1、49.1CHAPTER 49BIOMEDICAL APPLICATIONS OF CRYOGENIC REFRIGERATIONPreservation Applications 49.1Research Applications 49.6Clinical Applications 49.7Refrigeration Hardware for Cryobiological Applications . 49.8HE controlled exposure of biological materials to subfreezingTstates has multiple practical a
2、pplications, which have been rap-idly multiplying in recent times. Primary among these applicationsare long-term preservation of cells and tissues, the selective surgicaldestruction of tissue by freezing, the preparation of aqueous speci-mens for electron microscopy imaging, and the study of biochem
3、i-cal mechanisms used by a multitude of living species to withstandthe rigors of extreme environmental cold. Some of the applicationsare restricted to the research laboratory, but clinical and commercialenvironments are increasingly frequent venues for activities in low-temperature biology. The succ
4、ess of much of this work depends onthe design and availability of an apparatus that can control temper-atures and thermal histories. This apparatus can be adapted and pro-grammed to meet the specific needs of particular applications.This chapter briefly describes many of the principles driving thepr
5、esent growth and development of low-temperature biologicalapplications. An understanding of these principles is required tooptimize design of practical apparatus for low-temperature biolog-ical processes. Although this field is growing in both breadth andsophistication, this chapter is restricted to
6、 processes that involvetemperatures below which ice formation is normally encountered(i.e., 32F), and to an overview of the state of the art.PRESERVATION APPLICATIONSPrinciples of Biological PreservationSuccessful cryopreservation of living cells and tissues is coupledto control of the thermal histo
7、ry during exposure to subfreezingtemperatures. The objective of cryopreservation is to reduce thespecimens temperature to such an extent that the rates of chemicalreactions that control processes of degeneration become very small,creating a state of effective suspended animation. An Arrheniusanalysi
8、s (Benson 1982) shows that temperatures must be main-tained well below freezing to reduce reaction kinetics enough tostore specimens injury-free for an acceptable time (usually mea-sured in years). Consequently, one of two types of processes is typ-ically encountered: either the specimen freezes or
9、it undergoes atransition to a glassy state (vitrification). Although both of thesephenomena may lead to irreversible injury, most of the destructiveconsequences of cryopreservation can be avoided.A change in chemical composition occurs with freezing as watersegregates in the solid ice phase, leaving
10、 a residual solution that isrich in electrolytes. This process occurs progressively as the solidi-fication process proceeds through a temperature range that definesa “mushy zone” between the ice nucleation and eutectic states (Kr-ber 1988). If this process follows a series of equilibrium states, the
11、liquidus line on the solid/liquid phase diagram for a system of thechemical composition of the specimen defines the relationshipbetween the system temperature and the solute concentration. Thefraction of total water that is solidified increases as the temperatureis reduced, according to the function
12、 defined by applying the leverrule to the phase diagram liquidus line for the initial composition ofthe specimen (Prince 1966). This relationship has been worked outfor a simple binary model system of water and sodium chloride andhas been used to calculate the thermal history of a specimen ofdefined
13、 geometry during cryopreservation (Diller et al. 1985). Asexplained later, the osmotic stress on the cells with a concurrentefflux of intracellular water results from chemical changes. The crit-ical range of states over which this process occurs correspondsclosely to the temperature extremes defined
14、 by the mushy zone. Athigher temperatures there is no phase change, so osmotic stress doesnot exist. At lower temperatures, the permeability of the cell plasmamembrane is reduced significantly (as described via an Arrheniusfunction), and the membrane transport impedance is so high that nosignificant
15、 efflux can occur. Thus, the specimens chemical historyand osmotic response are coupled to its thermal history as definedby the phase diagram properties.The property of a cell that dictates response to freezing is the per-meability of the plasma membrane to water and permeable solutes.The permeabili
16、ty determines the mass exchange between a cell andits environment when osmotic stress develops during cryopreserva-tion. The magnitude of permeability decreases exponentially withabsolute temperature. Thus, resistance to the movement of chemicalspecies in and out of the cell becomes much larger as t
17、he temperatureis reduced during freezing. Because the osmotic driving force alsoincreases as temperature decreases, in general, the balance betweenthe osmotic force and resistance determines the extent of mass trans-fer that occurs during freezing. At high subfreezing temperatures(generally defined
