Changing Paradigms in Biopreservation

8/12/2019 Changing Paradigms in Biopreservation

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1Institute of Biomedical Technology, State University of New York at Binghamton, Binghamton, New York.2Department of Biological Sciences, Binghamton University, Binghamton, New York.3Cell Preservation Services, Inc., Owego, New York.

Changing Paradigms in Biopreservation

John M. Baust,13

Kristi K. Snyder,13

Robert G. VanBuskirk,13

and John G. Baust1,2

The field of cryopreservation has a long and successful history of in-depth study and progress. Advances in ourknowledge base and our ability to cryopreserve cells have been consequential and have led to its widespreadintegration into academic, clinical, and agricultural settings. While many cell systems are successfully cryopre-served today, there remains significant cell loss associated with cryopreservation. Moreover, even today somecell systems remain uncryopreservable from a practical perspective. This is due to the diversity of post-freezeresponses of individual cells to the various stressors experienced during the freeze-thaw process. In 1998, sev-eral independent groups reported on the direct involvement of apoptotic and necrotic cell death following cryo-preservation (Baust, et al., 1998 and Borderie, et al., 1998). In addition to those reports, a substantial literature base

describing the modulation of cell death through the use of various protease inhibitors, free radical scavengers,media formulations, and other novel compounds exist. These studies have identified diverse molecular-based,cellular responses to cryopreservation and have further demonstrated the significant improvements in cell sur-vival through the modulation of molecular events. Numerous studies have reported on the molecular-basedphenomena of cryopreservation-induced delayed onset cell death, yet our understanding of the pathway activa-tion, progression, control, and the downstream effect on cell function remains in its infancy. To this end, mod-ulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understandingof the activation pathways and are proving to be a critical next step in the evolution of the cryopreservationsciences. This review provides an overview of the current literature on the mechanisms of cell death associatedwith cryopreservation failure.

Introduction

T is experiencing rapid expan-sion1due in part to the growing interest in personalizedmedicine, and drug discovery. The recent successes in celltherapy reported by Geron coupled with the lift in restric-tion on stem-cell research, interest should continue to grow.As such, increasing demands have been placed on the pres-ervation sciences to improve the viability and function ofcomplex and sensitive cells including stem cells and engi-neered cells and tissues. These demands have now stretchedtraditional preservation sciences to a limit.2 As a result,cryobiology has morphed its focus into the disciplines of

cell and molecular biology to drive continued scientificadvancement.3

Underlying this shift is the discovery of the activation ofapoptosis during and following preservation.4In 1998, Baustet al.5reported the involvement of apoptosis contributing to

cryopreservation failure. Since that time, numerous studiesinto the molecular-based cell death following cryopreserva-tion have been reported.4,626Emphasis over the past 10 yearsto adopt the evolving cellular and molecular approaches tofurther the understanding of cryopreservation failure hasresulted in a series of studies, many of which are reviewedand expanded upon in this article.1,15,18,19,2731

Understanding biopreservation

Interdisciplinary efforts to advance the effective-ness of cell, tissue, and organ preservation have led to the

development of the scientific specialtybiopreservation.Biopreservation, an interdisciplinary approach, incorporatesthe fields of cryobiology, engineering, cellular and molecu-lar biology, including cell signaling, genomics, proteom-ics, metabolomics, systems biology, and computer sciences.

BIOPRESERVATION AND BIOBANKINGVolume 7, Number 1, 2009

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availability and biological reducing power in a cell, to namea few.53When considering the full context of the oxidativestressors presented to a cell in the cold, in conjunction withthe generation of free radicals, it is clear that low-tempera-ture exposure provides multiple routes for the initiation of amolecular-based stress response.

