title: Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly.
abstract: Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.
authors.affiliation: Institute of Bioscience, University Putra Malaysia, Kuala Lumpur, Malaysia.
authors.forename: Parvin
authors.initials: P
authors.lastname: Salehinejad
authors.forename: Noorjahan Banu
authors.initials: NB
authors.lastname: Alitheen
authors.forename: Abdul Manaf
authors.initials: AM
authors.lastname: Ali
authors.forename: Abdul Rahman
authors.initials: AR
authors.lastname: Omar
authors.forename: Maryam
authors.initials: M
authors.lastname: Mohit
authors.forename: Ehsan
authors.initials: E
authors.lastname: Janzamin
authors.forename: Fazel Sahraneshin
authors.initials: FS
authors.lastname: Samani
authors.forename: Zahra
authors.initials: Z
authors.lastname: Torshizi
authors.forename: Seyed Noureddin
authors.initials: SN
authors.lastname: Nematollahi-Mahani
mesh_terms: D015720:5'-Nucleotidase / Q000096:biosynthesis; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D000071063:Endoglin; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D019089:Stem Cell Factor / Q000096:biosynthesis; D018800:Thy-1 Antigens / Q000096:biosynthesis; D014357:Trypsin / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology; D059631:Wharton Jelly / Q000166:cytology*
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; C064206:ENG protein, human; D000071063:Endoglin; D018960:Hyaluronan Receptors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011956:Receptors, Cell Surface; D019089:Stem Cell Factor; D018800:Thy-1 Antigens; D017493:Leukocyte Common Antigens; D015720:5'-Nucleotidase; D006821:Hyaluronoglucosaminidase; D014357:Trypsin; D017364:Collagenases