Issue 9, 2023

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation

Abstract

Cell therapies such as allogenic CAR T-cell therapy, natural killer cell therapy and stem cell transplants must be cryopreserved for transport and storage. This is typically achieved by addition of dimethyl sulfoxide (DMSO) but the cryoprotectant does not result in 100% cell recovery. New additives or technologies to improve their cryopreservation could have major impact for these emerging therapies. L-Proline is an amino acid osmolyte produced as a cryoprotectant by several organisms such as the codling moth Cydia pomonella and the larvae of the fly Chymomyza costata, and has been found to modulate post-thaw outcomes for several cell lines but has not been studied with Jurkat cells, a T lymphocyte cell line. Here we investigate the effectiveness of L-proline compared to D-proline and L-alanine for the cryopreservation of Jurkat cells. It is shown that 24-hour pre-freezing incubation of Jurkat cells with 200 mM L-proline resulted in a modest increase in cell recovery post-thaw at high cell density, but a larger increase in recovery was observed at the lower cell densities. L-Alanine was as effective as L-proline at lower cell densities, and addition of L-proline to the cryopreservation media (without incubation) had no benefit. The pre-freeze incubation with L-proline led to significant reductions in cell proliferation supporting an intracellular, biochemical, mechanism of action which was shown to be cell-density dependent. Controls with D-proline were found to reduce post-thaw recovery attributed to osmotic stress as D-proline cannot enter the cells. Preliminary analysis of apoptosis/necrosis profiles by flow cytometry indicated that inhibition of apoptosis is not the primary mode of action. Overall, this supports the use of L-proline pre-conditioning to improve T-cell post-thaw recovery without needing any changes to cryopreservation solutions nor methods and hence is simple to implement.

Graphical abstract: Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation

Article information

Article type
Research Article
Submitted
13 Jun 2023
Accepted
17 Jul 2023
First published
27 Jul 2023
This article is Open Access
Creative Commons BY license

RSC Med. Chem., 2023,14, 1704-1711

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation

A. Murray, P. Kilbride and M. I. Gibson, RSC Med. Chem., 2023, 14, 1704 DOI: 10.1039/D3MD00274H

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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