Tools and Technologies II
Stem cells provide the starting material for cell replacement in tissues that are damaged as a result of disease, infection, congenital abnormalities, or trauma. Human pluripotent stem cells (PSCs) can generate multiple cell types and thus hold the greatest promise for future therapeutic applications. As PSCs differentiate in vitro into a mixture of different lineages, purification of the differentiated cells, as well as separation from their tumor-forming progenitors, is essential before PSC derivatives can be used in cellular therapies. For “cellular therapy” or “stem cell products”, sorting and purification processes that adhere to Good Manufacturing Process (GMP) are absolutely required. In the field of regenerative medicine, therefore, there is an urgent need for a robust cell culture strategy (or “process”) that allows the reproducible generation of a well-defined population of cells from a defined starting population of pluripotent stem cells in a GMP-compliant manner. This type of process-based approach is the only one likely to be acceptable to the FDA for INDs (Investigational New Drug) proposing immune-matched, patient-specific stem cell therapies. The research teams propose to develop a highly innovative Barcode-Activated Cell Sorter (BACS) chip and system that can be used to purify large numbers of differentiated cells for therapeutic purposes. The BACS platform combines the technologies of Barcoded Magnetic Beads (BMB), differentiated cell biomarkers, and a microfluidic magnetic sorter to rapidly capture, identify, and concentrate millions of specific cells for downstream applications. The BACS chip is disposable, easily replaceable, and always operates in a "sterile" mode. The system will uniquely enable specific cell types to be identified and selected in parallel from a highly heterogeneous cell culture without physical manipulation. No contamination of cells or sample carry-over will occur in the system. This novel BACS technology will be especially suitable for isolation of differentiated PSCs for cell-based human therapies.
Statement of Benefit to California:
Stem cells provide the starting material for cell replacement in tissues that are damaged as a result of disease, infection, congenital abnormalities, or trauma. For “cellular therapy” or “stem cell products”, sorting and purification processes that adhere to Good Manufacturing Process (GMP) are absolutely required. The research teams propose to develop a highly innovative Barcode-Activated Cell Sorter (BACS) chip and system that can be used to purify large numbers of differentiated cells for therapeutic purposes. The success of the project will lead to the development of a commercially viable prototype with more barcoded beads representing different cell lineages and lineage members. This will enable rapid and high-confidence lineage purification and cell sorting for many different human cell-based therapies in a GMP-compliant manner. The success of the proposed project will lead to the mass production of stem cells in the State of California. Since [REDACTED] are located in the California, the production facility will be also in California. Although stem cell industry is still in the embryonic stage, in the foreseeing future, it will be matured into the development and product industry. High demand of high quality and high purity stem cell products is expected. To meet the growing demand, the company will require hiring more scientists, system engineers, biologists, QC, production, sales, and marketing peoples.
This proposal is focused on the development of a novel technology for sorting pluripotent stem cells (PSCs) and their differentiated progeny under Good Manufacturing Practice (GMP) conditions. The applicant proposes to combine several technologies, including Barcoded Magnetic Beads (BMB) and microfluidic magnetic sorting, to generate a novel Barcode-Activated Cell Sorter (BACS) platform. The translational bottleneck identified is the lack of GMP-compliant methods to purify large numbers of cells of a desired phenotype from a heterogeneous mixture of cells. There are three Specific Aims: (1) to design and fabricate the BACS platform; (2) to produce and characterize human PSCs, tissue-specific precursor cells and terminally differentiated cells; and (3) to use these cells to assess the performance of the BACS platform. The reviewers agreed that this proposal addresses a significant bottleneck to the translation of stem cell therapies, as current methods for cell sorting are either inefficient or incompatible with GMP. They also appreciated the novelty of both the BMB and microfluidic magnetic bead sorting technologies. Reviewers agreed that, if successful, the BACS platform could have a significant impact on the field, although they noted that even 99% specificity in purifying differentiated cells may not be sufficient for clinical applications, due to the potential tumorigenicity of residual PSCs. Reviewers raised a number of concerns with the research plan and its feasibility. They noted that the project is completely dependent on the development of specific reagents to recognize different cell populations. The applicant lists a number of antibodies but does not address their specificities, or the feasibility of producing these reagents in a GMP compliant manner. Reviewers also were concerned that the proposed technology is focused on sorting beads, while ignoring free-floating, potentially contaminating cells that will detach or remain separate from beads. In addition, they noted that there is very little discussion of typical cell sorting issues such as aggregation (of both cells and beads) or coincidence exclusion, which becomes a major issue at higher sorting speeds. Reviewers found the lack of preliminary data on cell sorting to be a major weakness. They noted that the small molecule sorting data presented have little relevance to the complexity of cell separation. Reviewers also found some of the performance goals stated in the proposal to be unrealistic or insufficient improvements over existing technologies. For example, the increase in processing speed relies on many cells bound per bead, an approach unlikely to yield high purity and minimal contamination. Reviewers found the goals of 99% specificity and 95% efficiency to be unrealistic and noted that the proposed numbers of cells to be sorted are one to three orders of magnitude less than would be required for therapeutic purposes. Finally, reviewers noted that there is little discussion of how cells would be separated from beads after sorting and whether the sorting process might affect cell functionality. They would have appreciated the addition of functional tests of cells after sorting to the research plan. The reviewers found the Principal Investigator (PI) and research team to be generally well qualified, with an excellent track record of developing and utilizing BMB technology. However, they did note that the team appears to have little demonstrated experience with cell sorting. Reviewers appreciated that the team includes both engineers from industry and biologists from academia, but would have liked more detail about how the collaboration would function. Overall, while reviewers appreciated the novelty and significance of this proposal, they raised serious concerns about the research plan. The reviewers were not convinced of the project’s feasibility due to a lack of relevant preliminary data and unrealistic performance goals.