Cryopreservation

Cryopreservation represents the storage of biologicalmaterial at ultra subfreezing temperatures (80C) forextended periods (weeks to years). Cryopreservation proto-cols begin with hypothermic exposures, extend through thehypothermic continuum, and reach equilibrium in the glassystate (vitrified). This journey is reversed during the thawingprocess. It is essential to recognize that despite the presenceof extracellular ice, cells that are structurally preserved(avoidintracellular ice formation) remain in a state of deepeninghypothermia until reaching the vitrification state (Tg) ofthe preservation medium. During this period, solute levelscontinue to elevate due to freeze concentration.54Cell func-tion, while suppressed and uncoupled, does not cease until

vitrification has been achieved.55

In order to reduce the prob-ability of intracellular ice formation during freezing, cryo-protective agents (CPAs) are added during the initial coolingphase. CPAs include a diversity of penetrating (membranepermeable) and nonpenetrating agents, such as DMSO, glyc-erol, dextrans, sugars, and so on, often contained within a

buffered electrolyte media.15,32,33

With the first reports of glycerol serving as a protectivesolute and its application to freezing of avian spermatozoa56and human erythrocytes (RBC),57 mammalian cryopreser-vation research began a decade of advancements that cul-minated with the addition of DMSO to the preservationcocktail mix.58 By focusing on two highly differentiatedcellular products (RBC and spermatozoa) with fixed life

spans, the full spectrum of the impacts of preservation stresson the complex biology of normal functioning cells was ob-scured. In effect, these model systems provided a cloakthat obscured the spectrum of events associated with post-thaw, cryopreservation-induced delayed onset cell death. Asmethodological developments proceeded with nontermi-nally differentiated mammalian systems, many cell typesproved refractory to cryopreservation. Even those cells thatare successfully preserved often demonstrate significantpost-thaw death (3070%) within 2448 h.8

Following addition in the cryoprotective cocktail, coolingcontinues at a given rate (1C/min is typical). A seedingstep (ice nucleation) is included in the 2 to 6C range toprevent excessive undercooling (supercooling) of the cell

and the cryopreservation cocktail. If cooling rates are toorapid, inadequate cellular dehydration occurs and the proba-

bility of lethal intracellular ice formation increases, resultingin cell rupture and early-stage necrosis upon thawing.8,14,23,25If cooling rates are too slow, it is believed that the extendedexposure to the freeze-concentrated solutes (now multimo-lar levels) will result in toxic solution effects.5963

As temperature is lowered below the freezing (melting)point of the preservation medium, controlled slow coolingis again utilized to reduce sample temperature to 40 to80C followed by transfer to ultra low-temperature storage(ie, liquid nitrogen immersion, liquid nitrogen vapor, or lessthan135C mechanical storage). This temperature range is

Through this integration, biopreservation represents thesimultaneous management of numerous, lethal conditions(physical and biochemical), with the expectation of normalrecovery. Efforts to sustain living biologics in a dormantstatesupportive of reanimation have included either hypother-mic (refrigerated) or frozen storage.27Hypothermic storageinvolves maintenance at temperatures in the range of 0Cto ~32C, typically between 2C and 10C. Cryopreservation

is defined as the long-term maintenance of biologics at tem-peratures below 80C and typically below 140C (belowthe reported range of the nominal glass transition tempera-tures of pure water).

What is striking about the developments within the dis-tinct subdiscipline of biopreservation is the relative isolationof cryopreservation studies from organ-based hypothermicstorage research. Studies within the hypothermic stor-age area have focused primarily on improving tissue andorgan preservation in support of transplantation, target-ing ion balance, buffering capacity, free radical scavenging,oncotic support, and the provision of nutrients.2,5,24,28,29,32,33Methodological developments falling under the cryopres-ervation rubric link principles relating survival of cells in

solution to cooling. In other words, cryopreservation hasfocused primarily on the physical parameters associatedwith freezing events during the preservation process2,14,34atthe expense of understanding that a chill-freeze continuumexists (hypothermic continuum) that impacts survival.20Thisdisconnect has contributed, in part, to the limitations ofobtaining complete survival of normally functioning cellsfrom cryogenic storage (ie, cell in =cell out).

The hypothermic continuum

Nearly all biopreservation procedures begin with a re-duction in temperature from 37C to most typically the0 to 10C range. A maintenance target of 4C is common.

