A protein-free solution as replacement for serum in trypsinizationprotocols for anchorage-dependent cells

Many animal cell culture media require serum as a component necessary for supporting the growth and survival of the culture. However, serum is of unknown composition, which may vary from lot to lot, and is very protein-rich. Therefore, serum-free media have been developed for the investigation of nutritional demands of animal cells in vitro and the production of therapeutic proteins with animal cells. Despite the fact that there are many published serum-free media for anchorage-dependent animal cells, only little work has been done on the development of serum- and protein-free subcultivation techniques. However, the serum-free subcultivation of animal cells is often a cumbersome attempt resulting in clumping and loss of the major part of the culture. Here we describe a new subcultivation technique for anchorage-dependent cells. Anchorage-dependent cells were first detached from the culture surface by trypsinization, suspended in phosphate buffered saline, 0.02% (w/v) EDTA, 75 mg/l phenol red, and 1% (w/v) pluronic F-68, and centrifuged to separate the cells from trypsin. The use of a trypsin inhibitor was not necessary. Cells were then resuspended in medium and seeded into new tissue culture vessels. No interference of the subcultivation process with the viability of the cells was observed as judged from clonal growth assays.     

组织块消化法原代培养大鼠输精管平滑肌细胞及鉴定

目的 建立一种稳定高效、存活率高的大鼠输精管平滑肌细胞体外分离及培养方法,为相关研究提供理想的细胞模型。方法 采用组织块消化法对大鼠离体输精管平滑肌细胞进行原代培养。采用形态学观察、HE染色、抗α-SMA免疫细胞化学荧光染色对细胞进行鉴定,台盼蓝染色计算细胞存活率及细胞总数,胰酶消化传代并绘制生长曲线。结果 对照实验表明胶原酶Ⅱ消化效果优于胶原酶Ⅰ,最佳消化时间为60min,同时控制吹打次数、沉淀时间及培养环境,获得了理想的平滑肌细胞总数及存活率。平滑肌细胞呈典型的梭形、长条形,可传代,且出现平滑肌细胞培养典型的“峰-谷”状特征,生长曲线为“S”型。经抗α-SMA免疫荧光鉴定,培养细胞为平滑肌细胞,纯度为(92.6±4.3)%。结论 采用组织块消化法成功建立了大鼠输精管平滑肌细胞的体外分离及培养方法。     

小鼠肝细胞的简易高效高纯培养及鉴定

目的改进一种简易、高效的乳小鼠肝细胞培养法,并对所培养的细胞进行鉴定。方法原代培养采用改进的组织块贴壁方法,以少量培养基孵育1~2h。传代培养采用0·25%胰蛋白酶消化随后以小牛血清培养为单层细胞。整个过程无需离心。结果成功地用这种简便的方法获得了纯度较高的肝细胞并进行了传代和鉴定。结论使用此方法培养肝细胞简单、高效、快捷,适合大多数实验室培养非大规模肝细胞。