Applications and Optimization of Cryopreservation Technologies to Cellular Therapeutics

LATEST ADVANCES IN ADDRESSING BIOPRESERVATION CHALLENGES

EXPERT INSIGHT Applications and optimization of cryopreservation technologies to cellular therapeutics

Barry Fuller, Jordi Gonzalez-Molina, Eloy Erro, Joana De Mendonca, Sheri Chalmers, Maooz Awan, Aurore Poirier, Clare Selden

Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryopreservation itself poses many biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various processes involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena.

Submitted for Review: 24 April 2017 u Published: 6 Jun 2017 The explosion of interest in cell ther- such consideration is product stor- to meet these demands. This brief apies over recent years has necessarily age (cryobanking), which is often review will discuss the salient top- focused attention on the processes required to sustain cell therapy de- ics within this field and include the that will enable product delivery in livery to the end user facilities at the history of cryopreservation, what we reliable, regulatory-compliant and required time, providing a quality currently understand about the bio- robust ways. These considerations assured product with required safe- physical and biological processes that sometimes introduce new challeng- ty and potency characteristics [1,2]. allow successful cell recoveries after es that may not have been import- Cryopreservation in its various forms storage, and additional challenges ant in the original laboratory studies is one of the main facilitatory tech- for scale up and regulatory oversight on that particular cell therapy. One nologies for cell therapies to be able when the technology is applied.

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HISTORY OF the same era, such as Maximov [4] CRYOPRESERVATION provided evidence that in plants ca- The widely accepted term to de- pable of overwintering in northern scribe preservation of living cells, Russia, the tissues went through a capable of biological reanimation, seasonal hardening process, which is cryopreservation. In reality, cell was accompanied by accumulation biopreservation can be achieved of solutes such as sugars. Chambers across a range of reduced tempera- and Hale [5] at the turn of the 20th tures, which enhance survival by Century provided additional evi- reducing biological activities, but dence from light microscope stud- with limitations dependent upon ies that there was an osmotic effect the chosen modality. The common of freezing directly on the plant conception of cryopreservation is cells that was accompanied by lysis storage of living cells at the deep on thawing. Over the subsequent cryogenic temperatures provided by 50 years, others continued to ex- liquid nitrogen or the associated va- plore the biophysical principles pour phase (ranging from -196o to and biological effects of the water– approximately -170oC). ice phase transition; for example The challenge for biology of Luyet [6] made many pertinent ob- course is the phase change of wa- servations on ice crystal structures, ter that occurs at the rather in- changes in these brought about by conveniently high temperature alterations in the kinetics of cool- (for biopreservation purposes) just ing or the presence of solutes in below 0oC. There have been many the aqueous medium, and the ef- studies over the past few hundred fects on living cells. This collected years on the biological effects of knowledge base undoubtedly influ- freezing, and the allied phenome- enced Polge [7] and his colleagues non of freeze tolerance in overwin- in their studies in 1949 on freezing tering species (most often in the of reproductive cells (notably fowl plant kingdom). The growing un- spermatozoa to enhance animal derstanding of freezing effects fol- breeding in the period post the Sec- lowed the development of micro- ond World War) which resulted in scopes capable of directly observing the first clear evidence of recovery the freezing process; for example of functional cells after deliberate Molisch [3] described the freezing deep cryogenic exposure (in their process in plant tissues, which im- case to -79oC using solid carbon mediately highlighted one of the dioxide – liquid nitrogen was not central problems with ice forma- readily available at that time). The tion – the fact that ice, derived as key to their success was the expo- the phase change of pure water, re- sure of the sperm to glycerol ahead sulted in exposure of the cells to a of the cooling process, which Polge residual hypertonic environment as later acknowledged was partly by solutes (originally dissolved in the good fortune, but nevertheless (and aqueous environment) are excluded with the benefit of hindsight), com- from the ice crystal lattice. In simple bined all of those previous studies terms, the cells experienced a lethal into one successful outcome. That osmotic stress that could be detect- success also fuelled a global effort ed at the structural level very quick- to better understand and maxi- ly after thawing. Other scientists of mize the opportunities provided in

