Cryopreservation and Cold Chain 2024

Innovation in cryopreservation & cold chain management

Cell & Gene Therapy Insights 2024; 10(1), 1407–1422

DOI: 10.18609/cgti.2024.184

Published: 22 November 2023
Commentary
B Fuller, R Fleck, G Stacey

Successful cryopreservation depends to a large extent on how the cell water compartments respond to ultra-low temperature cooling, which in itself is necessary to inhibit all molecular interactions for long-term biopreservation. Biophysical principles dictate that water will undergo ice nucleation during cooling, which will cause severe cell injury in a number of complex ways, which can be mitigated by how the cryo-cooling is undertaken. The ice burden can be reduced by adding appropriate biocompatible solutes, called cryoprotectants (CPA), which act in a colligative fashion to interfere with the water-to-ice transition as deep cooling progresses, until the temperature range where the whole mixture enters a low temperature ‘glassy’ state (Tg) whence all other molecular interactions are inhibited. Optimisation of cell survival can also be achieved by controlling the kinetics of both cooling and warming rates during cryopreservation, which limit ice crystal growth until final melting temperatures are reached and normal cell biology can resume in the liquid aqueous state. 

The interest in and application of biologics (biological products derived from cells, agents, and small biomolecules) continues unabated in the current decade. These all require a range of integrated technologies, one of which is the ability to control biological time, both within production schedules and for distribution of validated products to end users. This is the so-called cold chain whereby unwanted molecular processes can be halted either for short periods (liquid storage: hours up to a few days) or for greatly extended time (cryopreservation: weeks, progressing forward to years) depending how ‘cold’ is defined and applied. True long storage by cryopreservation can offer significant advantages such as matching supply and demand of cell therapies, which is often dictated by patient disease and treatment course, and availability of specialist staff facilities. Cryopreservation can also avoid wastage and facilitate timely batch testing to meet quality assurance and release criteria. The current understanding of the scientific principles underpinning both approaches have been reported recently but of course, the clear difference between the methods is that cryopreservation requires cooling to deep subzero temperatures where the inescapable property of aqueous solutions (including the intracellular environment) to nucleate ice (Elliot-Fuller) dominates cellular damage beyond other adverse effects associated with low temperatures. The often-reported limited stability of cell therapies such as chimeric antigen receptor (CAR)-T cell products additionally points to benefits of cryopreservation. Currently, amongst the growing portfolio of cell therapies, many CAR-T cell products which have progressed to clinical application have relied on the cryo-cold chain. Although there have been as yet few studies comparing delivery of fresh versus frozen CAR-T cell products, the evidence supports the concept that thawed cryopreserved products perform with comparable efficacy to fresh cell products from the same production batches.

The debate about the suitability of cryopreservation strategies often centers around the undeniable fact that there are losses in cell numbers, reductions in early post-thaw viability indices, and in some cases, a delay in patient overall response rates. The discussions can become further complicated where cryopreservation is used at different stages in the production pathway, and/or for the batch banking of the starting materials (e.g., peripheral blood mononuclear cells [PBMC]) and subsequent transduced cell products ready for patient delivery. The focus of our current review is therefore an assessment of the current understanding of the cellular impacts of the various biophysical stresses of cryopreservation, and what novel ideas are being proposed to mitigate these.

