Ethier S P
J Natl Cancer Inst. 1985 Jun;74(6):1307-18.
A newly developed culture system was used to examine the proliferative potential of rat mammary epithelial (RME) cells in vitro. RME cells were obtained by enzymatic dissociation of mammary tissues of 45- to 50-year-old virgin female LEW rats. The tissues were dissociated to small aggregates (10-50 cells per aggregate) separated from stromal cells and plated at a density of 10(5) cells per 60-mm tissue culture dish. The cells were grown in Ham's medium F12 supplemented with 5% fetal bovine serum, insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, and cholera toxin. Plating of 10(5) cells as small aggregates resulted in the attachment of 1,000-1,500 aggregates per plate. When grown on tissue culture plastic, approximately 1-2% of these aggregates gave rise to rapidly proliferating epithelial colonies. Individual colonies expanded with a population-doubling time of 24-34 hours and grew for about 3 weeks. Although these cells grew well in primary culture, they were not subculturable. When RME cells were plated onto dishes coated with type I collagen, the number of rapidly proliferating epithelial colonies per dish increased fivefold to tenfold. Cells grown on type I collagen-coated dishes expanded with a population-doubling time of approximately 27 hours and after 2 weeks in primary culture were nearly confluent. Unlike cells grown on plastic, RME cells grown on type I collagen were readily subculturable and serial subculture resulted in the cells undergoing 15-20 population doublings (5-6 passages) before exhibiting any loss of growth potential. Continued feeding of senescent cultures resulted in the emergence of discrete RME cell foci that retained proliferative potential and that eventually developed into rapidly growing cell strains. Exposure of primary cultures to the carcinogen N-methyl-N'-nitro-N'-nitrosoguanidine (CAS: 70-25-7) enhanced the proliferative potential of RME cells in early passages and in later passages either delayed or eliminated the "senescent" phase of cell growth. Carcinogen treatment of RME cells also facilitated the establishment of rapidly growing cell strains with long-term growth potential (greater than 20 passages).
一种新开发的培养系统用于体外检测大鼠乳腺上皮（RME）细胞的增殖潜能。RME细胞通过酶解45至50岁处女雌性LEW大鼠的乳腺组织获得。将组织解离成小聚集体（每个聚集体含10 - 50个细胞），与基质细胞分离，并以每60毫米组织培养皿10⁵个细胞的密度接种。细胞在补充有5%胎牛血清、胰岛素、氢化可的松、表皮生长因子、催乳素、孕酮和霍乱毒素的Ham's F12培养基中生长。以小聚集体形式接种10⁵个细胞，每平板可附着1000 - 1500个聚集体。当在组织培养塑料上生长时，这些聚集体中约1 - 2%会形成快速增殖的上皮集落。单个集落以24 - 34小时的群体倍增时间扩展，并生长约3周。尽管这些细胞在原代培养中生长良好，但它们无法传代培养。当将RME细胞接种到包被有I型胶原的培养皿上时，每个培养皿中快速增殖的上皮集落数量增加了五倍至十倍。在I型胶原包被的培养皿上生长的细胞以约27小时的群体倍增时间扩展，原代培养2周后几乎汇合。与在塑料上生长的细胞不同，在I型胶原上生长的RME细胞易于传代培养，连续传代培养导致细胞在出现任何生长潜能丧失之前经历15 - 20次群体倍增（5 - 6代）。对衰老培养物持续传代培养会导致离散的RME细胞集落出现，这些集落保留增殖潜能，并最终发展成快速生长的细胞株。将原代培养物暴露于致癌物N - 甲基 - N'-硝基 - N'-亚硝基胍（CAS：70 - 25 - 7）可增强早期传代RME细胞的增殖潜能，在后期传代中要么延迟要么消除细胞生长的“衰老”阶段。对RME细胞进行致癌物处理还促进了具有长期生长潜能（超过20代）的快速生长细胞株的建立。
