The Trehalose Myth Revisited: Introduction to a Symposium onStabilization of Cells in the Dry State

John H. Crowe,*,† Lois M. Crowe,*,† Ann E. Oliver,*,† Nelly Tsvetkova,*,†Willem Wolkers,*,† and Fern Tablin*,‡

*Biostabilization Program, †Section of Molecular and Cellular Biology, and ‡Department of Anatomy, Physiology, andCell Biology, University of California, Davis, California 95616, U.S.A.

the dryr the past. We re-

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Cryobiology 43,89–105 (2001)doi:10.1006/cryo.2001.2353, available online at http://www.academicpress.com on

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This essay is an introduction to a series of papers arising from a symposium on stabilization of cells instate. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Ovetwo decades a myth has grown up about special properties of trehalose for stabilization of biomaterialsview many of such uses here and show that under ideal conditions for drying and storage trehalose has fspecial properties. However, under suboptimal conditions trehalose has some distinct advantages andremain the preferred excipient. We review the available mechanisms for introducing trehalose into the cyof living cells as an introduction to the papers that follow.© 2001 Elsevier Science (USA)

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Stabilization of cells by freeze-drying hbeen one of the major goals of cryobiology decades, but success has been limited mainprokaryotes (e.g., 32, 99, 100, 110). The firstports of preserving mammalian cells in a dstate—red blood cells (62, 147)—soon ledcontroversy (56, 148), as a result of whichconsensus developed that freeze-drying mmalian cells was probably not possible. Hoever, with the development of new technologand the application of molecular techniques (66, 167), this field has taken on a new lifemajor symposium devoted to this topic was hat the annual meetings of the Society for Cobiology in Edinburgh, July, 2001, with a vietoward learning from the controversial aspeand advancing the field. This essay is an induction and overview for that symposium.

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TREHALOSE PRODUCTION AND STRESS

Nearly everybody working in this field using the sugar trehalose as a protective mcule. This sugar is accumulated at high conctrations—as much as 20% of the dry weighby many organisms capable of survivi

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89

eceived October 30, 2001; accepted November 14, 20his work was supported by Grants HL57810 an8171 from NIH, 98171 from ONR, and N6601-00-C8 from DARPA.

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complete dehydration, spread across mamajor taxa in all kingdoms (reviewed in 34). Fexample, baker’s yeast cells, which have bethe subject of the most intensive investigatiodo not survive drying in log phase of growth ado not contain significant amounts of trehalobut in stationary phase they accumulate sugar and may then be dried successfully viewed in 7). Until relatively recent years, trehalose was thought to be a storage sugar in thcells, and the correlation between survival in tdry state and its presence was believed to belated to repair functions during rehydration, prviding a ready energy source. Evidence is acmulating that trehalose production may beuniversal stress response in yeasts; it can eprevent damage from environmental insusuch as ethanol production during fermentatin yeasts (59, 107, 111, 112, 141). In fact, ovproduction of trehalose in some yeast strainsincreasing synthesis (84, 146) and decreasdegradation (131) is being used to increaethanol tolerance and thus to boost industethanol production. Along the same lines, thalose has been shown to inhibit toxic effectstoluene in a bacterium (82). Trehalose prodtion may be more widespread as a stresssponse than had been previously appreciathe analogue of trehalose in higher plants been thought to be sucrose (reviewed in 76),

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several higher plants have recently been

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ported to produce trehalose in responsedrought stress, either by the plant itself or bsymbiont (54, 58, 60, 61, 80).

TREHALOSE AND STRESS PROTEINS

Trehalose is synthesized after heat shocyeasts (77), an effect so pronounced that halose was initially thought to be an analogheat shock proteins in yeasts. Subsequentlhas emerged that trehalose is produced in cbination with stress proteins (e.g., 46, 50, 13The synergy appears to confer resistance to mtiple stresses, including dehydration (132, 1143, 145). Similarly, Clegg and colleagues hashown that in cysts of the brine shrimp Artemiatrehalose is produced in response to heat sh(28), along with a small stress protein (16The trehalose seems to influence molecchaperone activity by the stress protein, accoing to Viner and Clegg’s data, but it is possibthat the stress proteins themselves may bevolved in stabilizing membranes and proteinsthe dry state; Sales et al. (135) recently pro-duced some evidence that a small stress proin yeasts is located at the plasma membraneappears to be involved in protection against hydration damage and ethanol stress—proties similar to those seen for trehalose. In keing with this proposition, Torok et al. (158)showed that a small stress protein, hsp17, stlizes membranes during heat stress, by directeraction with the bilayer. The mechanism stabilization is unknown. Potts and his cleagues (72) discovered a protein that thcalled water stress protein produced in laquantities by the dehydration-tolerant alga Nos-toc, but its function is unknown, a common feture in this field. Possibly, the best-studied strproteins related to dehydration resistance arefamily known as dehydrins, found in bohigher (29, 30) and lower (102) plants. Theproteins were first described in relation to dedration resistance (30, 162, 165), but subquently they have been shown to be produce