Current understanding & limitations of cryopreservation

It is of course well known that for stable long-term biopreservation, the temperature range needs to be pushed below −40 oC, and into deep cryo-cooling beyond −80 oC [9]Getreu N, Fuller B. Stopping the biological clock—merging biology and cryogenics in applied cryobiology. IOP conf. series: Mat. Sci. and Eng. 502 (2019) 012003, IOP Publishing. 1–11.  [10]Bojic S, Murray A, Bentley BL, Spindler R, Pawlik P, Cordeiro JL, Bauer R, de Magalhães JP. Winter is coming: the future of cryopreservation. MC Biol. 2021; 19(1), 56. . In some ways, the temperature range for storage is dictated by the widespread availability of a suitable cryogenic environment; specialist electrical freezers can operate down to about −135 oC, whilst vapor phase or liquid phase of nitrogen provides temperature control from about −170 oC to −196 oC (depending on the working phase chosen) [2]Meneghel J, Kilbride PG, Morris J. Cryopreservation as a key element in the successful delivery of cell-based therapies—a review. Front. Med. (Lausanne) 2020; 7, 592242.  [11]Stacey GN, Connon CJ, Coopman K, et al. Preservation and stability of cell therapy products: recommendations from an expert workshop. Regen. Med. 2017; 12(5), 553–564.  [12]Kilbride P, Meneghel J. Freezing technology: control of freezing, thawing, and ice nucleation. Meth. Mol. Biol. 2021; 2180, 191–201. . Many recent reviews have discussed the biophysical aspects of cryopreservation [11]Stacey GN, Connon CJ, Coopman K, et al. Preservation and stability of cell therapy products: recommendations from an expert workshop. Regen. Med. 2017; 12(5), 553–564.  [13]Baboo J, Kilbride P, Delahaye M, et al. The impact of varying cooling and thawing rates on the quality of cryopreserved human peripheral blood T cells. Sci. Rep. 2019; 9(1), 3417.  but it is worth outlining some of these to set them in the context of new approaches to cryopreservation. Biological stability is crucial, along with the equally-important need to recover the full range of cellular and molecular functions upon return to normal physiological temperatures, which for most clinical and biotechnological applications will be around 37oC [14]Kilbride P, Lamb S, Gibbons S, et al. Cryopreservation and re-culture of a 2.3 liter biomass for use in a bioartificial liver device. PLoS One 2017; 12(8), e0183385.  [15]Gurruchaga H, Saenz Del Burgo L, et al. Advances in the slow freezing cryopreservation of microencapsulated cells. J. Control Release 2018; 281, 119–138.  [16]Kilbride P, Meneghel J, Lamb S, et al. Recovery and post-thaw assessment of human umbilical cord blood cryopreserved as quality control segments and bulk samples. Biol. Blood Marrow Transplant 2019; 25(12), 2447–2453. . However, we should remember that in other scenarios such as aquatic species biopreservation, recovery to physiological temperature will be to about
10–18oC [17]Paredes E, Adams SL, Vignier J. Cryopreservation of sea urchin sperm and early life stages. Meth. Cell Biol. 2019;150: 47–69.  depending on the species. For any cell to survive cryopreservation, the supreme question is how to deal with ice formation within cellular aqueous compartments. Ice crystals, and the localized associated dehydration as water molecules join growing ice crystal fronts, cause catastrophic, multi-focused injuries [18]Yu G, Hubel A. The role of preservation in the variability of regenerative medicine products. Regen. Eng. Transl. Med. 2019; 5(4), 323–331. , which can disrupt the plasma membrane, intracellular organelles, endoplasmic reticulum, and other membrane-bound compartments. Consistent biological survival almost invariably requires addition of chemical agents termed cryoprotectants (CPA) [19]Fuller BJ. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo. Letters 2004; 25(6), 375–388. , which stabilize molecular and ultrastructural moieties within the cells when the water relationships are severely perturbed by the growing ice phase [20]Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiol. 2017; 76, 74–91.  during slow cooling freezing, which is commonly used. Equally, but in contrast to slow freezing, with the cryogenic storage technique of vitrification (where cooling rates are high and ice formation is deliberately suppressed [21]Fahy GM, Wowk B. Principles of cryopreservation by vitrification. Meth. Mol. Biol. 2015; 1257, 21–82.  [22]Fahy GM, Wowk B. Principles of ice-free cryopreservation by vitrification. Meth. Mol.Biol. 2021; 2180, 27–97. ), survival depends upon stabilizing properties of high concentrations of cryoprotectants [23]Yong KW, Laouar L, Elliott JAW, Jomha NM. Review of non-permeating cryoprotectants as supplements for vitrification of mammalian tissues. Cryobiol. 2020; 96, 1–11. . The injuries resulting from the biophysical changes both during cryopreservation and warming can become additive, leading to progressive and delayed onset cell death in the early post-thaw phase [24]Baust JM, Snyder KK, Van Buskirk RG, Baust JG. Assessment of the impact of post-thaw stress pathway modulation on cell recovery following cryopreservation in a hematopoietic progenitor cell model. Cells 2022; 11(2), 278. . The combination of all of these processes constitutes the science of cryobiology [25]Fleck R, Fuller B. Cell preservation. In: Medicines from Animal Cell Culture. (Editors: Stacey G and Davis J). John Wiley Press, 2007. Chapter 21; 417–431. [26]Pegg DE. Principles of cryopreservation. Meth. Mol Biol. 2015; 1257, 319. .

Figure 1Processes during temperature transitions in cryopreservation. depicts what happens during the temperature transitions of cryopreservation. Liquid water in the aqueous compartments is shown in blue droplets at the top of the image. Cooling temperatures are depicted on the X axis, and the concentration of added CPA are shown on the Y axis. Three different kinetic approaches to cooling (hyper rapid, slow, and slow-to-moderate) are shown which are pragmatic descriptions dictated by available procedures for cryopreservation depending largely by sample size and methods for applying the cryogens needed for cooling.

As cooling proceeds the ice nucleation temperatures for a particular mixture are reached and water enters a super-cooled state (represented by the pink zone) containing potential ice nuclei shown as pale clear droplets. During slow cooling (middle green curve) the ice burden increases significantly shown by ice crystals in the white zone. The stable ‘glassy’ state in the grey zone is reached once the Tg threshold has been passed, and thereafter long-term biopreservation is assured. As the concentration of added CPAs are increased (Y axis), the Tg is shifted upwards, and Tg can be reached with a lesser ice burden from the cooling process. Controlled slow cooling with moderate added CPA concentrations (between 5–20 wt g%) has been one of the traditional approaches to cell cryopreservation, an approach widely termed ‘two-step cooling.

Hyper-rapid cooling (dark green curve) is an experimental approach to achieve Tg using very specific approaches to handling the cryogens used for cooling. It is currently being refined for more user-friendly approaches.

Rapid to moderate cooling (light green curve) is the other main current approach, which depends for success on the use of relatively high added CPA concentrations (about 40–60 wt g%) and rapid cooling approaches. This is the approach widely termed ‘vitrification’ and has found widespread applications where small bio-specimens are being preserved (such as mammalian embryos or plant shoot tips). The Tg range is elevated and can avoid cell injury by the speed of attaining the ‘glassy’ state (grey zone). Truly stable ‘glassy’ states can only usually be achieved with addition of very high CPA concentrations (>80 wt g%) but these are often toxic to cells.

Given that in most cases cryopreserved cells end up within the unstable ‘glassy’ range (middle of lower grey section), control of the warming processes is also essential. The temperature transition out of the Tg range can result in a process called devitrification (buff coloured zone), where water molecules start to become mobile and can aggregate on pre-existing ice crystals. Thus the ice burden can increase (so-called freezing during warming) which can also add to the total injury profiles of the preserved cells. On this basis, warming is usually applied to be as fast as logistically achievable.

