天天操夜夜拍丨国产精品超清白人精品av丨天天爽天天噜在线播放丨国产精品久久久久久久福利竹菊丨色综合天天天天做夜夜夜夜做丨国内自拍xxxx18丨白人と日本人の交わりビデオ丨午夜激情在线观看丨成人18视频丨久久66热人妻偷产精品丨视频一区二区三区在线观看丨操操操插插插丨久久的久久爽亚洲精品aⅴ丨91精品一区二区三区在线观看丨aaa女人18毛片水真多丨99re在线视频精品丨超碰免费在丨中文字幕人妻无码专区app丨午夜香蕉视频丨又大又粗欧美黑人aaaaa片丨久久夫妻视频丨乌克兰性欧美精品高清丨亚洲欧美福利视频丨少妇高潮久久久久久一代女皇丨97国产高清

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin-0.53mM EDTA solution.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. (Or neutralize with medium containing 10% fetal bovine serum).
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. Seed cells on flasks precoated with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin.
7. Incubate cultures at 37°C.

Kahn CR, Rhim J. Human corneal epithelial cell lines with extended lifespan. US Patent 5,585,265 dated Dec 17 1996

Walker TL, Kahn CR. Human corneal epithelial cell lines with extended lifespan. US Patent 5,672,498 dated Sep 30 1997