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This chapter covers the key physical, biological and practical challenges encountered when developing cryopreservation protocols for larger biological structures and examines areas where cryopreservation has been successful in scaling to larger structures. Results from techniques being used in attempts to overcome these challenges are reviewed together with the indicators for future development that arise from them. The scale-up of cryopreservation to tissues with diverse functions and cell types makes the control of freezing and thawing more challenging. Technology may—or may not—be available depending on the size of the material involved. To meet the challenge there must be innovation in technology, techniques and understanding of damage-limiting strategies. Diversity of cell structure, size, shape and expected function means a similarly diverse response to any imposed cryopreservation conditions and interaction with ice crystals. The increasing diffusion distances involved, and diversity of permeability properties, will affect solutes, solvents, heat and cryoprotectant (CPA) transfer and so add to the diversity of response. Constructing a single protocol for cryopreservation of a larger sample (organoids to whole organs) becomes a formidable challenge.
UCL Great Ormond Street Institute of Child Health, UK
Susan Ross
UCL Great Ormond Street Institute of Child Health, UK
Tessa Crompton
UCL Great Ormond Street Institute of Child Health, UK
*Address all correspondence to: peter.kilbride@cytiva.com
1. Introduction
Historically, the predominant application of cryopreservation was in agriculture and reproductive medicine, starting with stored spermatozoa in the 1950s and oocytes being widely cryopreserved beginning in the late 1980s [1, 2, 3]. In the past decade, a revolution in tissue engineering has changed the landscape of cryopreservation and there is now a growing and critical need for successful cryopreservation of somatic cells not only as low volumes of cell suspensions but also in larger quantities and, increasingly, as part of a complex cell network. In such a network, different cells may have a range of different functions and structural requirements [3, 4, 5, 6]. These larger subjects can contribute directly to a therapeutic treatment or can be cryopreserved as tissue from which cells can be isolated to begin a manufacturing process [5, 7, 8]. A new demand has, therefore, been created for cryopreservation of larger subjects ranging from cell spheroids and organoids to tissue slices and, eventually, entire organs [3, 4, 6, 7, 8, 9, 10, 11]. The potential benefits of cryopreservation of these multicellular and differentiated structures range from facilitating population-wide biopsy studies to supporting large-scale manufacturing and providing economies of scale within organoid preservation. Realising these benefits would support a sizable fraction of the needs of regenerative medicine and would advance progress towards organ cryopreservation, a key and as yet unmet need in transplantation technology.
The first steps towards large-volume cryopreservation must necessarily exploit the knowledge gained from the widespread, successful cryopreservation of cell suspensions [12, 13]. This success stems from the level of control of pre-treatment, cooling, warming and recovery that can be exerted over the cells [14, 15]. Appropriate control is supported by a specific technology, including programmable freezers and mathematical modelling, and benefits significantly from the relative uniformity of cell size, shape and cytoplasmic content of the majority of cell types of interest [15, 16, 17, 18, 19]. The important diffusion distances for solutes, solvents and heat are short for these suspended cells with little cell-to-cell differences and so provide relatively uniform responses to imposed conditions. Responses to applied cryoprotectant (CPA), whether physiological, osmotic or related to toxicity are also relatively uniform within a single cell type [20]. Additional complications that are introduced by a relatively large bulk volume of suspension, such as heat transfer across the sample, can be modified by altering the geometry of the sample, e.g., by flattening a cryobag containing suspended cells during cooling and warming [6, 8, 16].
While there is a promise with ice-free techniques, also known as vitrification, these have been covered in other reviews and so will not be examined here [21, 22].
Significant challenges that arise when moving up to the cryopreservation of large, coherent cell masses are caused directly by the size and volume of the tissues concerned. As noted above, in a cell suspension the diffusion distances between the cytoplasm and surrounding medium are effectively constant for each cell, ensuring relatively uniform responses to imposed physical and chemical diffusion gradients, such as external cooling and CPA addition. For larger cell masses such as organoids, a much greater range of diffusion distances exists because cells further towards the centre of the structure are increasingly distant from the external medium [6, 8, 17, 18, 23, 24]. Rates of diffusion for these cells are further complicated as diffusion within the overall cell mass will involve transfer across adjacent cells and intracellular spaces, with a range of differing properties before the external medium is reached [4, 24].