Whilst cryopreservation has become almost routine over the past four decades to promote cold chain logistics across a wide range of applications in medicine and biology, there are widely acknowledged limitations to it as it is currently employed (Figure 2Traffic Lights: Green—very close or in use in certain areas; Amber—background science established but requiring some further development (3–5 years); Red—basic science principles requiring further in-depth evaluation, enhanced equipment technologies and regulatory approval needed (5–8 years). CPA: Cryoprotectant.A visual representation of the different approaches to cryo-cooling, the sizes and containers used, the kinetics of cooling and warming relevant to each, and the biological impacts on cells and their environment. The traffic light scheme depicts which are in most widespread usage at the moment. The limitations of each approach are also described. Given the pace of research into cryopreservation, there will likely be mitigation for some of these challenges in the next few years.Traffic Lights: Green—very close or in use in certain areas; Amber—background science established but requiring some further development (3–5 years); Red—basic science principles requiring further in-depth evaluation, enhanced equipment technologies and regulatory approval needed (5–8 years). CPA: Cryoprotectant. & Figure 3Traffic Lights: Green—very close or in use in certain areas; Amber—background science established but requiring some further development (3–5 years); Red—basic science principles requiring further in-depth evaluation, enhanced equipment technologies and regulatory approval needed (5–8 years).Translational status (on the basis of closeness to widespread applicability) of the main current fields of innovation in cryobiology.Traffic Lights: Green—very close or in use in certain areas; Amber—background science established but requiring some further development (3–5 years); Red—basic science principles requiring further in-depth evaluation, enhanced equipment technologies and regulatory approval needed (5–8 years).) [27]Stéphenne X, Najimi M, Sokal EM. Hepatocyte cryopreservation: is it time to change the strategy? World J. Gastroenterol. 2010; 16(1), 1–14.  [28]Yong KW, Wan Safwani WK, Xu F, Wan Abas WA, Choi JR, Pingguan-Murphy B. Cryopreservation of human mesenchymal stem cells for clinical applications. Curr. Meth. Challenges. Biopreserv. Biobank. 2015; 13(4), 231–239.

Current biobanking standards address the need for strategic management of known and adventitious microbial contamination hazards, requiring emergency planning and procedures to be in place to manage integrity, cleanliness, and biosecurity of long-term storage systems for frozen viable material [29]International Organization for Standardization (ISO). ISO 20387: 2018. Biotechnology–biobanking–general requirements for biobanking (Aug 2018). . Emergency response to vessel failure is also crucial to prevent total loss of frozen biological resources [30]Day JG, Childs KH, Stacey GN. Implications of a catastrophic refrigeration failure on the viability of cryogenically stored samples. Protist. 2022; 173(6), 125915. , as well as strategic approaches to avoid risk including planning, cleaning, and maintenance to assure stability and avoid spread of microbial contaminants into experimental work. Unfortunately, it is not uncommon for institutions to lack cleaning regimes for storage vessels, and it has been recognized for some time that there is a need for vessels that are more readily decontaminated or are designed to reduce or remove contamination [11]Stacey GN, Connon CJ, Coopman K, et al. Preservation and stability of cell therapy products: recommendations from an expert workshop. Regen. Med. 2017; 12(5), 553–564. .

The non-frozen cool chain—its relevance in the overall process

Non-frozen cool chains have often been actively pursued by cell therapy manufacturers based on the significant effectiveness of this approach from historical tissue shipment practices [31]Baust JM, Van Buskirk R, Baust J. The storage of cells, tissues and organs in gel-based media. BioLife Solutions Inc. US Patent WO 01/050851. (2002).  [32]Wheatley SP, Wheatley DN. Transporting cells over several days without dry-ice. J. Cell Sci. 2019; 132(21), jcs238139. . This approach is simple and inexpensive, relying on maintenance of cells in the liquid state at any convenient temperature above the freezing point, but is hampered by the short useful shelf-life of only about 2–3 days. Currently, across the cell therapy sector, there is still a general lack of understanding, expertise, and investment in necessary infrastructure for global application of cryo-technologies which is challenging for routine application of cryopreservation. The non-frozen cool chain can allow early startups to move more easily from laboratory settings into the clinic, or allow a simpler ‘hub and spoke’ delivery process for cell shipment from a central manufacturing process to end-user clinics nearby—for example, to linked hospital groups in major cities. However, growth of microbial contamination may not be fully inhibited in the chilled liquid state and thus, the serious potential hazard of patient infection must be managed. Such short shelf lives also create difficulties for completion for industry standard sterility and mycoplasma testing, which cannot be fully completed within the use-by date of the products. Accordingly, there has been much effort to establish guidance to facilitate rapid test methods for non-frozen products, which is now being implemented in European regulatory guidance for Good Manufacturing Practice [33]European Commission. Annex 1 Manufacture of sterile medicinal products, rules governing medicinal products in the European Union volume 4 EU guidelines for good manufacturing practice for medicinal products for human and veterinary Use. (Aug 22, 2022). and an ISO standard for method selection and validation is under development [34]International Organization for Standardization (ISO). ISO/FDIS 24190. Biotechnology-analytical methods-risk-based approach for method selection and validation for rapid microbial detection in bioprocesses.. Short shelf life is also very challenging for healthcare providers to ensure patient availability in readiness to receive the products within validated shelf life, especially when patients must be conditioned for treatment as in CAR T treatment. Failure to assure such fine coordination can mean increased cost and delayed therapy. A range of technologies for shipment of non-frozen materials are under development including gels (e.g., agarose, alginate) and liquid cell suspensions liquids (e.g., HypothermasolTM, Wisconsin solution) some of which have been used in delivery of cell therapy preparations [35]Choudhery MS, Petrenko Y, Chudickova M, et al. Clinically relevant solution for the hypothermic storage and transportation of human multipotent mesenchymal stromal cells. Stem Cells Int. 2019; 2019, 5909524.  [36]Al-Jaibaji O, Swioklo S, Shortt A, Figueiredo FC, Connon CJ. Hypothermically stored adipose-derived mesenchymal stromal cell alginate bandages facilitate use of paracrine molecules for corneal wound healing. Int. J. Mol. Sci. 2020; 21, 5849.  [37]Freitas-Ribeiro S, Reis RL, Pirraco R. Long-term and short-term preservation strategies for tissue engineering and regenerative medicine products: state of the art and emerging trends. PNAS Nexus 2022; 1 (4), 2752–6542. . However, extending the maximum storage/shipment times beyond a few days has proven to be challenging. Further improvements in such approaches may be achieved through the use of apoptotic inhibitors [38]Bissoyi A, Pramanik K. Role of the apoptosis pathway in cryopreservation-induced cell death in mesenchymal stem cells derived from umbilical cord blood. Biopreserv. Biobank 2014; 12(4), 246–254.  [39]Baust JM, Van Buskirk R, Baust JG. Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell. Dev. Biol.—Animal 2000; 36, 262–270., but use of such supplementary storage excipients will require additional regulatory approval. It is likely that over the next 5–10 years, many liquid storage delivery platforms will be replaced by cryogenic preservation platforms.

