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 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, PCNA protein analysis, β-galactosidase staining, flow cytometry and western blot analysis.

Results

Cells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. 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 method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing.

Conclusion

The method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.

中文翻译：

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一种新改进的原代细胞培养方法：皮肤成纤维细胞连续贴壁传代培养的组织块

背景

成纤维细胞（FB）已被广泛用作研究生物过程和细胞病理生理机制的典型体外细胞模型。然而，FBs在细胞培养过程中经过多次传代后容易出现衰老现象。因此，非常需要一种新的细胞培养方法来增强细胞的活力。

客观的

探索一种基于皮肤FB的细胞培养新方法。

方法

取BALB/c新生小鼠真皮组织块，随机分为实验组和对照组。实验组采用新改良的培养方法，即组织块连续贴壁传代培养（CASTB）法；对照组采用传统继代培养方法。收集第1、5和10代细胞并通过组织学/免疫组织化学和蛋白质印迹分析进行鉴定。通过应用细胞生长曲线、CCK-8测定、Ki67测定、PCNA蛋白分析、β-半乳糖苷酶染色、流式细胞术和蛋白质印迹分析来分析细胞活力、增殖、衰老和凋亡。

结果

两种培养模式下的细胞均呈纺锤形/不规则形，波形蛋白阳性表达。随着传代次数的增加，对照组细胞生长速度逐渐降低，而实验组细胞生长速度无变化。CASTB方法显着促进细胞生长和增殖。而且，随着传代数的增加，实验组的细胞凋亡和衰老趋势明显低于对照组。

结论

CASTB方法优于传统继代培养，可提供大量原代FB，效率和成功率较高，值得进一步推广应用。