Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

来自接受取卵的女性的人原代颗粒细胞 (GCs) 可以被培养并用作研究人卵巢功能的细胞模型。在体外，它们迅速变化，最初类似于排卵前卵泡细胞，然后是黄体细胞。它们来自个体患者，其不同的病史、生活方式和年龄导致异质性。因此，细胞很少能与细胞实验完美匹配，或者，如果有的话，只能少量匹配。我们推断人类 GC 的冷冻保存可能有助于改善这种情况。以前的研究表明这种方法的可行性，但报告了人类 GC 的低存活率，并且仅部分评估了对人类 GC 功能的影响。我们在从卵泡液中分离后测试了人类 GC 的慢速冷冻方案（使用 FCS 和 DMSO）。我们将冷冻保存和随后解冻的细胞与来自同一患者的新鲜、非冷冻保存的细胞进行了比较。大约 80% 的人类 GC 在冷冻/解冻中存活下来。在细胞形态、培养存活率或线粒体转录水平方面没有发现差异（COX4，OPA1 ， TOMM20），类固醇生成（CYP11A1，CYP19A1 ）或细胞 - 细胞接触基因（GJA1）在培养 1-5 天的细胞中的两组之间。蛋白质组学分析显示总共 5962 种蛋白质的丰度没有统计学上的显着变化。两组产生的孕酮基础水平相当，并且对 hCG 的反应相似，孕酮升高。总之，我们的结果表明这是一种快速且容易获得的人类 GC 冷冻保存方法。我们预计它将允许未来的大规模实验，从而可能改善人类卵巢细胞的细胞研究。