Horizon scanning to improve cryopreservation outcomes

As outlined above, the collective understanding of the essential biophysical controls which are needed to support cell recovery from the cryogenic excursions has improved in the past decade [1]Buriak I, Elliott G, Fleck R, et al. Preservation and storage of cells for therapy: fundamental aspects of low temperature science. In: Cell Engineering and Regeneration. Reference Series in Biomedical Engineering (Editors: Gimble JM, Marolt Presen D, Oreffo ROC, Wolbank S, Redl H). Springer, Cham. 2022; 1–60. [2]Meneghel J, Kilbride PG, Morris J. Cryopreservation as a key element in the successful delivery of cell-based therapies—a review. Front. Med. (Lausanne) 2020; 7, 592242.  [9]Getreu N, Fuller B. Stopping the biological clock—merging biology and cryogenics in applied cryobiology. IOP conf. series: Mat. Sci. and Eng. 502 (2019) 012003, IOP Publishing. 1–11. , but there are still many gaps in the fine details. These can be subdivided here for the purpose of this current commentary.

(i) Manipulating ice nucleation, crystal growth & total fraction

Addition of traditional CPA prior to cooling impacts the total ice fraction, but we are beginning to understand there are additional manipulations which can be beneficial. Ice re-crystallization inhibitors (IRI) are molecules of specific composition and structure which can interfere with water molecules joining growing ice fronts. These can be synthetic agents which have been developed based on knowledge of freeze avoidance or survival in the natural world [40]Jahan S, Adam MK, Manesia JK, Doxtator E, Ben RN, Pineault N. Inhibition of ice recrystallization during cryopreservation of cord blood grafts improves platelet engraftment. Transfusion 2020; 60(4), 769–778. [41]Ampaw AA, Sibthorpe A, Ben RN. Use of ice recrystallization inhibition assays to screen for compounds that inhibit ice recrystallization. Meth. Mol. Biol. 2021; 2180, 271–283.  [42]Ma Q, Shibata M, Hagiwara T. Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: effects of AF(G)Ps concentration and heat treatment. Biosci. Biotechnol. Biochem. 2022; 86(5), 635–645.  [43]Georgiou PG, Marton HL, Baker AN, Congdon TR, Whale TF, Gibson MI. Polymer self-assembly induced enhancement of ice recrystallization inhibition. J. Am. Chem. Soc. 2021; 143(19), 7449–7461. . The agents modify ice fractions in the mixtures by this limiting water molecule binding to ice on a kinetic basis, but are particularly important in the rewarming phase, where water molecules in the frozen matrix regain molecular mobility as temperatures reach about −40oC and warmer. This ice re-crystallization can induce additional injuries, which may be mitigated by effective IRI but in some cases the IRI molecules may confer additional protection mechanisms [44]Meister K, DeVries AL, Bakker HJ, Drori R. Antifreeze glycoproteins bind irreversibly to ice. J. Am. Chem. Soc. 2018; 140(30), 9365–9368. .

The physical events following the water–ice transition include an increase in volume. Since most cryopreservation procedures are performed in vials or bags with free head spaces, these volume changes are without significant consequences. Increasing pressure during cryogenic cooling can itself inhibit water molecules joining the ice mass. However, this property can be manipulated by isochoric cryopreservation by which the volume expansion is constrained, increasing pressure and facilitating the ‘glassy’ transition in the sample. The challenge is to control the effect reproducibly by developing novel technologies, with promising initial results [45]Ukpai G, Năstase G, Șerban A, Rubinsky B. Pressure in isochoric systems containing aqueous solutions at subzero Centigrade temperatures. PLoS One 2017; 12(8), e0183353. .

The mobility of water molecules can be modified by oscillating and high magnetic field strengths [46]Kojima S, Kaku M, Kawata T, et al. Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field. Cryobiol. 2013; 67(3), 258–263. , similar in concept to manipulation of water in other fields such as magnetic resonance imaging. This approach has been studied for application of magnetic fields during cryo-cooling as low-frequency oscillating electric and magnetic field cryopreservation [46]Kojima S, Kaku M, Kawata T, et al. Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field. Cryobiol. 2013; 67(3), 258–263.  [47]Kobayashi A, Kirschvink JL. A ferromagnetic model for the action of electric and magnetic fields in cryopreservation. Cryobiol. 2014; 68(2), 163–165. . The fundamental principles of this which might enhance cell survival remain a matter of debate [48]Wowk B. Electric and magnetic fields in cryopreservation. Cryobiol. 2012; 64(3), 301-3; author reply 304–305. , but commercial cryo-coolers have been built and tested based on this principle (ABI Corporation, Chiba, Japan). As more knowledge is accumulated, this may become a helpful technology in improving the cold chain.

