Cryopreservation

Introduction

In 1949 Polge, Parks and Smith¹ reported on the “chance” discovery of glycerol's cryoprotective function during their efforts to preserve avian spermatozoa in the frozen state. In the following year, Smith² extended these observations by successfully cryopreserving human red blood cells (RBCs) in glycerol. These two reports identified key elements that would play a crucial role in the evolution of the field of biopreservation including the need for a cryoprotective agent (CPA), the process by which cells could successfully be exposed to penetrating CPA and the manner of freezing and thawing. In 1959 Lovelock and Bishop³ first described the use of dimethyl sulfoxide as a CPA with its advantage of enhanced permeability versus glycerol for many cell types. In the following decades incremental advances were made focusing on changes in and study of the carrier media containing the CPAs as well as the mechanisms of cell cryoinjury and cryopreservation. Most notable was the Mazur et al. report⁴ in 1972 which put forth the “Two-factor Hypothesis” to describe the interrelationships between cooling rates and survival as influenced by either toxic “solution effects” experienced at sub-optimal slow cooling rates or lethal intracellular ice present at high cooling rates. In effect, these studies established a biophysical foundation upon which cryopreservation experimentation rested for nearly four decades. Other notable developments were discoveries by Fahy et al.⁵ and Rall and Fahy⁶ in the mid-1980s. This group reported on the novel vitrification strategy of cell preservation in which high concentrations (approaching 8 molar) of a cryoprotectant mixture could be titrated over a concentration gradient to create a medium that when “frozen” was devoid of ice even at liquid nitrogen storage temperatures. Beginning in 1998 a series of studies revealed that perturbations in the cell's proteome and genome during and following the cryopreservation process would significantly impact survival. This effect was observed regardless of the cryopreservation protocol utilized, “optimized” or other.^7–11

With the continued development of cellular-based technologies (e.g., bioreactors with stem cells, tissue engineering, etc.), there is a need for improved methods of preservation that meet the requirement for rapid return to normophysiological function. These methodologies must protect the genome and proteome thereby avoiding stress-related genetically selectivity,¹² a criterion not met by most preservation processes, even today. In addition, the use of animal-derived products, proteins, serums, etc., raises concern over contamination with non-native components (i.e., prions, etc.,).¹³ Efforts to maintain mammalian cells in a dormant state capable of “on demand” restoration have centered primarily on either hypothermic (refrigerated) storage or preservation in the frozen state.¹⁴ Hypothermic storage often includes processes that maintain cells at temperatures above 0°C but below a normothermic temperature range (32–37°C). Advances in the hypothermic storage research have depended on improvements in organ preservation media in support of cell and tissue storage and organ transplantation including those factors related to ion balance, buffering capacity, free radical scavenging, oncotic support and nutrients.^15–17 Cryopreservation may be defined as the maintenance of biologics at sub-freezing temperatures, below −80°C and typically below −140°C. As stated above, Mazur's “Two-Factor Hypothesis” established the principles and a priori evidence of lethal conditions that need to be avoided if successful structural preservation is to be accomplished, a supposition influenced by Lovelock's classic demonstration of the relationship between survival and sodium chloride content in human RBCs.¹⁸

Throughout much of its development, cryopreservation research has focused less on integration of the cellular fundamentals that emerged from organ-based, hypothermic storage research and more so on physical factors, events and engineering princioples.¹⁹ Cryopreservation research has had as its central focus on the physical parameters associated with the water-to-ice phase change (i.e., water flux) during the preservation process with less attention focused on the impact of the hypothermic continuum experienced by cells during freezing until a glassy storage state is achieved.²⁰ Cells that are structurally preserved (avoid intracellular ice formation) remain in a state of deepening hypothermia until attaining the vitrification state of the preservation medium.^11,21 Importantly, during this thermal excursion, solute levels continue to elevate due to freeze concentration.²¹ Cell function, while suppressed, does not cease until the intracellular glass transition (Tg) temperature is reached.²²

A Long Cold Journey

Biopreservation is now recognized as an integrative specialty defined to include processes that suppress biological aging while supporting post-preservation restoration of function. Successful cryopreservation incorporates relevant engineering principles with developments in cellular and molecular biology. Biopreservation represents the simultaneous application and management of numerous, often poorly defined (and not fully recognized), lethal conditions with the expectation of normal recovery.²⁰