18、by the mushy zone), the osmotic force dominatesand extensive transport occurs. At low subfreezing temperatures, theresistance dominates and the chemical species are immobilizedeither inside or outside the cells. The amount of mass exchangedacross the membrane is a direct function of the amount of ti
19、me spentin states for which the osmotic force dominates the resistance. Thus,at slow cooling rates, the cells of a sample dehydrate extensively, andat rapid cooling rates, very little net transport occurs. The absolutemagnitude of the cooling rate that defines the slow and rapid regimesfor a specifi
20、c cell depends on the plasma membrane permeability. Acell with high permeability requires a rapid cooling rate to preventextreme transport. The converse holds for cells with low membranepermeability: they require prolonged high-temperature exposure toeffect significant accumulated transport.When ver
21、y little transport occurs before low temperatures arereached, water becomes trapped within the cell in a subcooled state.Chemical equilibration is achieved with extracellular ice by theintracellular nucleation of ice. This phenomenon is referred to asintracellular freezing. In this process, a substa
22、ntial degree of liquidsubcooling occurs before nucleation, so the resulting ice structureis dominated by numerous, very small crystals. Further, at lowtemperatures, the extent of subsequent recrystallization is minimaland the intracellular solid-state surface energy is high.The preparation of this c
23、hapter is assigned to TC 10.4, Ultralow-TemperatureSystems and Cryogenics.49.2 2010 ASHRAE HandbookRefrigerationAt slow cooling rates and at high subfreezing temperatures, bothextensive dehydration of cells and an extended period of exposure toconcentrated electrolyte solutions occur. There is clear
24、 evidence thatsome combination of dehydration and exposure to concentrated sol-utes leads to irreversible injury (Mazur 1970; Meryman et al. 1977).Recently, Han and Bischof (2004a) showed that eutectic solidifica-tion during freezing can also contribute to cellular injury. Mazur(1977) also demonstra
25、ted that freezing at cooling rates that are rapidenough to cause intracellular ice formation causes a second mecha-nism of irreversible cell injury. These processes are illustrated in Fig-ure 1, which shows that each extreme of the cooling process duringfreezing produces a potential for damaging cel
26、ls. Figure 1 alsoimplies that an intermediate cooling rate should minimize the aggre-gate effects of these injury processes and define the conditions atwhich optimum recovery from cryopreservation can be achieved.Experimental data have been obtained for the survival of a largenumber of cell types fo
27、r freezing and thawing as a function of thecooling rate. Nearly without exception, the survival function followsan inverted V profile when plotted against cooling rate (Figure 2).This plot has been described as the survival signature of a cell; itillustrates the tradeoff between competing heat and m
28、ass transferprocesses that govern the cryopreservation process. Solution concen-tration/osmotic effects lead to slow cooling rate injury. In this state,there is adequate time for transport of water out of the cell before suf-ficient heat transport occurs to lower the temperature enough to drivethe m
29、embrane permeability to nearly zero. Conversely, at rapid cool-ing rates, the cell temperature is lowered so quickly that there isinsufficient time for dehydration, and injury is caused by formationof intracellular ice. The magnitude of the optimum intermediatecooling rate is a function of the magni
30、tude of the membrane transportpermeability. Higher permeabilities result in higher optimum cool-ing rates. Thus, the optimum thermal history for any cell type mustbe tailored for its unique constitutive properties.For most cell types, the bandwidth of cooling rates for optimumcryopreservation surviv
31、al is small, and the highest achievablesurvival is unacceptably low. Fortunately, for practical clinicalapplications, the spectrum of working cooling rates can be broad-ened and the maximum survival increased by adding a cryoprotec-tive agent (CPA) to the sample before freezing. Although a widerange
32、 of chemicals exhibit cryoprotective properties, as summa-rized in Table 1, the most commonly used include glycerol,dimethyl sulfoxide (DMSO), and polyethylene glycol. Numeroustheories have been postulated to explain the action of CPAs. In sim-plest terms, they modify the processes of solute concent
33、ration and/or intracellular freezing (e.g., Lovelock 1954; Mazur 1970). Intro-ducing CPAs to cell systems results in a major modification of thephase diagram for the system (Fahy 1980). In particular, the rate ofelectrolyte concentration with decreasing temperature may bereduced by nearly ten times,
