Development of a Novel Technology for Cryopreservation of Human Embryonic Stem Cells in Adherent State
Tools and Technologies I
Cryopreservation and storage (cryobanking), and shipment of biomaterials are one of the key components of any cell-based application including stem cell and regenerative medicine. However, human embryonic stem cells are proven to be very sensitive to stresses associated with CP. The recovery of fully functional (pluripotent) human stem cells is extremely low in comparison to other types of cells and ESC species, it rarely exceeds 25% and very often is as low as 5-10%. The action of cold temperature is a severe stress but this is not only related to the ‘standard’ CP procedures. It includes a variety of physical, chemical and thermodynamical factors, and each of them can reparably damage the precious and slow growing hESCs. It makes an art of cryopreservation - cryobiology which involves complex and multidisciplinary areas and from which the problem can only be successfully solved if such a multidisciplinary approach is used. We indeed will use such an approach: 1. We will measure crucial and to this day unknown cryobiophysical characteristics of hESCs, which will allow us to systematically search for better protocols for cooling, warming and the addition and dilution of cryoprotective agents (CPAs), in other words, we will elucidate how the cells and their membranes interact with the most commonly used antifreezes. 2. In parallel, we will study the fundamental molecular cryobiology of hESCs. By using specific and precise tools of molecular biology and genetics, we will identify why this type of cell is especially vulnerable to CP, what type of damage is introduced to the cells at different stages of CP, and what should be done to minimize the damaging effects. One of the causes of damage is associated with a procedure of detachment (taking out) of hESCs from the surface they normally grow and separating the cells into smaller clusters prior to freezing, and centrifugation. We think that these causes of damage make cells more susceptible to CP per se, and we will develop a method of CP that would avoid these deleterious effects. We shall simply keep the cells in their “natural environment” prior to freezing - that is, adherent to their growth surface. 3. We will develop a special system that “make cells happy” even after such a severe “journey to the Antarctica’s pole of cold”. We will assemble an interdisciplinary team of highly skilled cryobiologists, biophysicists, molecular biologists, geneticists, and engineers to execute this project. As a result, a novel, revolutionary technology of CP of hESCs will be developed that will substantially improve the quality and yield of hESCs after cryopreservation.
Statement of Benefit to California:
Cryopreservation and storage (cryobanking), and shipment of biomaterials are one of the key components of any cell-based application including stem cell and regenerative medicine. However, human embryonic stem cells (hESCs) are proven to be very sensitive to stresses associated with CP. The recovery of fully functional (pluripotent) stem cells is extremely low in comparison to other types of cells and ESC species; it rarely exceeds 25% and very often is as low as 5-10%. California has positioned itself through the leadership of CIRM as a leader in stem cell biology and regenerative medicine, so it is expected that a dramatic increase in the use of hESCs and their derivatives will be beneficial. Therefore, efficient cryobanking of hESCs will benefit all people of California, and especially those who will need SC transplantation in the future in addition to any other medical discoveries gleaned from SC studies. On the other hand, the scientific community of California would benefit from such development because it would improve the quality of the scientific materials, facilitate material exchange between different centers, etc.
This proposal focuses on the development of a novel technology for the cryopreservation of human embryonic stem cells (hESCs). In the first aim the Principal Investigator (PI) proposes to perform a through cryobiophysical characterization of hESCs to allow optimization of cryopreservative solutions and methods. In the second aim s/he proposes to profile the molecular biological changes hESCs undergo during cryopreservation and compare and contrast “cryotolerant” and “cryosensitive” cell lines. Finally, the PI proposes to develop a novel cryopreservation system to improve survival and maintenance of pluripotency of cryopreserved hESCs. The reviewers felt that this proposal addresses an important roadblock in the field but weren’t convinced that this approach would yield benefits over others being pursued. They expressed doubts about the feasibility of the proposal, commenting that its scope is too broad, and raised a number of questions about the research design and preliminary data. The reviewers found the research team to be well qualified but were concerned about the allocation of the budget. The reviewers’ major concerns about this proposal related to its scope and feasibility. They expressed doubt that the research team could complete all three aims within a two year timeframe. Reviewers were concerned about the lack of integration between the work proposed in each individual aim and apparent disconnect among the different teams of researchers conducting each of the three aims. In addition, reviewers raised a number of concerns about the experimental plan and design and commented that very little experimental detail is provided for both Aims 2 and 3. Questions were raised about the methodologies to analyze phenotype and viability of the adherent pluripotent hESC following cryopreservation. In addition it was difficult to analyze the preliminary data quantitatively because no error bars or statistical analysis was provided. Finally, they noted that there is very little mention of potential limitations of the proposed studies or alternative approaches that may be pursued. Although Aim 3 mentions a few alternatives, no clear plan is described for when to use them, or what criteria would be used for their evaluation. The reviewers praised the research team as well qualified to carry out the work described in the proposal. The PI is experienced in cryopreservation and has assembled a team with a broad range of expertise. Reviewers raised concerns about the budget, namely that it’s heavily weighted toward personnel (85% of the requested funds are for salary/consultants) and light on supplies. In addition, the roles of Key Personnel are not adequately described, making it difficult to evaluate the appropriateness of the budget. Finally, the subcontract budget seems excessive, constituting almost half of the direct costs with no breakdown of expenses provided. Overall, while this proposal addresses a significant roadblock in stem cell biology, the reviewers found it overly broad and unfocused and raised serious questions about its feasibility.