Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (14): 2161-2165.doi: 10.3969/j.issn.2095-4344.3092

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Morphological characteristics of in vitro cultured articular chondrocytes from Sprague-Dawley rats at passage number from 0 to 3

Yang Fan1, 2, Liu Baoyi2, Liu Jiahe2, Yang Jiahui2, Qin Kairong1, Zhao Dewei1, 2   

  1. 1School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; 2Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • Received:2020-04-01 Revised:2020-04-09 Accepted:2020-05-16 Online:2021-05-18 Published:2020-12-30
  • Contact: Zhao Dewei, MD, Chief physician, Professor, School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • About author:Yang Fan, MD candidate, Physician, School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81672139 and 81371942 (both to ZDW); the National Key Research & Development Plan of China, No. 2016YFC1102000 (to ZDW); the Scientific Innovation Foundation of Dalian, No. 2018J11CY030 (to ZDW)

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Abstract: BACKGROUND: Common methods for obtaining chondrocytes include mechanical-enzymatic digestion, sequential digestion of pronase and collagenase, sequential digestion of trypsin and type II collagenase, and simple type II collagenase digestion.
OBJECTIVE: To investigate the isolation, extraction and identification of knee joint chondrocytes from Sprague-Dawley rats, and to observe the morphological characteristics of chondrocytes from primary to passages 3.
METHODS: We used single-step digestive approach (type II collagenase digestion) to isolate and obtain chondrocytes from the knee joints of Sprague-Dawley rats in vitro, and then established a culture system in vitro. Cell proliferation assay and inverted phase contrast microscope were used to observe morphohistology of the cells. Toluidine blue staining, type II collagen as well as proteoglycan immunofluorescence staining were used to identify the characteristics of chondrocytes. The experimental protocol was approved by the Ethics Committee of Zhongshan Hospital of Dalian University.
RESULTS AND CONCLUSION: Microscopic observation and cell proliferation assay both showed satisfactory cell morphology and proliferation at passages 2-3, and the capacity of the cells decreased gradually. Moreover, toluidine blue staining, type II collagen and proteoglycan immunofluorescence staining revealed superior characteristics of passage 3 chondrocytes cultured in vitro. These findings indicate that chondrocytes can successfully be isolated by the single-step type II collagenase digestion. Cell proliferation and passage cultivation can also be achieved. And dedifferentiation capacity of chondrocytes is improved gradually after the passages 3 cultured in vitro. Passage 3 cells cultured in vitro have superior characteristics for the use in experimental researches.

Key words: chondrocyte, enzyme digestion, cell culture, rat, experiment

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