(ii) Cryoprotectants & their acceptability in cell therapies

The selection, efficacy, and limitations of both cell permeating and extracellular molecules which enhance cell survival have been reviewed in the cryobiology field [19]Fuller BJ. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo. Letters 2004; 25(6), 375–388.  [20]Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiol. 2017; 76, 74–91. . As a generalization, cell cryo-survival requires some degree of intracellular distribution of cell permeating agents such as dimethyl sulfoxide (DMSO), or ethylene glycol, glycerol (to name a few). Other classes of compounds (e.g., sugars, oligosaccharides, or polymers) may modify water-ice interactions but to a large extent remain in the extracellular fluid, and are considered secondary or adjunct CPAs. There are always exceptions to these definitions where successful recoveries of specific cell types have been reported [49]Pi CH, Yu G, Petersen A, Hubel A. Characterizing the ‘sweet spot’ for the preservation of a T-cell line using osmolytes. Sci. Rep. 2018; 8(1), 16223.  [50]Pi CH, Yu G, Dosa PI, Hubel A. Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells. Biotechnol. Bioeng. 2019; 116(3), 631–643., but these are often associated with other manipulations of the physics of the cooling processes, which may not be easily applied to routine cell therapy applications. DMSO has been widely used for hematopoietic stem cell cryopreservation for several decades with good efficacy, whilst acknowledging potential limitations concerning adverse patient events [1]Buriak I, Elliott G, Fleck R, et al. Preservation and storage of cells for therapy: fundamental aspects of low temperature science. In: Cell Engineering and Regeneration. Reference Series in Biomedical Engineering (Editors: Gimble JM, Marolt Presen D, Oreffo ROC, Wolbank S, Redl H). Springer, Cham. 2022; 1–60.. The toxicity of the agent itself to cryopreserved cell populations is generally low and can be mitigated by careful handling protocols, including time and temperature of cell exposure to DMSO and washing away the residual CPA before patient delivery the cells [51]Awan M, Buriak I, Fleck R, et al. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen. Med. 2020; 15(3), 1463–1491.  [52]Awan M, Erro E, Forster-Brown E, et al. Dimethyl sulfoxide for cryopreservation of alginate encapsulated liver cell spheroids in bioartificial liver support; assessments of cryoprotectant toxicity tolerance and dilution strategies. Cryobiol. 2022; 106, 79–83. . Novel approaches to formulate DMSO-free CPA are areas of intensive study [53]Yao X, Matosevic S. Cryopreservation of NK and T Cells Without DMSO for Adoptive Cell-Based Immunotherapy. BioDrugs 2021; 35(5), 529–545.  [54]Ekpo MD, Boafo GF, Xie J, Liu X, Chen C, Tan S. Strategies in developing dimethyl sulfoxide (DMSO)-free cryopreservation protocols for biotherapeutics. Front. Immunol. 2022; 13, 1030965.  but as yet, these have not been widely applied in the current cohort of cell therapy products and will need to meet patient-orientated regulatory requirements. At present, it is often possible to reduce the concentration of DMSO used in cryopreservation by introducing secondary CPA such as polymers or oligosaccharides [55]Petrenko YA, Jones DR, Petrenko AY. Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium. Cryobiol. 2008; 57(3), 195–200.  [56]Naaldijk Y, Johnson AA, Friedrich-Stöckigt A, Stolzing A. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants. BMC Biotechnol. 2016; 16(1), 85. .

(iii) Improved cryopreservation by vitrification

Vitrification (VF) methodologies, in contrast to those of slow freezing, attempt to vitrify both the surrounding bulk media and the cell/tissue components. A vitreous state is one where water reaches an amorphous, glassy, metastable state. Vitrification is a promising approach for cryopreservation (CP) of biological materials as it is simple, robust, and cell agnostic. In its simplest form, vitrification relies on rapid single-step sample cooling by direct immersion into liquid cryogen (e.g., liquid nitrogen)—accurately described as kinetic vitrification (K-VF). However, it is challenging to achieve in practice with pure water requiring cooling rates in the order of −107 oC/s [21]Fahy GM, Wowk B. Principles of cryopreservation by vitrification. Meth. Mol. Biol. 2015; 1257, 21–82.  [22]Fahy GM, Wowk B. Principles of ice-free cryopreservation by vitrification. Meth. Mol.Biol. 2021; 2180, 27–97. . In practice, the critical cooling rate (CCR) for K-VF achieves the ‘glassy state’ throughout the entire sample volume. Unfortunately, the high CCR often limits the volume of material which can be cryopreserved, e.g., oocytes, embryos, sperm, and human embryonic stem cells limited to very small (in the 0.5–10 mL range) sample volumes [57]Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022; 6(8), 579–593. Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022; 6(8), 579–593. . Moderation of CCR can be achieved by addition of high CPA concentrations (more than 4 M) but the toxicity profiles need careful attention [57]Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022; 6(8), 579–593. Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022; 6(8), 579–593. . Nevertheless, successful protocols are currently being developed, e.g., for pancreatic Islets of Langerhans [58]Zhan L, Rao JS, Sethia N, et al. Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation. Nat. Med. 2022; 28, 798–808..