For numerous mammalian cell types (including cells derived from blood, liver and ovaries), cryopreservation of suspensions is straightforward with limited loss of cell viability and function [12]. Moving up in size to single cell-type spheroids this success is often continued, as in the case for liver spheroids [8, 25, 26]. As biological structures become more complicated, e.g., cell organoids composed of several different cell types, success is more limited with a strong, negative influence of size [4, 6]. Many smaller, immature organoids, typically consisting of no more than a few hundred cells can be cryopreserved [27] but for larger, mature organoids in their final state for therapeutic use, cryopreservation success is more limited. Table 1 summarises some successful strategies for a range of such tissue types.
Biological Sample Type
Cryo-preparation and state
CPA
Cooling rate/method
References
HepG2 liver spheroids
HepG2 spheroids in alginate, spheroids of a few 100 s cells
A summary of some tissue types currently cryopreserved in the presence of ice successfully, and the methods used to achieve results.
For even larger tissues and whole organs, success is largely limited to those which can operate as discrete units when dissected, for example, ovarian tissue and thymic slices [31, 32, 34, 35]. These can be removed from the body and cut into smaller functional units, which can each be successfully cryopreserved, thawed and transplanted independently. Mammalian organs lacking this ability such as the heart and kidneys cannot, as yet, be cryopreserved successfully [9, 10]. A famous 1978 paper on the subject started with the line ‘Attempts to preserve viable kidneys by freezing in the presence of cryoprotective agents have been notoriously frustrating’—a statement no less true today than it was 45 years ago! [36]. The ability to cryopreserve elements of structure and function in excised tissues also has clear medical benefits when applied to biopsy samples. For microscopic investigation where the function is not required then structure/tissue architecture is of primary concern [37]. Conversely, when functional assessment is required, then balanced and optimal cellular performance must take precedence over structure. This indicates an interesting, and valuable, halfway house for cryopreservation where success can be measured in terms of either the structural integrity or function of recovered material [37, 38].
Cell therapies and regenerative medicine treatments require methods that offer successful cryopreservation, are practical and meet regulatory requirements if they are to form parts of medical devices and/or require cGMP manufacture [14, 26, 39, 40]. This can become an issue for larger samples if novel, larger sample containers need to be devised to facilitate effective processing, including CPA treatment, cooling and warming. For example, only cryobags and hermetically sealed cryovials are permissible for cGMP therapies. The latter enables simplified aseptic filling operations and typically has thickened plastic walls to prevent damage at low temperatures. These thick walls limit the heat transfer rates achievable and so may influence the design of the cryopreservation protocol [41]. On the contrary, cryostraws, commonly used in reproductive medicine, have internal diameters in the order of 1–2mm that increase their surface-to-volume ratio for more efficient thermal transfers of the sample and so they are only suitable for the smallest spheroids and organoids [42]. Regulations of course vary between regions, but broadly align when the manufacture and use can take place over multiple jurisdictions, requiring compliance with all regulatory regimes [14, 40, 43].
It is important to accommodate such practical difficulties into the initial design of the cryopreservation protocol as retro-adapting methods for clinical delivery once they have been developed are lengthy and costly and can delay (and in some cases prevent) a treatment gaining widespread use. Other issues such as a need for automation during processing may also have an impact [14, 40].
2.2 CPA loading and unloading
An early event where the extended diffusion pathways of larger structures are evident is in the loading and unloading of permeating CPAs such as Me2SO [6, 11, 17, 18, 24, 34]. Following the addition of a permeating CPA an initial, cellular response of exposed cells is to shrink due to the osmotic gradient the CPA exerts [34]. As the CPA then permeates into the cell, the gradient is diminished and cell volume recovers to a significant extent [34]. In larger structures, exposure to the gradient, and the responses to it, will be delayed for those cells embedded deeper in the structure [44]. This generates a risk of insufficient CPA protection if cooling proceeds before CPA equilibration is reached in the central regions of the structure. However, an extended incubation time in the CPA to ensure deep equilibration can lead to damaging levels of toxicity for more peripheral cells. The larger and more complex the structure the more challenging this issue becomes, with both extracellular channels, cell membrane parameters, viscosity, temperature and physical distance all playing a role [19, 24, 44]. A similar issue, but reversed in direction, is encountered on warming and subsequent CPA removal [18].