response to chilling (29, 79), freezing (16175), wounding (133), osmotic stress (154), achemical insults (33). Despite intensive stud

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functions of these proteins in response to these stresses is still in the realm of correlatiand a function has not been assigned. It selikely that dehydrins might be produced alowith sugars (sucrose or trehalose) in stresplants, particularly in view of the fact thadrought tolerance and sucrose production strongly influenced by the plant hormone ascissic acid (e.g., 81, and references thereDehydrin production is similarly stimulated bABA (25). Genes for several dehydrins habeen cloned (e.g., 153), thus presenting opptunities to study functions of these proteinsdehydration stress, both in vivo and in vitro.Even though we will concentrate in the rest

ET AL.

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this essay on trehalose, we believe that stproteins will ultimately be a part of the effort stabilizing cells in the dry state.

TREHALOSE AND BIOSTABILITY

Beginning in the early 1980s we establishthat biomolecules and molecular assemblasuch as membranes and proteins can be slized in the dry state in the presence of thalose. When comparisons were made wother sugars trehalose appeared to be clearlperior (reviewed in 36).

Since that time an astonishing array of apcations for trehalose have been reported (smarized in Table 1), ranging from stabilizatioof vaccines and liposomes to hypothermic sage of human organs. Other studies suggethat it might even be efficacious in treatmentdry eye syndrome (115) or dry skin (123) in hmans. According to one group, trehalose inhibbone resorption in ovariectomized mice (12apparently by suppressing osteoclast differention (173). Another group reported that trehaloinhibits senescence in cut flowers (126).

The point that we want to make is that a mhas grown up about trehalose and its properas a result of which it is being applied, somtimes rather uncritically, to a myriad of biologcal and clinical problems. We revisit this myand ask whether trehalose really has any of

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special properties to which it has been linked.At least half the applications listed in Table 1

deal with fully hydrated cells, and thus the solu-

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Note. In most of these studies other sugars or polymers were tested in addition to trehalose. This is by no means a complete , spray-

tion properties of trehalose are particularly revant. Since we are concerned principally wdry cells here, we point out only in passing ta considerable body of evidence on solutproperties of this molecule is developing (9,20, 31, 85, 105, 108, 109, 117, 118, 130, 14Among the most intriguing are findings that thydrated radius of trehalose is anomaloularge—at least 2.5 times that of the other sugtested (145). This would seem to be in goagreement with the report of Lin and Timash(104) that, unlike other sugars, trehalose istally excluded from the hydration shell of thproteins studied. This effect would, in turn, psumably maximize the stabilization of proteiby the preferential exclusion mechanism (1168), a possibility that warrants further invesgation. In fact, it seems possible that manythe properties reported for trehalose for sta

list, but represents only a sampling of what is being donedried; hs, hypothermic storage.

THE TREHALOSE MYTH REVISITED 91

TABLE 1Some Novel Applications for Trehalose

Application Treatment References

Enzymes and other proteins f, ad, fd, sd 1, 12, 21, 22, 70, 95, 120, 1Vaccines and antibodies f, fd, ad 8, 48, 49, 53Nanoparticles fd, sd 24, 44, 137Membranes fd, f 55, 57, 136, 36Liposomes fd 35, 68, 73, 88, 113, 161DNA and DNA–lipid complexes fd 4, 101, 172Bacteria and yeasts fd 31, 99, 100, 107, 110Nucleated mammalian cells ad, f 15, 43, 52, 66, 115Mammalian blood cells f, fd 19, 62, 128, 147, 167Mammalian organs hs 10, 11, 74, 91, 92, 169

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lization of biomaterials in bulk water or durinfreezing (cf. Table 1) might be related to this aparent anomaly.