Cooling represents a change in the energy state of a system.In effect, kinetic energy necessary to support the chemicalreactions that define the metabolome is reduced resulting inthe uncoupling and shunting of biochemical reactions.35,36These biochemical imbalances cause the depletion of ade-nylates (ATP), and disrupt membrane-mediated transport.With the progressive drop in temperature, cells experiencerapid gains in calcium,16,37,38the loss of potassium,17,38,39andintracellular acidosis (pH levels approaching 4). In addition,changes in cell and organelle membrane characteristics have

been reported as phase changes in the lipid domains29,4042from a liquid-crystalline to the solid-gel state occur.29,41,42Asa result, membranes become leaky, thereby contributingto transmembrane ionic imbalances.

These events occur with minor changes in the kineticenergy levels. One measure of the advantage total changein metabolism is Q10, which in mammalian systems calculatesto an ~50% decrease in oxygen consumption (metabolism)for each 10C decrease in temperature.29,4245 Accordingly,the oxygen consumption of a cell at 5C is ~6% of that at37C.4650Q10represents a simplification, as it does not reectindividual reactions but an average of regulatory and non-regulatory enzymatic processes and hence the net of uncou-pling/recoupling and shifts in metabolic pathways.29,40,41,51Q10has been observed to increase dramatically with the onset offreezing.52Accordingly, hypothermia impacts energy status,macromolecular reactivity and stability, adenylate levels,


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UPDATE ON THE MODERN STATE OF CRYOPRESERVATION 5

inuence ice formation/growth within a cell continue to aidour understanding of the cryopreservation process. The pro-cess, which depends on CPAs, has provided for the effectivecontrol of intracellular ice formation.54,5658,69,7685

Necrosis

Necrotic cell death has also been investigated and reportedin numerous cases of cryopreservation failure.4,70,71,86,87

Traditionally, necrosis, or pathological cell death, is usedto describe cellular murder.73,85,88 Necrosis is an energy-independent form of cell death characterized by cell andorganelle swelling, loss of membrane integrity, lysosomalrupture, and random DNA fragmentation, ultimately result-ing in cell lysis (Fig. 1B).78,79,85,89,90As a result, cytokines arereleased causing the activation of immune and inamma-tory responses in vivo.73,79,85,88,90 The initiation and progres-sion of necrosis often occurs rapidly, in a response to severecellular stress resulting in the activation of detrimental in-tracellular signaling cascades. Necrosis has been shown to

be activated in response to ischemia, osmotic shock, severethermal stress, ionic dysregulation, toxic agents, and so on.Many of these necrotic activating stressors are linked to

cryopreservation.

Apoptosis

Apoptosis plays an integral role in the homeostatic main-tenance of cell number and tissue size in complex organ-isms.91Apoptotic processes are also a critical line of defensecontrolling the daily deletion of damaged cells. Kerr et al.89

coined the term apoptosis in 1972 referring to cells under-going a form of cell death described as shrinking necrosis.Following this report, a distinct field of investigation describ-ing, characterizing, and unraveling the associated processes(genes, proteins, cascades, time course, and morphology)

ideal due in part to it falling below the reported glass tran-sition temperatures (Tg) of pure water.64The glass transitiontemperatures for cryoprotective mixtures vary substantiallyand have been reported to be in the 115 to 90C range.Below Tg, system viscosity increases exponentially yieldingcessation of all measurable molecular translational motion.Hence, the presumption is that molecular interactions (ie,metabolism) halt during the sub-Tgstorage interval.65Prior

to reaching the Tg, chemical reactivity continues at reducedrates yielding the potential for sustained free radical damage.It is for this reason that long-term storage at 80C (>612months) is ill-advised, even for biologics such as serum ormacromolecules. With the transition through Tg, the hypo-thermic continuum effectively ends. Structural preservationis afforded to these cells, but a clear inability to manage thepreservation-induced stresses is apparent. When one con-siders the stress factors associated with cryopreservation, itcreates a relatively clear picture of the critical involvementplayed by the cells biology in responding to freezing.Accordingly, a focal shift in investigations in cryopreser-vation has occurred centering around cell stress response

biology.