360 DOI: 10.18609/cgti.2017.038 expert insight biology and medicine for extend- results in the often observed slight ed biopreservation, culminating in change in density of ice over liquid the definition of the term ‘cryobi- water. The stabilisation of the net- ology’ to encompass the necessary work also results in the well-known collaboration between biologists, release of energy detectable as the engineers and physicists to better latent heat of ice formation. The understand the processes, and the ice crystal lattice cannot maintain establishment of the International previously accommodated solutes, Society for Cryobiology in 1963. which become excluded into the residual liquid volume surrounding the growing ice interface, which in turn depresses the freezing point of CURRENT UNDERSTANDING the residual water so that cells are OF CRYOPRESERVATION exposed to progressively higher solutes in a progressively smaller liquid The water–ice phase water space. Thus ‘freezing’ is not an transition instantaneous event in most practi- As is universally accepted, liquid cal applications, even though it may water is the essential component for appear so to the naked eye. almost all biological processes [8], Residual mobile water, all-be- and its removal during the forma- it as a tiny fraction of the origi- tion of ice poses extreme challeng- nal water volume, can be detected es. There have been many excellent down to surprisingly low tempera- reviews on this topic, but a decade tures [11], contributing to the pro- ago Mazur [9] and Muldrew and gressive osmotic stress experienced colleagues [10] provided excellent by the cells. Multiple biological discussions on the topic with spe- targets for this type of injury have cific relevance to cryobiology. These been discussed, including destabil- are beyond the remit of the current isation of cell membranes, change discussion, but as a brief summary, in the intracellular milieu includ- water exists in the liquid state in a ing pH, chemical and structural random but self-associating matrix changes to organelles and to pro- (on an extremely brief timescale) teins; it is fair to say that we still through interactions of hydrogen do not fully understand all the bi- bonding, which for biology enables ological consequences [9,10]. What solvation of essential ions and sol- is clear is that in almost all cases, utes, and structural stability of many cell membranes and intracellular macromolecules. During cooling, macromolecules to some degree energy within the system is removed hinder the kinetics of the forma- and water molecule self-association tion of ice crystals, such that ice leads to longer-lived intermolecular preferentially initiates and grows connections that result in ice nuclei. in the aqueous external solution In this process, central water mole- (i.e., the supporting culture medi- cules hydrogen bond with four sur- um in cell therapies), providing the rounding others that at the point of osmotic driving force for water to freezing repeat indefinitely through- leave the intracellular environment out the aqueous milieu to yield hex- and effectively shrinking the cells, agonal ice with which we are all fa- which can be observed in real-time miliar. The open lattice nature also by cryomicroscopy [12].

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Cryoprotectants: solutes However, it should be noted that in that enhance survival both these (and subsequent cases), during freezing the CPA solutes need to be applied in concentrations far higher than Given this understanding of events for other solutes normally present during freezing, we can begin to in the cell media solutions, which in understand the relevance of glycerol itself introduces complex biological in Polge’s original successful experi- challenges. ments [7], and the observations that The identification of these over-wintering species frequently freeze-protecting solutes led by the accumulate sugars or other poly- 1970’s to the term ‘cryoprotectant’ ols [13]. It became clear that such (or CPA) to be assigned to solutes solutes are structurally effective in that could enhance cell recovery, hydrogen bonding, which in turn and an attempt to describe cryopro- implies that they can interact in tectants in classical terms of phar- reversible ways to hydrogen bond macology [17]. A whole range of with water molecules. This like- water-modifying agents were listed wise imparts upon them proper- as CPA, but by the 1980’s the list ties to modulate ice formation on of widely used CPA with good effi- a kinetic basis, such that as cooling cacy had been refined down to ap- progresses, less ice is formed at a proximately seven agents (Table 1). given sub-zero temperature, often It became understood that some of to a greater degree than would be the CPA (such as DMSO or glyc- predicted from classical depression erol) would cross cell membranes of freezing point effects of includ- and provide intracellular protec- ed solutes. For example, starting tion, whilst other agents such as in an isotonic culture medium (ef- sugars or polymers could provide fectively close to 0.15M sodium CPA effects in cell systems where chloride), salt concentrations will little intracellular permeation was reach approximately 3.51 molal in taking place, which has been at- the residual liquid fraction by freez- tributed to their ability to produce ing to –5°C, whereas the presence partial dehydration and limit harm- of 1M cryoprotectant (CPA), such ful intracellular ice formation (see as glycerol, mitigates the rise in salt section below). This has led to the concentration such that even when pragmatic classification of CPA as freezing to below -30°C, less salt either cell-permeating or non-per- is present [10]. This process is de- meating agents [15]. Another im- scribed in cryobiological terms as portant point is that CPA must also the colligative effects imparted by remain in solution at very low tem- CPA. Glycerol was quickly applied peratures during the cooling process to studies on freezing other import- – solutes that precipitate out at high ant cell types, such as red blood subzero temperatures cannot effec- cells for transfusion. In this era, tively modify ice formation during Lovelock and Bishop described the cryopreservation. Whilst wishing freeze-modifying effects of dimtheyl to avoid over-simplification, it is a suphoxide [DMSO], which equally ‘rule of thumb’ that nearly all nucle- acts in a colligative fashion, and has ated mammalian cells require intra- now become perhaps the most wide- cellular protection during cryopres- ly applied protective solute [14]. ervation; non-permeating CPA can

362 DOI: 10.18609/cgti.2017.038 expert insight

ff TABLE 1 Common cryoprotectant (CPA) identified by widespread***, moderate**, or infrequent choice of agent.