ORIGINS OF THE TREHALOSE MYTH

Myth 1: Trehalose Is “Special” for StabilizingDry Biological Membranes

The first model membrane investigated w

sarcoplasmic reticulum (SR), isolated from lobster muscle (reviewed in 36). These membranhave a characteristic morphology, with in

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tramembrane particles, representing the CATPase, on the cytoplasmic facing half of tbilayer but not on the luminal face. This charateristic morphology provides a marker for strutural integrity. Further, the ability to transpoCa21 and coupling between Ca transport aATP utilization provided a physiological assafor integrity. When these membranes were drwithout trehalose and then rehydrated, we fouevidence of extensive damage: they fused, foing vesicles an order of magnitude larger ththe original vesicles; the intramembrane pacles were displaced from their original positioin the cytoplasmic monolayer and were foundboth monolayers in equal amounts; the tramembrane particles were aggregated; ability to transport Ca was lost; coupling btween ATP utilization and Ca transport was loBy contrast, when the membranes were drwith trehalose both the morphology and ttransport capabilities were maintained intavery similar to those properties seen in fresprepared vesicles.

When we compared the ability of a variety sugars to preserve the SR during drying,found that trehalose was without question surior to all other sugars tested. This observatwas the beginning of what has grown into t

in this field; f, freezing; ad, air-drying; fd, freeze-drying; sp

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trehalose myth. That is not to say that other sug-ars did not also stabilize the SR; indeed sucrose,for example, worked about as well as trehalose,

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but much higher concentrations of the suwere required. Some years later, however,obtained evidence that these SR membrahave a mechanism for translocating trehalacross the bilayer. When we measured the saccessible to trehalose compared with thatcessible to sucrose, we found that trehaloseaccess to the aqueous interior of the vesicwhile sucrose at comparable concentratidoes not. We still do not know the mechaniby which trehalose crosses the bilayer, buview of the fact that trehalose is a blood sugathe lobsters from which the SR was made that trehalose is synthesized in the cytoplasmhepatic cells, it seems likely that a carrier this sugar is present. At any rate, the fact trehalose penetrates into the interior of the vcles may explain its superior properties in pserving these membranes in particular. We sgest that other sugars such as sucrose mpreserve the membranes at concentrations slar to those seen with trehalose if they had cess to the aqueous interior.

Myth 2: Trehalose Preserves Dry PhospholipVesicles More Effectively Than Do OtherSugars

In the initial studies, from the mid-1980s (335, 37), liposomes were prepared from a lipwith low Tm, POPC.1 A fluorescent marker, carboxyfluorescein, was trapped in the aqueousterior. When the liposomes were freeze-drwith trehalose and rehydrated, the vesicles wseen to be intact, and nearly 100% of tcarboxyfluorescein was retained. It quickemerged that stabilization of POPC liposomand other vesicles prepared from low-meltinpoint lipids, had two requirements: inhibitionfusion between the dry vesicles and depressof Tm in the dry state. In the hydrated state,Tm

92 CROW

for POPC is about21°C and rises to about70°C when it is dried without trehalose. In thpresence of trehalose,Tm is depressed in the

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1Abbreviations used: POPC, palmitoleoylphosphatidycholine; DPPC, dipalmitoylphosphatidylcholine; HES, hydroxyethyl starch.

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dry state to220°C. Thus, the lipid is main-tained in liquid crystalline phase in the dry staand phase transitions are not seen during redration.

The significance of this phase transition duing rehydration is that, when phospholipids pathrough such transitions, the bilayer becomtransiently leaky. [The physical basis for thileakiness has recently been investigated in sodetail by Hayset al. (69)]. Thus, the leakagethat normally accompanies this transition mube avoided if the contents of membrane vesicland whole cells are to be retained. During dring, this need not be a problem sinceTm is notaffected until all the bulk water has been removed. But during rehydration, it is a seriouproblem; the membranes are placed in waand will undergo the phase transition in thpresence of excess bulk water, thus allowinleakage. In addition, and perhaps even more iportantly, phase separation of membrane coponents can occur in gel phase, an event thaoften irreversible.

