Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol |
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| Authors: | Katherine N MacDonald Sabine Ivison Keli L Hippen Romy E Hoeppli Michael Hall Grace Zheng I Esme Dijke Mohammed Al Aklabi Darren H Freed Ivan Rebeyka Sanjiv Gandhi Lori J West James M Piret Bruce R Blazar Megan K Levings |
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| Institution: | 1. School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada;2. BC Children''s Hospital Research Institute, Vancouver, BC, Canada;3. Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada;4. Department of Surgery, University of British Columbia, Vancouver, BC, Canada;5. Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA;6. Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada;7. Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada;8. Department of Surgery, University of Alberta, Edmonton, AB, Canada;9. Department of Pediatrics, University of Alberta, Edmonton, AB, Canada;10. Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada |
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| Abstract: | Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)–compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products. |
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| Keywords: | cell therapy cryopreservation Good Manufacturing Practice regulatory T cell T-cell manufacturing tolerance |
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