Stem cells are typified by their ability to differentiate into a wide range of specific cell types, including nerve cells, brain cells, and other cells that do not typically regenerate in the human body. This pluripotency holds great promise for regenerative medecine, possibly enabling the repair of damaged or mis-developed tissues in adults and children. A great promise for stem cells is the production of very large quantities replacement heart muscle, skin cells, nerve cells, or other types of cells that are needed to repair these tissues. Controlling into what type of cells stem cells differentiate, and separating those that have differentiated, from those that have not differentiated, are a key elements in using stem cells as therapeutic elements. Control is presently being explored using a wide range of different chemical and physical environments, and is proving a highly complex and challenging area of research. Distinguishing and purifying the results of these control experiments, both in research and in clinical application, will clearly be a significant and important part of the eventual therapeutic use of stem cells. A multiplexed, microfluidic, all-electronic stem cell sorter, which we propose to develop under this seed grant, would enable the rapid and highly specific separation of differentiated and non-differentiated stem cells into separate groups, producing very highly purified samples of each particular type. These would then be available for further experimentation, or for direct therapeutic use. The entire microfluidic sorter is based on technology that would allow this process to be performed in a very inexpensive and disposable instrument, making the sorter readily and cheaply available to both the researcher and the biomedical industry. It is based on a very novel and possibly disruptive technology, quite distinct from the methods presently used for these applications. This all-electronic approach would provide a route to much simpler and faster sorters, and more rapid results, than present methodologies.
Statement of Benefit to California:
The research proposed here will focus on the development of an all-electronic, microfluidic cell sorting technology, which will involve the combination of microfluidics, all-electronic biosensing, biomolecular labeling, and cell culture. The end product will be a small, hand-held unit that includes disposable components, that enables a rapid, highly specific way to sort and quantify cells in a fluid sample. Technology that will enable uniquely labeling stem cells based on surface protein expression, sorting labeled cells in a highly specific and rapid fashion, and quantifying the numbers of cells sorted into each channel, will be developed and used in creating this instrument. The benefits to California are multi-dimensional. First, the technology produced through this research will have a direct impact on stem cell research and possible clinical use, an area to which California has clearly made a strong commitment, so that efforts in these areas pursued within California would benefit directly. Second, the technology produced here will likely form the basis of a commercial enterprise to produce, market, and further develop this technology. This enterprise will most likely be based in California, and if successful would provide employment, new technology, and tax revenue to the state. Third, the participation of four researchers from quite diverse field, bringing highly distinct expertise in engineering biophysics (PI Cleland), biochemistry (co-PI Reich), molecular biology (collaborator Laird) and stem cell culture (collaborator Wesselschmidt), will provide a unique combination of backgrounds and technologies that will likely generate new ideas and more applications for this type of instrumentation. Fourth, the research efforts and education of the postdoctoral and graduate students will generate two new professionals in this cross-disciplinary area, two students that will doubtless continue to work on closely related areas of work and further develop the concepts and technology that will enable future progress.
SYNOPSIS: The investigator proposes to develop a microfluidic stem cell sorter capable of digitally labeling, sorting and purifying a diverse stem cell population. In the application, he represents that such a system would be potentially superior to existing FACS technology. He proposes to attach digital barcodes to antibodies, which will bind specifically to surface molecules on the stem cell population. This project is focused principally around developing this new electronic cell sorting technology. SIGNIFICANCE AND INNOVATION: The development of an electrically-based cell sorting instrument would be terribly exciting and would be a wonderful complement to existing optically based flow sorters. The barcodes developed by this team are a wonderful step toward this goal, as is the high-frequency impedance detection system. However, this proposal lacks innovation and significance with respect to stem cell biology. STRENGTHS: The main strength of this proposal is the instrument itself, which shows great promise and is a worthwhile pursuit. The supporting strengths of the proposal are the high information storage capability of barcodes, and the simple microfluidic + electronic detection of the barcodes. The investigator is knowledgeable regarding the technology that he proposes to develop. WEAKNESSES: There are several major weaknesses to this proposal that should be addressed to increase the likelihood of funding. Primarily, the proposal is heavily focused on instrument development, with much less information given on specific experiments with stem cells. For example, there is no discussion of the antibodies to be utilized, or the characteristics of the desired subset of stem cells that the PI wishes to recover with this new methodology. Indeed, the use of stem cells seems quite incidental to the project. Another major weakness is that the barcodes themselves have several challenges for implementation. Focusing on these early on, rather than on instrument development, would enhance the proposal. Questions to address include: once the barcodes are attached to big bulky cells, how do the authors intend to ensure that they flow across the detector in the oriented along the flow? More fundamentally, how do the barcodes lead to higher multiplexing than conventional fluorescent labels? In isolating (stem) cells with flow, one wishes to look at multiple surface markers. If one functionalizes each barcode with a different label and then binds those to cells, the cells may have many different barcodes on their surface. How does that impact the ability to resolve a unique signature? In other words, if a cell has two different barcodes on it, in various orientations, how does one detect both of those codes? Also, how big are the barcodes? If each "bit" is 1um, then one barcode will be as big as a cell, which may be a problem when trying to label cells with multiple barcodes. Working out these and related issues would seem to be a more important short-term goal than the other goals laid out in the work plan. With respect to the technical details, the inclusion of the osmotic flow weakens the proposal. The key advance here is the electronic labeling/sorting. If the authors wish to make that technology have impact soon, they should choose a simple "off-the-shelf" pumping approach. Pressure-driven flow is fine for most applications and won't suffer from the issues that electrokinetic flow has at high ionic strengths (i.e., cell-culture media or PBS). Also, the timescale for flow change due to change of the forcing function is limited by momentum diffusion from sidewalls to the middle of the fluid, which takes ~L^2/D where D is the dynamic viscosity of the liquid. For a typical 100-um channel in water, this results in a time scale of ~2 ms, which suggests a 500Hz actuation rate, much smaller than 100MHz. There were two minor issues of note. First, there is no explanation of how to deal with the overlap with the NSF proposal. Since both proposals involve instrument development, the more common outcome of dual funding would be a reduction in funds due to overlap. Second, Figure 2 needs controls to prove that they are truly functionalizing surface with biotin/SA. Since SU-8 is highly autofluorescent, how do the authors know that the fluorescence they see is not due to autofluorescence, and that the bead binding they see is not due to non-specific interactions? DISCUSSION: There was no discussion further following the reviewers' comments.