Other technical challenges to K-VF include the propensity of liquid nitrogen to vaporize and ‘boil’ around the plunged sample (known as the Leidenfrost effect), which reduces the effective cooling rate [59]Steponkus PL, Myers SP, Lynch DV et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345, 170–172. below CCR. Additionally, vitrified samples need to be stored below the effective glass transition temperature range (Tg), which for most biologicals is below approximately 120 oC. During sample recovery and warming, once Tg has been passed, ice nuclei within the sample can rapidly promote injurious ice crystal growth even at intermediate subzero temperatures. Thus, warming must also be rapid, introducing the concept of a critical warming rate (CWR). Sample storage and transport also need to be stable and below Tg to avoid devitrification [57]Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022; 6(8), 579–593. . As a proof of principle for K-VF, some studies have been reported for CPA-free systems, but so far are only amenable to very small sample volumes of near single cell thickness [60]Isachenko V, Rahimi G, Mallmann P, Sanchez R, Isachenko E. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Androl. 2017; 5(6), 1055–1063. .

Attempts to increase sample volume whilst maintaining low vitrification CPA concentrations have largely focused on achieving higher rates of cooling than can be achieved by direct plunge into LN. Use of intermediate cryogens (e.g., propane or ethane) with large temperature differences (more than 90 oC) between solidification and boiling points prevent the Leidenfrost effect and thus, increase cooling rate [59]Steponkus PL, Myers SP, Lynch DV et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345, 170–172.. Employing specialist cell supports which allow multiple individually separated cells to be plunge frozen in LN as small volumes (droplets) has been successful in reaching both CCR and CWR. Pancreatic islets supported on a nylon mesh, with excess CPA wicked away prior to freezing, increased cooling and warming rates by roughly an order of magnitude [58]Zhan L, Rao JS, Sethia N, et al. Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation. Nat. Med. 2022; 28, 798–808.. A comparable droplet approach is also supported by successful vitrification of plant genetic resources [61]Benelli C, Carvalho LSO, El Merzougui S, Petruccelli R. Two advanced cryogenic procedures for improving Stevia rebaudiana (Bertoni) cryopreservation. Plants (Basel) 2021; 10(2), 277.. Hyper-kinetic VF can be achieved with rapid (jet) delivery of pressurized LN, potentially allowing volumes up to 4000 mL containing 15% glycerol CPA solution to be cooled at a CCR of up to 10,000 oC/s [62]Katkov II, Bolyukh VF, Sukhikh GT. KrioBlastTM as a new technology of hyper-fast cryopreservation of cells and tissues part 1 thermodynamic aspects and potential applications in reproductive and regenerative medicine. Bull. Exp. Biol. Med. 2018; 164(4), 530–535. . Comparable approaches to achieve vitrification are routinely employed in electron microscopy to preserve cell and tissue ultrastructure [63]Fleck RA. Low temperature electron microscopy, (Editors: W Wolkers & H Oldenhof) methods in cryopreservation and freeze-drying, lab protocol series methods in molecular biology. Springer, Berlin. 243–274., but application to cell therapies will require significant technological development.

Novel approaches to increase the rate of warming are also being actively developed to meet CWR requirements not achievable by simple surface warming. With joule heating–based platform technology, biosystems are rapidly rewarmed by contact with an electrical conductor [64]Zhan L, Han Z, Shao Q, Etheridge ML, Hays T, Bischof JC. Rapid joule heating improves vitrification based cryopreservation. Nat. Commun. 2022; 13(1), 6017. . Other approaches use radiofrequency-excitable iron oxide nanoparticles in the CPA mix to provide uniform and fast rates of warming throughout large vitrified volumes (up to 80 mL), which reduces thermal mechanical stress and prevents rewarming phase ice crystallization [65]Manuchehrabadi N, Gao Z, Zhang J, et al. Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. Sci. Transl. Med. 2017; 9(379), eaah4586. .

(iv) The move to centralized cryopreservation facilities for greater efficiency in the cold chain

In some ways, the cold chain for cell therapies in the 2020s has built upon the expertise which evolved over decades into a robust patient treatment using cryopreserved bone marrow and associated progenitor cells. Highly specialized hematology units provided their own in-house cryo-expertise. Today, as commercialized or regionalized health service groupings grow at pace in cell therapy production, it is inevitable that there will be increasing pressure to move towards a contract development and manufacturing organization scheme as a hub and spoke model for the cryopreservation process. Such schemes are starting to evolve; for example, a centralized production and cryopreservation organization for bone marrow in the organ donor pathway is now functional in the USA [66]Johnstone BH, Woods JR, Goebel WS, et al. Characterization and function of cryopreserved bone marrow from deceased organ donors: a potential viable alternative graft source. Transplant Cell Ther. 2023; 29(2), 95.e1–95.e10. There will be advantages, including a cost reduction at scale, with focused safety and regulatory oversight, an ability to train and accredit specialist translational cryobiologists, and harmonize protocol and data management. The potential disadvantages include a need for improved delivery options for cryopreserved product with high traceability, safety, and training of the receiving institutions who may not have deep cryobiology expertise but who need to store (in some cases for short periods), thaw, and manipulate the product in robust and traceable ways for patient delivery. There may also be advantages at scale for specialist off-site cryo-storage utilization. As is currently happening in many countries, this requires collaborations and information exchange between cell therapy producers and the end-user units, two-way conversations with the relevant regulatory bodies, and a higher-level organizational network. This is very much a work in progress for the
coming years.

(v) Improved reproducibility & traceability in the cold chain

Wider adoption of novel cell therapies will trigger requirements to meet the types of stringent health standards already in place in other treatment pathways—for example, those in hematopoietic stem cell transplants and associated cryopreservation. In the UK, as one example, these activities are licensed by the Human Tissue Authority [HTA], and are accredited and registered by Joint Accreditation Committee ISCT-Europe & EBMT (JACIE) [67]National Health Service (NHS) blood and transplant. Stem cell processing and cryopreservation. NHS.. The agencies will differ in different countries, but will function with a broadly similar remit.