Tissue architecture can provide additional complications for CPA treatments. For example, mature organoids may contain a central cavity devoid of cells, or with a different cellular composition [27, 45] and sufficient time for CPA diffusion into this cavity is necessary to prevent further CPA diffusion from the innermost cells into the cavity following cellular equilibration. This would result in an overall CPA loss from the inner cells, compromising the chances of achieving the required level of their post-thaw cell survival to maintain organoid integrity. Chondrocyte and cartilage samples, typically cryopreserved with bone attached, provide a further example. As commonly used CPA cannot pass through bone, this further limits the surface area for diffusion of water and CPA, restricting diffusion pathways and transfer speed [17, 23].
Several methods have been employed to alleviate CPA loading and unloading difficulties that may prove to be applicable if modified for larger structures. One such method involves adding an initial CPA concentration to the external medium that is higher than that considered necessary for successful cryopreservation. As CPA diffusion is driven by concentration gradients, this higher concentration external to the biological sample will increase the CPA diffusion rate and thereby reduce the required incubation time. When the tissue is calculated to be sufficiently protected, the extracellular CPA concentration can be reduced to its equilibrium value [17, 23, 44]. Such methods are more often used with systems preserved through vitrification (ice-free cryopreservation) but are equally useful to overcome CPA loading issues in slow-cooling techniques. However, the high concentration of CPAs, at the relatively high incubation temperatures employed, can cause significant cytotoxic responses in sensitive cells near the outer surfaces of a larger structure. The temperature could be reduced to lower CPA toxicity, but as viscosity is temperature dependent [46, 47], any lowering of temperature would increase incubation times to achieve the required level of diffusion, thereby negating any benefit of the lowered temperatures. Using a mixture of different CPAs can reduce the concentration, and so toxicity, of any one given CPA can also be used to mitigate this problem. Such techniques are common in large-volume vitrification and may help in slow-cooled systems with long incubation times [20, 48, 49].
When working with entire organs in which the circulatory system is intact, the blood vessels can be perfused to reduce CPA distribution time and ensure homogenous CPA loading [35]. Perfusion is an established technique in major surgery and organ analysis [50] and the replacement of blood or stabilising solutions with CPAs can effectively reach areas of tissues difficult to reach by diffusion or surface-induced effects alone [21, 31, 35, 51]. This has shown to be effective in some cases [21, 31, 33, 51], yet most studies focus on the very high CPA concentrations required for vitrification that are currently less applicable to larger structures using slower cooling rates. The systems involved may be susceptible to vasculature cryoinjury, with damage to small blood vessels during cooling, sufficient to prevent effective CPA removal resulting in necrotic areas after thawing due to CPA toxicity. These methods are also limited to tissues with the full circulatory system—immune privileged tissues without vasculature cannot benefit from this technique—and require specific technical skills to perfuse the organs successfully.
Extracellular CPAs, which can help dehydrate cells and protect cell membranes pose particular problems for larger structures as they will only protect the outermost cells of the structure, or ones that can be reached through extracellular liquid channels. Innovative methods to exploit the potential benefits of perfusion to slow cooling techniques are required.
2.3 Diffusion of heat and intracellular water
In a suspension of separated cells undergoing cryopreservation, the diffusion distance for heat, water and solutes between individual cells and the external medium is no larger than the radius of a cell. Additionally, diffusion of water and intracellular CPAs is influenced by membrane permeability to these compounds and the cell surface area to volume ratio. These factors will vary in differing, but limited ways when small-cell aggregates are present. Having relatively uniform characteristics means that the cellular responses of single cells, and small-cell aggregates, to imposed thermal or chemical gradients will be similarly uniform, providing the level of control needed for successful cryopreservation. As noted above, the consequence of working with larger, multicellular structures is that the diffusion pathways are extended and depend on the dimensions of the cell mass. They will also involve transfer across a number of cells and extracellular space [11, 24, 44]. The location of individual cells within the cell mass and their type—each with their specific membrane permeability coefficients and surface area to volume ratios—will influence their response to any imposed diffusion gradient over time and so the level of overall control of heat and water and diffusion of CPAs will be diminished.