Cell permeating agents Sugars (which may permeate Polymers cells to a degree depending on molecular size) Dimethyl sulphoxide*** Sucrose*** Polyethylene glycol (PEG)*** Ethylene glycol*** Trehalose*** Hydroxy ethyl starch*** Propylene glycol*** Raffinose** Polyvinyl pyrrolidone (PVP)** Glycerol** Mannitol** Ficoll** Methanol* Glucose* Serum proteins (mixture)** Ethanol* Galactose* Milk proteins (mixture)** Particular CPA mixtures are often selected for specific cell preservation strategies. This list is not exhaustive and a wider discussion on CPA can be found in [15,16]. Oligosaccharides tend to act as non-permeating osmotically acting CPA, whereas monosaccharides may permeate mammalian cells to a degree depending on cell type. provide additional benefits to mod- problems for generalized cell func- ulate ice crystal growth in the extra- tion. Having high concentrations cellular environment (and thereby of a particular solute in the intra- help mitigate the osmotic effects cellular environment will impact of ice formation), but they cannot on almost all normal biological normally provide primary cryopro- processes before or after cryopreser- tection [15]. This statement has to vation, and this interference can at be moderated to some degree when some point become toxic, leading considering cryoprotection afford- to cell injury even before ice forma- ed by sugars such as trehalose and tion. This is equally true in osmotic sucrose, which can be used to en- terms because, even though perme- tirely replace agents such as DMSO ating CPA may be chosen for use, for some cell types; however, in this they are needed in relatively high situation it is clear that the sugars concentrations and they will take are needed both extra- and intra- an identifiable time to cross the cell cellularly, which can be achieved by membrane, much slower than for pre-cryopreservation culture for 24 water movement itself. Thus when hours [18,19]. Nevertheless, these exposing cells to CPA at concentra- additional effects of agents such as tions of 0.5M or above, there is an polymers and proteins should not initial osmotic shrinkage of cells as be discounted when considering water leaves, to be balanced by a cell how to develop a CPA protocol for volume re-equilibration as CPA and a given cell therapy, and can explain associated water molecules enter the to some degree why for laboratory intracellular space [20]. Transmem- scale cryopreservation, many groups brane CPA permeation is largely a have used high protein concentra- simple physico-chemical process, tions such as foetal calf serum in the the kinetics of which mean that at freezing media in their cryobanking ambient temperature for example, activities. CPA permeation into mammalian It will be clear by now that CPA oocytes as a model system requires have important roles as water-mod- some 10 minutes to approach equi- ifying agents during freezing stress, librium [21]. The process is gener- but those same properties can cause ally faster at higher temperatures,

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for example in mammalian cells was generally found to be associat- cultured at 37oC, but higher tem- ed with lethal cell injury in the vast peratures tend to exacerbate any majority of cases [26]. At an opti- chemical toxicities for the CPA. mal slow cooling rate, cells could Low temperatures for CPA expo- be dehydrated with their sensitive sure (such as 10oC or below) can molecular and ultrastructural com- mitigate chemical toxicity but also ponents protected by the added prolong exposure time to achieve CPA. However, if very slow cooling good intracellular permeation, of- (around -0.1oC min-1 or less) was ten by a factor of two or more [22]. instigated, cells were exposed to the Whilst some of these factors can be extreme ice-related dehydration for predictively modelled once variables intolerably long times, even in the such as cell membrane permeabili- presence of CPA, and again, lethal ty coefficients are known for water injury would ensue. This undoubt- or CPA [23], there is currently no edly is an oversimplification of the absolute substitute to performing complex biophysical processes oc- prospective investigations to define curring during cryopreservation, the best CPA protocols to maintain many of which remain to be fully good potency for specific cell ther- elucidated, but the two-factor hy- apies during the cryopreservation pothesis does tend to fit the ob- process. Recently the application of served outcomes in many cases over algorithm-based objective optimis- the intervening years. This is where ation of cryoprotectant protocols is the often quoted ‘-1oC min-1 cool- providing valuable information on ing rate’ has its origins; the ‘-1oC’ these important areas for cell ther- is not a mystical number, rather it apy cryopreservation [24]. is a reflection of the time required to achieve the optimal cell dehy- The kinetics of cooling for dration for cell survival during the successful cryopreservation cryopreservation process. It is ex- and storage considerations tremely important to understand Successful cryopreservation was that this is the safe cooling kinetic also found to be influenced by the for the biomass itself when cooling kinetics of the cooling process it- cells, not just the cooling profile self. As often practiced today, slow of the holding chamber, produced cooling (where slow as a relative by whatever equipment is used to term applies to cooling rates of be- control cooling process. Controlled tween about -0.3oC min-1 and -2oC cooling can be provided by a num- min-1) were found by empirical ob- ber of cooling technologies based servation to relate to good success. around liquid nitrogen vapour Mazur and colleague provided a (e.g., Planer PLC; Cryomed™) or hypothesis based on these obser- electrical Stirling Engine Systems vations, which he developed over (e.g., Asymptote PLC). In most many years [25]. It can be described cases, cooling small volumes in as Mazur’s two-factor hypothesis such equipment (such as tradition- and explained visually in Figure 1. al 1.8 ml cryovials) will allow the To survive cryopreservation, cells vial contents to closely track the are required to be optimally de- changing temperature conditions hydrated to avoid intracellular ice within the machine. However, clin- formation; intracellular freezing ical scale cell therapies (discussed

364 DOI: 10.18609/cgti.2017.038 expert insight

ffFIGURE 1 Schematic of Mazur’s 2-factor hypothesis.

