Quaroni A
J Cell Biol. 1985 May;100(5):1611-22. doi: 10.1083/jcb.100.5.1611.
Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.
通过超微结构、生化和免疫学标准，在悬浮器官培养中维持一个多月的胎鼠小肠段中，证实了肠上皮细胞的成熟和分化。在5-7天的时间里，胎儿肠段演变成球状结构，其表面覆盖着单层柱状上皮，超微结构上类似于哺乳期绒毛细胞。结构内部存在松散的间充质细胞、细胞碎片和胶原蛋白。培养6天后，杯状细胞大量出现且发育良好，而在第18天的胎儿肠中并不存在杯状细胞。还观察到了肠内分泌细胞。使用针对体内绒毛和隐窝细胞的单克隆抗体进行的免疫荧光研究以及各种酶分析表明，培养的上皮细胞的分化和成熟水平与体内哺乳期肠黏膜相似但不完全相同。在培养物中未观察到隐窝和隐窝细胞标志物。向培养基中添加糖皮质激素可诱导蔗糖酶-异麦芽糖酶的产生，但未能促进体内断奶时肠上皮特有的大多数功能变化。通过肠细胞角蛋白的特异性表达，在源自器官培养物的外植体中鉴定出上皮细胞。原代培养上皮细胞中存在的分化特异性标志物，在纯上皮细胞群体的选择和传代培养后丢失。这些结果表明，维持上皮细胞的分化状态需要间充质和/或细胞外基质成分。本文所述的胎儿肠器官培养在研究肠上皮发育和分化过程中涉及的细胞和分子相互作用方面，比传统的器官和单层培养技术具有显著优势。
