Damage to lamellar keratinocytes, an essential cellular component of the epidermal layer of hoof tissue, can have a detrimental effect on hoof health and the overall production value of dairy cows. In this study, we described four digestion methods: trypsin method, collagenase method, Dispase II combined with trypsin method, and Dispase II combined with collagenase method, compared their isolation and culture efficiency, observed primary cell and subculture cell growth, and according to the difference of digestion time and apposition time of fibroblasts and keratinocytes, lamellar keratinocytes were purified by differential digestion method as well as differential velocity adherent method at each passaging and identified specific keratin 14 of keratinocytes using immunofluorescence. The results revealed that the lamellar keratinocytes isolated using Dispase II combined with the collagenase method increased rapidly, the number of cells in the offspring 4 generation was high, and the cells demonstrated positive expression of keratin,which facilitated the subsequent purification.
Keywords: bovine hoof; cultivation; immunofluorescence; isolation; lamellar keratinocyte.