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Changes in red cells following rapid freezing with extracellular cryoprotective agents
CRYORIOLOG~,
9, 262-267 (192)
Changes
in Red Cells Extracellular H. T. MERYMaS
Following Rapid Freezing Cryoprotecdve Agents’ .ASDM. HOH~BI,O\VER
The recovery of human red cells following slow freezing requires the presence of a high (Boncentration of a penetrating cryoprotective agent such as glycerol. However, if the freezing rate is relatively rapid a variety of nonpenetrating compounds will provide good cryoprotection at low concentrations. Glucose (17)) glucose-lactose (al), polyvinylpyrrolidone (PVP) (3: 18)) hydroxyethyl starch (HES) (8) and a polypeptide ext.ract of peptone (2) have all been rcported cryoprotective for human red cells. dll but the peptone extract have been tested clinic~lly and shown to yield cells that survive in 11izlo.The freezing rates which produce optimum recovery vary depending on the cryoprotectivr agent but all require rapid or relatively rapid freezing and require liquid nit,rogen temperature for storage. The freezing of cells wit,h PVP and HES for transfusion has been particularly attractive since the optimum freezing rates with these cryoprotectants are the least demanding, making sterile processing in closed cont,ainers feasible. On t,he premise that intravenous administration of either of these two cryoprotectants would be acceptable, considerable effort has been espended in the hope of developing a one-step freezing method following which the thawed cell suspension could be administ,ered without. further treatment (5, 8), t,hus circumventing the need for post,thaw washing of the cells. The mechanism by which freezing injures living cells has been extensively investigated in our laboratory (14, 15). When cells are suspended in :I solution of nonpenet,rating solute and the concrntration is progressively inrreased (as by freezing out water) the cells are dehydrated and reduced in volume. In all animal cells we have studied no adverse effects are seen until a volReceived June 12, 1972. ‘Contribution No. 213 from tllfa American XaI ional Red Cross Blood R.csearch Laboratory. SupIlortcd in part by Office of Naval Research Contract N00014-70-C-0026 and National Institutes of Hrnlth Grant 1 ROl CrM17959-01.
tune is reached corresponding t,o the loss of approximately 65% of cell liquid. Beyond this point, there is an influx of extracellular solution with lysis on return to isotonic suspension. For red cells, t,he minimum tolerated volume i,; reached at about) 4.5 times isotonic. dccording to this “minimum cell volume” model, cryoprotective agents such as glycerol and dimethylsulfoxide function on a simple antifreeze basis as previously proposed by Lovelock (11). -it concentrat,ions of 2 to 4 M, they reduce the amount of ice formed suffirientlg to prevent a damaging courentration of extracellular solutr. It is, of course. essential that, these agents penct rate the cells otherwise their high osmot,ic potential will in itself he destructive through ~11 volume reduction. The sugars and polymers used to protect rrd cells during rapid freezing are, on the contrary. nominally nonpenetrating and are effectivr in low concentrations. It has been shown t,hat, at the freezing rates which yield maximum crll recovery, ice is ext,racellular (12) and there is no evidence that any freezable wat,er remains unfrozen (13). This leaves no alternative but to conclude that, with rapid freezing, cells are subjected to concentrations which at slower fooling rates would be hemolytic. A possible explanation of cryoprotection b! nonpenetrating agents was suggested by our studies (22) of cryoprotection of the grana from spinach leaf chloroplasts by t.he nominally nonpenetrating sugars and sugar alcohols. We found that grana exposed to increasing concentrations of salt in the absence of sugars were, like red cells, progressively reduced in volume and that beyond approximately 1200 mosm loss of membrane furl&ion occurred. In the presence of sugars, however, the grana failed to reduce further in volume beyond about 800 mosm but instead permitted an influx of extracellular solut,ion. On return to isotonicity, t,here was an efflux of the excess solute and at no time during this sequence of events was there any evidence of irreversible 262
Copsright, 111 rights
0 1972 by Academic Preuu. of reproduction in any form
Inc. reserved
with
RED
CELL
mrmbranc damngc. These observations demonstrated :I new mechanism for cryoprotcction c*ompatible with the minimum volume hypothe‘Ii. .I number of compounds were invrst igatcd to tlrtrrminc tliPir cryoprotectivc capacity for the q)in;tch granum. Sorbitol was found :I:: effect i\-c :I; sucrose. Both PVP and HE8 protected gran:1 from freezing injury as did an extract of peptone. We were struck by the fact that all of these compounds had also been reported protrctier for red cells during rapid freezing. Changes in red ccl1 sodium-potassium ratio II:IW been reported by Doebbler and Rinfret (4) following rapid freezing with PVP! by Knorpp catnl. with HES (9) and by Shrago and Dnnilinu (20) after freezing with polyethyleneoxide. The I)ossihility arose that in red cells these cryoprotcctive agents were cxnabling recovery by a leak mcclianism similar to that seen in spinach grana. A series of experiments was uridertakcn to detcrmint whether an exchange of extracellular and intracellular electrolytes might be a necessary component of the recovery of red cells following rapid freezing with rxtracellular cryoprotcctivr :lgellts.
