Purpose

Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components.

Methods

Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS.

Results

Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, β-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells.

Conclusions

LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.

中文翻译：

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使用人卵巢细胞外基质的层粘连蛋白成分在无异种条件下培养人卵巢组织。

目的

我们的目的是确定人类卵巢细胞外基质 (ECM) 成分，这些成分将支持人类卵巢组织的体外培养并与未来可能的临床应用兼容。我们对层粘连蛋白的卵巢表达进行了表征，并选择了三种层粘连蛋白三肽用于培养实验，以与基质胶进行比较，基质胶是一种未定义的基于动物的 ECM 成分混合物。

方法

使用皮质组织样本 ( n  = 6)、商业卵巢 RNA ( n  = 1)、卵泡液颗粒细胞 ( n  = 20) 和单细胞 RNA 测序数据，在转录物和蛋白质水平上确定 12 个层粘连蛋白基因的表达. 选择层粘连蛋白 221 (LN221)、LN521、LN511 和它们的混合物与 Matrigel 一起使用 17 名患者的组织进行为期 7 天的培养实验。在培养结束时，通过对连续组织切片进行评分和计数来评估卵泡，使用原位 TUNEL 测定法测量细胞凋亡，通过 Ki67 染色进行增殖，以及使用 UPLC-MS/MS 通过量化培养基中的类固醇来评估内分泌功能。

结果

卵巢皮质中大约一半的细胞表达至少一种层粘连蛋白基因。总体上表达最多的层粘连蛋白α链是LAMA2和LAMA5，β链LAMB1和LAMB2，以及γ链LAMC1。在培养实验中，LN221 与 Matrigel 相比提高了卵泡存活率 ( p  < 0.001)，而在 LN521 上培养的组织具有更高比例的次级卵泡 ( p  < 0.001)。LN511 和层粘连蛋白的混合物不支持导致较低的卵泡密度和较高的细胞凋亡的培养物。所有培养物都产生类固醇并含有增殖细胞。

结论

LN221 和 LN521 有望为人类卵巢组织培养提供无异种生长底物，这可能有助于进一步开发体外卵泡发生以用于临床实践。该系统还可用于识别化学物质对卵巢的不利影响。