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

项目总结 冷冻保存的脑细胞可以无限期地储存在液氮中，并在需要时进行复苏 与人类健康相关的研究。为了防止或减少冰冻对细胞造成的损害， 用于低温保存的介质中添加了低温保护剂。二甲亚砜(DMSO)是一种 一种易于穿透生物膜的低温保护剂，在以下方面优于所有其他低温保护剂 细胞解冻后的活性。因此，所有商品化的冷冻保存制剂都使用DMSO 细胞。然而，用DMSO超低温保存的细胞表达与凋亡相关的基因， 这些细胞中有80%可能在接种后不久就死于凋亡。因此，这些因素的相关性 用于严谨研究的冷冻保存细胞调查与人类健康有关的现象是有问题的。 此外，所有冷冻保存脑细胞的制造商都从从男性解剖的混合组织中获得这些细胞 以及雌性啮齿类动物的胚胎。因此，这些准备工作不能用来研究任何与性别有关的问题， 当美国国家卫生研究院正式要求所有联邦资助的研究比较和对比实验 在雄性和雌性动物和细胞中的发现。为了解决这些问题，Spot Cells LLC正在开发一种 专利细胞冷冻保存和复苏协议(CCRP)，其目的是消除 二甲基亚砜对深低温保存细胞的不利影响。Spot Cells LLC将使用CCRP生成 冷冻保存的特定性别的脑细胞制剂。为此，该公司已确认在 初步实验探讨了冷冻保存不同性别脑细胞的可行性。技术 CCRP的创新之处在于它消除了与细胞凋亡相关的离子干扰，这种干扰是由 二甲基亚砜。Spot Cells LLC假设，使用CCRP创建的细胞培养将与 从新鲜获得的(不是冷冻的)脑组织中获得的培养物，因此将适用于严格的 针对与性别有关和与人类健康无关的现象的研究。这一期望将 在此SBIR中对原代培养的小鼠皮质神经元进行测试。这项研究有两个具体目的：1) 验证CCRP是否能抵消细胞凋亡，以及2)验证CCRP对基因表达的影响。的产品 这个SBIR将是CCRP，CCRP介质，以及特定性别的脑细胞制剂，使用 CCRP。在第二阶段，Spot细胞将进行进一步的研究，使CCRP适应来自 啮齿动物大脑的其他部分。该公司将演示CCRP在创建2D和3D时的适用性 细胞培养。Spot Cells的长期目标是将CCRP实施到所有冷冻保存的细胞制剂中 二甲基亚砜。因此，该公司将调整CCRP，以适应正在开发的细胞制剂 治疗应用，包括用于生殖的人类诱导多能干细胞、精子和卵母细胞 医学，以及个体化药物的干细胞库。这些CCRP的全球联合市场- 预计到2025年，相关申请将超过900亿美元。

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.