title: [Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells].
abstract: 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)×10(5), (4.03 ± 0.24)×10(5), (4.91 ± 0.33)×10(5) and (5.94 ± 0.40)×10(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.
  
  
  CONCLUSIONS
  The collagenaseII/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Ling-ying
authors.initials: LY
authors.lastname: Liu
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China. Email: cjk304@126.com.
authors.forename: Jia-ke
authors.initials: JK
authors.lastname: Chai
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Hong-jie
authors.initials: HJ
authors.lastname: Duan
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Yu-sen
authors.initials: YS
authors.lastname: Hou
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Hui-nan
authors.initials: HN
authors.lastname: Yin
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Yong-hui
authors.initials: YH
authors.lastname: Yu
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Dai-feng
authors.initials: DF
authors.lastname: Hao
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Feng
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Burns and Plastic Surgery, Affiliated First Hospital, General Hospital of PLA, Beijing 100048, China.
authors.forename: Jun-dong
authors.initials: JD
authors.lastname: Du
mesh_terms: D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D014470:Umbilical Cord / Q000166:cytology*