X4TERIALS
AND
1IETHODS
Fresh human erythrocytes collected by s;tandard blood bank procedure into -4CD solution were used throughoutJ. The cells were mixed with cq-oprotectants as described below iu ratios comparnble to t,hose reported optimum for reco\-cry and i?~ llivo survival. The freezing rate producing optimum cell recovery was empirically drtt~rmined for well cryoprotecti\-c agent. I’VI’ and HES. One volume of packed cells was mixed with 5 vol of 10% PI’P. 0.6% iYaC1 at pH i or with 14% HES, O.i6% NaCl at pH i. Both of these cryoprotectant solutions had :IU osmolality of approximately 340 mosm. Small :Iliquots of there suspensions were frozen in thin walled polyethylene tubing, I/* in. in diameter. The tubes were frozen by direct immersion into liquid nitrogen and the freezing rate varied until optimum by wrapping t,he tube with cloth. Cells in PVP were frozen at 3.5”/sec, in HES at Z.i”/ sec. The frozen cells were thawed by agitating the bare plastic tube in a 37°C water bath. La&w-glucose. Whole blood was mixed with an qua1 volume of 15% lactose and 10% glurosc
263
FREEZIKG
:It pH i (21 j This suspension was frozen in I/Z in. polyethylene tubing at a freezing rate of al)proximately 20”/sec and the bare tubing was thawed by agitation in a 37°C water bath. Glucose nltd ,VaCZ. Packed cells were misrtl with a11 equal \-olumc of either 0.9% NaCl or 0.9’4~ NaCl plus 15% glucose at pH 7. The cellwere frozen at approximntel\; lOO”/scc by spraying thr suspension directly onto the surface of liyuid nitrogen (li) The cells were thawed 1,~ recovering small amounts of droplets in :I teaspoon, allowing the liquid nitrogen to evaporatr from t,hem and then dulnping them into :I beaker containing either saline or saline plus glufose at 37°C. Following thawing, aliquote of each rell puspension were assayed for percentage hemolysis by comparison of the supernatant hrmoglobin concent rat ion and the hemoglobin concent ration of the entire suspension. Hemoglobin assays were conducted using an automated hcmoglobinometer’ standardized by duplicate determinat ions using the cynnmethemoglobin method. IIF tracrllular and extracellular sodium and potassium determinations were made by flamr pliotomct,ry.3 To rompensate for possible varinrions in the number of red cells assayed due to ctxll volume changes or sampling errors, the hcmoglobin concentration of the dilute lit~hium suxpension actually measured in the flame photomctcr was assayed. The cation concentrations mcnsured were then adjusted by multiplying them by the ratio of the prefreczr hemoglobin measuremenf to t,he postthaw value. 2 .~i-di~~lio~~~liogl~c~ric acid (2,3-TIP(:) I\-:I~ :IMJYY~ using the sprc~tro~,liotonipt ric mrt hod of Kcitt (6). This was modified according to the recommendations of Dr. Norman Fortier of thr Blood Research Laboratory, U.S. Naval Hospital, Chelsen! MA, in which pyrophosphxte W:IS used to stimulate the 2,X-DPG phosphatase ncti\-ity of the phosphoglycrrate mutase using :I buffer containing (per ml of reaction mixture), 1.37 &ml ATP, 10.3 PM sodium pyrophosphate, 02 ,ULI\IN4DH, 10.3 PM Tris and 1.55 PM magnesium chloride brought, to pH 7.6 with 1.0 N HCl. ‘Model Ion. M. ’ Model ton. M.
231, 1nntrumcnt:~tion
Lnhoratories.
Ih+
143. Instrumentation
Lnhorntorics,
Ros-
264
MERYMBN
ASD
HORSL~I,OWEK
RESULTS
TABLE
Table 1 summarizes the results obtained. 111 all samples, cell potassium was severely depleted and replaced by whatever the extracellular solute may have been. In situations where t,he extracellulnr solute wad predominately sodium chloride, the total amount of intracellular cation following thawing was approximately that prior to freezing even though the ratios of sodium to potassium may have been radically changed. Cells frozen in PVP and HES were also somewhat depleted of 2,3-DPG. Cells frozen in PVP and HES also tended to hemolyze more when mashed in saline following thawing. In fact, it was never possible to wash these cells free of supernatant, hemoglobin no matter how man) washes were conduct,ed, suggesting that some mcmhrane instability remained following thawing. Optimum recovery is obtained with PVP and HES at subst,antinlly slower rates of freezing than are necessary when saline or saline-glucose is used. The loss of 2,3-DPG from cells frozen with PVP and HES may reflect the longer period of time that these cells are exposed to hypertonic solutions at temperatures high enough to permit diffusion. Alternatively it may be that PVP and HES exert a direct effect on the cell membrane rendering them more permeable to TABLE
1~ Hemo lysis
‘Na
I
K’Na~K i-
~.
.-~--
- ’
10% P\‘P 0.6% NaCl 147; HES 0.767, NaCl
13
211 79 117 12;
15”/c Lactose 1oyc (:lucose
0.90/
ples
I
i
4.4
13
6.1
3
3 8
4
39
NaCl
11Data represent
72
an arithmetical
mean.
4
Freezing solution
GlucoseNaCl (;lucoseKC1 0.9yc NaCl SucroseNH&l
Thawing
‘La
solution Na
K
(:lucose-KC1 Sucrose~NH~Cl (:lucose-NaCl
24 15 15 18
108 106 103 118
NaCl KC1 NaCl KC1
10 15 14 6
(ilucose-NaCl
Na
K
54 86 xl 33 I 93 64 21 18
io 6i 33 ~ 98 40 67 8 , 77 .n Cells were frozen by the droplet method. (:lucose-NaCl indicates 15yc glucose, 0.9% NaCl added 1:l to red cells for freezing or to isotonic saline for thawing. YucroseeNH&l indicates 12% sucrose plus 0.1 M NH&l added to maint,ain normal ionir concentration (16). 82 100 93 91
high molecular weight, components. Davies et al. (1) have reported that red cells hypot’onicall\ stressed in the presence of PVP lose some of their hemoglobin instead of lysing completely as in salt suspension, suggesting an alteration of membrane characteristics by PVP. One inescapable conclusion is that red cells recovered following rapid freezing either with or without an estracellular cryoprotective agent have experiembed an influx of extracellular solute and efflux of intracellular solute, suggesting that a transient reversible increase in permeabilky may bc an event inevitably associated with cell survival following rapid freezing. The question was raised mhet.her this reversiblc incrcnsc in permeability occurs only during freezing and thawing or whether there might be :t brief interval following thawing during which the ccl1 membrane remains relatively permeable to solutes. To t’cst this possibility cells were suspended in solutions of sodium chloride or potasaium chloride or in sucrose-ammonium chloride and rapidly frozen by t,hc droplet method. The cells were thawed in a variety of thawing solut,ions as indicated in Table 2. The intracellular sodium ‘and potassium were assayed both prior to freezing and following thawing. It is apparent, from this data that cation exchange occurs both during the freeze-thaw cycle and after the thswing is complctcd. In all cases where cell.; wrre
frozen in sodium chloride, cell sodium is elevated postthaw even though the thawing solution may have been sodium-free. This implies an entry of sodium during the freeze-thaw cycle, presumably during the hypertonic phase of freezing. On the other hand, all cells show a postthaw elevation of the cation present in the thawing medium compared to a experiment in which that cation was absent from the thawing solution. The question also arose whether t’he increase in membrane permeability was to low molecular weight compounds only or whether high molecular weight compounds could equally well cntcr t,he cells. To test this cells were frozen and thawed in a 20% (200 mosm) solution of P\X’ alone, without any other solute. The initial postthaw recovery wxs 97%. When the cells wrrc assayed for cation, only i% of the original electrolyte content of the cells remained. Under the microscope the cells appeared normal in volume. implying that PVP had entered the cells. -4lthough these cells remained intact following thawing they were pnrtic~ularly uiistablc on ,AI~),sclclurnt washing.
