Abstract
Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.
Highlights
Adoptive and regulatory T cell (Treg) therapies offer promising new options for the treatment of various clinical indications originating from a compromised immune system (Bluestone and Tang, 2018; Raffin et al, 2019; Roemhild et al, 2020; Waldmann, 2021)
Freezing media based on 10% DMSO and 10% serum are commonly used for cryopreservation of T cell products (Moll et al, 2016)
Optimizing cryostorage and freeze-thawing procedures is an essential task for developing successful cell therapy products that can be incorporated into clinical practice (Galipeau, 2013; Moll et al, 2014, 2016)
Summary
Adoptive and regulatory T cell (Treg) therapies offer promising new options for the treatment of various clinical indications originating from a compromised immune system (Bluestone and Tang, 2018; Raffin et al, 2019; Roemhild et al, 2020; Waldmann, 2021). All these innovative approaches face the same regulatory and logistical challenges, some of which are new and still rapidly changing (Hickson et al, 2021) Regulatory requirements or their interpretation vary between major markets, such as North America and Europe, especially in the early clinical phases, and between countries within Europe. The ability to manufacture “off the shelf ” products helps to reduce manufacturing costs drastically thereby increasing the access to such therapies (Abou-El-Enein et al, 2016, 2017a,b; Fritsche et al, 2020)
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