Understanding Cell Death

A generic listing of cell-based stress factors serves as atemplate to guide the design of improved preservation meth-ods, assuming adequate structural preservation. There arewell-noted differences in the sensitivity of various cell typesto preservation processes.66Van Buskirk et al.20reported onthese variations in three cell types indicating a possible needfor cell-matched preservation media and protocols. Thisstudy suggested that distinct cell types manage their stressresponse through differing molecular pathways. Given this,the new challenge facing biopreservation is the integrationof a molecular-based logic to develop an in-depth under-

standing of a cells round-trip excursion through the hypo-thermic continuum.

Modes of cell death

It is now understood that multiple paths of cell death areassociated with cryopreservation failure occurring hours todays post-thaw (Fig. 1).15,16,25Descriptions of the preservationprocess have been previously discussed with an emphasison the importance of the effect of a cells response to low-temperature exposure.1,27,67 In general, increases in cellularstress results in the activation of apoptotic and necroticcascades leading to increased cell death and as such man-agement of this stress response plays a critical role in pres-

ervation outcome.

Physical events related cell death

Ice-related cell rupture is most commonly associatedwith cryopreservation (Fig. 1A). Thousands of studies have

been dedicated to increasing the understanding of the con-trol and prevention of ice-related cell rupture since Polge etal.56published on the use of glycerol as a CPA in success-ful cryopreservation. As a result, a plethora of studies have

been devoted to understanding and preventing intracellu-lar ice formation to facilitate successful cryopreservationoutcome.8,58,60,63,65,6877Numerous studies on compounds that

IntracellularIce Formation

A

B

C

D

CellularStress

Apoptotic Cascade

ATP Loss

2

Necrosis

Apoptotic CellDisassembly

Cell Lysis

Necrosis

FIG. 1. Cell death pathways associated with cryopreser-vation failure. Diagrammatic representation of the variouspaths of death that a cell may undergo as a result of cryo-preservation stresses: (A) physical ice rupture, (B) necrotic

cell death, (C

) apoptotic cell death, or (D

) secondary necrosis.(Adapted from Baust et al., 2002,14Baust JM, 2007.67)


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also been noted as a consequence of genomic and proteomicalterations in cells.114,117120Gene mutations, either expressionalterations or deletion, often result in the inability of a cellto progress properly through classical apoptotic cascades,thereby switching to necrosis. The transitional nature of celldeath pathways in response to similar stressors creates anextremely complex environment to characterize.

Apoptosis in Cryopreservation

Apoptosis following cryopreservation has now beendocumented in a wide variety of cellular systems. Studiesidentifying post-thaw apoptosis have appeared in a myriadof systems including renal cells, fibroblasts, hepatocytes, pe-ripheral blood mononuclear cells (PBMC), cord blood, sper-matozoa, oocytes, ovarian tissue, vascular tissue, and soon.6,8,12,15,17,121123

Molecular-based cell death

Recently, it has been determined that cell death follow-ing cryopreservation is linked with apoptotic and secondary

necrotic mechanisms.3

Many stress phenomena associatedwith cryopreservation and a sampling of known apoptoticinitiating stressors. A simple comparison reveals the pleth-ora of commonalities between cryopreservation stress andapoptotic activation. A retrospective review of the stressesassociated with cryopreservation intuitively suggests theinvolvement of apoptotic processes. In 1995, Jurisicova etal.118 reported observations of apoptosis in preimplantedhuman embryos and identified programmed cell death(PCD) as a contributing factor to post-cryopreservationembryo demise. That same year, a number of other studiesalso described the reduction of cell stress in both cryopres-ervation and hypothermic storage resulting in improved cellsurvival.69,124130 These studies described the utilization of

