Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (1): 45-50.doi: 10.3969/j.issn.2095-4344.1869

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Ammonium chloride lysis method versus non-lysis method for isolation of human adipose-derived stem cells

Li Zifei, Wang Qian, Luan Jie, Mu Dali, Liu Chunjun, Xin Minqiang, Fu Su, Xu Boyang, Liu Wenyue, Chen Lin, Cheng Hao, Wang Chenglong, Kang Deni, Li Shangshan, Qi Jun   

  1. Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China
  • Received:2019-01-07 Revised:2019-01-17 Accepted:2019-03-12 Online:2020-01-08 Published:2019-12-11
  • Contact: Luan Jie, MD, Chief physician, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China
  • About author:Li Zifei, MD candidate, Plastic Surgery Hospital, chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China
  • Supported by:
    Innovation Fund for Medical Science (CIFMS), No. 2017-I2M-3-006

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Abstract:

BACKGROUND: A general standard has not been established for the extraction and purification of adipose-derived stem cells (ADSCs). An erythrocyte lysis step for stromal vascular fraction is the commonly used method in the procedure for ADSCs isolation. However, there is a lack of evidence on whether this step will have adverse effects on human ADSCs (hADSCs).

OBJECTIVE: To test the efficiency of two hADSCs isolation methods, which are erythrocyte-lysis method based on ammonium chloride and non-lysis method. Moreover, the biological characteristics of the hADSCs isolated by the two methods were also compared.

METHODS: After collagenase digestion of lipoaspirate, erythrocyte lysis buffer was used to purify stromal vascular fraction in erythrocyte-lysis method, while in non-lysis method the buffer was not used. A MuseTM cell analyzer was used to assess living cell counting and proportion of stromal vascular fraction in both methods. Then hADSCs were cultured to the second passage for next testing. Cell morphology was observed under light microscope. Cell phenotype was detected by flow cytometry. Cell counting kit-8 was used to evaluate cell proliferation. Oil red O staining and alizarin staining were used to evaluate adipogenic and osteogenic ability of hADSCs after adipogenic and osteogenic induction. This study was approved by the Ethics Committee of the Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, and informed consents were signed by all participants.

RESULTS AND CONCLUSION: (1) Compared with the erythrocyte lysis group, hADSCs obtained in the non-lysis group contained a larger number and a larger percentage of non-erythrocyte living cells. (2) The two groups of hADSCs were spindle-shaped and arranged as a fish shape. (3) The cell phenotypes of both groups met the phenotypic requirements for human mesenchymal stem cells. (4) The cell proliferation in the non-lysis group was faster than that in the erythrocyte lysis group, while there was no difference in the adipogenic and osteogenic abilities between the two groups. In conclusion, the use of erythrocyte lysis buffer reduces the isolation efficiency of hADSCs and inhibits cell proliferation. The non-lysis isolation method does not affect phenotypes, adipogenic and osteogenic ability of hADSCs. Therefore, it is not recommended to implement erythrocyte lysis during the isolation of hADSCs.

Key words: erythrocyte lysis buffer, cell isolation, adipose-derived stem cells, cell viability, cell phenotype, cell proliferation

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