Lucas-Clerc C, Massart C, Campion J P, Launois B, Nicol M
Laboratoire de Biochimie Médicale A, UER Médicale et Pharmaceutique, Rennes, France.
Mol Cell Endocrinol. 1993 Jul;94(1):9-20. doi: 10.1016/0303-7207(93)90046-m.
In this experiment, various conditions for embedding cultures of human pancreatic islets in type I collagen gel were studied in an attempt to maintain the highly differentiated functions of islet cells and particularly insulin secretion over a long period of time. The islets isolated by a collagenase digestion technique were plated either on or within the collagen gel and refed with either Eagle's minimum essential medium (5.5 mM D-glucose) or RPMI 1640 medium (11 mM D-glucose) supplemented with 10% FCS and antibiotics. The comparison between the two culture media showed that embedded islets cultured in RPMI had a higher basal insulin secretion rate, survived longer than their MEM counterparts, but exhibited impaired response to an acute glucose test contrasting thus with islets cultured in MEM. The secretory behaviour of islets was also related to the different morphological modifications occurring during culture. Islets directly embedded within the collagen gel more or less maintained their spherical structure and highest secretory capacities. When overlaid with a second layer of collagen, well established monolayers of human islet cells grown on collagen underwent a gradual and complete reorganization into a three-dimensional islet-like structure with a striking reinforcement of their secretory activity. Both cultures were able to survive more than 8 weeks, thus proving the usefulness of such a new model for long-term culture. In contrast, standard cultures on culture treated plastic dishes on which islets cells rapidly established wide monolayers, exhibited a rapid and definitive decline in insulin secretion with a survival not exceeding 14 days. In the light of these different culture conditions, possible mechanisms responsible for disturbance of hormonal release and their implications for in-vitro study of isolated islets functions are discussed. In conclusion, this work is a new example of the permissive effects of collagen matrices on the establishment or maintenance of tissue-like structures in vitro, suggesting the definition of a new model for the study of human pancreatic islets in long-term culture.
在本实验中，研究了将人胰岛培养物包埋于I型胶原凝胶中的各种条件，以期长期维持胰岛细胞的高度分化功能，尤其是胰岛素分泌功能。通过胶原酶消化技术分离得到的胰岛，接种于胶原凝胶上或凝胶内，并用添加了10%胎牛血清和抗生素的伊格尔最低限度基本培养基（5.5 mM D-葡萄糖）或RPMI 1640培养基（11 mM D-葡萄糖）进行再培养。两种培养基的比较表明，在RPMI中培养的包埋胰岛基础胰岛素分泌率更高，存活时间比在MEM中培养的对应物更长，但对急性葡萄糖试验的反应受损，这与在MEM中培养的胰岛形成对比。胰岛的分泌行为也与培养过程中发生的不同形态学改变有关。直接包埋于胶原凝胶内的胰岛或多或少保持其球形结构和最高分泌能力。当覆盖第二层胶原时，在胶原上生长的成熟人胰岛细胞单层会逐渐完全重组为三维胰岛样结构，其分泌活性显著增强。两种培养物均能存活超过8周，从而证明了这种新模型用于长期培养的有效性。相比之下，在经培养处理的塑料培养皿上进行的标准培养中，胰岛细胞迅速形成广泛的单层，胰岛素分泌迅速且最终下降，存活时间不超过14天。鉴于这些不同的培养条件，讨论了导致激素释放紊乱的可能机制及其对分离胰岛功能体外研究的影响。总之，这项工作是胶原基质对体外组织样结构的建立或维持具有允许作用的一个新例子，提示了一种用于长期培养人胰岛研究的新模型的定义。
