Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (10): 1825-1828.doi: 10.3969/j.issn.1673-8225.2010.10.024

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Culture and preparation of dog bone marrow mesenchymal stem cell sheet in vitro

Jing Heng, Tan Shuai, Gao Zhen-hua, Chen Li-qiang, Li Ning-yi   

  1. Department of Oral and Maxillofacial Surgery, Hospital Affiliated to Medical College, Qingdao University, Qingdao   266003, Shandong Province, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: 李宁毅，教授，青岛大学医学院附属医院口腔颌面外科，山东省青岛市 266003 ningyili342@ 163.com
  • About author:Jing Heng, Studying for master’s degree, Department of Oral and Maxillofacial Surgery, Hospital Affiliated to Medical College, Qingdao University, Qingdao 266003, Shandong Province, China jingheng1982@126. com
  • Supported by:

    the National Natural Science Foundation of China, No. 30872896*;
    the Natural Science Foundation of Shandong Province, No. Y2008C77*

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Abstract:

BACKGROUND: There are some disadvantages in harvesting and transferring cells in the traditional tissue engineering technique, and it is difficult to form dense tissues, which significantly limits the development of tissue engineering.

OBJECTIVE: To explore the culture and fabrication of dog bone marrow mesenchymal stem cell (BMSC) sheet in vitro.

METHODS: Bone marrow was extracted from dogs following anesthesia. BMSCs were isolated with the method of density gradient centrifugation in vitro. BMSCs at passage 4 at a density of 1×109/L were incubated in the temperature-responsive culture dishes with a diameter of 3.5 cm, and cultured in an incubator at 37 ℃, 5% CO2 and saturated humidity. The temperature of the incubator was changed from to 37 ℃ to 20 ℃ to prepare BMSCs cell sheet for 20 minutes. Cell morphological changes and cell sheet formation were observed under an inverted microscope.

RESULTS AND CONCLUSION: Dog BMSCs following 24 hours of primary culture presented ellipse or polygonal shape. Most cells adhered at hour 72, and cell colonies were visible at day 7. Cells showed long spindle and completely confluence at day 12, with unclear boundary. BMSCs in the temperature-responsive culture dishes presented short spindle shape, and gradually separated from the dish bottom, forming entire cell sheet containing extracellular matrix at 20 ℃. These verified that dog BMSCs can be effectively obtained with method of density gradient centrifugation. Complete cell sheet layer can be fabricated with temperature-responsive culture dishes.

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