PMID- 24351605
OWN - NLM
STAT- MEDLINE
DCOM- 20140710
LR  - 20181202
IS  - 0376-2491 (Print)
IS  - 0376-2491 (Linking)
VI  - 93
IP  - 32
DP  - 2013 Aug 27
TI  - [Comparison of different methods for the isolation of human umbilical cord 
      mesenchymal stem cells].
PG  - 2592-6
AB  - OBJECTIVE: To explore the most appropriate method for the isolation of human 
      umbilical cord mesenchymal stem cells (MSCs) through a comparison of different 
      methods. METHODS: Fifteen umbilical cord specimens from full-term healthy fetus 
      with caesarean birth were completely rinsed with phosphate buffer saline (PBS) 
      and sliced into 1 mm(3) tissue blocks after removal of umbilical vessels and 
      external membrane. These tissue blocks were averagely divided into 4 groups after 
      washing and centrifuge. Then four methods for the isolation of human umbilical 
      cord MSCs were compared: an explant culture and three enzymatic methods of 
      collagenaseII, collagenaseII/trypsin and collagenaseII/hyaluronidase. The count 
      of living cells was evaluated by trypan blue dye exclusion test. Cell morphology 
      was observed under inverted microscope. The expressions of cell surface markers 
      CD105, CD90, CD73, CD31, CD44, CD45, human leukocyte antigen-I (HLA-I) and human 
      leukocyte antigen class IImolecules (HLA-DR) were detected by immunofluorescent 
      staining. Cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 
      5-diphenyltetrazolium bromide (MTT). RESULTS: The human umbilical cord MSCs were 
      successfully isolated by four isolated methods. However the isolation method used 
      profoundly altered the cell number and proliferation capacity of isolated cells. 
      Isolated cells using four methods were counted at (5.44 +/- 0.21)x10(5), (4.03 +/- 
      0.24)x10(5), (4.91 +/- 0.33)x10(5) and (5.94 +/- 0.40)x10(5) respectively. More cells 
      were obtained with collagenaseII/hyaluronidase than other three methods (all P < 
      0.05). Cells out of tissue blocks were observed at Day 9-11 and cells were 
      observed at Day 2 with three types of enzyme digestion. The fusion time of cells 
      were (18.5 +/- 3.5), (8.0 +/- 1.0), (7.5 +/- 1.5) and (3.5 +/- 0.5) days respectively. 
      The fusion time of cells obtained with collagenaseII/hyaluronidase was lower than 
      other methods (all P < 0.05). Cell morphology: polygonal, irregular and of large 
      volume for explant culture; relatively short and small for collagenaseII and 
      collagenaseII/trypsin methods; thin spindle for collagenaseII/hyaluronidase 
      method. Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were 
      expressed in all groups while there was no expression of CD31, CD45 or HLA-DR. 
      And the cells obtained with collagenaseII/hyaluronidase method were in a higher 
      cell proliferation rate and activity compared to other methods. CONCLUSION: The 
      collagenaseII/hyaluronidase method is optimal for the isolation of human 
      umbilical cord MSCs than other methods.
FAU - Liu, Ling-ying
AU  - Liu LY
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Chai, Jia-ke
AU  - Chai JK
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China. Email: cjk304@126.com.
FAU - Duan, Hong-jie
AU  - Duan HJ
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Hou, Yu-sen
AU  - Hou YS
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Yin, Hui-nan
AU  - Yin HN
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Yu, Yong-hui
AU  - Yu YH
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Hu, Quan
AU  - Hu Q
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Hao, Dai-feng
AU  - Hao DF
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Feng, Guang
AU  - Feng G
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Li, Tao
AU  - Li T
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
FAU - Du, Jun-dong
AU  - Du JD
AD  - Department of Burns and Plastic Surgery, Affiliated First Hospital, General 
      Hospital of PLA, Beijing 100048, China.
LA  - chi
PT  - Comparative Study
PT  - Journal Article
PL  - China
TA  - Zhonghua Yi Xue Za Zhi
JT  - Zhonghua yi xue za zhi
JID - 7511141
SB  - IM
MH  - Cell Culture Techniques
MH  - Cell Separation/*methods
MH  - Humans
MH  - Mesenchymal Stem Cells/*cytology
MH  - Umbilical Cord/*cytology
EDAT- 2013/12/20 06:00
MHDA- 2014/07/11 06:00
CRDT- 2013/12/20 06:00
PHST- 2013/12/20 06:00 [entrez]
PHST- 2013/12/20 06:00 [pubmed]
PHST- 2014/07/11 06:00 [medline]
PST - ppublish
SO  - Zhonghua Yi Xue Za Zhi. 2013 Aug 27;93(32):2592-6.