Cryopreservation of placenta tissue allows isolating viable mesenchymal and hematopoietic stem cells

V Nikulina,M Kuchma,T Bukreieva,I Zahanich,V Kyryk,G Lobintseva,V Shablii

doi:10.1016/j.jcyt.2019.03.485

V Nikulina, M Kuchma + Show 5 more

https://doi.org/10.1016/j.jcyt.2019.03.485

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Background & Aim Cryopreserved placenta tissue could be prospective source of mesenchymal and hematopoietic stem cells for cell therapy and regenerative medicine. The aim of our study was to determine methods of placental cryopreservation with high stem cells recovery. Methods, Results & Conclusion Chorions (n=4) were isolated from full-term human placentas of healthy donors. One part of tissue from every specimen was seeded as a native control and the other part was frozen. Nine variants of cryopreservation medium composition were selected by combination of different concentration of dimethyl sulfoxide (5%, 7.5%, 10%) and sucrose (0.1 M, 0.2 M) as penetrating and non-penetrating cryoprotectants. The samples were frozen in cryogenic vials in a controlled rate freezer (IceCube, Austria). Placenta-derived multipotent cells (PDMCs) were subcultured from native and thawed chorion explants. Colony forming units (CFU) assay was carried out on passages 0, 2, 6 for PDMCs isolated from native and thawed tissue. Hematopoietic stem cells (HSCs) of full-term human placental tissue were obtained by enzymatic method with following depletion of dead cells. Immunophenotyping was performed on BD FACSAria (USA). MethoCult (StemCell Technologies, Canada) was used for differentiation assays. XY karyotype of PDMCs indicates their foetal origin. The results of flow cytometry analysis revealed CD90+ CD105+ CD73+ CD34− CD45− PDMCs phenotype. PDMCs were able to differentiate into osteogenic and adipogenic lineages. CFU assay showed that number of native tissue-derived colonies on passage 0 was significantly higher in comparison with thawed tissue for the nine variants of cryoprotectants. However, number of CFU for fresh-isolated cells on passages 2 and 6 was not significantly differ in comparison with PDMCs from thawed tissue. Flow cytometry analysis showed that the HSCs content among CD45+ cells of the total light-density cell fraction from the cryopreserved and native placental tissue did not significantly differ. CFU study demonstrated the presence of myeloid progenitors, quantitative analysis revealed difference in efficiency of HSCs preservation between certain variants of cryoprotectants. The best variant reached up to 50% of CFU recovery compared to native sample. Controlled cryopreservation of placental tissue under GMP requirements allows to obtain proliferative active stem cells with suitable for clinical issues characteristics.

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