Biopreservation begins with a reduction in temperature typically from 37°C to the 0–10°C range. A hold temperature of 4°C is common for a brief interval (nominally 10 minutes) to allow for cryoprotectant equilibration. The selection of the hold temperature is less related to a cellular rationale and more to the fact that liquid water reaches its maximum density at 4°C. With cooling, a change in the energy balance of the cell occurs due to heat flow from the biologic. This loss of kinetic energy of molecules results in the uncoupling and re-coupling (shunting) of biochemical reactions.²³ The metabolome experiences imbalances that cause the failure of aerobic production of ATP, disruption of membrane-mediated transport (i.e., rapid gains in calcium, the loss of intracellular potassium and gain of sodium) and intracellular acidosis with pH approaching 4.^23–26

While select mammalian species have evolved mechanism supportive of whole-body hypothermia (i.e., hibernators)^27,28 or seasonally adaptive heterothermy,^29,30 few human cells in vivo tolerate low temperature exposure beyond a few hours. Hypothermia without manipulative intervention yields progressive cell injury during each of its three phases (cooling, maintenance in the cold and rewarming). In addition to metabolic imbalances measurable changes in cell and organelle membrane lipid domains occur in a cell. These structural characteristics (transitions) result in a change in membrane fluidity from the liquid-crystalline state to the solid gel state^31–34 yielding a “leaky” membranous state. A cascade of damaging events may follow including: activation and leakage of lysosomal and lipoprotein hydrolases,^35,36 activation of calcium-dependent phospholipases^37,38 and the release of free fatty acids,³⁹ activation of the apoptotic cascade^40–44 and disruption of the cytoskeletal matrix.^45–47 Along with the generation of free radicals, the oxidative stressors attendant to hypothermia may result in the onset of apoptosis or gene regulated cell death.^{11,12,20,48–51}

Cryopreservation of Cells

Cryopreservation protocols begin with hypothermic exposures which persist through the period of active extracellular ice growth until equilibrium is reached in the glassy-state (vitrified). This journey of deepening hypothermic stress experienced by a cell has been termed the hypothermic continuum.⁵² CPA exposure represents the second step in the preservation process introducing a diversity of penetrating (membrane permeable) and non-penetrating agents contained within a carrier media to the hypothermic cell.⁵³ Incubation in the cryoprotective cocktail lasts between 10–30 minutes at 4°C followed by cooling at a nominal (“optimal”) cooling rate (ranging from 1 to 10°C·min⁻¹ is common for many mammalian cells). Seeding (ice nucleation) at a temperature close to the equilibrium freezing point of the cryoprotective medium (−2 to −6°C) supports the gradual growth of extracellular ice and limits “supercooling” of the system. This supports the osmotic efflux of water from the cell and the equilibration of cryoprotective agents across the cell membrane.⁵⁴ As extracellular ice formation continues, freezing reduces the availability of freezable water in the cell while freeze concentration of solutes increases the intracellular viscosity. When cooling rates are too rapid, cellular dehydration is inadequate increasing the probability of lethal intracellular ice formation.⁵⁵ Non-optimal freezing effects are recognized post thaw by increased cell rupture and early stage necrosis occurring over the first few hours post-thaw.^7,9,11,56 If cooling rates are too slow, prolonged exposure to multimolar levels of the freeze concentrated solutes results in cell toxicity (solution effects).^4,57 An indication of “solution effect” toxicity is the appearance of delayed necrosis peaking 6–12 hours post-thaw as well as apoptosis 12–36 hours post-thaw (cell type dependent)^58–60 followed by a secondary bout of necrosis related to the interplay between the progression of the modes of cell death.¹¹

Cooling rate control (CRC) is accomplished by devices which support microprocessor controlled injection of liquid nitrogen to achieve active controlled rate cooling, or by passive methods often using insulated alcohol baths placed in a −80°C freezer. Active CRC devices monitor a representative sample vial, straw or bag and follow a pre-established program to achieve a desired cooling profile. Profiles are typically set to maintain a standard rate of cooling (e.g., −1°C/min for a specific cell type) over a prescribed temperature range and include a “seeding” event, nucleation through a thermal shock administered by a surge in cryogen to “flatten” the temperature rebound resulting from the latent heat of fusion of ice formation. Active CRC devices also provide records of the cooling profile. Passive CRC devices contain the sample surrounded by, but isolated from, an alcohol bath or a thermal-insulation material, and when placed in a freezer (−80°C), a curvilinear rate (e.g., approximately −1°C/min for a given cell type) is achieved in the samples. In passive CRC process seeding is often accomplished via mechanical agitation to create a nucleation event at a prescribed time during the cooling period.

