SERIAL CULTIVATION OF STRAINS OF HUMAN EPIDERMAL KERATINOCYTES - FORMATION OF KERATINIZING COLONIES FROM SINGLE CELLS
SERIAL CULTIVATION OF STRAINS OF HUMAN EPIDERMAL KERATINOCYTES - FORMATION OF KERATINIZING COLONIES FROM SINGLE CELLS
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DOI：
10.1016/s0092-8674(75)80001-8
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发表时间：
1975-01-01
期刊：
影响因子：
64.5
通讯作者：
GREEN, H
中科院分区：
文献类型：
--
作者：
RHEINWALD, JG;GREEN, H
人类二倍体表皮细胞在连续培养中成功生长。为了启动集落形成，它们需要成纤维细胞的存在，但成纤维细胞的增殖必须得到控制，这样表皮细胞群才不会过度生长。这两种情况都可以通过在正确的地点使用致命照射的3T3细胞来实现。当胰蛋白酶化的人皮肤细胞与3T3细胞一起镀时，人成纤维细胞的生长受到很大程度的抑制，但表皮细胞从单细胞生长成集合体。每个菌落由角质形成细胞组成，最终形成层状鳞状上皮，其中分裂细胞局限于最低层。在培养基中加入氢化可的松，因为在继代和继代培养中，氢化可的松使菌落形态更加有序和独特，并以略高的速度保持增殖。在这些培养条件下，有可能分离出无活成纤维细胞的角质形成细胞克隆。像人类二倍体成纤维细胞一样，人类二倍体角质形成细胞似乎具有有限的寿命。7株菌株的培养寿命为20 ~ 50个细胞代。直接取自皮肤的表皮细胞的电镀效率通常为0.1-1.0%。在随后从新生儿开始的培养物的转移中，电镀效率上升到10%或更高，但大多数情况下在1-5%的范围内，并在培养寿命结束时急剧下降。老年人角质形成细胞的电镀效率和培养寿命较低。
Human diploid epidermal cells have been successfully grown in serial culture. To initiate colony formarion, they require the presence of fibroblasts, but proiiferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct denairy. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largety suppressed, but the epidermal cels grow from singte ceils into cotonies. Each colony consists of keratinocytes ultimerely forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the coiony morphology more orderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isotate keratinocyte clones free of viable fibroblasts.Like human diploid fibroblasts, human diploid keratinocytes appear fo have a finite caiture lifetime. For 7 strains studied, the culture lifetime ranged from 20–50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1–1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1–5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.