Automation and closed system manipulation in cell therapy pathways are acknowledged as important steps to permit product consistency, traceability, and regulatory-compliant release [68]Ball O, Robinson S, Bure K, Brindley DA, Mccall D. Bioprocessing automation in cell therapy manufacturing: outcomes of special interest group automation workshop. Cytother. 2018; 20(4), 592–599. . The multiple steps involved in cryopreservation, storage, and recovery have proven a challenge to automation, but these hurdles are gradually being surmounted [69]Kim KM, Huh JY, Kim JJ, Kang MS. Quality comparison of umbilical cord blood cryopreserved with conventional versus automated systems. Cryobiol. 2017; 78, 65–69.  [70]Cunningham AW, Jones M, Frank N, Sethi D, Miller MM. Stem-like memory T cells are generated during hollow fiber perfusion-based expansion and enriched after cryopreservation in an automated modular cell therapy manufacturing process. Cytother. 2022; 24(11), 1148–1157., and the importance of these approaches is likely to grow in future. Best practice guidelines for storage and handling of cryopreserved cells are increasingly being updated [71]Simione F, Sharp T. Best practices for storing and shipping cryopreserved cells. In Vitro Cell Dev. Biol. Anim. 2017; 53(10), 888–895. . Traceability is being enhanced by use of RFID labeling (supplied by commercial companies) e.g., [72]Barcode Technologies. CryoGenic UHF RFID Label Tags for CryoGenic Liquid Nitrogen −200 °C (−392 °F) Storage. [73]HID. Beyond Cool: RFID disentangles cryopreservation storage and management.  for cryo-vials or product bags. Remote cloud-based monitoring of samples in cryogenic storage and associated artificial intelligence medical enhancements are becoming an expected standard [74]Advanced Therapy Treatment Centres. UK review and recommendations on cryopreservation of starting materials for ATMPs. [75]Cytiva. VIA Capsule™ System.Cytiva. VIA Capsule™ System.; the development of electrically powered non-nitrogen-based cryogenic transport systems [75]Cytiva. VIA Capsule™ System. will likely enhance this, alongside automated monitoring of dry thawing systems [76]Röllig C, Babatz J, Wagner I, Maiwald A, Schwarze V, Ehninger G, Bornhäuser M. Thawing of cryopreserved mobilized peripheral blood—comparison between waterbath and dry warming device. Cytother. 2002; 4(6), 551–555.  [77]Jestice K. Dry or wet thawing: dare to compare. CryoLetters 2020; 41 (3), 154–184.  [78]Kilbride P, Meneghel J, Creasey G, Masoudzadeh F, Drew T, Creasey H, Bloxham D, Morris GJ, Jestice K. Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender. PLoS One 2020; 15(10), e0240310. .

One area of concern in current cryopreservation pathways has been the continued application of protocols developed in some cases more than 20 years ago. Such protocols have enabled successful cryopreservation applications in a number of areas such as stem cell cryo-banking and have been accepted by relevant regulatory bodies as standard. However, whilst promising new technology may appear even after extensive uptake in research laboratories, the path to implementation can be fraught with challenges. In translation to public health practice, there may be a reluctance to consider a new technology. In part, this may simply be because hard-pressed and resource-stretched service staff may not wish to contemplate the time and expense that will be needed to develop the validation including patient data required for regulatory acceptability. This concern is not unfounded; however, some public health systems are beginning to provide support for development and adoption of novel medical technology (e.g., NIHR UK) and some also look to the use of such technologies in the veterinary field, where early adoption may be possible in association with experimental treatments [79]Arzi B, Webb TL, Koch TG, et al. Cell therapy in veterinary medicine as a proof-of-concept for human therapies: perspectives from the north american veterinary regenerative medicine association. Front. Vet. Sci. 2021; 8, 779109. .

The cost of implementing new approaches and technologies must also be seriously considered and planned in order to ensure appropriate validation plus careful and efficient ‘change control’ during the transition to new technology. Where there are historical data that can be utilized, validation can be based on collaborations of multiple labs working in consort. This was exemplified by collaborative multicenter studies such as those run to enable a switch to alternative preservation methods to reduce the DMSO load in therapeutic cellular products [80]Morris C, de Wreede L, Scholten M, et al. Chronic malignancies and lymphoma working parties of EBMT. Should the standard dimethyl sulfoxide concentration be reduced? Results of a european group for blood and marrow transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide. Transfusion 2014; 54(10), 2514–2522.  [81]Morris C, Scholten M, de Wreede L, et al. DMSO reduction strategies reduce DMSO induced post autologous transplant morbidity in patients with lymphoma and myeloma—results from an EBMT non interventional prospective study. Blood 2010; 116(21), 2397..

