Tools and Technologies I
Stem cells have been characterized and isolated based on a range of criteria, including surface proteins, morphology, and dye exclusion, mostly visualized by either histology or flow cytometry. Often a combination of techniques or markers are used to define a stem or progenitor cell, exemplified by multiple cytometric parameters commonly used to characterize hematopoietic or embryonic stem cells. Developmental studies and clinical application depends on characterization and isolation of stem cells from complex cell mixtures. With the exponentially growing knowledge of the multitude of stem and progenitor cells that exist in diverse and often complex organs or cultures, there has been a corresponding increase in the need for novel markers and separation technologies to precisely distinguish specific cell populations. Our proposal to develop a DEP system is significant in that it applies a novel technology to the problem of cell separation and characterization. DEP separates cells based on size and intrinsic electrical properties, and we propose can be developed as a stand-alone system as well as in conjunction with traditional separation or characterization technologies. Preliminary data are presented showing feasibility of DEP separation of cells in biological media and solutions with retention of cell viability. The specific goal of our research is to scale up existing prototype instruments and to develop methods separation methods based on intrinsic cellular properties as well as use of known surface epitopes as currently done in traditional histology and flow cytometry.
Statement of Benefit to California:
Cell separation and characterization is the basis for pinpointing stem cells within an organ or cell culture. Thus, the need to define and purify cells underlies the entire stem cell research and clinical enterprise. With exponentially growing knowledge of the multitude of stem and progenitor cells that exist in complex tissues and cultures has come an increased need for methods and markers to define and purify stem and progenitor cells. In part, this can be addressed by development of additional cell surface markers, as well as genomic or proteomic profiling. However, new separation technologies that introduce novel parameters, as proposed in this application, will contribute to solving the problem. Preliminary data using small-scale instruments indicate feasibility of the technology. The goals are the development of a large-scale instrument and matching methods and reagents for stem and progenitor separation. Benefits to California would accrue through: 1. Support of the stem cell research enterprise. 2. Intellectual property that would be transferred to California companies. 3. Potential founding of new companies to develop commercializable versions of the instrument and/or technology. California has many models for companies that offer cell separation technologies and instruments, including: Beckman Coulter, Becton Dickinson, Invitrogen, as well as many smaller companies that would be capable of developing commercial versions of the prototype instruments proposed in this application. Therefore, we anticipate that our goal of developing DEP cell separation technology will find applications in basic research, biotechnology and clinical settings through prototype instruments and through the production of commercial instruments.
This proposal focuses on the development of a dielectrophoretic (DEP) technology platform for the separation of stem and progenitor cells. The proposed device builds on earlier work by members of the group using arrays of circular electrodes to separate cells based on their electrical properties prior to molecular analysis. This proposal extends that work mainly by increasing the operating scale, by increasing the numbers of electrodes, and by adding a coating to the electrodes to prevent deleterious electrochemical interactions with the liquid, allowing operation at higher conductivity and higher frequency. The applicant will apply the technology to sorting subpopulations of cells from neonatal heart tissue (fibroblasts, cardiomyocytes, endothelial cells) and subpopulations of differentiated hESCs, with a goal of separating 105-106 stem cells from a complex mixture. The applicant will also investigate the use of metal-conjugated antibodies as a means of enhancing separation by combining label-free electrical separation with methods using characterized cell epitopes. Finally, the applicant will measure the efficacy of the proposed techniques by comparison to traditional phenotyping methods. The reviewers agreed that the goals of the proposal are important ones for the field but that the potential impact is limited by the chosen approach. They questioned the feasibility of the project and the rationale for several aspects of the research design. The praise for the well-qualified research team could not overcome the concerns surrounding feasibility and design of the project. While reviewers felt the potential impact of this proposal is high, problems with the approach reduce the likelihood of achieving the objectives of the proposal. Cell sorting is currently restricted in both efficacy and throughput by the use of cell surface marker-based techniques. DEP technology is an innovative approach to the problem and could provide label-free sorting techniques, which would be a huge advance for the field. However, one reviewer would have appreciated more discussion of why DEP is superior to fluorescent-activated or magnetic cell sorting. This reviewer also noted that metal-conjugated antibodies will be used in the third aim to aid separation and wondered if magnetic sorting would be quicker, gentler and cheaper. Reviewers expressed concern about the feasibility of the project, and raised a number of specific technical issues. One reviewer questioned the rationale for increasing the voltage of the device. Higher voltages may allow for higher flow rate and thus higher throughput, but the maximum operating voltage of the device will be limited by the induced transmembrane potential (Vtm) of the cells near the electrodes (or the coating). Demonstration that higher voltages are obtainable without causing excessive Vtm, at least by mathematical modeling and simulation, would strengthen the proposal. This reviewer also noted that higher voltages will lead to higher heating, which may limit operation. The proposal suggests that this problem will be mitigated by the electrode coating, which will disperse the field lines. However, lower field intensities will directly impact the DEP force. Since both heating and DEP force scale with the square of the voltage, varying voltage cannot increase the force without increasing temperature. This reviewer was not convinced of the benefit of using a high voltage with a thick electrode coating versus a lower voltage with no coating. Another reviewer questioned whether higher voltages would increase shear forces on cells with potentially deleterious effects. A reviewer also questioned the rationale of operating at high conductivity. This reviewer noted that the primary reasons to separate at low conductivity are that it maximizes the DEP contrast between the cells and media allowing for use of both positive DEP (pDEP) and negative DEP (nDEP). Operating at physiological buffer conductivity (or higher) will only allow nDEP, which would seem to be incompatible with the proposed circular arrays that retain cells against flow via pDEP. The applicant presents data that nanoparticles experience nDEP at high conductivity, but nanoparticle electrical properties are dominated by surface conductance, which is not the case for cells. For these reasons, the reviewer questioned the utility of higher conductivity in the proposed system. A reviewer also noted that the DEP device, as proposed, cannot distinguish cell separation by electrical properties from separation by size. Given typical 2x variations in cell size across a population, the reviewer recommended that applicants either develop a size-independent separation scheme or perform additional measurements to ensure that their separation is not merely size-based. Another reviewer felt that not enough detail was provided regarding the scale up of the system. It was also not clear to this reviewer how the desired cell population, once isolated, will be recovered from the DEP device. The reviewers agreed that the assembled research team is experienced and well-qualified to carry out the project. One reviewer found the budget to be excessive, containing too much travel and a large, unjustified subcontract. Overall, while this proposal uses an innovative technology to address an important roadblock in stem cell research, the reviewers raised a number of serious technical issues they felt diminished the proposal’s feasibility and therefore, potential impact.