Some dehydration may occur in response to CPA treatment in the initial phase of the preservation protocol but the greater part occurs once the ice has formed in the system [41, 44]. This is referred to as cryodehydration. During controlled, slow cooling the extracellular solution commonly falls below its melting point, entering a supercooled state, before ice forms by spontaneous, or induced nucleation [52, 53, 54]. When ice nucleates there will be a temperature discontinuity (an exotherm) within the system related to the release of latent heat of freezing accompanied by a sharp increase in the osmolality of the extracellular medium as water molecules, and only water molecules, become components of ice crystals [55]. Biological material is excluded from the crystal lattice [52]. The nucleation event initiates protective cryodehydration, as described above, but if supercooling is extreme prior to nucleation then the large and immediate osmotic shock delivered once ice forms can be damaging to the sample. The overall size of a sample (tissue mass plus cryomedium) influences ice nucleation and the larger the volume the earlier ice nucleates [52, 56].
Once nucleation has taken place, cells in suspension become entrapped in channels between ice crystals and cellular dehydration is primarily limited by their membrane permeability to water [54, 57, 58]. This protective cryodehydration during cooling is essential as cells retaining a high intracellular water content are more likely to experience lethal intracellular ice formation (IIF) than their more dehydrated counterparts [11, 59, 60]. Cells that have a high membrane permeability to water can survive relatively rapid cooling as water is able to leave the cell quickly enough to prevent IIF. However, at lower temperatures cell permeability decreases, the level of this reduction being cell type dependent. The lower the permeability the slower cooling must proceed to ensure sufficient dehydration occurs, with 1°C/min after ice nucleation being a typical value for somatic mammalian cells in suspension [41, 54].
Larger structures will become embedded in the matrix of ice crystals after nucleation. In a cell spheroid, for example, not all the cells are at the outer surface and so, rather than dehydrating directly into the cryoprotective medium, some cells will transfer water to those in physical contact with them that generate an osmotic gradient, and only those at the outer surface of the sphere will interact directly with the extracellular medium. The overall dehydration rate for the spheroid is, therefore, slower than would be observed for single cells in suspension and, inevitably, the fastest acceptable cooling rate for cryopreservation of the biological sample will also be lower. However, as the slowest cooling rate is essentially defined by the sensitivity of the cell type to CPA toxicity, this remains unaltered, resulting in a narrowing of the range of acceptable cooling rates for successful post-thaw survival [59]. In some instances, a lower recovery rate than is seen in suspensions can be the consequence of the slower rate for the complex system—the highest survival after optimisation being lower than the value achieved in suspensions. In HepG2 liver cell spheroids, the optimal cooling rate falls to 0.3°C/min from 1 to 2C°C/min for a cell cluster of a few hundred cells [8]. This problem becomes more pronounced in organoids containing multiple cell types where dehydration will be limited by the cells with the lowest membrane permeability—the maximum cooling rate becoming increasingly slower as the biological structure becomes larger and more complex. The solution to this dehydration problem is likely to be to lower the cooling rate, where this does not impact post-thaw cell functions. Most somatic mammalian cells can tolerate a relatively low cooling rate, down to 0.1–0.3°C/min, which is usually sufficient for dehydration to occur. T cells for example have shown similar optimal survival at rates of 1°C/min and as low as 0.1°C/min [41], and ovarian tissue samples are typically cooled at rates of 0.2–0.3°C/min [31, 61, 62, 63, 64]. As can be seen in Table 1, most spheroid and organoid cryopreservation methods currently use passive coolers, where control of the cooling rate is limited and producing rates in the vicinity of/of approximately 1°C/min—moving to controlled rate freezers with lower and more precise rates would allow for more precise control over cell dehydration [15].