Translational insight—what’s coming soon

The importance of the cryogenic cold chain for delivery of the relevant functional biomass in cell and tissue therapies will continue to grow in order to control and stop biological time—i.e. time to select cells at their optimal functional status phenotype for the particular therapy, time to avoid wastage when product cannot be immediately used, time to validate and provide batches against release criteria, and time to deliver to patient cohorts at distance and to dovetail with the clinical logistics for treatment. However, improvements in functional recovery are still clearly needed, which will depend on better cryo-technologies. One area for improvement which seems within grasp is the avoidance of injury from ice recrystallization and redistribution during transit upon warming through higher sub-zero temperatures [82]Poisson JS, Acker JP, Briard JG, Meyer JE, Ben RN. Modulating intracellular ice growth with cell-permeating small-molecule ice recrystallization inhibitors. Langmuir 2019; 35(23), 7452–7458.  using novel synthetic agents such as ice recrystallization inhibitors, antifreeze proteins, or polyampholytes [83]Capicciotti CJ, Poisson JS, Boddy CN, Ben RN. Modulation of antifreeze activity and the effect upon post-thaw HepG2 cell viability after cryopreservation. Cryobiol. 2015; 70(2), 79–89.  [84]Sun Y, Maltseva D, Liu J, et al. Ice recrystallization inhibition is insufficient to explain cryopreservation abilities of antifreeze proteins. Biomacromolecules 2022; 23(3), 1214–1220. [85]Ishibe T, Gonzalez-Martinez N, Georgiou PG, Murray KA, Gibson MI. Synthesis of Poly(2-(methylsulfinyl)ethyl methacrylate) via oxidation of Poly(2-(methylthio)ethyl methacrylate): evaluation of the sulfoxide side chain on cryopreservation. ACS Polym. Au. 2022; 2(6), 449–457. . Some success has also been achieved in vitrification techniques without addition of any CPA, but this has only been achieved using extremely high cooling rates and very small samples [86]Wang M, Todorov P, Wang W, Isachenko E, Rahimi G, Mallmann P, Isachenko V. Cryoprotectants-free vitrification and conventional freezing of human spermatozoa: a comparative transcript profiling. Int. J. Mol. Sci. 2022; 23(6), 3047.  with a specific cell type (spermatozoa). Wider application will require further cryo-technological developments. Similarly, the ability to control the ice nucleation temperature and avoid supercooling may have a more widespread application in the future [12]Kilbride P, Meneghel J. Freezing technology: control of freezing, thawing, and ice nucleation. Meth. Mol. Biol. 2021; 2180, 191–201. . Development of inert crystalline materials as catalysts for ice nucleation have recently been investigated for this purpose [87]Daily MI, Whale TF, Kilbride P, Lamb S, John Morris G, Picton HM, Murray BJ. A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format. J. R. Soc. Interface 2023; 20(199), 20220682. .

Use of water-modifying CPA which can better stabilize the ice/residual aqueous and glassy states has led to investigation of natural deep eutectic solutes (NADES [88]Jesus AR, Meneses L, Duarte ARC, Paiva A. Natural deep eutectic systems, an emerging class of cryoprotectant agents. Cryobiol. 2021; 101, 95–104.  [89]Jesus AR, Duarte ARC, Paiva A. Use of natural deep eutectic systems as new cryoprotectant agents in the vitrification of mammalian cells. Sci. Rep. 2022; 12(1), 8095.) which may limit ice associated cryo-injury. The combination of these solutes can promote high depression of aqueous melting points compared to those recorded for individual solutes, through co-operative molecular interactions which enhance the ‘glassy’ transition at higher sub-zero temperatures during cry-cooling. Similar application of a better understanding of fundamental cryobiology is leading to the reappraisal of older concepts such as super-cooling (biopreservation at high subzero temperatures without ice nucleation), high subzero freezing (preservation in the presence of a stabilized ice/aqueous mix), and isochoric freezing (in which the biophysical principle of volume expansion following the water/ice transition can be channeled to increase pressure in a closed system and favor transition to the ‘glassy’ state [90]Taylor MJ, Weegman BP, Baicu SC, Giwa SE. New approaches to cryopreservation of cells, tissues, and organs. Transfus. Med. Hemother. 2019; 46(3), 197–215. ). Stable vitrification of larger samples may also be possible by a process known as liquidus tracking, where higher conditions of CPA can be progressively added during the cooling process, thus limiting the exposure of the cells to these higher, toxic CPA levels [91]Puschmann E, Selden C, Butler S, Fuller B. Liquidus tracking: large scale preservation of encapsulated 3-D cell cultures using a vitrification machine. Cryobiol. 2017; 76, 65–73. . However, this is not yet close to routine cell therapy application. All or any of these approaches may make valuable future contributions to the cryo-based cold chain.

In a different direction, cryobiology is already merging with nanotechnology to yield better warming control by modulating heating by oscillating magnetic fields via radio frequency induction [64]Zhan L, Han Z, Shao Q, Etheridge ML, Hays T, Bischof JC. Rapid joule heating improves vitrification based cryopreservation. Nat. Commun. 2022; 13(1), 6017.  [92]Khosla K, Zhan L, Bhati A, Carley-Clopton A, Hagedorn M, Bischof J. Characterization of laser gold nanowarming: a platform for millimeter-scale cryopreservation. Langmuir 2019; 35(23), 7364–7375. . Significant further development will be needed to move this into general applications, but the ability to consistently warm products of various sizes with defined monitoring will be a bonus. All of these improvements will require investment in research and development. They will be driven not only by enhanced end-product function but also by the need to demonstrate and deliver better regulatory oversight of cryopreservation, which is likely to grow in demand as more cell therapies reach the clinic.

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Affiliations

Barry Fuller
Division of Surgery & Interventional Science, UCL Medical School,
Royal Free Hospital Campus,
London, UK

Roland Fleck
Fleck Centre for Ultrastructural Imaging,
King’s College London, London, UK

Glyn Stacey
International Stem Cell Banking Initiative, Barley, UK
and
National Stem Cell Resource Centre,
Institute of Zoology,
Chinese Academy of Sciences, Beijing, China
and
Innovation Academy for Stem Cell and Regeneration,
Chinese Academy of Sciences, Beijing, China

Authorship & Conflict of Interest

Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

Acknowledgements: None.

Disclosure and potential conflicts of interest: Fuller B discloses he is Lead Professor in UNESCO Chair in Cryobiology.

Funding declaration: The authors received no financial support for the research, authorship and/or publication of this article.

Article & copyright information

Copyright: Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.

Attribution: Copyright © 2023 UCL. Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.

Article source: Invited; externally peer reviewed.

Submitted for peer review: Feb 27, 2023; Revised manuscript received: Mar 30, 2023; Publication date: Nov 22, 2023.