The mechanism by which trehalose depresTm in dry phospholipids has been a matter some debate, with at least three alternative,not necessarily mutually exclusive, hypothesin the literature (34, 94, 152). Discussion of thmechanism is beyond the scope of the presessay.

These effects were reported first for trehalo(reviewed in 35). When we compared the effecof other sugars and polymers on the presertion, we found that, with vesicles made fromlipids with low Tm, trehalose appeared to be sinificantly superior to the best of the additivetested. Oligosaccharides larger than trisaccrides did not work at all (35).

Other sugars, particularly disaccharides, dprovide good stabilization of POPC vesicles the dry state, but much higher concentratiothan trehalose were required, at least accordto initial reports. However, as freeze-dryintechnology improved, the differences betwedisaccharides tended to disappear, and the m

ET AL.

eventually got modified to encompass disaccha-rides in general. Nevertheless, the observationthat trehalose was significantly more effective at

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low concentrations under suboptimal conditiofor freeze-drying requires explanation, whiwe provide later.

Myth 3: Disaccharides Are Required forStabilization of Liposomes

DPPC is a lipid with saturated acyl chainand thus an elevatedTm (41°C). When it isdried without trehaloseTm rises to about110°C; with trehalose presentTm rises to about65°C (reviewed in 34). Thus, DPPC is in gphase at all stages of the freeze-drying andhydration process, and one would expect tinhibition of fusion might be sufficient for thestabilization. In other words, any inert soluthat would separate the vesicles in the dry stand thus prevent aggregation and fusion shostabilize the dry vesicles. That appears tothe case; a high-molecular-weight (450,00HES has no effect onTm in dry DPPC, but pre-serves the vesicles, nevertheless. With scning electron microscopy the dry vesicles aseen to be embedded in a matrix of HES (F1), with no change in diameter from thosethe freshly prepared vesicles. By contraDPPC vesicles dried without HES showemassive fusion (Fig. 1) and leaked all the

THE TREHALOSE

FIG. 1. Scanning electron micrographs. (a) DPPCHES. The freshly prepared vesicles were 100 nm in drevealing the liposomes. (b) DPPC vesicles freeze-d

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Myth 4: Polymers Like HES May PreserveVesicles with High Tm, but not Those withLow Tm

It is true that HES alone will not stabilize dPOPC vesicles, but a combination of a low-mecular-weight sugar and HES can be effectEven glucose and HES are effective (37). His the apparent mechanism: glucose depreTm in the dry lipid, but has little effect on inhibiting fusion, except at extremely high cocentrations. On the other hand the polymer no effect on the phase transition, but inhibits sion (Fig. 2). Thus, the combination of the twmeets both requirements, while neither alodoes so (37). A glycan isolated from the descation-tolerant alga Nostoc apparently has similar role in conjunction with oligosacchrides (72). Recent results from Hincha et al.(73)have shown that certain polymers from desiction-tolerant higher plants will by themselvboth inhibit fusion and reduce Tm in dry phos-pholipids such as egg PC. The mechanismhind this effect is still unclear.

There is little about trehalose and its effeon preserving dry phospholipid vesicles thaconsistent with “special properties.” Once

YTH REVISITED 93

rentim-

contents when rehydrated, as a result of thefusion.

preservation was achieved, it became appathat many routes can lead to the same end. S

vesicles (one is indicated by an arrow) freeze-dried withiameter. The dry sample was broken open by mild abrasion,

ried with no excipient, illustrating massive fusion.

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FIG. 2. Fusion, retention of trapped carboxyfluorescein, and effects on Tm in POPC vesicles freeze-dried withit lu-em

ilar observations on the stability of protei

HES, glucose, or mixtures of the two. Phase transcose contents, and the values shown are for an avT

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dried with sugars and polymers have been mby Carpenter and his group, with similar concsions (e.g., 3, 5, 70).