A cell with CPA protection subjected to cooling at different rates. Maximum functional recovery is achieved with an optimal cooling rate providing reversible dehydration occurring over the high subzero temperature range. If cooling is too slow, irreversible injurious dehydration can take place, for example the mitochondria and endoplasmic reticulum are structurally compromised. If cooling is too fast, cells do not have time to optimally dehydrate, and residual intracellular water can form ice, which is again injurious and can compromise structure of organelles. For many nucleated mammalian cells, ‘optimal’ cooling equates to rates of around -1oC min-1. below) often require cryopreserva- Cryobiology has provided information at much larger volumes, where tion on the physico-chemical status it is a much greater challenge to of the frozen matrix which indicates ensure that the biomass cools at that in the range -120 to -130oC, a appropriately survivable low rates glassy transition occurs that finally (discussed below) because of the and completely solidifies the mixed nature of heat and mass transfer in matrix of ice, solutes, CPA and bio- the large volumes. mass in the samples. This tempera- The question then arises as to what ture range relates to the total solute the desired end temperature should concentration of the mixture, but be for success, and the requirement for all practical purposes in cell ther- for temperature control over that apy the quoted figures are relevant. range. It has now been established It has been established empirically from a number of studies that for in different systems however, that true long-term stability, temperatures practical benefits for slow cooling be- below -100oC become essential [27]. yond about -60oC to -80oC are not

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detectable, so cryopreservation pro- by about -60oC, the residual solution tocols often terminate the imposed in the sample, containing now high- control of slow cooling at these ly concentrated solutes and CPA, temperatures [25,28]. Thereafter, the becomes so highly viscous that the samples can be cooled below the glass glass transition event (Tg) becomes transition to close to the storage tem- highly probable on further cooling. perature in liquid or vapour phase Within this matrix, the biomass is nitrogen (for example to -160oC) present as extremely dehydrated cells at faster, pragmatically convenient, that contain no ice [30]. A schematic (but still controlled) rates of about of a slow cooling process is provided -10o or -20oC min-1 [29]. Effectively in Figure 2.

ffFIGURE 2 Steps in a slow cooling protocol.

Cells usually need permeation with CPA (green – top left) and often a secondary CPA (sucrose, orange), which is often achieved at temperatures between ambient to +4oC (top left). In many cases, a secondary CPA such as sucrose is added , which also starts cell dehydration. The mixture is cooled (red line) down to the ice nucleation temperature (often around -7oC) and ice nucleation starts (white icons). Active ice nucleation (called seeding) may be applied. A hold time (approximately 10 minutes depending on sample) can be introduced to allow dissipation of latent heat which may confound cooling control. Thereafter, slow controlled cooling produces responsive dehydration as ice crystal volume grows. This benefit of this is maximized by reaching approximately -50oC, and thereafter the sample can be cooled to below the glassy transition range (approx. -120oC) either at the same rate or more quickly (for practical considerations) and held in cryogenic storage. The cryopreserved sample thus comprises biomass (orange) and glassy matrix (grey blocks), mixed with ice already present.

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The value of liquid nitrogen as more complex systems, storage at the storage medium of choice will -80oC for prolonged periods may be now be apparent because the be more problematic for exam- temperature provided is far below ple, in work on liver cell spheroid that of the glass transition tem- cryo-storage it was shown that sig- perature range, imparting a degree nificant cryo-attrition occurred over of safety to the process. The sta- 1 year of storage [36]. For each cell ble cryogenic temperatures (below therapy, the ease of application for -150oC) that can be obtained (with storage at -80oC needs to be bal- modern storage containers fitted anced by an understanding of prod- with appropriate monitoring and uct stability. The advent of electrical if needed, autofill devices) are suf- freezers operating in the -120oC ficiently far enough below the glass range has opened this possibility as transition range to yield true long- a storage temperature for cell ther- term biopreservation on a scale apies, which is predicted to have probably greater than required for successfully long storage times, but most cell therapies. It has been often little published information on that discussed that below Tg and in the is currently available for prospective highly viscous environment, there studies comparing these technolo- is insufficient thermal energy for gies with liquid nitrogen storage. any chemical reactions or molecu- Given the expected stability of lar diffusion to take place. The only storage below Tg, the occasional potential harmful process to impact report on negative effects of storage cryogenic storage has been suggest- duration [37] are difficult to under- ed to be background ionising radia- stand on a physico-chemical basis. tion, and it has been estimated that However, during practical storage it would take in excess of 104 years of cell products, there is always a for cells at cryogenic temperatures potential for fluctuations in storage to accumulate lethal injury [1]. In temperature if cryogen levels are al- practical terms, there have been lowed to vary widely (e.g., during reports of cryopreserved cells, such intermittent delayed times of filling as sperm or embryos, retaining nor- with liquid nitrogen) or if samples, mal functional processes after up to held in racks or trays, are removed about 30 years cryo-storage [31,32]. from storage whilst accessing specif- Likely of more relevance to cell ic products on day of requirement. therapies, cells from umbilical cord Even though the products may ap- blood have been used effectively af- pear visibly ‘deep frozen’ during ter 11 years cryo-storage [33]. this process, it has been shown that Historically, storage at -80oC us- significant temperature upshift (e.g. ing electrical freezers has been used from -135oC to -60oC) could occur for some cells such as red blood within a few minutes of lifting racks cells [34]. However, it will be clear out from storage [38]. Repeated tem- from above discussions that storage perature cycling through the range above Tg is likely to result in slow for Tg in this fashion led to a de- but progressive cryo-attrition. In crease in cell functional recoveries. practical terms, a storage shelf-life The choice of storage in liquid or has often been imposed. The FDA vapour phase nitrogen for cell ther- has approved -80oC storage of red apies has trended towards vapour blood cells for 10 years [35]. For phase storage. The possibility of