We conrlude from this study that, the rccovery of unhemolyzed red cells following rapid freezing and thawing is associated with an influx of extraccllular solute possibly during the hypertonic phase of freezing, a subsequent loss of intracellular solute on return to isotonicity and a brief interval following thawing during which t’he cell membrane remains permeable. That such cells can survive following reinfusion in GUO demonstrates the reversibility of the event. Since over half the red cells suspended in sodium chloride in the absence of any cryoprotective agent arc unhemolyzed following rapid freezing, the cryoprotective agent is presumably not essential for the development of reversible membrane leak. The cryoprotect8ive agents do reduce the rate of freezing necessary to ncliievc~ optimum recovery. It is notable t,hat the same group of conpounds that are cryoprotective for red cells and for spinach grana also produce alterations in red cell permeability under hypotonic stress (16). One of the common properties of t,hese compounds is their ability to stabilize macromolecules against conformational changes, possibly through the modification of water structuring
around nonpolar groups (7). Both PVP and HES have much higher stabilizing capacity t’hnn do the sugars, and it is possible that their effect on membrane permeability is more pronounced. permitting the passage of larger molecules suck11 as dipho~phoglycerate or the polymers themsrlrrs. It may also be the prcscnce of int racclhlur PVP and HES that causes the <11bs(~(Illont instability to suggessive saline washes. Robinson (19) has rcportcd that hamster 41s in culture develop vesicles following freezing and t,hawing and are killed. However, when the SIP+ pending medium is replaced following thawing by a medium in which hnmstcr cells h:~v(: prcviollely been grown, veniculntion does not OCC~I and 100% survival is seen. This clearly implies that events following thawing can prevent tht development of irreversible changes and permit the healing of freeze-thaw damage. It is entirrl! possible that extracellular rryoprotectants ma? be performing a similar function particularly in view of the fact, that all known cs;traccllul:~I c,r~oprotc~c,tnnt~ :II’(~ strong IIi;1c~romolec’nl:rr. St;]l)ilizcra. -1 poattliaw effect of P\‘P on frozc,n red ct,ll,. is demonstrated by the data ill Table 3. It ii clear from these experiments that the prescncae of PVP after thawing can substantially redurcl post,thnm hemolysis. Such events as the loss of lipid component’s from the membrane (10) ma! be prevented through the stabilizing capacity of the PVP. The possibility that PVP and similar extracellular compounds may exert their cryoprotective effect after freezing and thawing must be considered as a real possibilit,y. From the clinical standpoint, a primary goal of the rapid freezing method has been to achier-e a one-step procedure, eliminating the extensive po&thaw processing which is required by the glycerol method. However, it would appear that at least some cell washing will still be nccessnry. Although Knorpp et a,l. (9) hnvc reported much less potassium loss than seen in our csperimcnta, his average of about 35 mmolcs of free potasAm/unit, would be clinically undesirable where multiple transfusions are involved. Knorpp et nl. (9) also found the viscosity of cells in 14% HES such that the removal of the HES and resuspension of the cells is desirable in order to facilitntr administration. It remains to be seen whet,her the amount of washing necessary to provide a clinirally nrceptnhle product will be sufficientlp
MERYMAN
AND HORNBI,OW~;R TABLE
3
EFFECTIVENESS
OF POLYVINYLPYRKOLIDONE IN ERYTHI~OCYTE FI~EEZING ADDED EITHER BEFORE FREEZING OR DURING THAWING” ____-~~~~
WH~:X
Erythrocyte suspending solution
Thawing solution
5yoHemolysis
Total hemolysis following resuspension in isotonic saline
Isotonic saline Isotonic saline 2oyo PVP, 0.37, NaCl 207o PVP, 0.3c/ NaCl
Isotonic saline 20% PVP, 0.37, NaCl Isotonic saline 20% PVP, 0.3% Nack;
39.6
44.1
8.3 0‘3 3.2
27.3
10.0 9.7
a Data are based on an arithmetical average of four experiments each. Human red cells were rapidly frozen either in an isotonic solution of 20(x PVP or in isotonic saline. Since the optimum freezing rate in the presence of PVP is slower than in saline, the former preparations were frozen as pellets by spraying directly onto liquid nitrogen. These procedures were found empirically to give optimum recovery for the respective preparations. Cells frozen with PVP were thawed by shaking with an equal volume of thawing solution. Cells frozen as pellets were thawed by immersion in thawing solution. After thawing, the resulting hemolysis was measured, the cells were centrifugally sedimented, separated and resuspended in isotonic saline following which any additional hemolysis was measured. As shown, cells frozen with PVP showed slightly more hemolysis when thawed in saline compared to a PVP solution. However, cells thawed in saline hemolyzed less on saline resuspension, the total hemolysis following the two thawing methods being roughly the same. Cells frozen in isot’onic saline, however, showed almost four times the hemolysis when thawed in saline compared to cells thawed in PVP solution, indicating that the PVP was exerting some sort of protective effect after thawing was completed. That some of this protective effect may have been simply mechanical support given to damaged cells is suggested by the substantial additional hemolysis when cells thawed in PVP are resuspended in saline. Even so, t,he total hemolysis is substantially less when cells are frozen in saline and thawed in PVP compared to cells both frozen and thawed in isotonic saline. (From “Cryobiology” (H. T. Meryman, li:d.), p. 64. Academic Press, London, 1966.) reduced to compensate for t,he practical problems of rapid freezing and thawing and the adverse economics of storage in liquid nitrogen. In any event, the prevention of freezing damage t,lirough the use of extracellular cryoprotectants llrovides :I wholly different approach from the conventional glycerol procedures and may provide some insight into the nature of freezing injury and its reversibility. SUMMART Cells rapidly frozen and thawed both in the presence and absence of extracellular cryoprotectants lose pot,assium in exchange for extracellulnr solute without hemolysis and with subsequcnt in zlivo survival. There is some loss of 2,3-diphosphoglycerate from cells frozen with polyvinylpyrrolidine and hydroxyethyl starch as cryoprotectants. At least a portion of the cation exchange occurs following thawing, suggesting a transient loss of normal membrane semipermeability. The cryoprotectants reduce the freezing rate necessary for optimum recovery and may
enhance the transient membrane permeability. They may also serve to stabilize membrane components against irreversible postthaw changes.
