title: Freezing of Fresh Wharton's Jelly From Human Umbilical Cords Yields High Post-Thaw Mesenchymal Stem Cell Numbers for Cell-Based Therapies.
abstract: Some cord blood banks freeze entire pieces of UC (mixed cord, MC) which after post-thaw yields mixed heterogeneous populations of mesenchymal stem cells (MSCs) from all its microanatomical compartments. Freezing of such entire tissues results in sub-optimal post-thaw cell recovery because of poor cryoprotectant diffusion and intracellular ice-formation, heat and water transport issues, and damage to intercellular junctions. To develop a simple method of harvesting pure homogeneous MSCs for cord blood banks, we compared the post-thaw behavior of three groups of frozen UC tissues: (i) freshly harvested WJ without cell separation; (ii) MSCs isolated from WJ (WJSC); and (iii) MC, WJ, and WJSC produced high post-thaw cell survival rates (93.52 ± 6.12% to 90.83 ± 4.51%) and epithelioid monolayers within 24 h in primary culture whereas post-thaw MC explants showed slow growth with mixed epithelioid and fibroblastic cell outgrowths after several days. Viability and proliferation rates of post-thawed WJ and hWJSC were significantly greater than MC. Post-thaw WJ and WJSC produced significantly greater CD24(+) and CD108(+) fluorescence intensities and significantly lower CD40(+) contaminants. Post-thaw WJ and WJSC produced significantly lesser annexin-V-positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation compared to MC. qRT-PCR analysis of post-thaw MC showed significant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK 1) compared to WJ and WJSC. We conclude that freezing of fresh WJ is a simple and reliable method of generating large numbers of clinically utilizable MSCs for cell-based therapies.
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Kent Ridge, 119228, Singapore.
authors.forename: Chui-Yee
authors.initials: CY
authors.lastname: Fong
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Kent Ridge, 119228, Singapore.
authors.forename: Arjunan
authors.initials: A
authors.lastname: Subramanian
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Kent Ridge, 119228, Singapore.
authors.forename: Arijit
authors.initials: A
authors.lastname: Biswas
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Kent Ridge, 119228, Singapore.
authors.forename: Ariff
authors.initials: A
authors.lastname: Bongso
mesh_terms: D017304:Annexin A5 / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D000077022:Survivin; D046509:Tissue Culture Techniques; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D059631:Wharton Jelly / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D017304:Annexin A5; D015703:Antigens, CD; C494826:BAX protein, human; C000595707:BCL2 protein, human; C107489:BIRC5 protein, human; D015415:Biomarkers; D003451:Cryoprotective Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D051034:Inhibitor of Apoptosis Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D000077022:Survivin; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C515851:ROCK1 protein, human; D054460:rho-Associated Kinases
keywords: CHARACTERIZATION; CRYOPRESERVATION; FREEZE-THAW CELL SURVIVAL RATES; HUMAN UMBILICAL CORD; MIXED CORD; WHARTON'S JELLY; WHARTON'S JELLY STEM CELLS