The formation of extra embryonic endoderm (XEN) occurs early in embryonic development. The cell types that develop from the XEN remain poorly studied in ruminant species because of the lack of suitable cell culture model systems. The goal of this work was to establish a protocol for producing XEN cell cultures from bovine blastocysts. Previous work identified fibroblast growth factor 2 (FGF2) as a facilitator of bovine XEN development. Further refinements in culture conditions studied here included exposure to 20% fetal bovine serum and FGF2 replenishment. These modifications yielded an endoderm outgrowth formation incidence of 81.6 ± 5.5% compared to 33.3% ± 5.5% in bovine serum albumen-supplemented (BSA) controls. These cells resembled XEN when examined morphologically and contained XEN transcripts (GATA4, GATA6) as well as transcripts present in visceral (BNIP1, VEGFA) and parietal (CXCR4, THBD, HHEX) XEN. Two XEN cell lines were maintained for prolonged culture. Both lines continued to proliferate for approximately 6 wk before becoming senescent. These cultures maintained an XEN-like state and continued to express GATA4 and GATA6 until senescence. An increase in abundance of visceral and parietal XEN transcripts was observed with continued culture, suggesting these cells either undergo spontaneous differentiation or retain the ability to form various XEN cell types. Stocks of cultured cells exposed to a freeze-thaw procedure possessed similar phenotypic and genotypic behaviors as non-frozen cells. To conclude, a procedure for efficient production of primary bovine XEN cell cultures was developed. This new protocol may assist researchers in exploring this overlooked cell type for its roles in nutrient supply during embryogenesis.

中文翻译：

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技术说明：提高产生牛胚外内胚层细胞的效率。

胚外胚层（XEN）的形成发生在胚胎发育的早期。由于缺乏合适的细胞培养模型系统，从XEN产生的细胞类型在反刍动物物种中的研究仍然很少。这项工作的目的是建立一种从牛胚泡生产XEN细胞培养物的方案。先前的工作将成纤维细胞生长因子2（FGF2）鉴定为牛XEN发育的促进剂。在这里研究的培养条件的进一步改进包括暴露于20％的胎牛血清和FGF2补充。与牛血清蛋白补充（BSA）对照的33.3％±5.5％相比，这些修饰产生了81.6±5.5％的内胚层增生形成率。当进行形态学检查时，这些细胞类似于XEN，并含有XEN转录本（GATA4，GATA6）以及内脏（BNIP1，VEGFA）和顶壁（CXCR4，THBD，HHEX）XEN中的转录本。维持两个XEN细胞系以延长培养时间。两条线在衰老前持续增殖约6周。这些文化保持XEN样状态，并继续表达GATA4和GATA6直到衰老。继续培养观察到内脏和顶叶XEN转录物的丰度增加，表明这些细胞要么自发分化，要么保留形成各种XEN细胞类型的能力。暴露于冷冻-解冻过程的培养细胞库存具有与非冷冻细胞相似的表型和基因型行为。总而言之，开发了有效生产原代牛XEN细胞培养物的方法。这项新协议可能有助于研究人员探索这种被忽略的细胞类型，以了解其在胚胎发生过程中在营养供应中的作用。