Cryopreserved preparations of male and female brain cells to study gender-related issues

PROJECT SUMMARY Cryopreserved brain cells can be stored in liquid nitrogen indefinitely and revived when needed to conduct research related to human health. To prevent or reduce the ice-induced cell damage caused by freezing, the media used for cryopreservation are supplemented with cryoprotectants. Dimethyl sulfoxide (DMSO) is a cryoprotectant that easily penetrates biological membranes and outperforms all other cryoprotectants in terms of post-thaw viability of the cells. Therefore, DMSO is used in all commercial preparations of cryopreserved cells. However, cells cryopreserved with DMSO express genes associated with apoptosis, and as many as 80% of these cells may die by apoptosis shortly after plating. Consequently, the relevance of these cryopreserved cells for rigorous studies investigating phenomena related to human health is problematic. Further, all manufacturers of cryopreserved brain cells obtain them from a mixed tissue dissected from male and female rodent fetuses. Therefore, these preparations cannot be used to study any gender-related issues, while the NIH formally requests that all federally-funded research compares and contrasts experimental findings in male and female animals and cells. To address these problems, Spot Cells LLC is developing a proprietary Cell Cryopreservation and Resuscitation Protocol (CCRP) whose purpose is to obliterate the detrimental effects of DMSO on cryopreserved cells. Spot Cells LLC will use the CCRP to generate cryopreserved gender-specific preparations of brain cells. To this end, the company has confirmed in preliminary experiments the feasibility of cryopreserving gender-specific brain cells. The technological innovation of CCRP is that it eliminates ionic disturbances that are associated with apoptosis and caused by DMSO. Spot Cells LLC posits that cell cultures created using the CCRP will be indistinguishable from the cultures obtained from freshly obtained (not frozen) brain tissue and, therefore, will be suitable for rigorous studies addressing gender-related and unrelated phenomena relevant to human health. This expectation will be tested in this SBIR on primary cultures of murine cortical neurons. The study has two specific aims: 1) Verify that CCRP counteracts apoptosis, and 2) Verify the impact of CCRP on gene expression. The product of this SBIR will be the CCRP, CCRP media, and the gender-specific preparations of brain cells produced using the CCRP. In Phase II, Spot Cells will conduct further research to adapt the CCRP to brain cells derived from additional parts of rodent brains. The company will demonstrate applicability of the CCRP to create 2D and 3D cell cultures. Spot Cells’ long term goal is to implement the CCRP into all cell preparations cryopreserved with DMSO. Accordingly, the company will adapt the CCRP for the cell preparations that are being developed for therapeutic applications, including human induced pluripotent stem cells, sperm and oocytes for reproductive medicine, and stem cell banking for individualized medicine. The combined global market for these CCRP- related applications is expected to exceed $90 billion by 2025.PROJECT NARRATIVE All commercial preparations of cryopreserved murine neurons contain dimethyl sulfoxide (DMSO), a cryoprotectant that promotes apoptosis and affects gene expression. Further, these preparations are derived from a mix of male and female tissue and, therefore, cannot be used for a research addressing gender-related phenomena relevant to human health. To address these problems, Spot Cells LLC will develop a proprietary method and cryopreservation medium that eliminates the deleterious effects of DMSO and use these to cryopreserve male and female brain cells for commercial availability.