Upon completion of freezing, samples are placed into long-term storage. During storage, the cell surrounded by a thin layer of vitrified cryoprotectant is maintained in a vitreous state encased in the mass of extracellular ice as long as the temperature remains above the glass transition temperature (Tg) of the solution. In addition to controlled rate cooling, high molar concentrations of cryoprotectant mixtures introduced in a stepwise manner can also be used to vitrify samples creating an ice free state both in the cell and extracellular matrix. This approach eliminates most ice crystal structure formation and has been reported to be of benefit when attempting to cryopreserve complex tissues and organs.^5,6,61–63 Retrieving samples from storage requires rapid thawing often accomplished by placing the sample in a 37–40°C stirred water bath until most of the ice melts. Once the ice has dispersed, elution of the cryoprotectant cocktail with cell culture media in a single-step or a step-wise (for high CPA concentrations) dilution process is used. Step-wise elution minimizes the volume excursions of the cell thereby preventing mechanical damage to the cell membrane and rupture. The effect of physicochemical changes that occur during cryopreservation has been extensively reported by Mazur.^21,55

Despite intensive research focused on improving cell preservation, not all mammalian cells cryopreserve “equally.” To highlight this issue, Lane⁵⁷ states that “Few scientific problems have proved as intractable as cryopreservation” and “…cryobiology has been straitjacketed by its need to conform to the intractable laws of biophysics. For all its successes, cryobiology has been stuck in a rut.” Further, Mazur²¹ has stated that “The problem today (with cryopreservation) is that applying basic principles of biophysics simply cannot solve many of the remaining challenges in cryobiology.” As traditional approaches to cell storage are applied to non-terminally differentiated mammalian cells, many of these native and engineered cell types prove refractory to cryopreservation. Even in “successfully preserved” cell systems, significant death (30–70%) is often observed within 24–48 hours post-thaw.¹¹ Structural protection is afforded to these cells, but mitigation of the preservation-induced stress response resulting in biomolecular-based cell death many hours post-thaw remains a critical issue. As such, it is often the case where the cryopreservation sciences have provided effective strategies for structural preservation of most mammalian cell types but, until recently, have lacked to the molecular-based tools necessary to understand and mitigate much of the post-thaw damage. Recent studies have linked numerous stress factors associated with cryopreservation to known initiators of molecular-based apoptotic cell death processes (Table 1).

Table 1.

Stress factors characteristic of cryopreservation

Hypothermia	Known initiators of apoptosis
Metabolic Uncoupling/Shunting	Biochemical Alterations/Inhibitions
Energy Deprivation	Energy Deprivation
Ionic Imbalances	Ionic Imbalances
Cellular Acidosis	Cellular Acidosis
Protease Activation	Protease (caspases) activation
Membrane Phase Transitions	Membrane Alterations
Free Radical Production	Free Radical Accumulation
Cytoskeletal Disassembly
Freezing
Water Solidification (Solute Concentration)
Cell Volume Excursions	Membrane Alterations
Hyperosomolality	Ionic Imbalances
Protein Denaturation	Biochemical Alterations

The combination of partial physical damage to a cell coupled with cell stresses experienced during the freeze-thaw cycle can result in necrosis. Further, “cross talk” between the apoptotic and necrotic cascades may also yield secondary necrosis. This complex series of events and factors demonstrates the critical involvement that a cell's “biology” plays in cryopreservation outcome.