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transfering infectious agents found traditionally been favored [9,43]. in commercially produced liquid In fact, for slowly cooled cryopre- nitrogen has been discussed and served cells, the impact of warming experimentally demonstrated [39]. kinetics is complex and not easy Vapour phase storage (with tem- to predict. It has been alternative- peratures usually in the region of ly argued that slow warming may -150o to -170oC) is attractive, but be beneficial to allow time for os- potentially capable of rapid tem- motic re-equilibration processes perature fluxes such as when open- to take place [25] when the highly ing the storage container. Happily, shrunken cells start to encounter the placement of commonly used more liquid water as the ice matrix aluminium racks within the storage melts at high subzero temperatures. tank acts as a ‘cold sink’ to stabilise In some experiments on particular the internal environmental tem- cell types, the impact of slow versus perature, and this can be further fast warming was either not signifi- stabilised by simple processes such cant or was related to the particular as adding a copper fin [40] within CPA used [44]. This raises another the storage tank. For optimal stor- potential warming factor – expo- age of cell therapies, cryo-bank ac- sure of the cells to toxic high CPA cess procedures should be subject to concentrations at high subzero tem- protocol management and routine peratures during slow warming. data record. Warming is an area which deserves greater future investigation. As in Considerations for warming cooling, terms such as ‘fast’ or ‘slow’ For successful recovery of potent warming are relative and should be cell therapies, the reversal of all the defined in any cell therapy protocol above described biophysical events for process repeatability. has to be performed in ways which do not compound injury. For the Cryopreservation and cell most part, cell therapies are tradi- product potency tionally subject to cooling, in the In many cases, the potency of thawed presence of significant ice content cell therapies may not equate to in the sample. It has long been rec- what might be predicted from the ognised that during the warming doses selected for cryopreservation. process, existing ice crystals may It has been known for some time undergo ice crystal growth and that a series of sublethal stresses can re-organisation (known as Ostwald accumulate in the cells during the ripening), which may impart fur- multiple steps of cryopreservation, ther injury depending upon the and may only be expressed gradually kinetics of the process [41]. In addi- after rewarming in a process known tion, if small intracellular ice nuclei as ‘cryopreservation cap’ or ‘cryo- were established during the cool- preservation-induced delayed onset ing process, but did not grow to cell death’ [45,46]. Cell death path- become injurious, these may grow ways such as apoptosis and necrosis during warming, again depending have been identified. There appears on the kinetics of warming, such to be a progression of expressed that ‘freezing during thawing’ can injury over the first 12–24 h post- be conceptualised [42]. For these thaw, and subsequently surviving various reasons, fast warming has cells may resume normal function

368 DOI: 10.18609/cgti.2017.038 expert insight including cell division [29]. Target- Cryogenic preservation ed molecular strategies have been in the absence of ice: suggested to mitigate against these vitrification injuries [47] but for clinical applica- The concept that biological cryo- tion these need to be regulatory ap- preservation could be achieved proved strategies. These post-thaw by avoiding ice injury with pro- injury markers also offer one way cesses that entirely inhibit crystal to audit effectiveness of cryopreser- growth has been considered for vation protocols or assess protocol many years; the original paper by change. ffFIGURE 3 A stylized liquidus curve for a notional CPA, showing the physical states achieved as cooling proceeds.

The initial aqueous sample can be viewed as an amorphous liquid for this purpose. At low starting CPA concentrations (say <20%), slow cooling (to the right of the image) produces ice (white icons)) plus liquid and residual liquid. As slow cooling proceeds and more ice is produced, the relative concentration of CPA in residual liquid increases (to the right of slow yellow curve), until increased concentration and decreased temperature drive the glassy transition (Tg). Tg occurs at higher subzero temperatures if higher CPA concentrations are used. It is possible to cool to a stable Tg using very high (>80%) CPA, but these are invariably cytotoxic. In practical cell cryopreservation by slow cooling, ice plus glassy matrix co-exist beyond approximately -120oC. During warming, devitrification (dashed yellow lines) tends to occur at lower temperatures with lower CPA, and ice crystals can grow. For ultra-rapid cooling (left side box, red), amorphous matrix is preserved from above 0oC throughout the cooling, at rates greater than -1000oC min-1 to reach Tg (dashed red curve) but this tends to be metastable with similar problems of devitrification on warming. The figure is a simplification of events for visual presentation; for details see Muldrew (2004), Fahy & Wowk (2015).