1. Davies, H. G.. Marxdrn. K. V. 13..&lling. Y. G., and Zade-Oppen, A. M. M. The t,fiect, ol some neutral macromolecules on the pattern of hypotonic hemolysis. Acta Physiol. Sccmo’. 74,577-593 (1968). 2. Davies, J. D. The role of peptides in preventing freeze-thaw injury. In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor. Eds.), pp. 213-232. Churchill, London, 1970. 3. Doebbler, G. F., Buchheit, R. G., and Rinfrct. A. P. Recovery and in viro survival of rabbit, erythrocytes. Nature (London) 191, 1405 (1961). 4. Doebbler, G. F., and Rinfret, A. P. A biochcmical basis of hemolysis of erythrocytes by freezing and thawing. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 21, 67 (1962). 5. Doebbler, G. F., Rowe, A. W., and Rinfrct, A. P. Freezing of mammalian blood and its constituents. In “Cryobiology” (H. T. Merr.
RED
CELI,
man, ed.), pp. 407-450. .\wdc~n~ic~ 1’1~s. London. 1966. 6. Kritt, A. S. Reduced nicotinamide adenine dinuclcotide-linked analysis of 2,3-diphosphoglyceric acid: spectrophotometric and fluorometric procedurrss. .I. Lab. Clin. Mrd. 77, 470-475 ( 1971). 7. Klotz. I. M. liolc of watrr Aructurtx in macwmolrcules. Feel. ~roc., FNJ. Amer. Sot. l:‘x/J. Viol. S15, S24-S33 (1965) 8. Iinorl)p. C. T., Mrrc~hant. W. It., Gikw 1’. R.. dpcnc~~r. H. H.. and Thompson. X. I\‘. H.vdro~yf~thyl starch : extracellular cryophylartic~ agrnt for erythroc~ytrs. Science 157, 13121313 (1967). 9. Knorpp. C. T.. dtnrk\vcather, XT. H.. Spwcer. H. II.. >md Weathcrhw. I,. The prcserwtion of crythrocytes at liquid nitrogen trmperatnrrs with hydroxyethyl starch: the wmol-al of hydroxyethyl starch from erythrocytes after thawing. Cryobiology 8,511-516 (1971). J. E. HaemolSsis by thermal shark. 10. LOVC~OC~, Bit. J. Haenantol. 1, 117-129 (1955). 11. Lo\-clock, J. E:. The mechanism of the proiwtive action of glycerol against hcmolysis 1~5 freezing and tha\ving. Biochin~. Rio$~?/s. Acttr 11, 28-36 (1953). 12. Nazur. P. Cryobiology: the freezing of biological systems. Science 168, 93%949 (1970). 13. Mcryman. H. T. Absenc*e of unfrozen frerzable water in rapidly frozen red cells. Cry)Dio2oqv 7,4-6 (1971). 11. Mrrymnn. H. T. Osmotic stress as a mcchanirm of frwzing injlvy. Cryohio/og!/ 8, 489-500 (19il).
F’REEZISC
x7
15. Mrryman. H. T. The c?cweding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury. In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor, Eds.). pp. 51-64. Churchill. London. 1970. lfi. Mcrymnn, H. T. Red crll hemolysis from frcezing: alterations in membrane l)ermcnbilit\. following osmotic stress. 112“Red Cell Mrmt ,r:me: Structure and Function” (G. A. .Jnmieson and T. J. Greenwalt: Eds.). 1111. 352-367. Lippincott: Philadelphia. 1969. 17. Merymnn, H. T.. and I&fig, E. Rapid frrczing rnttl thawing of whole blood, P~oc. Sot. I?‘x/j. Riol. df etl. 90, 587-589 (1955). 1s. Richards, V., Braverman, M.. Floridia, I< ., Persidsky, hr., and I,o\\mstein. J. Initial clinical csperiences Tyith liquid nitrogen prrserved blood. employing PVP as a protectircx additive. Amer. J. S~wg. 108, 313-322, 1964. 19. Robinson, D. M. Repair of freeze-thaw-inducrd damage in mammalian cells: possible rolcl of mcmbranc glycoprotrins. (‘ryol)iologq 8, 384 (1971). 20. Shmgo, hl. I., and Danilina. V. 5’. LOSS of potassium from crythroqtrs during freezing in Ilolyethyleneortide solutions. l+ob. (;rmn!ol. Pet&v. Krovi 16, 54-56 (1971). 21. Strumia. MI. M.. Col~wll. L. S.. and Strumia. I’. V. Prescrrntion of whole blood in frozrn stnt.e for transfusion. Science 128, 1002-1003 (1958). 22. Williams. R. J.. and Moryman. H. T. Freezing injury and resistance in spinach chloroplast grann. PInnt Physiol. 45, 752-755 ( 1970).
