Tools and Technologies I
To realize the full potential of human embryonic stem cells (hESCs), it will be critical to not only understand, but also control the highly orchestrated combinatorial and temporally restricted sequence of signals that direct their differentiation into specific tissues or tissue progenitors. Our understanding of the fundamental processes driving stem cell tissue specific differentiation remain in its infancy and current, empirically developed, techniques yield heterogenous cell populations. New technologies that can enable researchers to efficiently screen multiple conditions affecting growth and differentiation, as well as identify and select individual cells or colonies, would be a great benefit to understanding stem cell biology, and would greatly accelerate the development of methods to prepare stem cell progeny populations for use in regenerative medicine applications. We propose to develop a single platform technology that enables (1) rapid imaging and sorting of adherent hESC and progeny colonies, (2) high throughput studies of hESC growth conditions, and (3) automated, high volume culturing of cells. This will be built on work already developed by this team in micro/nanotechnology and instrumentation for cell biology. This project will employ three key technologies. The first technology, the micropallet array, is an array very small plastic pedestals on a glass (1" x 3") slide that permits individual stem cells to be grown in a pre-indexed location, allowing researchers to watch a colony develop over time using manual or automated imaging systems, while performing chemical or labeling experiments to identify the characteristics of each progeny population. Specific cells or groups of cells can then be selected and safely removed from the array for further study. This "sorting" capability is extremely powerful for stem cell research, and cannot be done by any method to date. The second technology, a superimposed microfluidic system to produce multiple growth conditions on a single micro-array slide. This technology uses "laminar flow" to produce parallel streams of growth media, each containing different kinds and amounts of signals. By including all the stem cells together in a single cassette and controlling the local medium around each cell, one can study the effects of micro-environment. Cells will grow close enough to communicate and share extra cellular chemistries, however, neighboring cells will be subjected to different stimuli in the form of chemical compositions in their media. The third technology will be the integration of microelectronics and computer control in the small cell culture cassette. Microelectronics will be used to control the temperature and flow rates of liquid media, as well as to monitor growth conditions and imaging of the plate. This will reduce the footprint necessary for performing stem cell studies, and allow one to perform remote, pre-programmed stem cell growth experiments.
Statement of Benefit to California:
This work will lead to three major benefits to the state of California: 1. Dramatic increase in throughput for stem cell research in California. With this technology development, automated high throughput experiments can be set up for pursuing large scale studies of stem cells. This will enable California researchers, both in academia and industry, to move research from "single researcher at a bench" style, to large scale automated research. The problems of stem cell research are so rich and diverse, that affordable, high volume research technologies will need to be developed. 2. Development of new techniques for studying stem cell differentiation. The technology proposed in this study will enable detailed studies of stem cell development and differentiation that are not possible using conventional technology, thus providing California researchers with key advantages over our out-of-state competitors. The first beneficiaries of this technology will be California scientists in academia and industry, which will advance the state of the art for stem cell research in California. 3. New economic opportunities for California biotech. The new approaches developed in this work will enable the development of new, high value therapies based on stem cell research. In addition, the techniques will provide for a wealth of opportunities for California companies to participate in the research ecosystem. Opportunities include the production of these technologies and their related materials, supplies, etc. for use by research and medical institutions, use of the technologies for developing advanced therapies, and use of these technologies for delivering therapeutic solutions to patients.
The goal of this proposal is to produce and test a new single platform technology for the rapid sorting, imaging and growth of stem cells with the goal of increasing throughput of experiments by as much as 10-fold. The applicants propose to engineer a micropallet array for the growth, imaging and isolation of stem cells and then to produce a microfluidic laminar flow chip that will allow cells to be exposed to 16 different culture conditions on one array. The final aim is to integrate the micropallets and laminar flow system within a cassette to allow environmental control and portability. The applicants comprise a team with a strong engineering background and previous experience with micropallet fabrication. While acknowledging that microtechnology offers tremendous potential for biological research, the reviewers felt that the proposed tools and their intended use do not substantially address the current roadblocks in stem cell research. As described, the micropallets will be used to grow, phenotype, and differentiate cells, but it was not clearly defined how the proposed tools represent improvement over current methods or commercially available systems. Reviewers felt that the real advantage of the micropallets would be for cell sorting, and most indicated that their enthusiasm would have been considerably higher if the pallets were used to study some aspect of stem cell biology rather than serve primarily as a surface for cell growth. The reviewers were impressed with the concept of the micropallets, although some felt that not enough technical details were provided to critique their fabrication, particularly with regards to potentially necessary adjustments in pallet geometry. The applicants gave careful consideration to the issues that are important for successful cell culture, and the reviewers appreciated the ambition with which they proposed to evaluate the cell phenotypes at both the molecular and functional level. One reviewer indicated that the proposed application of neural differentiation was not of the highest impact. The most serious concerns were raised over the utility and effectiveness of the laminar flow technology. Presumably, multiple posts will be exposed to the same laminar stream, and thus all cells could be exposed to whatever their neighbors are secreting, including factors from feeder cell layers. Reviewers were also concerned with the differential effects that each post and its adherent cells might experience depending on their position within the stream. The applicants did not address how the interface between the fluid and posts would be maintained nor did they offer insight as to how bubbles or other impediments would effect the operation. A reviewer noted that the laminar flow stream device has only moderate impact in this setting as the general approach for testing multiple growth conditions simultaneously has moved towards developing arrays with multiple miniaturized chambers where each condition to be tested is entirely sequestered from its neighbors. Thus, the device is less likely to be adopted. The principal investigator is an accomplished engineer, and there is no doubt that the proposed devices could be readily made. However, the reviewers were uniformly concerned that the overall team lacks the significant biological expertise that will be required to adapt these tools for use in cell culture. This particularly evident in the fact that the proposal did not address, nor offer potential solutions, to the challenges described above. The reviewers felt that these applicants would greatly benefit from a consultant that is familiar with the effects of fluid flow on cells. Overall, this proposal offers a cleverly engineered tool that in its proposed use, does not significantly impact the field of stem cell science.