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Review
. 2022;8(2):72-92.
doi: 10.1007/s40778-022-00212-1. Epub 2022 Apr 27.

Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics

Chasen Cottle  1 , Amanda Paige Porter  1 , Ariel Lipat  1 , Caitlin Turner-Lyles  1 , Jimmy Nguyen  1 , Guido Moll  2 , Raghavan Chinnadurai  1
Affiliations
Review

Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics

Chasen Cottle et al. Curr Stem Cell Rep. 2022.

Abstract

Purpose of review: Cryopreservation and its associated freezing and thawing procedures-short "freeze-thawing"-are among the final steps in economically viable manufacturing and clinical application of diverse cellular therapeutics. Translation from preclinical proof-of-concept studies to larger clinical trials has indicated that these processes may potentially present an Achilles heel to optimal cell product safety and particularly efficacy in clinical trials and routine use.

Recent findings: We review the current state of the literature on how cryopreservation of cellular therapies has evolved and how the application of this technique to different cell types is interlinked with their ability to engraft and function upon transfer in vivo, in particular for hematopoietic stem and progenitor cells (HSPCs), their progeny, and therapeutic cell products derived thereof. We also discuss pros and cons how this may differ for non-hematopoietic mesenchymal stromal/stem cell (MSC) therapeutics. We present different avenues that may be crucial for cell therapy optimization, both, for hematopoietic (e.g., effector, regulatory, and chimeric antigen receptor (CAR)-modified T and NK cell based products) and for non-hematopoietic products, such as MSCs and induced pluripotent stem cells (iPSCs), to achieve optimal viability, recovery, effective cell dose, and functionality of the cryorecovered cells.

Summary: Targeted research into optimizing the cryopreservation and freeze-thawing routines and the adjunct manufacturing process design may provide crucial advantages to increase both the safety and efficacy of cellular therapeutics in clinical use and to enable effective market deployment strategies to become economically viable and sustainable medicines.

Keywords: Cellular therapeutics; Cryopreservation; Effector T cells (Teff); Freeze-thawing; Functionality; Induced pluripotent stem cells (iPSCs); Mesenchymal Stromal/Stem Cells (MSCs); Natural killer (NK) cells; Regulatory T cells (Treg); Safety and efficacy; Stem cells (MSCs).

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Conflict of interest statement

