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Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield
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Gonzalez-Martinez, Natalia and Gibson, Matthew I. (2023) Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield. RSC Medicinal Chemistry, 14 (10). pp. 2058-2067. doi:10.1039/d3md00378g ISSN 2632-8682.
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Official URL: https://doi.org/10.1039/d3md00378g
Abstract
Emerging cell-based therapies such as CAR-T (Chimeric Antigen Receptor T) cells require cryopreservation to store and deliver intact and viable cells. Conventional cryopreservation formulations use DMSO to mitigate cold-induced damage, but do not address all the biochemical damage mechanisms induced by cold stress, such as programmed cell death (apoptosis). Rho-associated protein kinases (ROCK) are a key component of apoptosis, and their activation contributes to apoptotic blebbing. Here we demonstrate that the ROCK inhibitor fasudil hydrochloride, when supplemented into the thawing medium of T-cells increases the overall yield of healthy cells. Cell yield was highest using 5 or 10% DMSO cryopreservation solutions, with lower DMSO concentrations (2.5%) leading to significant physical damage to the cells. After optimisation, the post-thaw yield of T-cells increased by approximately 20% using this inhibitor, a significant increase in the context of a therapy. Flow cytometry analysis did not show a significant reduction in the relative percentage of cell populations undergoing apoptosis, but there was a small reduction in the 8 hours following thawing. Fasudil also led to a reduction in reactive oxygen species. Addition of fasudil into the cryopreservation solution, followed by dilution (rather than washing) upon thaw also gave a 20% increase in cell yield, demonstrating how this could be deployed in a cell-therapy context, without needing to change clinical thawing routines. Overall, this shows that modulation of post-thaw biochemical pathways which lead to apoptosis (or other degradative pathways) can be effectively targeted as a strategy to increase T-cell yield and function post-thaw.
Item Type: | Journal Article | ||||||||
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Subjects: | Q Science > QD Chemistry Q Science > QP Physiology Q Science > QR Microbiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Science > Chemistry Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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SWORD Depositor: | Library Publications Router | ||||||||
Library of Congress Subject Headings (LCSH): | Cryopreservation of organs, tissues, etc., T cells, Cold -- Physiological effect | ||||||||
Journal or Publication Title: | RSC Medicinal Chemistry | ||||||||
Publisher: | Royal Society of Chemistry | ||||||||
ISSN: | 2632-8682 | ||||||||
Official Date: | 1 October 2023 | ||||||||
Dates: |
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Volume: | 14 | ||||||||
Number: | 10 | ||||||||
Page Range: | pp. 2058-2067 | ||||||||
DOI: | 10.1039/d3md00378g | ||||||||
Status: | Peer Reviewed | ||||||||
Publication Status: | Published | ||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||
Date of first compliant deposit: | 12 September 2023 | ||||||||
Date of first compliant Open Access: | 13 September 2023 |
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