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Polge and colleagues [7] used the APPLICATIONS OF CRYO- term ‘vitrification’ to denote that PRESERVATION FOR CELL something unusual was happen- THERAPIES & CHALLENGES ing which allowed sperm survival FOR ROBUST PRODUCT (although their system does not DELIVERY match up with what we understand by ‘vitrification’ today). At around Historical and current cell the same time, Luyet and Gehen- therapy cryopreservation io made observations of an ice-free Cryopreservation of bone mar- solidification of protoplasm which row-derived hematopoietic pro- required ultra-rapid cooling (sev- genitor cells (HPC) is an example eral thousand oC min-1) [6]. Later, of a cell therapy product which Rall and Fay were able to translate has been applied widely since the the ideas into an achievable tech- 1960s [52,53]. The principles of slow nology for successful cryogenic cooling cryopreservation (around preservation of mouse embry- -1oC min-1), with DMSO at 10% os [48]. Vitrification as currently w/v as CPA and storage at either practiced requires very high con- -80oC or, less than -130oC have centrations of CPA (as much as six been routinely used [54]. Using sec- times those used in traditional slow ondary CPA such as hydroxyl eth- cooling cryopreservation), which yl starch has allowed reduction of cells can only tolerate for short pe- DMSO to 5% concentrations, with riods (usually seconds of exposure hydroxyl ethyl starch added at 6% time) before they become toxic, w/v [55]. Special cryopreservation and thus still requiring rapid cool- bags (e.g. Miltenyi Biotec or Maco- ing and warming (which in turn Pharma) with a range of fill volumes limits the volumes used to <1 ml). up to 250 ml allow maintenance of This combination of factors allows suitable cooling profiles and good Tg of the system to be reached warming rates when immersed in whilst suppressing ice crystal for- a waterbath at 37oC [56]. Post-thaw mation. Much has been learned manipulation of HPC has often about the biophysics underpin- been avoided by direct transfusion ning vitrification [49], and Figure 3 of the thawed cell product, but this depicts the important biophysical can occasionally lead to patient-re- characteristics. The technology has lated adverse events [54]. Some au- recently become widely applied in thors developed washing procedures reproductive cryo-banking for in- to dilute CPA before infusion, as a fertility treatment [50]. However, way to reduce any patient-related because of the volume limitations, adverse events following infusion of vitrification has not yet been de- DMSO, with acceptable cell prod- veloped into technologies for what uct survival [57]. might be considered more general As other cell therapies have en- cell therapy cryo-banking. Recent tered the arena within the broad studies have been made into devel- area of hematopoietic stem cell opment of vitrification technolo- replacement, slow cooling cryo- gies compatible with large volumes preservation has maintained its (see Puschmann et al [51] for a re- beneficial role, but with some ad- view), but these have yet to be ap- dition to detail in terms of protocol plied to deliverable cell therapies. management. For example, when

370 DOI: 10.18609/cgti.2017.038 expert insight cryopreserving umbilical cord With the recent arrival of manip- blood, Woods and colleagues [58] ulated T cell therapies, such as chi- and Hunt et al [59] investigated meric antigen receptor engineered steps to limit CPA toxicities when (CAR ) T cells destined for autolo- applying DMSO. There have been gous transfusion [64,65], cryopreser- several reports of mesenchymal vation will undoubtedly remain the stromal cells for expansion and important facilitatory technology in cryopreservation for clinical appli- the pathways for product manage- cations, such as in graft versus host ment, batch safety and potency val- disease [60], but the product poten- idation, and, in many cases, deliv- cy has come into question [61] and ery to end users. Cryopreservation cryopreservation was one of the has already been discussed in these steps under scrutiny. Further de- terms [64] but cryo-protocol details tailed studies indicated a range of are as yet sparsely reported and it is reversible and non-reversible effects likely that general slow cooling re- of cryopreservation in mesenchy- gimes originally developed for HPC mal stromal cells using slow cooling have been adopted. Here also, prod- with a range of DMSO concentra- uct depletion and post-thaw poten- tions and cell pre-treatments [62]. cy have been reported [66]. Other Post-thaw culture for 24 h was manipulated allogeneic immune cell found to be effective in reparation therapies (such as natural killer cell of many of the cryo-induced abnor- or regulatory T-lymphocyte thera- malities. A recent review of cryo- pies) face the same requirement for preservation practices across UK cryopreservation to meet ‘off the centres applying slow cooling cryo- shelf’ product delivery, but again preservation for peripheral blood post-thaw attrition in cell respons- stem cells identified differences in es has been highlighted [67]. There practices which were reported un- is an opportunity for much further der the umbrella term ‘cryopreser- work on fundamental cryobiology vation’ [63]. These combined studies and translational cryopreservation highlight an important point for all research in the CAR-T cell arena cell therapy cryopreservation activ- to improve potency. In the short ities; this is that cryopreservation is term, it may be possible to mitigate not a simple ‘standardised one op- against loss of potency post-thaw tion’ process, the various protocol by adjusting dose (delivered cell steps need to be well documented numbers) but this still leaves diffi- in standard operating procedures, cult questions if the impact of cryo- and the equipment involved should preservation on the therapeutic cell be applied with care and monitored population is not fully understood, frequently against documented per- and which may therefore vary be- formance criteria. The authors also tween batches. cautioned that straightforward as- says of cell viability post-thaw may Challenges for scale up for not correlate well with other more large volume cell therapies exacting tests of product poten- In the discussions so far, cryopres- cy, which again is something that ervation has been applied to single needs to be part of protocol testing cell products at a scale very similar and audit for any cell therapy cryo- to those used traditionally for hae- preservation practices. matopoietic stem cells. In other

