Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Prata, Karen de Lima; Santis, Gil Cunha De; Orellana, Maristela Delgado; Palma, Patrícia Vianna Bonini; Brassesco, María Sol; Covas, Dimas Tadeu

doi:10.3109/14653249.2012.677820

Cited by 41 publications

(27 citation statements)

References 16 publications

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“…3,33,34 In contrast, other studies 10,[35][36][37] have demonstrated that cryopreserved MSC exhibit equivalent viability and immunosuppressive potential to freshly isolated MSC from cell culture. Consistent with these findings, we found that freeze-thawed MEP displayed a viability of 95% and retained the ability to effectively suppress lymphocyte proliferation in vitro.…”

Section: Discussionmentioning

confidence: 60%

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

et al. 2016

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© Ferrata Storti Foundation IntroductionSince the first clinical trial of mesenchymal stromal cells (MSC) 9,10 Another important issue regarding the clinical application of MSCs is their culture under serum-free conditions. The majority of clinical studies have used MSCs that were expanded in media supplemented with fetal bovine serum (FBS). 1,[11][12][13][14][15] To avoid the risks associated with the use of FBS, 16 platelet lysate (PL) was proposed as a supplement to tissue culture media for MSCs. 17 Recently, several studies showed that MSCs that were expanded in PL exhibited the same efficacy as MSCs cultured in serum-containing media for the treatment of GvHD. 18-22To date, clinical studies have used MSCs that have been generated from several individual donors. Considering the aforementioned inter-donor heterogeneity and the need for a large number of "off-the-shelf" MSCs, the establishment of MSC banks appears to be an indispensable strategy for providing a continuous supply of MSCs with predictable potency. To our knowledge, there are few established MSC banks worldwide, and these MSC banks were generated by separately isolating, expanding, and freezing MSCs from up to 10 donors in FBS-containing media. [23][24][25][26] In the current study, we report for the first time the establishment of a serum-free and GMP-compliant MSC bank generated from pooled bone marrow mononuclear cells (BM-MNCs) of multiple donors as a novel strategy to circumvent donor-to-donor variability. Clinical-grade MSC endproducts (MEPs) derived from the MSC bank were thoroughly assessed for their proliferation, differentiation, and, in particular, for the allosuppressive potential in vitro. Importantly, 81 MEPs were administered as a rescue therapy to 26 pediatric patients with severe steroid-refractory aGvHD in seven transplantation centers. Safety and efficacy of MEPs was compared to MSCs generated from a single or several individual donors that have been used in the GvHDclinical studies reported thus far. Methods Generation of MSC bank and clinical-grade MEPsBone marrow was collected from 8 healthy volunteers (age 21-45 years old) after written informed consent and after the approval of the local Ethics Committee (n. 275/09). BM-MNCs were enriched from the bone marrow aspirate by using the Sepax II NeatCell process (Biosafe, Eysins, Switzerland) and frozen individually. After thawing and washing these BM-MNCs were pooled. This pool of BM-MNCs from 8 donors was used to generate MSCs over 14 days in culture. After their detachment, passage 1 mesenchymal stromal cells (MSC-P1) were washed and aliquoted into 209 cryovials (each containing 1.5x10 6 MSC-P1). Cryopreserved vials with MSC-P1 were referred to as the MSC bank.To generate clinical-grade MEPs, MSC-P1 aliquots from the MSC bank were thawed and after washing they were expanded in medium containing 10% PL till the end of passage 2. These MSCs were re-suspended in cryomedium (0.9% NaCl containing 5% HSA and 10% DMSO), distributed in cryobags (each containing 1-3x10 6 MS...

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Section: Discussionmentioning

confidence: 60%

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

et al. 2016

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“…Chieregato et al [132] followed a xero-free culture protocol and showed that addition of epidermal growth factor (EGF), bFGF and PDGF-BB to a medium supplemented with human platelet-poor plasma (hPPP) was able to expand hATMSCs better than FBS, without modifying the MSCs phenotype but enhancing their adipogenic and osteogenic differentiation potential. De Lima Prata et al [133] showed that P5-P7 hUC-MSCs can be cryopreserved in xeno-free dimethyl sulfoxide without significant reduction in viability while keeping MSCs immunomodulatory and karyotype properties. Julavijitphong et al [134] showed evidences supporting that human serum from umbilical cord blood (hUCS) could replace fetal bovine serum as culture reagent for hWJ-MSCs.…”

Section: Biosafety: New Scenes and Strategiesmentioning

confidence: 99%

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives

Fiore

Mazzolini

Aquino

2015

Stem Cell Rev and Rep

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Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.

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“…40,41 To evaluate the use of cryopreserved MSCs in these applications, their immunomodulatory ability is determined by the immunosuppressive effect on lymphocytes or peripheral blood mononuclear cells (PBMCs). [42][43][44][45][46] It has been reported that cryopreservation does not affect the ability of MSCs to suppress the proliferation of lymphocytes or PBMCs. 42,45,46 However, Francois et al 43 and Moll et al 44 suggested a potential impairment of cryopreserved MSC immunosuppressive phenotype directly after thawing, which is associated with the activation of heat-shock proteins (e.g., Hsp 27, Hsp 47, Hsp 56, and Hsp 90) that initiate a heat-shock stress on MSCs during the thawing process.…”

Section: Immunomodulatory Abilitymentioning

confidence: 99%

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

Yong

Zaman

Xufeng

et al. 2015

Biopreservation and Biobanking

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Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

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Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Cited by 41 publications

References 16 publications

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

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