compounds including vitamin E, EDTA, protease inhibitors,and free radical scavengers, all known inhibitors of apop-tosis, in preservation media (both hypothermic and cryo-preservation) to positively inuence cell survival. Reportsdetailing discrepancies in cell survival following frozenstorage of human keratinocytes observed apoptotic cellsinuencing post-thaw viability assessments.69The presenceof apoptotic cells in cryopreserved allograft heart valves fol-lowing transplantation has also been reported.131Althoughapoptosis was reported in association with these systems,it was not until 1998 that studies directly linked apoptosisto cryopreservation failure.5 Since then, there have beenmany studies looking to identify apoptotic involvement incryopreservation failure.4,7,8,10,14,17,19,23,26,116119,121,131135 In 2000,

Fowke et al.86

reported on apoptosis following cryopres-ervation in PBMC. The following year, Fu et al.6 and Yagiet al.7reported on the involvement of apoptosis followingcryopreservation in mouse and porcine hepatocytes, respec-tively. Additionally, Schuurhuis et al.123 and Lund et al.136documented apoptosis in PBMCs following thawing. Thepresence and contribution of apoptosis has also now beenreported in renal cells,4,5fibroblasts,8 blood cells,137139 cor-nea,140 stem cells,9,141 cord blood,10 lymphocytes, sperm,142ovarian tissue,143and oocytes.121,144These reports as well asothers continue to solidify the foundation of molecular-

based cell death following cryopreservation as a universalphenomena inuencing outcome.24,26,119,133,145

emerged.83,8698 These studies have led to the characteriza-tion of apoptosis as a highly conserved set of cellular pro-cesses among complex organisms ranging from nematodesto primates.93,94,99101

Apoptotic cell death is defined by three stages: initiation,execution, and termination (Fig. 1C). During each stage, aseries of specific events is activated as part of a complex cas-cade leading to cell death. Progression through each stage

requires energy input (ATP) throughout the process withoutwhich, cells may shunt to a necrotic cell death pathway.102This shunting has been termed secondary necrosis15,20,103andis discussed in the transitional cell death section further(Fig. 1D). Apoptosis has been shown to initiate as a resultof stresses including radiation, cytotoxic agents, nutrientdeprivation, excess or diminished gene products, anoxia,growth factor withdrawal, and temperature.4,8,11,20,90,96100,104108Following induction at any number of organelles, apoptosisproceeds through a cascade of events including caspase ac-tivation, mitochondrial release of cytochrome C, cell cyclearrest, externalization of membranous phosphatidylserine,or alterations in gene expression.89,91,96,101,102,104,105,109112 Theseevents lead to the termination stage where DNA is cleaved

into ordered fragments, the membrane blebs and apop-totic bodies form, and the complete disassembly of the celloccurs.

Transitional cell death

Molecular-based cell death has been perceived as ablack or white process, proceeding through apoptosis ornecrosis. At the intracellular signaling level, apoptosis isviewed as true organized molecular response while ne-crosis involves random molecular events. With that said,the cell death landscape has evolved substantially over thepast 10 years suggesting that apoptosis and necrosis repre-sent extremes on each end of the molecular-based cell death

continuum. Bras et al.113

have suggested that three types ofapoptosis occur. Type I: the conventional apoptosis does notinvolve lysosomes but relies on caspase activation; Type IIis characterized by lysosomal-linked autophagocytosis,whereas Type III is lysosomal-independent, necrosis-likeapoptosis marked by swelling of intracellular organelles. Infact, many of the caspases now appear to play roles in bothapoptosis and necrosis.114It is now thought that when a cellcommits to death, an apoptotic response is activated. Thisproceeds through cellular execution (classical apoptosis) or tothe point where the initiation stress becomes too great or en-ergy levels too low for continuation. At this point, cell deathshunts from apoptosis to necrosis for completion (secondarynecrosis)20,111,112,115,116(Fig. 1D). The vacillating nature of apop-

totic and necrotic cell death was demonstrated in Jurkat cellsby Leist et al.102as the apoptotic-induced population could beshifted to necrotic characteristics with the removal of energysubstrates. Conversely, the replenishment of energy returnedthe system to the apoptotic program, up to a nonreversiblepoint. This transitional cell death has been demonstrated ina number of studies and provides a basis for the cell deathcontinuum in cryopreservation. Common stressors such asnutrient deprivation, DNA damage, cytokine exposure, cy-totoxic agents, oxygen deprivation, and ionic imbalancemay result in both apoptosis and necrosis. The relative de-gree of the stress experienced by the cell determines themode of death. Observations of transitional cell death have


