The Utilization of Freezing Steps in Mesenchymal Stromal Cell (MSC) Manufacturing: Potential Impact on Quality and Cell Functionality Attributes
Open Access
- 16 July 2019
- journal article
- research article
- Published by Frontiers Media SA in Frontiers in Immunology
- Vol. 10, 1627
- https://doi.org/10.3389/fimmu.2019.01627
Abstract
Some recent reports suggest that cryopreserved and thawed mesenchymal stromal cells (MSCs) may have impaired functional properties as compared to freshly harvested MSCs from continuous cultures. A cryopreservation step in the manufacturing process brings important benefits, since it enables immediate off-the-shelf access to the products and a completion of all quality testing before batch release and administration to the patient. Cryopreservation is also inevitable in MSC banking strategies. In this study, we present the results from the MSC stability testing program of our in-house manufactured clinical-grade allogeneic bone marrow-derived MSC product that is expanded in platelet lysate and frozen in passage 2. The current manufacturing protocol contains only one freezing step and the frozen MSC product is thawed bed-side at the clinic. We can conclude superior viability and cell recovery of the frozen and thawed MSC product utilizing the validated freezing and thawing protocols we have developed. The MSC phenotype and differentiation potential was generally found to be unaltered after thawing, but the thawed cells exhibited a 50% reduced, but not completely abolished, performance in an in vitro immunosuppression assay. The in vitro immunosuppression assay results should, however, be interpreted with caution, since the chosen assay mainly measures one specific immunosuppressive mechanism of MSCs to suppress T-cell proliferation. Since at least two freezing steps are usually necessary in MSC banking strategies, we went on to investigate the impact of repeated freezing on MSC quality attributes. We can conclude that two freezing steps with a preceding cell culture phase of at least one passage before freezing is feasible and does not substantially affect basic cell manufacturing parameters or quality attributes of the final frozen and thawed product. Our results suggest, however, that an exhaustive number of freezing steps (≥4) may induce earlier senescence. In conclusion, our results support the utilization of frozen MSC products and MSC banking strategies, but emphasize the need of always performing detailed studies on also the cryopreserved MSC counterpart and to carefully report the cryopreservation and thawing protocols.Keywords
This publication has 40 references indexed in Scilit:
- Mesenchymal stem cell therapy and acute graft-versus-host disease: a reviewHuman Cell, 2014
- Do Cryopreserved Mesenchymal Stromal Cells Display Impaired Immunomodulatory and Therapeutic Properties?The International Journal of Cell Cloning, 2014
- Mesenchymal stem cells: immune evasive, not immune privilegedNature Biotechnology, 2014
- Treatment of Graft versus Host Disease with Mesenchymal Stromal Cells: A Phase I Study on 40 Adult and Pediatric PatientsTransplantation and Cellular Therapy, 2013
- Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservationJournal of Cellular Biochemistry, 2012
- Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensingCytotherapy, 2012
- Platelet-lysate-Expanded Mesenchymal Stromal Cells as a Salvage Therapy for Severe Resistant Graft-versus-Host Disease in a Pediatric PopulationTransplantation and Cellular Therapy, 2010
- Adult Human Mesenchymal Stem Cells Added to Corticosteroid Therapy for the Treatment of Acute Graft-versus-Host DiseaseTransplantation and Cellular Therapy, 2009
- Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II studyThe Lancet, 2008
- Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statementCytotherapy, 2006