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

> **NIH NIH R43** · SPOT CELLS, LLC · 2020 · $251,989

## Abstract

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, ...

## Key facts

- **NIH application ID:** 10080448
- **Project number:** 1R43NS115317-01A1
- **Recipient organization:** SPOT CELLS, LLC
- **Principal Investigator:** LECH Kiedrowski
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $251,989
- **Award type:** 1
- **Project period:** 2020-09-15 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10080448

## Citation

> US National Institutes of Health, RePORTER application 10080448, Cryopreserved preparations of male and female brain cells to study gender-related issues (1R43NS115317-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10080448. Licensed CC0.

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