REVIVAL OF THE MYTH: TREHALOSE WORKSWELL UNDER SUBOPTIMAL CONDITIONS

We implied above that trehalose works wfor freeze-drying liposomes under less than timal conditions. The same applies for storaunder conditions that would normally degrathe biomaterial. Leslie et al. (100) reported thabacteria freeze-dried in the presence of halose showed remarkably high survival immdiately after freeze-drying. Furthermore, thfound that the bacteria freeze-dried with thalose retained a high viability even after loexposure to moist air. By contrast, when bacteria were freeze-dried with sucrose tshowed lower initial survival, and when th

ere exposed to moist air viability deceaseapidly. More recently, Esteves et al., (53) re-orted that when immunoconjugates we

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freeze-dried with trehalose or other disacchrides all the sugars provided reasonable levelpreservation. However, when the dry sampwere stored at high relative humidities and teperatures, those dried with trehalose were stafor much longer than those dried with other suars. This finding is of some considerable signcance since such immunoconjugates, vaccinantisera, and the like are being shipped freeze-dried preparations to areas such asAmazon, where they would be exposed to htemperatures and humidities as soon as theyexposed to air.

Do Solution Properties Explain Stability?

Several workers have implied that studies the solution properties of trehalose may helpexplain the stability of biomaterials dried in ipresence (e.g., 20, 105). That may well be,we suggest that such properties have more to

ions show considerable heterogeneity at low to medium grage (data from 37).

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group when the system is carefully protecfrom water vapor (39). In such a state it is dicult to see how solution properties obtainfrom studies in excess water apply. That is nosay that the lipid and sugar might not formnonaqueous solution, each serving as cosolfor the other, but that is a different matter andany case, provides little insight into the probleof long-term stability.

Does Nonenzymatic Browning Contribute toInstability?

The Maillard (browning) reaction between rducing sugars and proteins in the dry state often been invoked as a major source of dam(e.g., 103), and the fact that both sucrose trehalose are nonreducing sugars may explaleast partly why they are the natural productscumulated by anhydrobiotic organisms. Hoever, the glycosidic bonds linking the monomin sucrose and trehalose have very different ceptibilities to hydrolysis (reviewed in 124When O’Brien (124) incubated a freeze-drimodel system with sucrose, trehalose, and cose at water activity 0.33 and pH 2.5, the rof browning seen with sucrose approached of glucose—as much as 2000 times faster tthat with trehalose. Thus, under less than omal conditions for storage, it is clear that thalose is preferred.

Do Glass Transitions Explain Stability?

Using liposomes as a model, we attemptefind a mechanism for long-term stability in tpresence of trehalose. As with the bacteria immunoconjugates, the dry liposomes expoto increased relative humidity rapidly leaktheir contents when they were dried with scrose, but not when they were dried with thalose (40, 151). The liposomes underwent tensive fusion in the moist air when dried wsucrose, but not with trehalose.

Examination of the state diagram for trhalose provides a possible explanation for

THE TREHALOSE

effect (see 27 for an excellent review and etended state diagram). Tg for trehalose is muchhigher than that for sucrose, a finding first re

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ported by Green and Angell (64). As a resuone would expect that addition of small amouof water to sucrose by adsorption in moist would decrease Tg to below the storage tempeature, while at the same water content Tg for tre-halose would be above the storage temperatThis proved to be the case. Furthermore,point out again that degradation does proceesamples below Tg, albeit at a slower rate. Withtrehalose, a sample at 20°C would be nea100°C below Tg. By contrast, one dried with sucrose would be only about 45°C below Tg.Under these conditions one would expect sample dried with sucrose to be degraded mrapidly.

Aldous et al. (2) suggested an additional interesting property of trehalose, which we weable to confirm. They proposed that since crystalline structure of trehalose is a dihydrasome of the sugar might, during adsorption water vapor, be converted to the crystalline hydrate, thus sparing the remaining trehalofrom contact with the water. Experimental oservation showed that this suggestion is correwith addition of small amounts of water thcrystalline dihydrate immediately appeared, aTg for the remaining glassy sugar remained uexpectedly high (40). More recently, Yoshii etal. (171) showed that trehalose readily convebetween the crystalline and the amorphoforms under certain conditions, so this procemight be reversible.

We stress, however, that the elevated Tg seenin trehalose is not anomalous. Indeed, trehallies at the end of a continuum of sugars tshow increasing Tg (40), although the basis fothis effect is not understood.

Is trehalose special? Under ideal conditiofor drying and storage, no, it is not. But undsuboptimal conditions it provides stability wheother sugars do not. This is not to say that halose is the magic bullet that will be preferrover all other excipients; indeed, as we dscribed above, combinations of polymers aother sugars may work just as well and m

YTH REVISITED 95

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even be preferable in some circumstances. Nev-ertheless, it is still the preferred first excipient tobe tested.