9, 262-267 (192)
Changes
in Red Cells Extracellular H. T. MERYMaS
Following Rapid Freezing Cryoprotecdve Agents’ .ASDM. HOH~BI,O\VER
The recovery of human red cells following slow freezing requires the presence of a high (Boncentration of a penetrating cryoprotective agent such as glycerol. However, if the freezing rate is relatively rapid a variety of nonpenetrating compounds will provide good cryoprotection at low concentrations. Glucose (17)) glucose-lactose (al), polyvinylpyrrolidone (PVP) (3: 18)) hydroxyethyl starch (HES) (8) and a polypeptide ext.ract of peptone (2) have all been rcported cryoprotective for human red cells. dll but the peptone extract have been tested clinic~lly and shown to yield cells that survive in 11izlo.The freezing rates which produce optimum recovery vary depending on the cryoprotectivr agent but all require rapid or relatively rapid freezing and require liquid nit,rogen temperature for storage. The freezing of cells wit,h PVP and HES for transfusion has been particularly attractive since the optimum freezing rates with these cryoprotectants are the least demanding, making sterile processing in closed cont,ainers feasible. On t,he premise that intravenous administration of either of these two cryoprotectants would be acceptable, considerable effort has been espended in the hope of developing a one-step freezing method following which the thawed cell suspension could be administ,ered without. further treatment (5, 8), t,hus circumventing the need for post,thaw washing of the cells. The mechanism by which freezing injures living cells has been extensively investigated in our laboratory (14, 15). When cells are suspended in :I solution of nonpenet,rating solute and the concrntration is progressively inrreased (as by freezing out water) the cells are dehydrated and reduced in volume. In all animal cells we have studied no adverse effects are seen until a volReceived June 12, 1972. ‘Contribution No. 213 from tllfa American XaI ional Red Cross Blood R.csearch Laboratory. SupIlortcd in part by Office of Naval Research Contract N00014-70-C-0026 and National Institutes of Hrnlth Grant 1 ROl CrM17959-01.
tune is reached corresponding t,o the loss of approximately 65% of cell liquid. Beyond this point, there is an influx of extracellular solution with lysis on return to isotonic suspension. For red cells, t,he minimum tolerated volume i,; reached at about) 4.5 times isotonic. dccording to this “minimum cell volume” model, cryoprotective agents such as glycerol and dimethylsulfoxide function on a simple antifreeze basis as previously proposed by Lovelock (11). -it concentrat,ions of 2 to 4 M, they reduce the amount of ice formed suffirientlg to prevent a damaging courentration of extracellular solutr. It is, of course. essential that, these agents penct rate the cells otherwise their high osmot,ic potential will in itself he destructive through ~11 volume reduction. The sugars and polymers used to protect rrd cells during rapid freezing are, on the contrary. nominally nonpenetrating and are effectivr in low concentrations. It has been shown t,hat, at the freezing rates which yield maximum crll recovery, ice is ext,racellular (12) and there is no evidence that any freezable wat,er remains unfrozen (13). This leaves no alternative but to conclude that, with rapid freezing, cells are subjected to concentrations which at slower fooling rates would be hemolytic. A possible explanation of cryoprotection b! nonpenetrating agents was suggested by our studies (22) of cryoprotection of the grana from spinach leaf chloroplasts by t.he nominally nonpenetrating sugars and sugar alcohols. We found that grana exposed to increasing concentrations of salt in the absence of sugars were, like red cells, progressively reduced in volume and that beyond approximately 1200 mosm loss of membrane furl&ion occurred. In the presence of sugars, however, the grana failed to reduce further in volume beyond about 800 mosm but instead permitted an influx of extracellular solut,ion. On return to isotonicity, t,here was an efflux of the excess solute and at no time during this sequence of events was there any evidence of irreversible 262
Copsright, 111 rights
0 1972 by Academic Preuu. of reproduction in any form
Inc. reserved
with
RED
CELL
mrmbranc damngc. These observations demonstrated :I new mechanism for cryoprotcction c*ompatible with the minimum volume hypothe‘Ii. .I number of compounds were invrst igatcd to tlrtrrminc tliPir cryoprotectivc capacity for the q)in;tch granum. Sorbitol was found :I:: effect i\-c :I; sucrose. Both PVP and HE8 protected gran:1 from freezing injury as did an extract of peptone. We were struck by the fact that all of these compounds had also been reported protrctier for red cells during rapid freezing. Changes in red ccl1 sodium-potassium ratio II:IW been reported by Doebbler and Rinfret (4) following rapid freezing with PVP! by Knorpp catnl. with HES (9) and by Shrago and Dnnilinu (20) after freezing with polyethyleneoxide. The I)ossihility arose that in red cells these cryoprotcctive agents were cxnabling recovery by a leak mcclianism similar to that seen in spinach grana. A series of experiments was uridertakcn to detcrmint whether an exchange of extracellular and intracellular electrolytes might be a necessary component of the recovery of red cells following rapid freezing with rxtracellular cryoprotcctivr :lgellts.
