Jin Hui, Yang Peng-Bo, Feng Gai-Feng, Jia Ning, Yang Wei-Na, Wang Wei-Xi
Department of Human Anatomy and Histo/Embryology, Xi'an Jiaotong University College of Medicine, Xi'an 710061, China.
Sheng Li Xue Bao. 2015 Feb 25;67(1):103-9.
The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.
本研究旨在观察胰蛋白酶消化对体外培养星形胶质细胞纯度的影响并优化培养方法。取新生Sprague Dawley（SD）大鼠的大脑皮质组织，用0.25%胰蛋白酶消化20、30或40分钟。然后将获得的单细胞悬液进行培养。细胞汇合后，进行恒温摇床处理。接着，将20分钟组和30分钟组各自再分为两组，即正常消化对照组和二次消化组，并对细胞进行传代和纯化。通过倒置相差显微镜和MTT法分别观察细胞生长和增殖情况。采用胶质纤维酸性蛋白（GFAP）免疫荧光法观察星形胶质细胞形态并评估不同阶段其纯度。运用流式细胞术分析检测纯化星形胶质细胞的凋亡率。结果显示，消化20分钟的细胞通常在接种后9天汇合。当消化时间延长至30分钟时，细胞生长加快，接种后7天汇合，同时星形胶质细胞形态正常，GFAP阳性率（70.2±4.0）%显著高于20分钟组（P<0.05）。与20分钟组相比，40分钟组GFAP阳性率更高，但细胞增殖较慢，细胞损伤更明显。恒温摇床处理后，二次胰蛋白酶消化可减少传代后污染细胞数量。传代1（P1）时二次消化组的GFAP阳性率高于相应对照组，30分钟+二次消化组在P1时的GFAP阳性率达到（98.1±1.7）%，与P3时20分钟+对照组相当。然而，两组凋亡率无显著差异。基于上述结果，我们得出结论：30分钟+二次胰蛋白酶消化可有效提高星形胶质细胞纯度，缩短原代培养和纯化时间，表明这是一种在体外快速有效获得高纯度星形胶质细胞的方法。
