人食管上皮细胞培养、冻存及复苏方法的实验研究

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

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。

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 resuscitation. 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 nonserum 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 morphology, characteristic of epithelial cells and stained positive for the epithelial cell marker cytokeratin, which indicated 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 cell survival rate was higher in cryoprotectant containing K-SFM than DMEM. Conclusions For the cultivation of hEECs, the non-serum medium K SFM is convenient with cells spread out more properly and higher survival rate.