Objective To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. Methods The primary porcine keratinocytes were cultivated with DKSFM(Defined keratinocyteSFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 005% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. Results The proliferation of the primry porcine keratinocytes cultured with the medium ofDKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFMcontaining 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 005% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygong and arranged like slab
stone. After 14 and 16 days,the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. Conclusion To culture primary porcine keratinocytes with the medium that DKSFMcontaining 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscafold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.

Citation： FAN Weijie,YANG Zhiming,LI Xiuqun,et al.. EXPERIMENTAL STUDY ON PORCINE KERATINOCYTES CULTURED AND PURIFIEQ RAPIDLY AND COCULTURED ON ACELLULAR AMNION IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2006, 20(3): 282-286. doi: Copy