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CAN WE USE WHAT WE HAVE LEARNED FROMMODEL SYSTEMS TO PRESERVE INTACT CELLS

IN THE DRY STATE?

Clearly, trehalose must be introduced into cytoplasm of a cell if it is to be effective at stalizing intracellular proteins and membranes ding dehydration. Current efforts are centearound this fundamental problem, as summrized below.

Trehalose Biosynthesis

Since trehalose biosynthesis is a simple twstep process, it seemed amenable to geneticgineering; two enzymes are involved, as shoin Fig. 3, the substrates for which are normmetabolites in virtually all cells, so substraavailability would not seem to be a probleThe genes were cloned some time ago, frbacteria, designated, otsA and B (83, 84, 15yeasts, designated tps 1 and 2 (e.g., 16, 84, 1and higher plants, also designated tps 1 an(18, 174). The first transfections into highplant cells showed reasonably high levels of pression and led to improved drought tolera(129). Subsequent investigations showed inhibition of trehalase activity increased trhalose production (61, 119). Even more cently, Sode et al. (144) have synthesized a trhalose derivative that is a potent inhibitor trehalase; it may be even more promising amechanism for increasing trehalose productSimilar transfections of the genes for sucrosynthesis into Escherichia colialso led to accumulation of sucrose and improved resistancedehydration damage (17).

The complete operon-containing gene coplex for trehalose synthesis has recently bcloned (93) and subjected to directed evoluti

96 CROWE

in an effort at improving trehalose production

FIG. 3. Classical pathway for synthesis of trehalobeen cloned. Alternative pathways are now known (s

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trols. Along the same lines, Seo et al.(138) pro-duced a fusion protein encompassing otsA B. The logic here is to get the enzymes in cproximity, which they suggest will increase thalose production. Bloom, Levine, and cleagues have taken up this approach in twork on transfection of mammalian cells, as scribed below, and in their presentations in symposium (63, 96).

High levels of expression in mammalian cewere only recently achieved. Guo et al.(66) en-gineered the genes for trehalose synthesismammalian cells in an adenovirus vector. Wmultiple infections, the trehalose biosyntheincreased. The cells, which were then dried level where free water was no longer detectaretained viability for 3 to 5 days. It seemed rsonable to suspect that the level of viabimight be improved by altering the storagedrying conditions, and, indeed, Gordon et al.(63) reports on such studies in this symposiOne of the difficulties in such studies has bin obtaining expression of the genes at high els, to produce large amounts of trehalose. et al. (96) report in the symposium improvmethods for overexpression that may hsolved this problem.

De Castro and Tunnacliffe (43) reported tthey did not obtain anyviable cells after a simlar transfection. While it may be true that transfected cells do not survive complete dryit is also possible that differences in the methfor drying may account for the widely differinresults. Chen et al.(26), Gordon et al.(63), andTunnacliffe et al. (159) report further studies this symposium aimed at resolving this madiscrepancy.

There is a pathway alternative to trehal

ET AL.

.synthesis involving conversion of maltose to tre-col-ntly

The results look very promising, with as muchas a fivefold increase in production over con-

halose, first reported by Panek and her leagues (127). This pathway was more rece

se. The genes encoding the enzymes for this pathway haveee text).

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investigated in some detail by several laborries, including cloning the genes involved (84, 86, 121) and investigations of the regulatpathways (116). There is at present no clearvantage to transfections with these genes cpared with the trehalose phosphate synthasephosphatase genes, at least for mammacells, but we nevertheless point out the existeof this pathway. It has been proposed amethod for industrial production of trehalo(170). Still other known pathways are summrized by De Smet et al.(45).