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areas, larger cell therapy volumes before returning to the pro- are required, e.g., cell therapy for grammed slow cooling protocol. organ support such as the bioarti- Organisation of water molecules ficial liver where multicellular units into ice nuclei can also be achieved in volumes of between 1–2 litres by certain types of physico-chemi- are required at clinical scale [68]. A cal surface properties, (often termed potential difference in approach to ice nucleating agents) for example, cryopreservation for multicellular crystalline cholesterol, included products (such as cell spheroids) in the CPA mix, and providing a under consideration is the impact more reproducible cell cryo-de- of controlling ice nucleation during hydration and often increased cell slow cooling. We have discussed the recoveries [29]. Other types of in- fact that optimal cryo-dehydration organic ice nucleating surfaces are is essential for cell survival, which under development [IceStart™, As- is achieved by the osmotic effects ymptote]. Another problem with of the growing ice content in the cryopreservation of large samples is sample. This implies that ice crys- maintaining consistent heat transfer tal initiation should be a repeatable throughout the sample volume for process during cooling to achieve homogenous cooling, since cooling a standardised process, but ice nu- devices work by surface cooling, cleation is a stochastic event, and which depends on the heat trans- on any one cooling run can occur fer properties of the container and unpredictably at different tempera- the cell mixture [73]. The appro- tures far colder than the equilibri- priate placement of thermocouples um melting temperature of the mix- in sample containers during exper- ture (where ice would first be able imental cryo-cooling runs allows to co-exist with liquid) in different assessment of differences between samples [69]. Controlled ice nucle- different sample compartments, or ation can be achieved by momen- individual samples, and the cooling tary application of a cold metal rod device chamber [29,73]. Any differ- to the outside of a container such ences can be mitigated by altering as a cryo-vial in the process known the cooling programme if required. as ‘ice seeding’ and has become The desired control of cooling can routine in reproductive cryopreser- be further disrupted by the release vation where there are only a small of latent heat of ice nucleation deep number of samples per run [70]. within large aqueous volumes [74]. This is a physical event, whereby Alterations of the cooling ramp local deep cooling applied to the of the cooling machine, such that surface of the container produces heat extraction is maximised over ice crystals on the inside wall – it a particular but defined range of has to be visually checked that ice temperatures where the latent heat is present internally in the vial and effect predominates is one way to not just ‘frosting’ on the external improve overall sample cooling surface. Ice nucleation can also be profiles [30]. induced across a group of vials be- When considering warming rates ing slowly cooled together by intro- for larger cryopreserved volumes, ducing a brief ‘shock cool’ step with similar concerns exist about ho- rapid cooling in the cryo-cooler for mogenous heat transfer; the usual approximately 2 minutes [71,72] process of immersing cryopreserved

372 DOI: 10.18609/cgti.2017.038 expert insight samples in a warming bath may availability to the patients. Similar leave core volumes in a frozen to the production process, recovery state for extended periods and not could be achieved by transferring achieving the desired rapid warm- these cell constructs into a biore- ing profile [73,74], thereby expos- actor which provides a dynamic ing cells to CPA at potentially toxic environment aiming to mimic the temperatures. Warming is one area perfusion condition which cells are of cryopreservation that requires subjected to in vivo, with more ef- much further study, and under- fective mass transfer properties [75]. standing the interactions between A number of different types of bio- optimal cooling and warming pro- reactors exist (fluidised bed, rotary files to maximise cell recovery and cell culture system) that can op- potency could benefit from this. timally support cell recovery and Commercially available contain- metabolism whilst minimising cell ers (cryo-bags) enable cryopreserva- damage [76–79]. Such systems can tion of biomass up to volumes of ap- be readily scaled-up for fast recovery proximately 250 mL and are filled to of large volumes of cryopreserved a recommended volume to produce cell therapies. a sample height of less than 5 mm. Cell encapsulation within hy- This thin envelope configuration drogels is widely used in some ensures that the contents of the bag cell-based therapies, as biocom- are cooled homogenously; the rela- patible materials can isolate ther- tive thinness of the bag also facili- apeutic cells from the host, avoid- tates fast warming rates. However, ing rejection, providing a physical the dimensions of the current cryo- support, and allowing a controlled bags cannot sustain volumes greater release of cells from biodegrad- than 250 mL whilst maintaining able materials. The process of less than 5 mm height. Increasing encapsulation typically uses liq- the height of the bag compromises uid precursors that become solid the heat transfer process and creates upon polymerization or polymer a temperature gradient within the chain crosslinking. Cryopres- biomass. At volumes greater than ervation of encapsulated cells 250 ml, maintaining the height at must ensure maintenance of the less than 5 mm requires a bag with chemical structure and physical impracticable dimensions (length characteristics of the biomaterial and width) to freeze with the cur- as they greatly influence cell be- rent accessible technology (i.e., con- havior and may potentially affect trolled rate freezers). Therefore, the the outcome of the therapy. Sev- design of the cryo-container is also eral studies have shown promising a factor under careful consideration protective effects of biomaterials when cryopreserving large volume on cell cryopreservation [80]. In a cell therapies, as it must support more mechanistic study, alginate the homogenous freezing and thaw- hydrogel was shown to inhibit de- ing kinetics of the biomass. vitrification during the warming Another important key aspect for process of vitrified stem cells [81]. successful cell therapy is the quick Altogether, these beneficial prop- recovery of the biological con- erties make encapsulation an en- structs after cryopreservation in a couraging strategy for cell therapy relevant time frame for immediate and the cryopreservation process.