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activation of caspase in human spermatozoa and Yagi et al.7in a porcine hepatocytes model. Expanding on these find-ings, Vogel et al.146reported numerous alterations in humanfibroblast protein levels following cryopreservation. In thisstudy, the authors further described the utilization of cel-lular proteomic fingerprinting in a diagnostic manner toassess the quality of biologics following preservation.

Initiation of cryopreservation-inducedmolecular death

While much research has been focused on identifyingand quantifying apoptosis following cryopreservation,few detailed investigations into the initiating stresses exist.Inherent in the process is the exposure of cells to numer-ous stressors, many of which can initiate a molecular deathresponse. Many of these factors include metabolic uncou-pling, production of free radicals, alternations in cell mem-

brane structure and uidity, dysregulation of cellular ionicbalances, release of calcium, osmotic uxes, and CPA expo-sure.14,15,24This list of stresses associated with cryopreserva-tion is by no means complete, but illustrates stress response

complexity and multiplicity of potential initiation points. Itis believed that the accumulation of sublethal stressors dur-ing the preservation process results in activation of apopto-sis followed by a shift to secondary necrosis. In an effort toprovide insight into the effect of the various stressors associ-ated with cryopreservation, studies have begun to focus onthe various cellular initiation sites of apoptosis. These stud-ies remain in their infancy, but have begun insight into thepathways associated with cryopreservation-induced molec-ular cell death, including the cell membrane, nucleus, andmitochondria.

Control of Cryopreservation-InducedMolecular Response

With the discovery of molecular responses in cells to thepreservation process, there have been a number of attemptsto control these events in an effort to improve preservationoutcome. These approaches vary and include alteration insolution design (cryoprotectant carrier media), and additionof protective agents for targeted apoptotic control (TAC).

Cryopreservation solution design

One shift in the approach to improving cryopreservationoutcome in recent years is that of carrier media formulation.Traditional cryopreservation media consists of a basal cul-ture media with serum protein and DMSO supplementa-

tion. While providing for physical protection through theDMSO and protein components, the basal solutions do notprovide adequate control or maintenance of an appropriatephysiological environment for cells during the cryopres-ervation process. These traditional solutions fall short inaddressing changes in solution pH, free radical production,energy deprivation, and so on. Further, culture media-basedsolutions designed for use at normothermic conditions donot provide the appropriate ionic environment necessary forcell maintenance during preservation,24as these media areconsidered extracellular-like with regards to ionic concen-trations (high Na+, low K+). Accordingly, the solution prop-erties of these historical preservation media do not provide

Cryopreservation-induced delayed onset cell death

Reviewing the l iterature, it can be concluded that molec-ular-based cell death (apoptosis) plays a critical role in cryo-preservation outcome in many systems. One critical aspectis the temporal component of post-cryopreservation celldeath.3To this point, evaluation of cell populations withina few hours post-thaw does not allow for the identification

of the full extent of apoptosis or necrosis (Fig. 2). Molecular-based cell death may take many hours to days to manifestfollowing thawing due to the chronological nature of the celldeath machinery. It is this temporal component that contin-ues to elude many investigators attempting to characterizemolecular cell death following preservation. In 2001, a re-port detailed the timing of cell death following cryopres-ervation, termed the phenomena cryopreservation-induceddelayed onset cell death (CIDOCD).8This study documenteda delayed peak in necrosis (6 h) and a subsequent peak inapoptosis (12-h) post-thaw. Due to the ordered temporal pro-gression of the cell death cascades, the nadir in cell survivalwas not observed until 24-h post-thaw. Subsequently, a seriesof investigations into the path of molecular cell death pro-

