Tools and Technologies II
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have tremendous potential for basic research and applied applications. However, a severe limitation is currently inefficient recovery after cell freezing. Cell freezing is essential for preserving cells (e.g. banking) for future use. This proposal seeks to apply modern cryobiological principles and methods to efficient preseration of stem cells. The results will accelerate research and large scale applications.
Statement of Benefit to California:
This proposal is a multidisciplinary collaboration to address a common problem in the stem cell field. Preservation of stem cells and their differentiated derivatives is essential but currently inefficient. or, in cases of some differentiated derivatives, have not been developed at all .The benefits to California include: 1. The new technology will find application in both basic and applied research settings across the state. All institutions doing stem cell research deal with preserved cells, so improving the protocols will benefit all stem cell scientits by accelerating their research. 2. The benefits extend to companies, such as pharmaceutical and biotech companies, since they rely on preserved samples of cells for their products and services. 3. Bringing the diverse people together (cryobiologists, engineers, and stem cell biologists) to address a stem cell problem forges new links in the academic community that should be capable of opening new areas of research. These new areas of research will be a important legacy of the stem cell initiative and will invigorate academic research.
The applicant seeks to develop a combination of tools that will enable optimized cryopreservation (CP) of undifferentiated human pluripotent stem cells (hPSC) and therapeutically relevant hPSC derivatives, i.e. neural progenitor cells (NPC), neurons and cardiac myocytes (CM). Cryopreservation (CP) of these cell types currently results in inefficient recovery, decreased cell viability and can cause phenotypic changes, thereby presenting a bottleneck to the translation of stem cell therapies. The Principal Investigator (PI) proposes to optimize existing suspension CP methods for hPSC as well as develop suspension CP protocols for the above-mentioned hPSC derivatives. The PI also plans to develop a device and methods to enable efficient, large-scale CP of these cell types as adherent cultures on multi-well plates. Reviewers agreed that the application addresses a significant translational bottleneck in the development of stem cell-based therapies and technologies. They did not find the proposed systematic optimization of suspension CP parameters especially novel and noted that, owing to cell intrinsic properties, the developed protocols would not extend to all cell types. However, they appreciated the applicant’s efforts to develop methods for freezing cells in adherent formats and noted some novelty in developing this approach. Published reports demonstrate improved viability of human embryonic stem cells and preservation of neuronal networks following CP in adherent formats, but these have not yet been implemented because of technical limitations on sterility and scalability. The applicant proposes to address these limitations by fabricating accessories for cooling and thawing cells in plates. The review group expressed mixed levels of enthusiasm for the presented preliminary data. They found the data that hPSC and NPC appear to tolerate freezing as adherent cultures solid support for further effort in this area. However, they questioned the practicality of this approach because of the difficulty of using plates with current cryopreservation and storage equipment. Further, culture plates do not form a tight seal and thereby present sterility challenges. Reviewers appreciated the improvements achieved by modifying suspension CP protocols for hPSC. However, some did raise concern that the applicant did not discuss the potential functional or regulatory implications of freezing cells in the presence of an apoptosis inhibitor. Several concerns were raised regarding the experimental plan. Reviewers noted that it lacked critical details, and overall was poorly written. For example, while plans to optimize CP of pluripotent cells are well described, reviewers were left uncertain how neural or cardiac lineage cells would be generated and purified and at what stage they would be frozen. One reviewer noted an absence of long-term storage studies, which are required to extend the developed CP methods to cell banking, an activity critical for therapeutic application of the proposed technology. While the PI proposes to gather a body of data intended to reveal the mechanisms of CP-induced cell death, the application fails to describe how the information from these studies will be applied to meaningfully improve CP protocols. Therefore, reviewers questioned the impact of this effort. A reviewer noted the plan does not describe the goals, structure, and utility of the proposed mathematical model. Finally, little information was provided regarding the design of the proposed customized CP equipment. Reviewers found the outcomes, milestones and timeline appropriate. The PI has assembled a strong team with expertise in the differentiated cell types addressed in the proposal. The PI is an expert in CP, has substantial experience in PSC stabilization and a good track record in the field. However, given the PI’s existing responsibilities, reviewers questioned this individual’s ability to commit the proposed 75% effort. Also, in the absence of knowing which CP method is best, the panel requested better justification for the expense of developing the proposed CP equipment customized for use with plates. In summary, the goal of this application is to optimize CP protocols for hPSCs and two differentiated derivatives. Although the application addresses a key bottleneck, deficiencies in the experimental plan and a lack of detail in critical areas of the application reduced reviewer enthusiasm. Thus, the application was not recommended for funding.