Ice formation can be physically damaging for cell suspensions when the cells become trapped in channels between crystals [58]. At higher temperatures, the channels are relatively wide, and the cells have minimal direct contact with ice crystals, minimising the potentially damaging effects of distortion, crushing and shear forces. As the temperature falls, more water molecules are locked away as ice and the channels reduce in size [54, 55, 58]. Larger samples are at an increased risk of direct contact with ice under these circumstances, resulting in damage that can impact negatively on recovery. Relatively delicate tissues such as spheroids and organoids can be crushed in this way. Extracellular ice also damages complex tissue structures by disrupting cell-cell contacts, and thereby damaging intercellular communications. Severing these connections is not only damaging to individual cells, it can also reduce the overall function and communication between the surviving cells tissue or organoid.
Different CPAs can be used, perhaps in combination, to help with dehydration difficulties with larger samples. Where lower cooling rates are not practically possible or biologically tolerable with only the permeating CPA Me2SO, then dehydration can be accelerated through the use of extracellular CPAs such as sugars [3, 20, 31, 64]. These CPAs decrease the osmotic potential in the extracellular space, and so can drive more rapid dehydration. This may offset the effect of a lower surface-to-volume ratio of spheroids and organoids relative to individual cells. The addition of different types of CPAs, such as apoptosis inhibitors to the cryopreservation and post-culture medium, has been shown to improve organoid survival in some systems [4]. Altering the size and shape of samples where original structure and integrity are not the priority can also improve the outcome. Ovarian tissue for example is often cryopreserved in strips to maximise the dehydration rates as these tend to be more effective than spheres due to the larger surface area they provide, and an increased surface area can improve biological outcomes [63, 64]. However, in many cell types, when it comes to large, mature organoids containing several cell types the problems faced by dehydration issues cannot easily be overcome. Intracellular ice can still form and be lethal and more research is required to increase dehydration rates, or perhaps lower the possibility of IIF even at relatively high cell hydration levels.
2.4 Ice nucleation and direct ice damage
A further issue with extracellular ice formation is the increased volume of ice crystals—when ice forms it expands to occupy approx. 12% more volume than the liquid state. In cell systems such as organoids, the formation of ice in the internal, liquid-filled cavity, can generate sufficient mechanical pressure on the cells lining the cavity to cause significant fractures. This can disrupt the organoid structure, yet individual cells may survive the cryopreservation procedure.
Intracellular ice is lethal for the cell in which it forms but in a cell suspension, where the cells have limited direct contact with each other, a frozen cell rarely nucleates others. A proportion of weakened or damaged cells in the suspension will experience intracellular freezing but this poses little risk for the greater cell population. However, in a larger structure where cells can be tightly pressed together and/or physically interconnected, ice that forms in one cell can spread to another [65]. This triggers a chain of intracellular freezing throughout the structure that can cause significant damage and cell mortality. Strong evidence of the damage that can be caused by ice comes from tissues and organs which survive cryopreservation, at least in part, with slow cooling. Excised ovarian and thymus tissues are notable in this regard and are dealt with in more detail below. The impact of this damage has been observed in thymus slices, cryopreserved at 1°C/min in 10% Me2SO [32].
A good example of this chain reaction of cell-to-cell ice growth is seen when considering the studies presented by Ross et al. [32]. In this work, histology was carried out (H&E staining) to detect viable tissue and areas of autolysis (indicating cell death); autolysis was seen over continuous areas with some completely devoid of surviving cells and other areas with almost total survival. Autolysed areas form in different places in different samples and so are not related to location in the tissue or placement in the vial in which it was preserved. In a thawed tissue, mass with limited intercellular connections, living and dead cells would be expected to be distributed relatively uniformly throughout the tissue. The aggregated areas of autolysed cells observed suggest there was a significant intercellular connection (as far as required for ice to spread) within the tissue and that once intracellular ice nucleation occurred in a small number of cells it spread rapidly to conjoined neighbours. When thawed, these slices were transplanted into an athymic mouse model where they were able to support T-cell development, showing preservation of function [32].