gression ensued. In 2002, Baust et al.14

reported on a genomicresponse following thawing in the form of up-regulation oftranscriptional activity of key apoptotic enzymes (caspase-3,-8, -9) in a delayed manner (1218 h post-thaw). Vogel et al.146also reported on the post-thaw activation of caspase-3 ina renal model and demonstrated substantial alterations inproteolytic activity throughout the initial 24-h recovery pe-riod. Schmidt-Mende et al.132 has reported post-thaw pro-tease activation in a bone marrow cell model as well. Thisstudy found a high level of intrinsic proteolytic activityfollowing preservation leading to the cleavage of variousapoptotic proteins. Further implicating caspase involvementfollowing cryopreservation, Paasch et al.134reported on the

Post-Thaw Recovery Interval (h)

True

SurvivalOnset and Progression of Delayed Cell Death

Population Regrowth Interval /

Functional Utilization Interval

Apparent

Survival

00

10

20

30

40

50

SampleViability

60

70

80

90

100

4 8 12 16 20 24 48

FIG. 2. Timing and progression of cell death followingcryopreservation. Representation of the progression of thetemporal sample survival status during recovery from cryo-preservation. Cell viability is typically seen as elevatedimmediately post-thaw and then progressively decreasesduring the initial 24 to 48 h of recovery as apoptotic andnecrotic events manifest, yielding a true survival that ismuch lower than initially observed. (Adapted from Baust

JM, 2005.122)


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during cold storage of rat liver prevented apoptotic induc-tion in endothelial cells as a result of cold ischemia.

More recently, focus in TAC-based preservation hasshifted to understanding and inhibiting the activity of Rhokinases as a means of improving cell survival.66,157159 In2008, Martin Ibaez et al.159reported on the benefits of Rho-associated kinase (ROCK) inhibition for the cryopreserva-tion of embryonic stem cells. In this study, ROCK inhibitor

was added to both the freeze and recovery medium. ROCKinhibition resulted in an increase in stem-cell survival, andreduced the level of stress-induced spontaneous differentia-tion while maintaining differentiation capacity of the cells.Li et al.158expanded investigations into the action of ROCKinhibition and has suggested that ROCK inhibition might notdirectly effect cold-induced apoptosis, but rather reduces thenegative effects of the cell dissociation and handling processassociated with preservation protocols, thereby reducing theoverall cell loss. While dissecting two interlinked portionsof the preservation process (preparation of sample for freez-ing and freezing), this study demonstrated the inuence ofRho kinase activity on stem-cell cryopreservation outcome.Further, this study again illustrates the inuence of the entire

process on cell state and ultimate outcome of the cell preser-vation process. Most recently, Heng and colleagues have con-tinued this line of TAC study, incorporating ROCK inhibitorsinto preservation media for bone marrow mesenchymal stemcells (MSC), and reported a marked increase in MSC sur-vival.66,157 Interestingly, as discussed earlier, Heng reportedthat the benefit of ROCK inhibition was not seen immediatelypost-thaw, but manifested by 24-h post-thaw. These findingsare consistent with previous reports in other cell systems suchas renal cells, fibroblasts, PBMCs, and liver cells among oth-ers.4,8,86,118,147,151Taken together, studies focused on TAC haveshown tremendous promise for cell- and application-specificdevelopment of improved cryopreservation processes.