Molecular-Based Cell Death Associated with Cryopreservation

Apoptotic activation in response to low temperature exposure has been documented in a variety of systems including renal cells, fibroblasts, hepatocytes, peripheral blood mononuclear cells, cord blood, spermatozoa, oocytes, ovarian-tissue, vascular tissue, et cetera.^{11,48,53,64–68} Comparison of the stressors associated with cryopreservation and those known to activate apoptosis reveals substantial overlap. An analysis of these stresses and the cryopreservation literature demonstrates the well documented involvement of apoptosis in cryopreservation failure and the benefits of cell stress response modulation to improve outcome.^51,69–76 Although apoptosis was described in association with these reports, it was not until 1998 that apoptosis and cryopreservation failure were directly linked.⁸ Over the last decade, there has been the emergence of numerous studies focused on understanding the role apoptosis plays in cryopreservation failure.^{7,9,11,49,50,65,66,68,70,77–89} The involvement of apoptotic cell death in cryopreservation has now been reported in renal cells,^7,8 fibroblasts,¹¹ blood cells,^90–92 cornea,⁹³ stem cells,^94–96 cord blood,⁷⁸ lymphocytes, sperm,⁹⁷ ovarian tissue⁹⁸ and oocytes^66,99 to name a few. One aspect of this molecular involvement is the temporal component of post-cryopreservation cell death.⁴⁴ Molecular-based cell death often takes hours to days following thawing to manifest following thawing as various pathways are activated. This results in a delay in necrotic (6 hours) and apoptotic (12-hours) activity and ultimately observable cell death.^{9,11,85,87,100} This temporal component continues to elude many investigators attempting to characterize the extent of molecular cell death following preservation in a variety of settings. While much research has been focused on identifying and quantifying apoptosis following cryopreservation, few have detailed the initiating stresses. While this area remains in the early stage, studies have, none-the-less, begun to provide insight into the pathways associated with cryopreservation-induced molecular cell death. These studies have implicated a host of initiation sites including the cell membrane, nucleus and mitochondria. These reports continue to solidify of the universal role molecular-based cell death exerts on cryopreservation.^{7,48,51,81,83,86–88,101,102}

Major Problems in Cryopreservation of Tissues and Organs

Tissue/organ cryopreservation is much more complicated and difficult than cryopreservation of individual cells. First, in addition to the cell cryoinjury caused by the intracellular ice formation (IIF) and “solution effects” as described above, there are several following major problems associated with organ cryopreservation: (a) vascular damage/rupture caused by ice formation/expansion in blood vessels (water moves into blood vessels from dehydrated cells).^116–118 To minimize this cryo-destructive effect, increased understanding and prediction of the fundamental mechanisms of ice formation and cell dehydration in tissues/organs are required. Although these biophysical events have been extensively studied in single cells with cryomicroscopy techniques,^106–108 experimental data in whole tissues are very limited. (b) Fracture of frozen tissue/organ caused by the thermal stress during the warming process. Thermal stress is one type of mechanical stress caused by non-uniform warming in a solid/frozen body (e.g., the fracture of glass when a surface is rapidly heated or cooled). To reduce the thermal stress, uniform heating is needed. Unfortunately, biological tissues have relatively low thermal conductivity and high specific heat. Therefore, the conventional heating method (e.g., heating in a stirred water bath) causes a large temperature gradient within the tissue, resulting in high thermal stress and tissue fracture. Pegg et al.¹⁰⁹ and Cui and Gao, et al.¹¹⁰ have developed a slow-cooling technique for relatively uniformly warming frozen rabbit carotid arteries to prevent the fracture. However, generally speaking, the slow warming may cause intracellular ice re-crystallization killing cells. A rapid and uniform heating technology is desired to prevent both potential lethal ice re-crystallization and thermal-stress-induced fracture. Apparently, rapid and uniform heating cannot be achieved by conventional heating methods. Recently, scientists have been developing single mode microwave resonance technology to achieve very rapid and uniform heating of frozen biomaterials.^111,112 (c) Problems associated with vitrification: a dramatically different approach to cryopreservation is to use either high concentrations of certain CPAs or ultra-rapid cooling rates (>10⁶°C/min) to induce the cell cytoplasm to form a glass (i.e., to vitrify cells/tissues) rather than to crystallize. Indeed, vitrification is an ideal approach for organ cryopreservation. However, ultra-rapid cooling rates are technically difficult to achieve for tissues/organs. Several of the CPAs that are effective in ameliorating slow freeze injury also act to promote glass formation, but the required concentrations are so high, e.g., 4–8 M,^113,114 that they can be very toxic to the cells/tissues.¹¹⁵ In addition, a vitrified organ is very brittle and can be easily fractured by thermal stress if not uniformly heated. (d) The lack of a single optimal cryoperservtion condition for all cell types in a tissue/organ. As we know, the optimal cryopreservation condition (e.g., cooling rate) is cell-type dependent due to the cell-type dependence of membrane permeability to water and CPAs, intracellular ice formation, osmotic tolerance limits, cryo-sensitivity and other physical/biological factors for cell cryoinjury. Because of many different cell types in a tissue or organ, it is difficult to define one single condition which is optimal for cryopreservation of all cell types. Up to now, cryopreservation (in either frozen or vitrified conditions) of large tissues and organs is not successful, in general.

