基于微生物转谷氨酰胺酶交联明胶水凝胶的三维培养脂肪干细胞
[THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL].
作者信息
Ren Xiaomei, Qian Hong, Xiao Zhenghua, Long Haiyan, Guo Yingqiang, Yang Gang
机构信息
Department of Medical Information Engineering, College of Electrical and Information Engineering, Sichuan University, Chengdu Sichuan, 610041, P. R. China.
Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China.
出版信息
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Dec 8;30(12):1532-1537. doi: 10.7507/1002-1892.20160316.
OBJECTIVE
To study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed.
METHODS
ADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment.
RESULTS
The result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (<0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly.
CONCLUSIONS
ADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.
目的
为研究种植于三维（3D）材料中的脂肪来源干细胞（ADSCs）的生长情况，构建了一种基于微生物转谷氨酰胺酶（mTG）交联明胶水凝胶的3D培养ADSCs系统。
方法
通过胶原酶消化和离心从Sprague Dawley大鼠的皮下脂肪组织中分离出ADSCs，并进行传代培养。首先将明胶与mTG混合合成mTG酶交联明胶水凝胶，然后将ADSCs包封于其中（二维环境）并在3D材料（三维环境）中培养。通过倒置相差显微镜观察细胞的形态和黏附情况。此外，进行苏木精-伊红（HE）染色和Masson染色以观察材料中细胞的分布。通过荧光显微镜和激光扫描共聚焦显微镜观察ADSCs在材料中的存活和死亡情况。使用扫描电子显微镜观察ADSCs在水凝胶表面的黏附情况。采用Alamar-Blue法检测ADSCs在水凝胶中的增殖情况。此外，对二维环境和三维环境中培养的细胞结果进行比较。
结果
二维培养结果显示，ADSCs在水凝胶表面生长良好，功能正常且黏附性良好。三维培养结果显示，ADSCs在3D培养的mTG酶交联明胶水凝胶中生长良好，并呈现三维形态。细胞明显向各个方向伸展。凋亡细胞数量极少。三维培养各时间点的细胞数量均显著少于传统培养细胞，差异具有统计学意义（<0.05）。但培养8天后，在mTG酶交联明胶水凝胶中培养的细胞增殖更快。
结论
ADSCs在由mTG酶交联明胶水凝胶构建的三维培养系统中能良好生长，黏附性好且活力高。
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