Yang N S, Kube D, Park C, Furmanski P
Cancer Res. 1981 Oct;41(10):4093-100.
We have developed a method for sustained growth of human mammary epithelial cells in monolayer cultures. Epithelial organoids derived from solid breast tissues were grown on the surface of thin (approximately 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum, and horse serum. To transfer the cultures, the collagen layers were dislodged and digested with collagenase. The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylene-diaminetetraacetate. Dissociated single cells were repleted with 75 to 95% efficiency onto collagen layers or tissue culture plastic surfaces. The dissociated cells could also be cryopreserved and reactivated with greater than 80% plating efficiency on collagen layers. Normal human mammary epithelial cells grown under these conditions progressed through 12 to 15 population doublings. The population-doubling times for normal and malignant mammary cells on collagen layers were 34 and 65 hr, respectively. After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen. Within the gels, the cells became organized into three-dimensional tubular structures. The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation, and cell transfer from monolayers.
我们已经开发出一种在单层培养中使人类乳腺上皮细胞持续生长的方法。源自实体乳腺组织的上皮类器官在富含激素、生长因子、胎牛血清和马血清的富集生长培养基中的薄（约1毫米）胶原凝胶层表面生长。为了转移培养物，将胶原层去除并用地胶原酶消化。然后使用胰蛋白酶-乙二胺四乙酸将释放到悬浮液中的细胞单层解离成单细胞。解离的单细胞以75%至95%的效率重新接种到胶原层或组织培养塑料表面。解离的细胞也可以冷冻保存，并以大于80%的接种效率在胶原层上重新激活。在这些条件下生长的正常人乳腺上皮细胞经历了12至15次群体倍增。正常和恶性乳腺细胞在胶原层上的群体倍增时间分别为34小时和65小时。达到汇合后，一些源自正常或恶性组织的培养物中的细胞穿透凝胶表面并生长到胶原中。在凝胶内，细胞组织成三维管状结构。胶原层的使用消除了培养人类乳腺上皮细胞生长中的一个主要问题，即有效解离和从单层转移细胞的困难。
