Acta Veterinaria et Zootechnica Sinica ›› 2023, Vol. 54 ›› Issue (7): 3091-3101.doi: 10.11843/j.issn.0366-6964.2023.07.039

• CLINICAL VETERINARY MEDICINE • Previous Articles     Next Articles

Comparative Study on Different Methods of Collection, Isolation, Culture and Identification of Feline Limbal Stem Cells

XU Huihao1,2,3, FENG Xueqian1, PIAO Xueling1, SHEN Xiaojun1, ZHENG Xiaobo1, YANG Heng1, LIN Jiahao3*, JIN Yipeng3*, LIN Degui3*   

  1. 1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
    2. Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, Chongqing 400700, China;
    3. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
  • Received:2022-12-10 Online:2023-07-23 Published:2023-07-21

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Abstract: The purpose of this study was to compare the effects of different isolation and culture methods on the culture of feline limbal stem cells (FLSCs) in vitro, and to establish the best isolation and culture system for the continuous and stable proliferation, normal structure and function of FLSCs. The limbal tissues of cats were collected under surgical microscope. Three different digestion methods which were the tissue adhesion method (T method), the mixed enzyme digestion method (N method) and the mixed enzyme digestion methods combined with tissue adhesion method (NT method) were used to separate FLSCs, to identify the expression of LSCs marker protein ABCG2, in order to screen the most suitable method for separation. Cells were cultured through three kinds of complete media which were BC, DLM, and SCM, the cell count and the cell morphology was observed every day for 7 days, and the positive markers of LSCs p63, vimentin and epithelial differentiation markers CK3, CK12 were selected to qualitatively analyze the 3rd, 4th, and 5th generation cells. The results showed that the expression of ABCG2 from cells separated by three methods were positive and that of NT method expressed the most positive, indicating that three methods can all be used to separate FLSCs, and the separation effect of NT method was the best. FLSCs can be cultured in all three media, with no difference in cell morphology. Cells proliferation speed were the fastest in group DLM. Compared with the other two groups, time of cell reaching the peak cell growth was shorter in DLM, and the difference was significant (P<0.05). The growth curve of FLSCs was showed a typical "S" type. The first 72 h of LSCs growth was in the lag phase, the 72-216 h was in the logarithmic growth phase, the 216-264 h was basically in the plateau phase, and the cells entered the decay phase at the 264th hour. The population doubling time of FLSCs was 38.9 h, and the average maximum proliferation concentration was 5.66×105 cells·mL-1. The 3rd, 4th and 5th generation FLSCs showed strong positive expression of vimentin and p63, but CK3 and CK12 didn't express. FLSCs can be isolated by NT method and cultured in DLM to establish a stable proliferation system of FLSCs in vitro.

Key words: feline, limbal stem cells, isolation method, culture system

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