 3, if the large-scale use of these chips is beyond the level of what a technician can understand or perform, it might not be attractive, as it would require specialized expertise that would make the whole purpose of this being widely used detrimental. There are some weaknessses to the proposal that the investigators should address to increase the likelihood of success. First, several research groups are starting to apply MSL to stem cell culture. Most groups are following identical or similar paths to that proposed here: start with self-renewal, looking at matrix, then growth factors, etc. The danger, then, is that another group will find the "answers" very soon and make the proposed research obselete. The investigators should be ready to adapt if such results come out by being able to modify their research plan to either look at differentiation or other issues in self-renewal. Second, 4-5 days seems short to assess self-renewal in hESCs. Markers of pluripotency may take days to down-regulate, or even several passages to become evident. Thus, the investigators should either validate a sensitive phenotypic marker that becomes apparent in 4-5 days or lengthen their assays. It would be a shame to get false positives because the assay is not stringent enough. Third, the investigators propose monitoring 13 or even 52 chips. This is not feasible to run in parallel without robotics to put chips on and off the microscope. Without robotics, the authors are either proposing to run chips sequentially (and thus have controls on each chip, rather than one dedicated control chip) or must run them in parallel but only monitor endpoint phenotype. This technical issue is not addressed in the proposal, but is crucial to the high-throughput claims made throughout. DISCUSSION: The strength of this platform is that the technology allows combination of factors to be tested on hESC. There was considerable discussion about the question of cell growth on PDMS. HESCs apparently do not grow well on it; most disturbingly, they turn off Oct 4. Although it was pointed out that the applicant has been able to get cells to grow for at least 4 days on PDMS, one question is whether or not the biology of the cells is changed.