A simple and serum-free protocol for cryopreservation of human umbilical cord as source of Wharton's jelly mesenchymal stem cells.

Mesenchymal stromal cells (MSCs) show promise in cell-based transplantations and regenerative medicine applications. MSCs from Wharton's jelly (WJ) of umbilical cord can be easily harvested and exhibit greater proliferative activity than bone marrow MSCs. It is important to develop a practical cryopreservation technique to effectively store umbilical cord for potential future applications. Successful cryopreservation would allow access to umbilical cord from the same donor for repeated WJ MSC-based transplantations. For therapeutic applications, one should be able to obtain clinically-relevant quality and quantity of MSCs from cryopreserved tissues. In this study, we optimised a serum-free formulation of 10% dimethyl sulfoxide (DMSO) and 0.2M sucrose for cryopreservation of umbilical cord tissue. Slow freezing and rapid thawing were adopted. MSCs harvested from WJ of cryopreserved umbilical cord could undergo robust expansion, differentiate to mesodermal lineages and express MSC-characteristic surface antigens. The cumulative cell yield, however, was less compared to corresponding fresh cord tissue.

Roy S ，Arora S ，Kumari P ，Ta M ... -  《-》

DMSO- and Serum-Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord.

The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post-thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [-1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post-thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog-Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin-V-positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv-Cock). Real-time PCR and Western blot analysis of post-thaw WJMSCs from Conv-Cock group showed significantly increased expression of pro-apoptotic factors (BAX, p53, and p21) and reduced expression of anti-apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog-Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC-based regenerative therapies. J. Cell. Biochem. 117: 2397-2412, 2016. © 2016 Wiley Periodicals, Inc.

Shivakumar SB ，Bharti D ，Subbarao RB ，Jang SJ ，Park JS ，Ullah I ，Park JK ，Byun JH ，Park BW ，Rho GJ ... -  《-》

Freezing of Fresh Wharton's Jelly From Human Umbilical Cords Yields High Post-Thaw Mesenchymal Stem Cell Numbers for Cell-Based Therapies.

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.

Fong CY ，Subramanian A ，Biswas A ，Bongso A ... -  《-》

Wharton's jelly mesenchymal stromal/stem cells derived under chemically defined animal product-free low oxygen conditions are rich in MSCA-1(+) subpopulation.

Devito L ，Badraiq H ，Galleu A ，Taheem DK ，Codognotto S ，Siow R ，Khalaf Y ，Briley A ，Shennan A ，Poston L ，McGrath J ，Gentleman E ，Dazzi F ，Ilic D ... -  《-》

Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton's jelly matrix.

Hendijani F ，Sadeghi-Aliabadi H ，Haghjooy Javanmard S 《-》