Improving Cryopreservation Outcome

Differences in the sensitivity of various cell types to cryopreservation processes are well known. In an article by Van Buskirk et al.²⁰ it was suggested that the basis for differing cellular survival is linked to individual cell stress response and the resultant differential activation of cell death processes. The discovery of molecular responses in cells to the preservation process has therefore resulted in a variety of attempts to control these events in an effort to improve outcome. These attempts have included alteration in solution design (cryoprotectant carrier media), addition of cryoprotective agent cocktails, and the incorporation of select compounds for the Targeted Control of Apoptosis (TAC) during the cryopreservation process.

The mitigation of the molecular-based stress responses to low temperature exposure and storage has been shown to be attainable with cryopreservation solution formulation that addresses both physical and cellular related events.⁴⁴ The concept of specialty preservation media has evolved out of the organ preservation specialties. The Belzer and Southard team^15,16 first developed ViaSpan^® (the University of Wisconsin solution) to support the transport of organs (pancreas, kidney and liver). ViaSpan^®, formulated for hypothermic storage, was the first solution designed to manage select putative stress factors (Table 1) and became the first “intracellular-like” preservation medium. In the decade that followed additional preservation solutions were developed (i.e., Celsior, HTK—Custodiol, HypoThermosol, Unisol and others). More recently, cryopreservation solution formulation has moved beyond the addition of a penetrating cryoprotective agent such as DMSO (5–15%) to cell culture media, buffered saline or these media plus serum or a protein component.⁵³ Now recognized as essential to optimization of the cryopreservation process is the maintenance of proper cold-dependent ion ratios, control of pH at lowered temperature, prevention of the formation of free radicals, oncontic balance, the supply of energy substitutes, etc.^17,44 Traditional media fall short in addressing changes in solution pH, free radical production, energy deprivation, etc. Accordingly, the basal properties of these historical preservation media often do not provide for protection at the cellular level.⁵³ In attempt to address this issue, the cryopreservation sciences have taken lead from the organ preservation and molecular biology arenas combining these knowledge bases to increasing cell survival. Media including Viaspan, CryoStor, Unisol, Adesta, Celsior and others, to name a few, when combined with CPAs for have been reported to improve cell survival to varying degrees. Improvements have been observed in systems including hepatocytes,^58,103 cord blood stem cells,¹⁰⁴ PBMC's,^67,105 fibroblasts,¹¹ keratinocytes,⁶⁹ blood vessels¹⁰¹ and engineered tissues.⁵⁶ In these studies, evaluation of the cryopreservation media was conducted and correlated with improvements in cell survival, function and growth. The improvement was not noted immediately post-thaw but not until following manifestation of the molecular-based events was the effect observed. It is now recognized that the integration of an intracellular-type solution with a penetrating cryoprotectant along with an understanding of the molecular responses of the cell at low temperature, provides for improved cryopreservation outcome.^44,53 The success of these solutions is linked to an in depth knowledge and understanding of the cell death pathways activated as a result of cryopreservation-induced cell stresses. To this end, studies have suggested that the improvement in cell survival and function was due to a reduction of both apoptosis and necrosis during post-thaw recovery although the mechanism of which remains unknown.^11,44,48,77

Closing Thoughts

Cell-based applications in cell therapy, regenerative and reparative medicine, biobanking and tissue engineering are now focusing on normal, predictable and timely return to function of the cells after cryopreservation. This is often not achieved with today's technologies and approaches. In order to address this issue, continued improvement in cryopreservation outcome will rely on the integration of cellular biology, molecular biology, biophysics, engineering and cryobiology. Furthermore, with the growing body of evidence suggesting that CPAs, such as DMSO, affect the cellular, proteome, genome and structures such as the mitochondria, the cell membrane and nucleus, it is obvious that successful preservation requires new strategies for the new definitions of success.

Traditionally, cryopreservation developments focused on structural preservation of cells through the inclusion of penetrating cryoprotectants and the management of ice and chemo-osmotic perturbations. New strategies improved preservation outcome through alteration of preservation solution to mitigate some of the detrimental effects of stress that contribute to the post-thaw launch of apoptotic and necrotic cell death cascades. The literature base utilizing the integrated approach to understanding and developing new approaches for preservation grows slowly. Current studies are now focused on linking the “management” of gene regulated stress dependent effects on a cell with the traditional cryopreservation approaches. In combination cell cryopreservation outcome will doubtlessly improve to meet the increased needs in biomedical applications.