Conflict of InterestR.C. is an inventor on a pending patent application (US Patent 20190099449 A1) related to this publication. The other authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Impact of cryopreservation and freeze-thawing on clinical cell therapies. (A) Cell therapy manufacturing and application with and without cryopreservation: Cell-based advanced therapy medicinal products (ATMPs), such as mesenchymal stromal / stem cells (MSCs) are typically generated from human donor tissue by selective in vitro expansion under environmentally controlled conditions, e.g., by employing advanced bioreactors. The cell batches are then typically stored in a cryopreserved state and subjected to good-manufacturing-practice (GMP) quality controls, in order to obtain batch release for clinical use. The therapeutic cells are then delivered to patients most commonly either by local or systemic injection [3, 4]. Predominantly freeze-thawed cells delivered shortly post cryoretrieval were used in the past. The optional clinical use of fresh cells has been mainly suspended in Europe since 2007, due to the adoption of the European Union Tissues and Cells Directive (EUTCD) although conditional exemptions can be warranted, e.g., under use of the European Hospital Exemption [•]; and (B) Clinical Experience and Performance of Fresh and Thawed MSC Therapeutics: Therapeutic cell engraftment, immune, and clinical response evaluation in animal models and patient cohorts suggest that the therapeutic activity of MSCs is not the result of long-term engraftment and tissue formation, but probably the result of a complex (immune)-modulatory action exerted within the first hours to days and weeks of infusion. Differences in transient engraftment / persistence (signal intensity of cells, %), different susceptibility to triggering of the instant blood-mediated inflammatory reaction (IBMIR) / innate and adaptive immune responses (adverse immune response, %), and different bioactivity and environmental responsiveness may provide an explanation, why fresh minimal expanded cells appear to have favorable therapeutic activity [12]
Fig. 2
Fig. 2
Cryopreservation and freeze-thawing of cell products. (A) Phase changes during freezing and thawing of clinical cell products: For permanent long-term cryostorage in a frozen state in a biobank the clinical cell product (e.g., mesenchymal stromal/stem cells; MSCs) has to be first harvested (e.g., from a suspension bioreactor) and then frozen under controlled conditions with the cells concentrated and resuspended in a suitable highly optimized cryomedia that prevents cell damage and supports robust thawing with amenable cell recovery, viability, and functionality for clinical use as an infusion or tissue injection product (cell infusion bag and cell injection syringe) with all steps being performed under good manufacturing practice (GMP) conditions; and (B) Temperature profile for freezing and thawing curves of cell products: A crucial aspects in cryopreservation of clinical cell products is the optimal temperature profile and speed of the freezing and thawing curve (e.g., freezing of MSCs is typically done in a controlled rate automated freezing device at < 1*C/min with appreciation of the seeding temperature to prevent harmful ice crystal formation and allowing for constant cooling, while thawing is typically done in 37*C water baths with optimal heat transfer at 45*C/min, which allows fairly rapid thawing to ambient temperature (e.g., room temperature 22*C). Once frozen, the cell product can be transferred to ultra low temperature cryostorage (Less than -135 to -196*C) and maintained in an inert state for years. However, temperature fluctuation spikes due to frequent sample retrieval can also slowly degrade / damage frozen products
Fig. 3
Fig. 3
Adverse immune reactions to systemically infused fresh and frozen cell products. Studies on the biodistribution and time course of MSC engraftment upon clinical delivery via systemic intravenous infusion to patients suggest that most cells are rapidly embolized within the lung microvasculature and destroyed shortly post infusion due to a number of antagonizing mechanisms, e.g., triggering of innate immune cascades and the associated thromboinflammation including activation of innate and adaptive effector cells, a sequence of events commonly summarized under the term “Instant Blood-Mediated Inflammatory Reaction” (IBMIR) [, •, 79, 145]. (A) Instant Immune Recognition of Infused Fresh vs. Freeze-Thawed MSCs: While freshly harvested cells display optimal cell membrane physiology, freeze-thawed cells readily derived from cryostorage can display a disturbed cell membrane physiology, depending on the specifics of the cryopreservation and cryorecovery procedures used at site. This makes freeze-thawed cells more prone to hemodynamic disruption and innate immune attack, here exemplified through complement activation and coating with sequential complement component 3 (C3)-activation products (e.g., C3b, iC3b, C3d) and the concomitant release of complement C3 and C5 anaphylatoxins (e.g., C3a and C5a) [•, 10, 11]. (B) Innate Effector Cell Engagement by Fresh and Freeze-Thawed MSCs: The complement opsonins and anaphylatoxins are potent activating ligands for innate effector cells, such as phagocytes and NK-cells, which can attack and damage therapeutic cells, e.g., through release of perforin and granzyme, leading to triggering of target cell apoptosis, membrane lysis, and cell death. Furthermore, depending on the initial tissue source MSCs display varying levels of procoagulant tissue factor (TF/CD142) [3, 146], which is a potent trigger of the coagulation cascade, and can thus promote embolization and sequestration of cellular therapeutics in the microvasculature, which is less evident for fresh cells. (C) Differential Release or Paracrine Mediators and Microparticles: Differential susceptibility of fresh and thawed MSCs to innate immune attack promotes their differential release of 1) Paracrine mediators (e.g., cytokines, chemokines, and small sized metabolites) and 2) MSC-derived microparticles (e.g., exosomes 70-150 nm, microvesicles 100 nm to 1um, and apoptotic bodies 1-5um) [147, 148]. The stronger innate attack and faster killing of freeze-thawed MSCs limits their active secretion of soluble paracrine mediators but augments their passive release of cell-derived microparticles. In turn, fresh MSCs exhibit longer in vivo persistence and paracrine secretion, but less microparticle secretion. Preclinical and clinical data suggest that an active response by metabolically active fresh cells elicits stronger beneficial immune modulation
Fig. 4
Fig. 4
Cryopreservation process and product optimization as central steps in designing next generation MSC therapeutics. A discrepancy in response rates between pre-clinical proof-of-concept studies and clinical trials with mesenchymal stromal / stem cells (MSCs) has been observed in the past [, ••, 72]. This discrepancy may be explained by the predominant use of fresh from culture derived MSCs in the pre-clinical proof-of-concept studies as contrasted by the predominant use of cryopreserved MSCs in advanced clinical trials. Major regulatory bodies (e.g., FDA and EMA) demand extensive testing of MSCs for safety and efficacy, thus often making the cryopreservation of MSCs essential to conform to the regulatory standards. The importance of MSC cryopreservation in the past, the present, and the future is evident in the horizontal time line. Four main aspects should be taken into consideration for the future use of MSCs. First, donor variability should be reduced by defined inclusion criteria. Second, cryopreservation should be optimized, e.g., a promising tool to boost immunomodulatory activity is the stimulation of MSCs with IFN-γ prior to initiating the cryopreservation process, which promotes activation / priming of the key immunoregulatory mediator indoleamine 2,3-deoygenase (IDO). Third, culture recovery of cryopreserved MSCs should be performed for 1–2 days prior to in vivo use to restore optimal cellular function. The latter two aspects support the fourth aspect: maintenance of MSC immunomodulatory activity post-transplantation. A new consensus protocol should be established for the clinical use of MSCs, which includes donor specifications, a standardized procedure of the freeze–thaw process of MSCs, requirements for advanced biobanking, and actual considerations on the process of MSC application / delivery in clinical use [–6, 33, 66]. Due to advances in the development of cardiovascular regenerator (CVR) systems that are compatible with standard dialysis units, cell culture and transplantation may potentially be performed using a single device [33]
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