In certain circumstances, supercooling techniques have been proposed as an alternative cryopreservation method that avoids ice and its associated lethal impacts. Supercooling involves cooling a sample to high sub-zero temperatures, typically between 0 and − 10°C, under conditions where ice is relatively unlikely to form thermodynamically. At such temperatures, biological activity is reduced and both structure and function can be protected for several days. Whilst such a short timeframe is limiting, this can be sufficient to overcome extreme time constraints associated with, for example, transport, quality checks and organ transplants [11, 62, 66, 67, 68].
2.5 Control of ice structure: a way forward?
Ice damage is generally accepted to be the most severe and the leading cause of cryopreservation-related injury and cell death in large biological tissues [11, 21, 60, 69] and can be considered the most difficult problem to overcome. However, ice crystal structure is not constant [41, 58] and new ways of manipulating ice growth may help reduce the damage it causes.
One of the simplest ways to change ice structure is by manipulating the cooling rate, especially in the high sub-zero zone where most ice forms (c. −5 to −40°C) [41, 47]. In Figure 1, the ice structure of a 10% Me2SO solution is shown for samples experiencing cooling at 10°C/min; cooled at 1°C/min and at 0.1°C/min. These rates were those recorded after ice was nucleated at −4°C. At very low rates of cooling where ice growth rates are also very slow, the ice has time to organise into large crystals—the most thermodynamically favourable state. Research is limited as to how different forms of macroscopic ice structure impact cryopreservation; however, slower rates of ice growth are known to inhibit damaging ice-recrystallisation on thawing and reduce the osmotic pressure on the cells as the rate at which water molecules are locked into any recrystallising ice is reduced [41, 70]. The ice structure is very different at 1°C/min, a typical cooling rate for cell suspensions, compared with cooling at the much slower 0.1°C/min.
Figure 1.
The structure of ice in a 10% Me2SO saline solution in a cryomicroscope at 10x magnification after controlled cooling at different rates to −100°C. samples were cooled, left to right, at 10°C/min, 1°C/min and 0.1°C/min. The extremely low cooling rate used in C results in a markedly different ice structure.
There are some indications that by using very low rates of cooling, more structure can be preserved. Figure 2 shows the whole mouse embryonic kidney, heart and liver cryopreserved at only 0.2°C/min in 12.5% Me2SO. As can be seen in the figure, these organs (2–5 mm max. dimension) had good post-thaw structure.
Figure 2.
Mouse embryonic kidney, heart, and liver after cooling at 0.2°C/min and storage in LN for >30 days. The overall structure of the organ (top), and histology (bottom, H&E stain) of the tissue indicate minimal cell and structural damage on cooling.
New developments in cryopreservation technology allow ultra-slow cooling rates and long cooling times, and so open the door to new ice structures—mammalian nucleated somatic cells tend to be robust to very slow rates of cooling. Many of the large tissues currently cryopreserved use very slow rates of cooling—ovaries at 0.2 or 0.3°C/min [31, 63, 64], liver spheroids at 0.3°C/min [8] and uterus at 0.2°C/min [33] —while this will help in dehydration and CPA diffusion as discussed above, the different ice structure in these ultra-slow cooling regimes likely plays a role.
Historically, the manipulation of cooling rates was seen as a key parameter to the successful cryopreservation of whole organs. A 1984 study found that extremely low rates of cooling, as low as 1°C/hr. in this case, resulted in better vascular resistance readings, tissue architecture observations, with ice seeming to have been localised to extracellular zones more at these slower rates of cooling [71]. Microscopic studies using freeze-substitution paint a similar picture [72]. Such slow rates of cooling have been scarer in recent years, partly due to the practical difficulties of applying low cooling rates at the time, and due to fewer needs for larger structure cryopreservation. Applying these exciting but somewhat neglected methods to modern tissue-engineered structures and organs, along with combining them with new cryoprotectant knowledge and technologies offers perhaps the best chance for widespread tissue preservation.