34、 and the eutectic state depressed by asmuch as 108 to 144F. These consequences greatly extend theregime of the mushy zone during solidification (Cocks et al. 1975;Jochem and Krber 1987).Although phase diagrams provide much information for under-standing the possible states that may occur during cryo
35、preservationof living tissues, their interpretation is limited by two major factors.First, the chemical complexity of living systems is far greater thanthe simple binary, ternary, or quaternary mixtures that are used tomodel their behavior. Second, and more importantly, the thermaldata used to gener
36、ate phase diagrams are usually obtained for near-equilibrium conditions. In contrast, most cryopreservation is exe-cuted under conditions far from the equilibrium state. Han andBischof (2004b) describe the significance of nonequilibrium phasechange in the presence of CPAs. For some situations, the g
37、oal is tomaintain a state of disequilibrium; this includes vitrification methodsFig. 1 Schematic of Response of Single Cell DuringFreezing as Function of Cooling RateFig. 1 Schematic of Response of Single Cell During Freezing as Function of Cooling RateTable 1 Summary of Cryoprotective Agents (CPAs)
38、Category CPAs CommentsPermeable Glycerol, ethylene glycol, DMSOLow molecular massOsmotically transportable across cellular membraneShrink/swell cellular responseImpermeable Sugar group: sucrose, raffinose, trehalose High molecular massPolymer group: polyvinyl pyrrolidone (PVP), polyethylene glycol (
39、PEG), hydryoxyethyl starch (HES)Osmotically untransportable across cellular membraneShrink-only cellular responseFig. 2 Generic Survival Signature Indicating IndependentInjury Mechanisms Associated with Extremes of Slow andRapid Cooling Rates During Cell FreezingFig. 2 Generic Survival Signature Ind
40、icating Independent Injury Mechanisms Associated with Extremes of Slow and Rapid Cooling Rates During Cell FreezingBiomedical Applications of Cryogenic Refrigeration 49.3that are applied to reach a solid glassy state that avoids ice crystal for-mation, latent heat effects, and solute concentration e
41、ffects. In manycases, the degree of thermodynamic equilibrium reached for the low-temperature storage state may differ significantly between the intra-cellular and extracellular volumes (Mazur 1990). The equilibrationcan be controlled by manipulating the thermal boundary conditionsof the cryopreserv
42、ation protocol and by altering the systems chem-ical composition prior to initiating cooling. Many of the same chem-icals used for cryoprotection may be added at higher concentrationsto decrease the probability of ice crystal formation at subzero tem-peratures and elicit vitrification (Fahy 1988).In
43、 addition to the thermal history of the interior of a specimen,the thermodynamic relations determining the release of the latentheat of fusion as a function of temperature in the mushy zone (Hayeset al. 1988) must be considered. A specimen of finite dimension hasa distribution of thermal histories w
44、ithin it during the freezing pro-cess (Meryman 1966). The pattern assumed for modeling the evo-lution of latent heat during freezing has a large effect on the coolingrates predicted as a function of local position in a specimen. Con-sequently, the anticipated spatial distribution of cell survival as
45、 aconsequence of the preservation process may depend strongly onthe model chosen for the thermodynamic coupling between thesystems thermal and osmotic properties. Hartman et al. (1991)applied this principle to evaluate how to choose the optimum loca-tion for a thermal sensor to record the most repre
46、sentative thermalhistory during the freezing of a specimen of finite dimensions. Hart-man et al.s analysis indicated that the geometric center of a sampleis a poor selection for positioning the sensor. A position approxi-mately one-third of the distance from the center to the peripherymore accuratel
47、y represents the integrated thermal history experi-enced by the mass during freezing.Preservation of Biological Materials by FreezingBiological materials are primarily cryopreserved by freezingthem to deep below freezing temperatures. Among clinical andcommercial tissue banks, freezing is the predom
48、inant method forpreservation. Following the discovery of the cryoprotective proper-ties of glycerol (Polge et al. 1949) and other CPAs, procedures forcryopreservation were developed for storing a variety of cells andtissues. Table 2 summarizes representative research efforts to pre-serve various cel
49、ls and tissues by freezing.A typical protocol for cryopreservation consists of the followingsteps:1. Place specimen in an appropriate container.2. Add CPA by sequential increments at reduced temperatures.Table 2 Spectrum of Various Types of Living Cells and Tissues Commonly Stored by Freezing (as of 1993)Tissue Comments ReferencesBlood vessels DMSO used for CPA; cooling rate 2F /min. Gottlob et al. (1982)Bone marrow stem cells DMSO is usual CPA; widely used in cancer therapy. McGann et al. (1981)Cornea DMSO is usual CPA. Armitage (1991)Erythrocytes Usual CPA is glycerol; high con
