Development of clinical grade protocols for cryopreservation and quality control of perinatal tissues and their derivatives

Autologous and allogeneic “off-the-shelf cell” products occupy an ever-increasing niche in the new therapeutic protocols and clinical trials. A large part of this division belongs to placental stem cells and fetal tissues, which are applied for therapy of burns, wounds and many diseases such as osteoarthritis, type 2 diabetes, hematologic malignancies, Crohn’s disease, Peyronie’s disease, idiopathic pulmonary fibrosis, etc. Our laboratory has a certain history of the cryopreservation techniques development for different tissues and cells [Preservation of parenchymal and stromal progenitors in a cryopreserved human fetal liver Shablii, V.A., Kuchma, M.D., Kyryk, V.M. et al. Cytol. Genet. (2012) 46: 41. doi: 10.3103/S0095452712010100]. We develop approaches for cryopreservation of placenta-derived stem cells and tissues that makes possible product manufacturing along with ATMP manufacturing requirements from batch to batch. For this purpose, we are determining an optimal conditions including cryoprotectant solutions and appropriate cooling parameters for controlled rate freezing. Developing approaches allows achieving high tissue recovery and stem cells propagation after thawing. The quality of our products is proven by cell viability assay based on membrane integrity (trypan blue, fluorescent dyes exclusion methods) and cell proliferation capacity determination (colony forming units assay – see Fig below, doubling time), immunophenotyping of thawed cells (by flow cytometry, immunofluorescence), gene expression analysis and in vitro functional testing (multipotency, cell migration assays). All together it allows launching the clinical-grade placenta cryobank as a stock of tissue for manufacturing of placenta-derived MSCs products according to GMP regulations. [Mesenchymal and trophoblast immunophenotype of multipotent stromal cells from human placenta Shablii V.A., Kuchma M.D., Kyryk V.M., Svitina H.M., Shablii Yu.M., Lukash L.L., Lobintseva G.S. Biopolym. Cell. 2014; 30(2):118-121. doi: 10.7124/bc.000889; Controlled rate freezing provides high recovery of placenta derived multipotent cells V. Nikulina, T. Bukreieva, I. Zahanich, M.Kuchma, V. Kyryk, G. Lobintseva, V. Shablii (2018); P24 – IPLASS2018 Abstract book, 5th IPLASS Meeting – International Stem Cell Conference, September 6-7, 2018, Bern, Switzerland].

 

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CFU from thawed tissue explant on passage 0, day 14, Romanowsky-Giemsa staining (A). There were no significant differences of CFU numbers (passage 0) for thawed tissue (all variants of cryoprotectant solutions ##1-9) and native (#10) (B). CFU of placenta-derived MSCs from thawed tissue on passage 2, day 9, Romanowsky-Giemsa staining (C). On passage 6, combinations #4 (10% DMSO + 0,2M Sucrose) and #8 (5% DMSO + 0,1M Sucrose) revealed in higher tissue recovery after thawing in comparison with the others, whereas it did not differ significantly from native (#10) (D).  

 

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