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[Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes.] [Article in Chinese]

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 ]iaotong University College of Medicine, Xi'an 710061,China

Abstract

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 SD rats was isolated and digested with 0.25% typsin for 20, 30, and 40 min, respectively. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each digestive group 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. 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 apoptosis rate of purified astrocytes. As a result, the cells being digested for 20 min usually reached confluence at 9 d after seeding. In this study, when the digestion time was extended to 30 min, cells grew faster and reached confluence at 7 d after seeding. At the same time, the morphology of astrocytes was normal and the GFAP positive rate reached (70.2 ± 4.0)% which was much higher than that of the 20 min group (P < 0.01). Compared to 20 min group, the GFAP positive rate was higher, but the cell proliferation was slower and cell injury was more obvious in 40 min group. After shaking at a constant temperature, two times of trysine digestion could decrease the number of contaminated cells after passage. The GFAP positive rate of two-time-digestion groups in passage 1 (P1) was higher than that of the control groups, and 30 min+ two-time-digestion group reached the highest level, about (98.1 ± 1.7)%, which was equivalent to that of the 20 min+control group in P3. However, the apoptosis rate in these two groups showed no significant difference. From above results, we could conclude that 30 min+two-time of trysine digestion effectively improved the purity of astrocytes and shortened the time of primary culture and purification. It would be a rapid and effective method to obtain astrocytes with high purity in vitro.

Key words: astrocytes; cell culture; typsin

Received: 2014-07-23　　Accepted: 2014-10-30

Corresponding author: 王唯析　　E-mail: wangwx@mail.xjtu.edu.cn

Citing This Article：

JIN Hui, Yang Peng-Bo, FENG Gai-Feng, Jia Ning, Yang Wei-Na, WANG Wei-Xi. [Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (1): 103-109 (in Chinese with English abstract).