胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较

小鼠胚胎成纤维细胞(MEFs)是目前国际上公认的人胚胎干细胞(hESCs)体外培养的饲养层细胞,但MEFs存在寿命短、动物源性污染等问题,需要探索适于hESCs体外培养且寿命长的人源性饲养层.采用胰蛋白酶消化法和组织块法分别原代培养人包皮成纤维细胞(hFFs),探索hFFs原代培养的最佳方法,为hFFs作为饲养层在hESCs研究中的应用提供可靠的科学依据;通过倒置显微镜下观察其生长状态和免疫细胞化学染色鉴定,结果显示两种原代培养方法获得的hFFs的形态及生物学特性均符合成纤维细胞;通过测定细胞生长曲线及MTT法检测,结果显示两种原代培养方法获得的不同代数的hFFs均具有较高的增殖活性,传代10余代仍能保持较好的细胞形态,可以制备成饲养层用于hESCs的研究.

Comparing Trypsin Digestion and Tissue Piece Method in Primary Culture of Human Foreskin Fibroblasts

At present,it is recognized internationally that mouse embryonic fibroblasts(MEFs) can be used as the feeder-layer of human embryonic stem cells(hESCs) in vitro culture.But MEFs have defects such as short life,animal-derived contamination.Therefore,human-derived feeder-layer which owns long life and is suitable for hESCs in vitro culture needs to be explored.Trypsin digestion and tissue piece method were adopted respectively to carry on primary culture of human foreskin fibroblasts(hFFs).The best primary cul-ture method of hFFs was explored and it will provide evidence for the application of hFFs in hESCs study.Morphology and characteristic of hFFs obtained by both primary culture methods were verified with inverted microscope and immunocytochemical stain.Results of cell growth curve and MTT method displayed high proliferative ability in hFFs obtained by both primary culture methods at different passages.Good morpholo-gy of hFFs can be maintained after 10 passages.Thus hFFs obtained by these two primary culture methods can be applied in the study of hESCs.