Biological properties of primary chondrocytes isolated by one-step digestion versus stepwise digestion in newborn rats

doi:10.3969/j.issn.2095-4344.0962

Abstract

Abstract:

BACKGROUND: In vitro culture of primary chondrocytes is one of the most important methods to study the bone growth. Currently, the main methods of primary chondrocyte separation are one-step digestion and stepwise digestion. However, the biological properties of chondrocytes isolated by these two methods have not been well documented.
OBJECTIVE: To compare the biological properties of chondrocytes in newborn rats isolated using these two methods and to provide experimental basis for the programmed study of cartilage development.
METHODS: Articular cartilage of newborn SD rats was removed under aseptic conditions. Chondrocytes were isolated by one-step digestion (type II collagen) and stepwise digestion (type II collagen and trypsin). Primary culture and subculture were performed. Cell morphology was observed under light microscope, cell proliferation was detected by cell counting kit-8 assay, and the expression of type II collagen of chondrocytes was analyzed by semi-quantitative immunofluorescence.
RESULTS AND CONCLUSION: A large number of chondrocytes were yielded by two digestion methods, but the purity of chondrocytes obtained by stepwise digestion method was higher than that by one-step digestion method. Under the light microscope, there was no significant difference in the cell morphology between the two groups. The growth of chondrocytes obtained by one-step digestion was faster than that by stepwise digestion method. Results from the semi-quantitative immunofluorescence analysis showed that the chondrocytes obtained by these two digestion methods showed the same ability to express type II collagen. These findings indicate that compared with one-step digestion, stepwise digestion is more suitable for primary chondrocytes isolation in newborn rats.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Chondrocytes, Cells, Cultured, Trypsin, Collagenases, Collagen Type II, Tissue Engineering

CLC Number:

R394.2

Deng Lin-xia, Yu Mu-xue, Pan Si-nian, Guo Chu-yi, Qiu Xiao-shan, Cao Zhi-yu. Biological properties of primary chondrocytes isolated by one-step digestion versus stepwise digestion in newborn rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4047-4052.

Figures/Tables 2

2.1 软骨细胞鉴定结果软骨细胞特异性表达Ⅱ型胶原蛋白，在荧光显微镜下呈红色，胞核呈蓝色，将胞质与胞核图片合成后可见典型的软骨细胞形态。 Ⅱ型胶原蛋白酶单独消化法所得的软骨细胞，Ⅱ型胶原蛋白表达量较少，且有少量细胞不表达Ⅱ型胶原蛋白，在显微镜下只见蓝色细胞核(图1A)。胰蛋白酶和Ⅱ型胶原蛋白酶分步消化法所得的软骨细胞，Ⅱ型胶原蛋白表达丰富，所有细胞均表达Ⅱ型胶原蛋白(图1B)，这提示分步消化法所得软骨细胞纯度较单独消化法纯度更高。 2.2 软骨细胞形态倒置相差显微镜下观察，单独消化法与分步消化法所得悬浮细胞肉眼可见无差异，圆球形，体积较小，大小均一，折光性较强；锥虫蓝染色显示，90%以上细胞不着色，为活细胞。 Ⅱ型胶原蛋白酶单独消化法分离的细胞培养6 h后开始贴壁，绝大部分24 h内贴壁，48 h生长加速，96 h后已经基本铺满皿底，细胞呈多形态，有多角形、扁圆形、多角形、长梭形(图2A)。胰蛋白酶和Ⅱ型胶原蛋白酶分步消化法分离的细胞培养9-12 h后开始贴壁，绝大部分24 h内贴壁，48 h后生长加速，96 h后细胞已经基本铺满皿底，细胞呈典型的“铺路石”状，兼见多角形、扁圆形、多角形，少数呈长梭形(图2B)。 2.3 软骨细胞的增殖能力 CCK-8法检测显示，培养 1-7 d时，两种消化法分离的细胞生长曲线都呈不典型“S”型。接种后1 d开始增长，2 d后呈指数增长，4 d后增殖速度减慢，约第6天细胞数达最大，第7天生长受到接触抑制的影响，细胞增殖进入平台期。分步消化法分离的细胞增殖速度略低于单独消化法分离的细胞，第3，4，5天差异有显著性意义(图3)。 2.4 免疫荧光半定量分析结果随机取10张不同培养时间的免疫荧光照片(图4-6)，通过Image J进行细胞核计数和荧光密度分析，第4天时，单独消化法比分步消化法所得软骨细胞数量更多，其差异有显著性意义；第2，6天时细胞数量基本一致，差异无显著性意义(图7)。两种方法所得软骨细胞的Ⅱ型胶原蛋白表达量都随培养时间的延长而增加，第4天时，单独消化法比分步消化法所得软骨细胞的Ⅱ型胶原蛋白表达量稍多，其差异有显著性意义，第2，6天时基本一致，差异无显著性意义(图8)。"

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