Sweat JMDunigan D D, Wright S D
Marine Mammal Pathobiology Laboratory, Florida Fish and Wildlife Conservation Commission, St. Petersburg 33711, USA.
In Vitro Cell Dev Biol Anim. 2001 Jun;37(6):386-94. doi: 10.1007/BF02577576.
The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.
西印度海牛（Trichechus manatus latirostris）是一种草食性海洋哺乳动物，在佛罗里达州沿海水域被发现。由于它们处于濒危状态，不允许进行动物实验。因此，从海牛尸检时收集的组织中培养并鉴定了一种细胞系。通过酶解和机械技术从肾脏组织中分离单个细胞，建立了原代细胞培养物。对原代海牛肾脏（MK）细胞进行传代培养以进行鉴定。这些细胞在形态上与上皮来源的细胞系相似。采用免疫细胞化学分析来定位上皮来源细胞共有的细胞角蛋白丝。在第二代时，上皮样细胞的平均群体倍增时间为48小时，最佳接种密度为5×10³个细胞/cm²，并且以高接种效率容易附着于塑料培养板。尽管上皮样细胞在前三代中生长速度较快，但克隆潜力较低。这些细胞在琼脂培养基中不形成集落，依赖血清，寿命有限，约为九代，并且具有细胞接触抑制。这些数据表明这些细胞是有限的（非连续生长），不具有转化特性，且来源于上皮。这些细胞现在被称为MK上皮细胞。