X4TERIALS
AND
1IETHODS
Fresh human erythrocytes collected by s;tandard blood bank procedure into -4CD solution were used throughoutJ. The cells were mixed with cq-oprotectants as described below iu ratios comparnble to t,hose reported optimum for reco\-cry and i?~ llivo survival. The freezing rate producing optimum cell recovery was empirically drtt~rmined for well cryoprotecti\-c agent. I’VI’ and HES. One volume of packed cells was mixed with 5 vol of 10% PI’P. 0.6% iYaC1 at pH i or with 14% HES, O.i6% NaCl at pH i. Both of these cryoprotectant solutions had :IU osmolality of approximately 340 mosm. Small :Iliquots of there suspensions were frozen in thin walled polyethylene tubing, I/* in. in diameter. The tubes were frozen by direct immersion into liquid nitrogen and the freezing rate varied until optimum by wrapping t,he tube with cloth. Cells in PVP were frozen at 3.5”/sec, in HES at Z.i”/ sec. The frozen cells were thawed by agitating the bare plastic tube in a 37°C water bath. La&w-glucose. Whole blood was mixed with an qua1 volume of 15% lactose and 10% glurosc
263
FREEZIKG
:It pH i (21 j This suspension was frozen in I/Z in. polyethylene tubing at a freezing rate of al)proximately 20”/sec and the bare tubing was thawed by agitation in a 37°C water bath. Glucose nltd ,VaCZ. Packed cells were misrtl with a11 equal \-olumc of either 0.9% NaCl or 0.9’4~ NaCl plus 15% glucose at pH 7. The cellwere frozen at approximntel\; lOO”/scc by spraying thr suspension directly onto the surface of liyuid nitrogen (li) The cells were thawed 1,~ recovering small amounts of droplets in :I teaspoon, allowing the liquid nitrogen to evaporatr from t,hem and then dulnping them into :I beaker containing either saline or saline plus glufose at 37°C. Following thawing, aliquote of each rell puspension were assayed for percentage hemolysis by comparison of the supernatant hrmoglobin concent rat ion and the hemoglobin concent ration of the entire suspension. Hemoglobin assays were conducted using an automated hcmoglobinometer’ standardized by duplicate determinat ions using the cynnmethemoglobin method. IIF tracrllular and extracellular sodium and potassium determinations were made by flamr pliotomct,ry.3 To rompensate for possible varinrions in the number of red cells assayed due to ctxll volume changes or sampling errors, the hcmoglobin concentration of the dilute lit~hium suxpension actually measured in the flame photomctcr was assayed. The cation concentrations mcnsured were then adjusted by multiplying them by the ratio of the prefreczr hemoglobin measuremenf to t,he postthaw value. 2 .~i-di~~lio~~~liogl~c~ric acid (2,3-TIP(:) I\-:I~ :IMJYY~ using the sprc~tro~,liotonipt ric mrt hod of Kcitt (6). This was modified according to the recommendations of Dr. Norman Fortier of thr Blood Research Laboratory, U.S. Naval Hospital, Chelsen! MA, in which pyrophosphxte W:IS used to stimulate the 2,X-DPG phosphatase ncti\-ity of the phosphoglycrrate mutase using :I buffer containing (per ml of reaction mixture), 1.37 &ml ATP, 10.3 PM sodium pyrophosphate, 02 ,ULI\IN4DH, 10.3 PM Tris and 1.55 PM magnesium chloride brought, to pH 7.6 with 1.0 N HCl. ‘Model Ion. M. ’ Model ton. M.
231, 1nntrumcnt:~tion
Lnhoratories.
Ih+
143. Instrumentation
Lnhorntorics,
Ros-
264
MERYMBN
ASD
HORSL~I,OWEK
RESULTS
TABLE
Table 1 summarizes the results obtained. 111 all samples, cell potassium was severely depleted and replaced by whatever the extracellular solute may have been. In situations where t,he extracellulnr solute wad predominately sodium chloride, the total amount of intracellular cation following thawing was approximately that prior to freezing even though the ratios of sodium to potassium may have been radically changed. Cells frozen in PVP and HES were also somewhat depleted of 2,3-DPG. Cells frozen in PVP and HES also tended to hemolyze more when mashed in saline following thawing. In fact, it was never possible to wash these cells free of supernatant, hemoglobin no matter how man) washes were conduct,ed, suggesting that some mcmhrane instability remained following thawing. Optimum recovery is obtained with PVP and HES at subst,antinlly slower rates of freezing than are necessary when saline or saline-glucose is used. The loss of 2,3-DPG from cells frozen with PVP and HES may reflect the longer period of time that these cells are exposed to hypertonic solutions at temperatures high enough to permit diffusion. Alternatively it may be that PVP and HES exert a direct effect on the cell membrane rendering them more permeable to TABLE
1~ Hemo lysis
‘Na
I
K’Na~K i-
~.
.-~--
- ’
10% P\‘P 0.6% NaCl 147; HES 0.767, NaCl
13
211 79 117 12;
15”/c Lactose 1oyc (:lucose
0.90/
ples
I
i
4.4
13
6.1
3
3 8
4
39
NaCl
11Data represent
72
an arithmetical
mean.
4
Freezing solution
GlucoseNaCl (;lucoseKC1 0.9yc NaCl SucroseNH&l
Thawing
‘La
solution Na
K
(:lucose-KC1 Sucrose~NH~Cl (:lucose-NaCl
24 15 15 18
108 106 103 118
NaCl KC1 NaCl KC1
10 15 14 6
(ilucose-NaCl
Na
K
54 86 xl 33 I 93 64 21 18
io 6i 33 ~ 98 40 67 8 , 77 .n Cells were frozen by the droplet method. (:lucose-NaCl indicates 15yc glucose, 0.9% NaCl added 1:l to red cells for freezing or to isotonic saline for thawing. YucroseeNH&l indicates 12% sucrose plus 0.1 M NH&l added to maint,ain normal ionir concentration (16). 82 100 93 91
high molecular weight, components. Davies et al. (1) have reported that red cells hypot’onicall\ stressed in the presence of PVP lose some of their hemoglobin instead of lysing completely as in salt suspension, suggesting an alteration of membrane characteristics by PVP. One inescapable conclusion is that red cells recovered following rapid freezing either with or without an estracellular cryoprotective agent have experiembed an influx of extracellular solute and efflux of intracellular solute, suggesting that a transient reversible increase in permeabilky may bc an event inevitably associated with cell survival following rapid freezing. The question was raised mhet.her this reversiblc incrcnsc in permeability occurs only during freezing and thawing or whether there might be :t brief interval following thawing during which the ccl1 membrane remains relatively permeable to solutes. To t’cst this possibility cells were suspended in solutions of sodium chloride or potasaium chloride or in sucrose-ammonium chloride and rapidly frozen by t,hc droplet method. The cells were thawed in a variety of thawing solut,ions as indicated in Table 2. The intracellular sodium ‘and potassium were assayed both prior to freezing and following thawing. It is apparent, from this data that cation exchange occurs both during the freeze-thaw cycle and after the thswing is complctcd. In all cases where cell.; wrre
frozen in sodium chloride, cell sodium is elevated postthaw even though the thawing solution may have been sodium-free. This implies an entry of sodium during the freeze-thaw cycle, presumably during the hypertonic phase of freezing. On the other hand, all cells show a postthaw elevation of the cation present in the thawing medium compared to a experiment in which that cation was absent from the thawing solution. The question also arose whether t’he increase in membrane permeability was to low molecular weight compounds only or whether high molecular weight compounds could equally well cntcr t,he cells. To test this cells were frozen and thawed in a 20% (200 mosm) solution of P\X’ alone, without any other solute. The initial postthaw recovery wxs 97%. When the cells wrrc assayed for cation, only i% of the original electrolyte content of the cells remained. Under the microscope the cells appeared normal in volume. implying that PVP had entered the cells. -4lthough these cells remained intact following thawing they were pnrtic~ularly uiistablc on ,AI~),sclclurnt washing.