Ice structure can also be effectively manipulated through the introduction of ice nucleation and ice-inhibiting particles, as well as cooling rates and CPA concentration [60]. Higher nucleation temperature tends to cause larger ice crystals as less of the freezable water solidifies at the initial point of nucleation, more supercooling—as is seen in the absence of ice nucleators—causes a smaller, more dendritic and ice structure. More viscous CPAs will slow the rate of ice crystals growth by inhibiting the diffusion of water molecules onto the crystal-liquid interface [19, 70]. In future, adapting parameters such as this may be able to reduce the damage caused by ice enough to allow for the preservation of a larger portion of a larger number of tissues.
3. Cryopreservation of larger structures: the special case of ovaries and thymus
The ovary consists of follicles at various states of maturity, in which immature oocytes reside. These follicles and slices of the mammalian ovary have been successfully cryopreserved [61, 63, 64, 73]. Ovarian tissue can be cryopreserved before cancer treatments which may damage the ovaries and can be thawed and transplanted when the patient wants to have a baby, allowing for natural conception [63, 64, 74, 75]. In human ovaries, the tissue is often cryopreserved in follicle-containing slices, which in addition to simplifying the physical problems of larger tissue cryopreservation, has the additional advantage that only a single slice has to be transplanted back at any one time, allowing for multiple pregnancies following separate thawing procedures. Ovarian tissue preservation can be particularly beneficial in pre-pubescent girls undergoing treatment where hormonal stimulation to produce mature oocytes for cryopreservation is usually not possible [63, 64, 74].
Carroll et al. [76] first published successful births in mouse ovarian follicles (a liquid membrane containing immature oocytes which are surrounded by layers of granulosa cells) in 1990. The method involved incubating the samples in Me2SO (1.5 M) and serum for 10–12 minutes, then seeding ice at −7°C and followed by a cooling rate of 0.3°C/min [76]. By 2014, Campbell et al. cryopreserved whole sheep ovaries, which were able to produce fertile offspring after thaw and re-transplantation [31]. For these larger tissues, the ovary was first perfused [35] using the blood vessel architecture with CPAs (Me2SO, calf serum, and extracellular CPA sucrose, for up to 60 minutes, and cooling proceeded at only 0.2°C/min). The success of these techniques shows that, with sufficient CPA incubation, appropriate cooling rates and controlled ice nucleation, then larger structures can be preserved with widespread success [61, 62, 63, 64, 75, 77]. It is observed that tissue that can be physically sliced and still function on transplant can also survive cryopreservation, and tissues that cannot be sliced and survive do not survive cryopreservation. This may indicate that physical damage due to ice disruption within tissues is certainly a central issue in the cryopreservation of larger tissue samples.
Another tissue that can be cryopreserved with success is the paediatric thymus. Thymus transplantation is carried out to treat paediatric diseases such as complete DiGeorge syndrome, in which infants lack a thymus [78]. Thymus is obtained from a donor and sliced into up to 30 pieces, approximately 1 mm thick. These slices are then cultured to deplete the donor thymocytes (large numbers of donor thymocytes could potentially cause an immune reaction in the host), leaving mainly stromal and epithelial cells for transplantation. Transplantation is done in the well-vascularised thigh where circulating recipient progenitor cells are able to populate the transplanted slices and undergo T-cell development, eliminating the need for more complicated chest surgery where the thymus usually resides [32, 78, 79].
Cryopreserving such tissues will allow for the creation of thymic tissue banks, giving a supply of tissue on patient demand and allowing for future recipient tissue or partial tissue matching, surgery at the optimal time and location for the recipient. The authors have found that these samples can be cryopreserved at 1°C/min in 10% Me2SO without the need for ice nucleation [32]. Rapid diffusion of water and solutes is facilitated by the slicing of the tissue pre-cryopreservation. The thymus does not have to be completely intact to fulfil its function of supporting T-cell development, so the areas of tissue that survive the freeze/thaw have sufficient capacity to restore the peripheral T-cell population in the mouse model [32].
