Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I

OBJECTIVE: Different strategies for epithelial cell isolation significantly affect the viability and physiological properties of primary cells. Trypsin digestion, a conventional method, causes collateral damage owing to its strong digestive potential. To better preserve the physiological properties of epithelial tissues, we aimed to develop a modified method (hyaluronidase and collagenase I combination) for primary cell isolation. METHOD: We used conventional and modified methods to compare cell viability, morphology and stemness. Additionally, we investigated the passaging stability of epithelial cells and their capacity for organoid formation. Finally, we compared the two methods for isolating urothelial, oesophageal, lingual, and epidermal epithelial cells. RESULT: Gingival epithelial cells obtained using the modified method had higher viability, better morphology and stronger stemness than those obtained using the conventional method. Additionally, primary cells obtained using the modified method were stably passaged. Regarding organoid culture, adopting the modified method led to a significant increase in the growth rate and expression of the stem cell markers cytokeratin (CK)‐19 and Ki‐67. Furthermore, the modified method outperformed the conventional method for isolating urothelial, epidermal, oesophageal and lingual epithelial cells. CONCLUSION: We demonstrated that the combination of hyaluronidase and collagenase I outperformed trypsin in preserving the physiological properties of primary cells and organoid formation. The modified method could be broadly applied to isolate different types of epithelial cells and facilitate studies on organoids and tissue engineering.