We conrlude from this study that, the rccovery of unhemolyzed red cells following rapid freezing and thawing is associated with an influx of extraccllular solute possibly during the hypertonic phase of freezing, a subsequent loss of intracellular solute on return to isotonicity and a brief interval following thawing during which t’he cell membrane remains permeable. That such cells can survive following reinfusion in GUO demonstrates the reversibility of the event. Since over half the red cells suspended in sodium chloride in the absence of any cryoprotective agent arc unhemolyzed following rapid freezing, the cryoprotective agent is presumably not essential for the development of reversible membrane leak. The cryoprotect8ive agents do reduce the rate of freezing necessary to ncliievc~ optimum recovery. It is notable t,hat the same group of conpounds that are cryoprotective for red cells and for spinach grana also produce alterations in red cell permeability under hypotonic stress (16). One of the common properties of t,hese compounds is their ability to stabilize macromolecules against conformational changes, possibly through the modification of water structuring
around nonpolar groups (7). Both PVP and HES have much higher stabilizing capacity t’hnn do the sugars, and it is possible that their effect on membrane permeability is more pronounced. permitting the passage of larger molecules suck11 as dipho~phoglycerate or the polymers themsrlrrs. It may also be the prcscnce of int racclhlur PVP and HES that causes the <11bs(~(Illont instability to suggessive saline washes. Robinson (19) has rcportcd that hamster 41s in culture develop vesicles following freezing and t,hawing and are killed. However, when the SIP+ pending medium is replaced following thawing by a medium in which hnmstcr cells h:~v(: prcviollely been grown, veniculntion does not OCC~I and 100% survival is seen. This clearly implies that events following thawing can prevent tht development of irreversible changes and permit the healing of freeze-thaw damage. It is entirrl! possible that extracellular rryoprotectants ma? be performing a similar function particularly in view of the fact, that all known cs;traccllul:~I c,r~oprotc~c,tnnt~ :II’(~ strong IIi;1c~romolec’nl:rr. St;]l)ilizcra. -1 poattliaw effect of P\‘P on frozc,n red ct,ll,. is demonstrated by the data ill Table 3. It ii clear from these experiments that the prescncae of PVP after thawing can substantially redurcl post,thnm hemolysis. Such events as the loss of lipid component’s from the membrane (10) ma! be prevented through the stabilizing capacity of the PVP. The possibility that PVP and similar extracellular compounds may exert their cryoprotective effect after freezing and thawing must be considered as a real possibilit,y. From the clinical standpoint, a primary goal of the rapid freezing method has been to achier-e a one-step procedure, eliminating the extensive po&thaw processing which is required by the glycerol method. However, it would appear that at least some cell washing will still be nccessnry. Although Knorpp et a,l. (9) hnvc reported much less potassium loss than seen in our csperimcnta, his average of about 35 mmolcs of free potasAm/unit, would be clinically undesirable where multiple transfusions are involved. Knorpp et nl. (9) also found the viscosity of cells in 14% HES such that the removal of the HES and resuspension of the cells is desirable in order to facilitntr administration. It remains to be seen whet,her the amount of washing necessary to provide a clinirally nrceptnhle product will be sufficientlp
MERYMAN
AND HORNBI,OW~;R TABLE
3
EFFECTIVENESS
OF POLYVINYLPYRKOLIDONE IN ERYTHI~OCYTE FI~EEZING ADDED EITHER BEFORE FREEZING OR DURING THAWING” ____-~~~~
WH~:X
Erythrocyte suspending solution
Thawing solution
5yoHemolysis
Total hemolysis following resuspension in isotonic saline
Isotonic saline Isotonic saline 2oyo PVP, 0.37, NaCl 207o PVP, 0.3c/ NaCl
Isotonic saline 20% PVP, 0.37, NaCl Isotonic saline 20% PVP, 0.3% Nack;
39.6
44.1
8.3 0‘3 3.2
27.3
10.0 9.7
a Data are based on an arithmetical average of four experiments each. Human red cells were rapidly frozen either in an isotonic solution of 20(x PVP or in isotonic saline. Since the optimum freezing rate in the presence of PVP is slower than in saline, the former preparations were frozen as pellets by spraying directly onto liquid nitrogen. These procedures were found empirically to give optimum recovery for the respective preparations. Cells frozen with PVP were thawed by shaking with an equal volume of thawing solution. Cells frozen as pellets were thawed by immersion in thawing solution. After thawing, the resulting hemolysis was measured, the cells were centrifugally sedimented, separated and resuspended in isotonic saline following which any additional hemolysis was measured. As shown, cells frozen with PVP showed slightly more hemolysis when thawed in saline compared to a PVP solution. However, cells thawed in saline hemolyzed less on saline resuspension, the total hemolysis following the two thawing methods being roughly the same. Cells frozen in isot’onic saline, however, showed almost four times the hemolysis when thawed in saline compared to cells thawed in PVP solution, indicating that the PVP was exerting some sort of protective effect after thawing was completed. That some of this protective effect may have been simply mechanical support given to damaged cells is suggested by the substantial additional hemolysis when cells thawed in PVP are resuspended in saline. Even so, t,he total hemolysis is substantially less when cells are frozen in saline and thawed in PVP compared to cells both frozen and thawed in isotonic saline. (From “Cryobiology” (H. T. Meryman, li:d.), p. 64. Academic Press, London, 1966.) reduced to compensate for t,he practical problems of rapid freezing and thawing and the adverse economics of storage in liquid nitrogen. In any event, the prevention of freezing damage t,lirough the use of extracellular cryoprotectants llrovides :I wholly different approach from the conventional glycerol procedures and may provide some insight into the nature of freezing injury and its reversibility. SUMMART Cells rapidly frozen and thawed both in the presence and absence of extracellular cryoprotectants lose pot,assium in exchange for extracellulnr solute without hemolysis and with subsequcnt in zlivo survival. There is some loss of 2,3-diphosphoglycerate from cells frozen with polyvinylpyrrolidine and hydroxyethyl starch as cryoprotectants. At least a portion of the cation exchange occurs following thawing, suggesting a transient loss of normal membrane semipermeability. The cryoprotectants reduce the freezing rate necessary for optimum recovery and may
enhance the transient membrane permeability. They may also serve to stabilize membrane components against irreversible postthaw changes.