Trehalose Transport

Panek and her colleagues have shown thtrehalose transporter is required in yeaststransport trehalose out of the cell during dry(41, 51). Since stabilization requires that halose be on both sides of the membraneviewed in 34), this is a way of meeting that quirement. The gene for this transporter has been cloned (67) and expressed at a high levyeasts (149), so there is no obvious reason it could not be incorporated into the cassetteready in use for trehalose synthesis (66,159), a treatment that might well improve vbility. It also provides an obvious route for intrducing trehalose into the cell. In the yeast frwhich the gene was cloned, the transporter iactive H1 symporter (38), but it may also act a passive carrier in the absence of a H1 gradientacross the membrane (6, 38), so the prospecusing it as a passive carrier in mammalian cseems reasonable. A related transporter fbacteria has been characterized extensivelya crystal structure was recently produced (47

A Pore for Permeation

Eroglu et al.(52) engineered an elegant poforming hemolytic protein, hemolysin, so ththe pore could be switched on and off. By sstituting a number of residues with histidinthe pore could be regulated; by adding micmolar quantities of Zn21 the pore could bclosed, and by removing the Zn21, the pore

THE TREHALOSE

could be reopened. Since the pore protein sptaneously inserts into membranes, Eroglu et al.found that they could simply incubate the ce

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owl inhy

al-6,--m ans

s ofllsomand.

e-

in its presence, add trehalose in the absencZn21, and introduce the sugar via the poUsing this procedure, they were able to obtvery high rates of survival of two lines of mammalian cells in the frozen state. However, threport in this symposium that when the cewere dried survival was disappointingly low, athough the cells were protected from damarelative to the controls (26).

Use of Phase Transitions

Beattie et al. (15) discovered that the insulinproducing cells from mammalian pancreas haa membrane lipid phase transition well abothe freezing point. Since membranes are knoto become transiently leaky during the phatransition, Beattie et al.used that leakiness to introduce trehalose into the cells, which were thsuccessfully frozen, and kept frozen, for etended periods. The thawed cells were traplanted into rats, where they were found to main viable for many months. These findinare being developed into a commercial produwhich, it is proposed, will provide a stabletransplantable device for treatment of diabete

Beattie et al. (15) did not attempt to dry thecell into which trehalose had been introducebut, more recently, Wolkers et al. (167) havedone so in another cell type, with remarkable sults. Wolkers et al. (167) have discovered thatrehalose can be introduced into the cytosolhuman blood platelets, probably by a tempeture-mediated endocytotic pathway. Thshowed that when the trehalose-loaded plateare freeze-dried an astonishing proportion svive—about 90%—all of which appear to bfunctional platelets upon rehydration. Thefindings are already under clinical investigatiin animal models, with a view toward clinica

MYTH REVISITED 97

tb-s,o-

testing in humans within the next 2 years. Morecent findings in this regard are reported Tablin et al.(155) in this symposium.

WHAT OTHER LESSONS LEARNED FROM NATUREMIGHT BE APPLIED TO CELL DRYING?

on-

lls

Under nonideal storage conditions, degrada-tive reactions that trehalose cannot prevent mayoccur. Oliver et al.(125) show in their contribu-

ueg

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tion to the symposium that a second molecfound at high concentrations in certain desplants, arbutin, may play a key role in inhibitinsuch degradations and that several other naproducts, particularly from plants, may be ivolved in anhydrobiosis and could be adapfor use in drying mammalian cells. Hoekstraetal. (75) show that amphiphiles that appear nmally to be in solution in the hydrated cells sert into the bilayer during drying. They discuthe possibility that this insertion may reduce Tm

by making defects in the bilayer, but they malso increase permeability. They discuss adaptive significance of this effect and whetit might be useful with mammalian cells. Fnally, Battista et al.(14) have identified genes Deinococcus radiodurans, a bacterium that isremarkably resistant to desiccation damage,are linked to desiccation resistance. Results

98 CROWE

en

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na-le

ssn

ac-

be.

S.: A

ui,r

olu-

.,

n-er-

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sented in this symposium suggest that thgenes are related to desiccation-linked gefound in higher plants.

CONCLUSIONS

Under ideal conditions for drying and stoage, trehalose is probably no more effective tother oligosaccharides at preserving biomatals. Nevertheless, under suboptimal conditiit can be very effective and is thus still a pferred excipient. There is growing evidencpresented in the symposium, that additiomodifications to the cellular milieu will probably be required if we are to achieve a stabfreeze-dried mammalian cell. Neverthelemost workers in this field have already co

tr

ible

ing of

cluded that the best approach is to start with halose, which provides the recurrent themethis symposium.

ACKNOWLEDGMENTS

itlm ac-

toen-

.Cryobiology43,133–139 (2001).

We gratefully acknowledge many useful discussions wour colleagues Fred Bloom, Malcolm Potts, Richard HeJohn Battista, and James Clegg.

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