Comparison of Different Methods in Primary Culture of Human Normal Esophageal Squamous Epithelium

Abstract: The purpose of this study was to compare the different methods in primary culture of human normal esophageal epithelial cells (HNEECs) and look for appropriate methods to acquire abundant and well growth esophageal epithelial cells for next experiment. The human normal esophageal epithelial cells were isolated from surgically excisional normal esophagus of patients with esophageal cancer. Tissue explant and enzyme digestion methods were used to clutrue HNEECs with DMEM/F12 mixed medium and with serum-free medium K-SFM. Gross examination， inverted microscopy observation and immunocytochemistry had been used to observe and identify growth， morphological characteristics of the regenerate esophageal epithelial cells. In DMEM/F12 mixture medium using tissue explant method， the regenerated esophageal epithelial cells， shaping slabstone appearance， grew fast with little fibroblasts pollution， and covered 70%~80% area of culture flask after 15~17 days， but the passage cells grew with more fibroblasts pollution. In the serum-free medium K-SFM using enzyme digestion method， the cells grew as well as the former， covered 70%~80% area of culture flask after only 10~12 days. Moreover， the cells can be used for cryopreservation， recovery and passage. The cells cultured with the other methods were not as good as the cells cultured with these two methods. About 90% of the regenerated cells， cultured with above methods， were cytokeratin positive staining with immunohistochemistry， which indicated that they were all epithelial cells. For primary culture of human esophageal epithelial cells， enzyme digestion method with the serum-free medium K-SFM is the best， but expensive. Tissue explant method with DMEM/F12 mixture medium is cheap with a long culture time， but not for passage. The two methods both can be choosen for different experiments.

CSTR: 32200.14.cjcb.2011.06.0011