人食管上皮细胞培养、冻存及复苏方法的实验研究

目的比较K-SFM无血清培养基及DMEM有血清培养基在人食管上皮细胞培养中的作用,并比较不同冻存方法对人食管上皮细胞复苏后存活率的影响,探讨适合应用于组织工程食管研究的人食管上皮细胞培养、冻存及复苏方法。方法人食管上皮细胞来源于食管癌患者手术切除的食管。采用组织块法和消化法培养,两种方法均分别加入K-SFM无血清培养基及DMEM有血清培养基。采用直接观察、锥虫蓝染色活力鉴定及细胞计数法绘制细胞生长曲线,对比细胞在不同培养基中的生长情况。分别以两种培养基配制的冻存液进行冻存,复苏后的细胞采用锥虫蓝染色法鉴定其存活率。结果细胞呈典型“铺路石”状排列,免疫组织化学检测细胞角蛋白呈阳性结果,证明培养的细胞为人食管上皮细胞。人食管上皮细胞在K-SFM无血清培养基中生长快,细胞形态好,不易被成纤维细胞污染。以K-SFM培养液配制的冻存液冻存的细胞复苏后的存活率明显高于以DMEM配制的培养液。结论应用K-SFM无血清培养基培养和冻存人食管上皮细胞的方法简便、培养效果好,适合推广应用。

Experimental study on the method for cultivation, frozen reservation and resuscitation of human esophageal epithelial cells

Objective To compare the effect of serum-free medium K-SFM with serum-contained medium DMEM on culture of human esophageal epithelial cells (hEECs) and the effect of different cryoprotectants on cell survival rate after resuscitation and searching for a better method for hEECs' cultivation, frozen reservation and re- suscitation. Methods HEECs were derived from surgically resected esophagi of patients with esophageal cancer. Using different methods of tissue nubbles and cultivation, compared the growth and proliferation of hEECs in non- serum medium K-SFM with serum-contained medium DMEM. Cryopreservation with cryoprotectants made up of two kinds of medium: non-serum medium K-SFM and serum-contained medium DMEM was taken to compare the cell survival rate after resuscitation by means of trypan blue stain. Results The cells displayed a cobblestone mor- phology, characteristic of epithelial cells and stained positive for the epithelial cell marker cytokeratin, which indi- cated that the cells were hEECs. With the non-serum medium K-SFM, the hEECs grewed faster with better growth shape and less liable to be contaminated by fibroblasts. The celt survival rate was higher in cryoprotectant containing K-SFM than DMEM. Conclusions For the cultivation of hEECs, the non-serum medium K-SFM is con- venient with cells spread out more properly and higher survival rate.