Summary

Many cell-based applications in regenerative and repar-ative medicine, biobanking, tissue engineering, and so onrequire normal, predictable, and timely return of cells follow-ing cryopreservation. This is often not the case with todaystechnologies and approaches. Additionally, there exists agrowing body of evidence suggesting that CPAs may alsohave a potentially negative impact on the proteome, genome,and fragmentome. Accordingly, it has become imperativethat new lines of investigation into cellular response to cryo-preservation commence. As our understanding continues togrow, advancements will continue to push the present-daylimits of successful preservation. Molecular-based study

has once again helped to propel cryopreservation forward.A union between the optimized structural protection andcellmolecular-based modulation is most likely to providethe next level of improvements in post-preservation out-come. The first generation of cryopreservation developmentsfocused on structural preservation of cells through theinclusion of cryoprotectants and ice management. Second-generation strategies, focusing on preservation media com-position, have integrated with first-generation strategies andimproved preservation outcome.15Current studies are nowfocused on linking the management of gene-regulatedstress-dependent effects on a cell (TAC) with that of first-and second-generation approaches.

for protection at the cellular level, thereby in many cases ex-acerbating cell death.15

In contrast to extracellular-like media, the development ofintracellular-like media has shown benefit in increasing cellsurvival. Reports on media such as ViaSpan (University ofWisconsin Solution), CryoStor, Unisol, Adesta, and Celsior,to name a few, have detailed varying levels of improvedsurvival when combined with CPAs for cryopreservation.

Successes with this approach have been reported in cellularsystems including hepatocytes,147,148cord blood,149stem cells,PBMCs,122fibroblasts,8 keratinocytes,14blood vessels,145 andengineered tissues.25In the majority of these studies, eval-uation of the cryopreservation media was conducted andcorrelated with improvements in cell survival, function,and growth. Interestingly, in most of these studies, the im-provement in viability and sample quality was not notedimmediately post-thaw; it was not until the molecular-basedevents fully manifested that the improvement was observed.Continuation studies have suggested that the improvementin cell survival and function was due to a reduction of bothapoptosis and necrosis during post-thaw recovery althoughthe mechanism of which is unknown.9,15,24

Application of targeted apoptotic control

As previously discussed, the processes and pathwaysassociated with the induction of apoptosis and necrosisare complex. The current state of knowledge relating to theextent and activation sites of these molecular-based eventscontinues to grow. While new cryopreservation media haveprovided for an improvement in cryopreservation outcome,the involvement of apoptosis in cryopreservation failure hasled to many studies investigating the feasibility of TAC.15In 2000, our group reported on the attempt to control theactivation of apoptosis following cryopreservation throughdirect caspase inhibition,4 which markedly improved cell

survival.8

Subsequent studies further demonstrated thatTAC could be used to modulate both the levels of post-thawapoptosis and necrosis.8Yagi et al.7reported on the benefitof TAC in improving hepatocyte cryopreservation, a signif-icant investigative milestone. These data provided a basisfor the hypothesis describing transitional cell death in cryo-preservation failure. A number of additional reports haveemerged describing incorporation of caspase inhibitors intocryopreservation media to improve cell survival.15

In addition to these studies, there have been numerousother reports on the inuence of TAC on cell survival in bothcryoprevention and hypothermic storage. In the mid- to late1990s, several reports on the utilization of protease inhibi-tors, free radical scavengers (vitamins), and ion chelation

emerged.124130,150While not specifically targeting apoptosisas the mechanism of death, these reports nonetheless clearlydemonstrated the benefit of this approach. With the specificidentification of apoptosis contributing to cryopreservationfailures5along with other studies,3,6,7,9 the benefits employ-ing TAC to improve cryopreservation outcome becameobvious. Subsequent independent studies from 2001 to 2003recognized this and reported the benefit of TAC using cas-pase inhibitors.3,68,26,151154 A report describes the benefitsof calpain inhibitors during cryopreservation to improvecell survival.155The Robilotto et al.155study was, in part, anextension of studies by Baust et al.3,4,8in combination withstudies by Sindram et al.156 that showed calpain inhibition


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Address reprint requests to:Dr. John G. Baust

Lead Professor and UNESCO Chair of CryobiologyInstitute of Biomedical Technology

Science 3 Suite 144State University of New York

Binghamton, NY 13902E-mail:[email protected]

Received 22 February, 2009/Accepted 16 March, 2009

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