An area of cryopreservation that is sometimes overlooked is that of biopsies. These small pieces of tissue, typically of the order of 1–3 mm3, are cryopreserved for reasons ranging from diagnostics and cell extraction to fundamental research [7, 29, 30, 80, 81]. Typical cryopreservation of these structures involves direct plunging into liquid nitrogen without the use of CPAs [80]—this may allow the recovery of some markers and DNA but living cells and faithful tissue architecture is lost. A particularly promising use of optimising biopsy preservation is their use for population-wide studies where biopsies are taken from many patients over many years and stored in biobanks [11, 82]. For the most effective use of such biobanks, preservation methods should allow tissue architecture to be preserved, together with undamaged DNA and protein content, and for viable cells to be available for regrowth. This would open up the possibility of extracting an increased range of data from the samples as well as future-proofing samples for examination by techniques not developed at the time of preservation.
Using current methods, even with the use of CPAs and some control in cooling, liver biopsies can have recovery of oxygen consumption and mitochondrial functions—something elusive with the whole organ [81]. Cryopreserving as tissue or at least as cell clusters may give better single-cell performance than tissue fully digested prior to cryopreservation [83].
Current preservation techniques can provide high level, tissue architectural preservation in organs as complex as the brain, and success has been reported in heart valves using ice-free methods (either vitrification or non-low temperature preservation) to preserve the structure [11, 21, 84, 85]. However, such methods tend to preserve only architecture and not viable cells. Accepting current technical limitations, the balance between preserving tissue architecture/structure or cellular function can be altered. Typically, the structure is the preferred option for biopsies with samples cryopreserved rapidly sometimes in the absence of CPA, resulting in near-total cell death. However, biopsies can also be used to extract living cells, typically for regenerative medicine and organoid culture [29, 30, 82], and slow cooling methods discussed above could allow for sufficient structural preservation as well as ensuring an acceptable recovery of some viable cells. A cryopreservation method where the structure is preserved but also allows for live cells to be extracted would enable considerably more data to be extracted from population-wide samples, markedly improving scientific efficiency and productivity. Overcoming these challenges with new techniques may require an initial focus on specific applications where known demand exists, for example, in biopsy preservation in cancer patients for extraction of tissue infiltrating lymphocytes. Success here might also provide valuable new knowledge relevant to the development of protocols for larger structure cryopreservation.
Cryopreservation is a rapidly developing field that is continually adapting to meet the challenges presented by ice and low temperatures when trying to preserve viability in larger tissues and structures. The larger structures become, the more challenging attempts at cryopreservation, using current techniques, becomes. It is possible that some methods, such as optimisation of known CPAs, may be approaching maximally optimised thanks to modelling (although the door to new CPAs and their reactions remains open), and most current knowledge gained from cell suspensions has already been applied. However, many relatively unexplored avenues of research are available and actively being explored to achieve a viable post-thaw outcome—combining these new techniques with the manipulation of ice structure from lower cooling rates shown to minimise ice damage [71, 72] is an obvious route forward.
There are also cryopreservation methods exploiting higher temperatures, such as supercooling discussed above. Taking samples below the appropriate glass transition temperature (as in conventional storage in liquid nitrogen) will provide dramatically extended storage time for samples, measured at least in decades. However, at a practical level, many applications may not need such a guarantee e.g. preparations for cell therapy or organ and tissue samples destined for application in the short term. Where storage of several weeks would suffice, for example, then storage at a relatively high temperature, where ice could be avoided or at least occupy a lesser fractional volume was harnessed, may provide significant benefit.
In some special cases, such as the thymus and ovaries, it is already becoming possible to cryopreserve mammalian organs and, in time, the number of these cases will doubtless grow through the development of new CPAs, new loading and unloading methods, and techniques to overcome the damaging effects of ice crystals. While ice-free methods offer a promising, but more distant avenue for cryopreservation, slow-cooling methods enjoy current success and will likely form the key to the delivery of many cell therapies, tissue-engineered constructs and other larger tissues in the future.
This work was supported by a grant from Great Ormond Street Hospital Children’s Charity; MMC was supported by a PhD studentship from the MRC; research at UCL GOSICH is supported by the NIHR BRC at GOSH and UCL.
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