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Considerations for GMP devices may be suitable for small transfer of cell therapy cryo- volumes and sample numbers of a preservation technologies particular product type, such as Mr Frosty™ (Thermo Fisher Scientific) Moving cell therapy cryopreser- or CoolCell®, (Biocision). Equip- vation to clinical application will ment capable of handling larger predictably require maintenance of volumes or more exacting cooling the same GMP-aligned steps that profiles are available based on liq- are important across the whole uid nitrogen technology (Planer cycle for product delivery. All pre- PLC; Cryomed™) or electrical Stir- and post-cryopreservation product ling engine principles (Asymptote manipulations must be performed Ltd). The importance of under- in ways that adhere to regulatory standing the true sample cooling requirements, using media formu- profile (which can be audited by a lations, CPA additives, containers recording system in a dummy vial) (such as cryo-bags), cryo-cooling as opposed to the selected chamber equipment, identifying labelling program has already been discussed and traceability documentation, above [29,74]. For storage, filling storage temperature monitoring and cryogenic transportation of and product delivery systems; all of cell therapy products, a number of equal importance [1]. The value of companies provide a range of regu- producing a comprehensive quali- latory compliant options [Panason- ty programme for a cryopreserved ic; Thermofisher Scientific; Praxair; cell therapy (in this case periph- Thames Cryogenic]. Liquid nitro- eral blood mononuclear cells) has gen is not a sterile product unless been discussed by Ducar and col- specifically treated, and thus proto- leagues [82]. DMSO has so far been cols for handling and storage need the most widely used CPA, which to be developed that avoid compro- must be of highest quality and endo- mising GMP environments [39,86]. toxin-free. One point of discussion Since nitrogen vapour can deplete has been the use of regulatory-com- room oxygen levels, air oxygen pliant protein sources (such as foetal monitors are required where the liq- bovine serum or human serum) [83] uid nitrogen is located and handled or xeno-protein free solutions [84] as part of general risk assessments as a CPA carrier media. Some and safety measures, which also in- commercially available GMP-com- clude personal safety equipment to pliant CPA solutions have recent- avoid ‘cold injury’ from accidental ly become available (CryoStor™, contact with the cryogen [87]. BioLife) [2]. For specific types of Technical innovations continue non-autologous cell therapies, the to come on stream to make cryo- importance of recording and testing preservation more robust in the of cell provenance throughout the context of regulatory oversight for development of master and working delivering cryo-banked cell thera- cell banks is an additional import- pies. Labelling systems that survive ant consideration [85]. cryogenic exposure are provided by A number of options are avail- a range of companies (GA Interna- able for cooling equipment when tional; Biosafe). Thawing devices applying controlled slow rate capable of imposing and recording cryopreservation. Passive cooling warming profiles for cryopreserved

374 DOI: 10.18609/cgti.2017.038 EXPERT INSIGHT product in either cryo-vial or cryo- reduce costs by avoiding wastage of bag formats are now available (Med- product which would otherwise be cision™; Asymptote). Secure closure ‘out of shelf life’. However, there vials and automatic fi ll systems remains a signifi cant opportunity can meet the demands for some to improve application of the cryo- cell therapies (e.g. Cook Regentec; preservation process itself by con- Wheaton®). Lyophilisation (freeze tinued research into both funda- drying) with ambient storage is mental and applied cryobiology. As being developed as an additional diff erent, novel, or large-scale cell biopreservation strategy for some therapies come on stream, they will nucleated cell products (Lyotech- likely require improved approaches nology, Osiris Th erapeutics Inc.). to cryogenic preservation. A number of companies are devel- oping sophisticated cryo-product FINANCIAL & COMPETING management systems and cloud- INTERESTS DISCLOSURE based information repositories that will help drive up standards of best Th e authors have no relevant fi nancial practice and information sharing involvement with an organization or entity between organisations. Much infor- with a fi nancial interest in or fi nancial mation in these areas of cryopres- confl ict with the subject matter or materials ervation is available via specialist discussed in the manuscript. Th is includes academic societies such as the In- employment, consultancies, honoraria, stock ternational Society for Cryobiology options or ownership, expert testimony, and International Society for Stem grants or patents received or pending, or Cell Th erapy. royalties. No writing assistance was utilized in the production of this manuscript.

TRANSLATIONAL INSIGHT Th is work is licensed under Cryopreservation of cell therapies a Creative Commons Attri- is an important part of product bution – NonCommercial – NoDerivatives 4.0 delivery and process management. International License When optimally applied, it can also

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378 DOI: 10.18609/cgti.2017.038