A Newly Improved Method of Primary Cell Culture: Tissue Block with Continuous Adhesion Subculture in Skin Fibroblast

Abstract

Abstract Background Fibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells. Objective To explore a novel method of primary cell culture based on skin FBs. Methods Dermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, β-galactosidase staining, flow cytometry and western blot analysis. Results Cells under two culture patterns showed vimentin positive expression via immunohistochemistry and western blot assay. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB remarkably promoted cell growth and proliferation. Besides, a lower apoptosis tendency emerged from the experimental group than the control goup with the increasing passages. Conclusion The method of CASTB may offer a large number of primary FBs with higher efficiency and success rate, which is worth of further popularization and application.