Funding opportunities

Stabilization of human embryonic stem cells at higher yields and temperatures than currently possible

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01042
Funds requested: 
$784 970
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Efficient preservation and banking of human embryonic stem cells (hESC) and derivatives are critical components of any plan for using these products to regenerate tissues in clinical settings. Banking hESC will provide sufficient time to derive products and/or send them to medical centers. Unfortunately, current methods for cryopreservation of hESC exposes the cells to rapid changes in water content, formation of dangerous ice crystals, exposure to chemicals that prevent ice crystals but are themselves soewhat toxic, and significant shifts in temperature. The result is that production of human embryonic hESC is inefficient, hampered by poor recovery due to cell death, loss of self-renewal capability, and changes in gene expression that indicate the nature of the cells has altered during cell processing. Some researchers have explored vitrification, (the transformation from a liquid into a highly immobile, noncrystalline, amorphous solid state, known as the ìglass stateî) as a method of preservation, whih has been promising; however, it has not been possible to adapt vitrification methods to produce large enough batches of hESC to keep up with increased culture and propagation methods. . There is great need to formulate effective methods, adaptable to manufacturing large stable batches of viable hESC for long-term preservation, This proposal addresses two types of technology development solicited by CIRM 1) creation and design of novel tools and technologies and 2) optimization, scale up and application of an existing tool or technology for which there is proof of concept. In this project, we will apply our novel patent-pending technology, [REDACTED] process to stem cell preservation. [REDACTED] dries sensitive biological material by vaporization (simultaneous sublimation, boiling, and evaporation) from a slush state several degrees below 0∞C. The [REDACTED] technology provides gentle, cost-effective and efficient industrial scale stabilization of sensitive biologicals, thereby allowing production of sensitive biological products not possible by existing methods of manuacture. For this project we will use [REDACTED] for dehydration and subsequent vitrification of five human stem cell lines. Our preliminary data indicates that utilizing the combination of [REDACTED] before vitrification and utilizing preservation solutions of low molecular weight polyols can produce effective hESC. Our approach for formulation of less toxic vitrification solutions has been used with high success to preserve drosophila fly embryos in liquid nitrogen with about 80% of the embryos hatching nto viable flies after preservation. Through the use of the novel [REDACTED] technology and subsequent vitrification utilizing low molecular weight polyols in the preservation solutions, we should be able to optimize the current inefficient vitrification process to produce quantities of viable hESC in keeping with current manufacturing requirements.
Statement of Benefit to California: 
Human embryonic stem cells (hESC) offer great promise in the development of medical treatments for a wide range of conditions. These include damage to the brain, spinal cord, skeletal muscles and the heart. Treatments that have been proposed follow either physical trauma (e.g. spinal cord injuries), degenerative conditions (e.g. Parkinson's disease), or even genetic diseases (in combination with gene therapy). Yet further treatments using hESC could potentially be developed thanks to their ability to repair extensive tissue damage. Though much success and potential has been demonstrated from research using adult stem cells, many are of the opinion that the differentiation potential of embryonic stem cells has greater usefulness. For example, embryonic stem cells are considered more useful for nervous system therapies because researchers have not been able to identify and isolate neural progenitors from adult tissues. Though there are not yet any ongoing therapies or even clinical trials for hESC, research toward efficient manufacture and preservation of these potentially valuable tools in the arsenal against disease is extremely important for successful research and product development of human tissue regeneration interventions. Improvements in hESC preservation methodology will facilitate banking and safety/efficacy testing for the wide range of diseases for which hESC therapy may be applicable. Potential applications of stem cell intervention are coronary heart disease, diabetes, stroke and head/spinal cord injuries. The number of Californians who die from these disease each year are: coronary heart disease (52,000), diabetes (27,000) stroke (16,000). Hospitalization rates for nonfatal head injury is 25,000 and spinal cord 1400, both of which could potentially be mitigated by hESC intervention. Through the "Preservation by Vitrification" preservation methods are being targeted toward hESC, methodologies could be developed that are applicable to preservation of adult stem cells. Besides research toward life-saving interventions, establishment of hESC efficient manufacture and preservation methods through a California company is likely to provide economic stimulus as the value of this technology is realized. In total, the impact of this research is far-reaching with respect to potential for individual lives saved, range of medical conditions alleviated, and the state’s economic health.
Review Summary: 
This application proposes to optimize and standardize cryoprotective tools and protocols for scalable long-term storage of hESC lines compatible with current manufacturing requirements. To achieve these ends, the Principal Investigator (PI) proposes to test a variety of conditions and parameters for improving the process of vitrification and enabling its use at larger scale. The best protocols and conditions will be selected for biological validation using several different hESC lines. The reviewers were enthusiastic about the proposed technology but were uncertain of its feasibility. The proposal was poorly written and difficult to understand. Moreover, the proposed goals and timelines appeared to be unrealistic. Finally, while there were strengths in the research design, the applicants did not provide sufficient detail about the methods to enable assessment of feasibility. The reviewers felt that if successful, the proposed technology would be valuable to the field regenerative medicine and would positively impact diagnostics, therapy development, and basic stem cell research. Current methods for cryopreservation are far from ideal and are particularly ill-suited for use at large scale. The proposed technology could provide key improvements that are critical for clinical translation and commercialization of hESC research. The reviewers commented favorably on the well chosen, pertinent list of variables to be tested. Additional strengths included the proposed use of multiple cell lines for validation, and the evident consideration for GMP compliance. However, the reviewers’ enthusiasm was substantially diminished by the PI’s omission of key details that were necessary to assess the validity of the methods. In addition, they considered several of the indicated timelines and goals to be unrealistic. Finally, the inclusion of preliminary data from drosophila rather than hESCs was disappointing and did not raise the reviewers’ confidence in the overall feasibility of this effort. The reviewers considered the applicants well qualified to conduct the described research. The PI has an excellent, proven track record with cryoprotection deliverables and has assembled a team with the appropriate expertise. However, absence of a letter of collaboration or support from a key hESC expert listed as a consultant on the project was noted. The proposed budget was not controversial, but some reviewers indicated that it might be somewhat higher than expected. One reviewer suggested that the equipment cost could be lowered if it were possible to eliminate the state of the art cell surface phenotype analyzer and use a standard model. Overall, while the proposed technology could be of great value to the field of stem cell science, the applicants failed to convince the reviewers of its overall feasibility.
Conflicts: 

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