1. Davies, H. G.. Marxdrn. K. V. 13..&lling. Y. G., and Zade-Oppen, A. M. M. The t,fiect, ol some neutral macromolecules on the pattern of hypotonic hemolysis. Acta Physiol. Sccmo’. 74,577-593 (1968). 2. Davies, J. D. The role of peptides in preventing freeze-thaw injury. In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor. Eds.), pp. 213-232. Churchill, London, 1970. 3. Doebbler, G. F., Buchheit, R. G., and Rinfrct. A. P. Recovery and in viro survival of rabbit, erythrocytes. Nature (London) 191, 1405 (1961). 4. Doebbler, G. F., and Rinfret, A. P. A biochcmical basis of hemolysis of erythrocytes by freezing and thawing. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 21, 67 (1962). 5. Doebbler, G. F., Rowe, A. W., and Rinfrct, A. P. Freezing of mammalian blood and its constituents. In “Cryobiology” (H. T. Merr.
RED
CELI,
man, ed.), pp. 407-450. .\wdc~n~ic~ 1’1~s. London. 1966. 6. Kritt, A. S. Reduced nicotinamide adenine dinuclcotide-linked analysis of 2,3-diphosphoglyceric acid: spectrophotometric and fluorometric procedurrss. .I. Lab. Clin. Mrd. 77, 470-475 ( 1971). 7. Klotz. I. M. liolc of watrr Aructurtx in macwmolrcules. Feel. ~roc., FNJ. Amer. Sot. l:‘x/J. Viol. S15, S24-S33 (1965) 8. Iinorl)p. C. T., Mrrc~hant. W. It., Gikw 1’. R.. dpcnc~~r. H. H.. and Thompson. X. I\‘. H.vdro~yf~thyl starch : extracellular cryophylartic~ agrnt for erythroc~ytrs. Science 157, 13121313 (1967). 9. Knorpp. C. T.. dtnrk\vcather, XT. H.. Spwcer. H. II.. >md Weathcrhw. I,. The prcserwtion of crythrocytes at liquid nitrogen trmperatnrrs with hydroxyethyl starch: the wmol-al of hydroxyethyl starch from erythrocytes after thawing. Cryobiology 8,511-516 (1971). J. E. HaemolSsis by thermal shark. 10. LOVC~OC~, Bit. J. Haenantol. 1, 117-129 (1955). 11. Lo\-clock, J. E:. The mechanism of the proiwtive action of glycerol against hcmolysis 1~5 freezing and tha\ving. Biochin~. Rio$~?/s. Acttr 11, 28-36 (1953). 12. Nazur. P. Cryobiology: the freezing of biological systems. Science 168, 93%949 (1970). 13. Mcryman. H. T. Absenc*e of unfrozen frerzable water in rapidly frozen red cells. Cry)Dio2oqv 7,4-6 (1971). 11. Mrrymnn. H. T. Osmotic stress as a mcchanirm of frwzing injlvy. Cryohio/og!/ 8, 489-500 (19il).
F’REEZISC
x7
15. Mrryman. H. T. The c?cweding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury. In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor, Eds.). pp. 51-64. Churchill. London. 1970. lfi. Mcrymnn, H. T. Red crll hemolysis from frcezing: alterations in membrane l)ermcnbilit\. following osmotic stress. 112“Red Cell Mrmt ,r:me: Structure and Function” (G. A. .Jnmieson and T. J. Greenwalt: Eds.). 1111. 352-367. Lippincott: Philadelphia. 1969. 17. Merymnn, H. T.. and I&fig, E. Rapid frrczing rnttl thawing of whole blood, P~oc. Sot. I?‘x/j. Riol. df etl. 90, 587-589 (1955). 1s. Richards, V., Braverman, M.. Floridia, I< ., Persidsky, hr., and I,o\\mstein. J. Initial clinical csperiences Tyith liquid nitrogen prrserved blood. employing PVP as a protectircx additive. Amer. J. S~wg. 108, 313-322, 1964. 19. Robinson, D. M. Repair of freeze-thaw-inducrd damage in mammalian cells: possible rolcl of mcmbranc glycoprotrins. (‘ryol)iologq 8, 384 (1971). 20. Shmgo, hl. I., and Danilina. V. 5’. LOSS of potassium from crythroqtrs during freezing in Ilolyethyleneortide solutions. l+ob. (;rmn!ol. Pet&v. Krovi 16, 54-56 (1971). 21. Strumia. MI. M.. Col~wll. L. S.. and Strumia. I’. V. Prescrrntion of whole blood in frozrn stnt.e for transfusion. Science 128, 1002-1003 (1958). 22. Williams. R. J.. and Moryman. H. T. Freezing injury and resistance in spinach chloroplast grann. PInnt Physiol. 45, 752-755 ( 1970).