Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation, the effect of glycerol on lysosomes, kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

CRYOBIOLOGY

17, 198-212 (1980)

Cryopreservation of Granulocytes for Transfusion: Studies on Human Granulocyte Isolation, the Effect of Glycerol on Lysosomes, Kinetics of Glycerol Uptake and Cryopreservation with Dimethyl Sulfoxide and Glyceroli,2 ARTHUR W. ROWE The Lindsley

F. Kimball

Research

AND LESLIE L. LENNY Institute of The New York Blood Center, 310 East 67th Street, New York, New York 10021

Considerable evidence now demon- viability and function better than do mature strates that the transfusion of leukocytes is granulocytes. Efforts to achieve cryoof value in the supportive therapy of pa- preservation of leukocytes have been contients with leukemia, agranulocytosis, and cerned primarily with freezing and storage sepsis (14, 36, 46, 47, 58). Recent advances of lymphocytes but, unfortunately, the in leukocyte procurement have led to the problem of granulocyte cryopreservation use of such techniques as continuous flow appears to be much more difficult than that centrifugation or filtration leukopheresis, in of lymphocyte preservation. Rowe et al. (44) and Rowe and Cohen addition to centrifugal batch separation. These techniques have now permitted the (39, 40) first reported in 1963 on the cryoroutine collection of normal granulocytes preservation of granulocytes and the use of from single donors in quantities sufficient phagocytic activity as a measure of functional viability of leukocytes preserved in for treatment of infections in granulocytopenic patients (20). dimethyl sulfoxide (Me,SO, also known as The feasibility of having on hand normal DMSO).” Since that time Cavins et al. (6, granulocytes in sufficient quantities to meet 7), Schwarzenberg et al. (46), Shohet et al. clinical needs is contingent upon some (48), Bouroncle (4), Kessel et al. (24), Skeel means of preservation. Since the storage et al. (50), Perry et al. (37, 38), Malinin (31), life of granulocytes at 4°C is very limited, it Crowley et al. (8), Lionetti et al. (28-30), is therefore necessary to preserve granulo- Knight et al. (25, 26), Bank (2), French et cytes by some method involving cryo- nl. (15, 16), Zaroulis et al. (59), and other preservation and low-temperature storage investigators (1,3,9, 19,21) have described (27, 33, 35). Perry (36) has noted that good the use of Me,SO as the additive of choice and extensive quantitative data on leuko- for preservation of granulocytes. When used for cryopreservation of leukocyte preservation are lacking, but available cytes, M%SO is usually employed at coninformation suggests that lymphocytes, monocytes, and the less mature prolifera3 DMSO has been the commonly used abbreviation tive hematopoietic precursor cells retain for dimethyl sulfoxide. In 1977, the Editorial Board of Received February 4, 1980; accepted March 17, 1980. ’ Part of a Symposium on Granulocytes presented at the 16th Annual Meeting of the Society for Cryobiology, Atlanta, Ga., September 30-October 4, 1979. 2 Supported by NHLI Contract l-H13-4-2951.

the journal Cryobiology agreed to encourage use of the more chemically correct abbreviation Me,SO instead of DMSO. This decision was based on a nomenclature policy statement issued by the IUPAC-IUB Combined Commission on Biochemical Nomenclature and the National Academy of Sciences-National Research Council. 198

OOll-2240/80/030198-15$02.00/O Copyright Au rights

@ 1980 by Academic Press, Inc. of reproduction in any form reserved.

CRYOPRESERVATION

centrations of 10 to 15% combined with a freezing rate of approximately 1°C per minute and subsequent storage at -80°C or in liquid nitrogen (LN,) at -196°C. Tests to demonstrate preservation of function of peripheral blood granulocytes after storage have included not only an in vitro evaluation of their ability to phagocytize and kill bacteria, but also an in viva evaluation of their circulation and clinical response when transfused into recipients (3, 18, 45, 51). Although Me&SO has been used as the cryoprotective additive of choice, the administration of leukocytes which have been preserved in Me,SO has been reported to result in adverse clinical side effects. These include nausea and vomiting, as well as an objectionable odor to patients (36). In large doses, Me&SO has caused pathologic eye changes in experimental animals. The routine clinical use of Me,SO-preserved cells has therefore been a subject of controversy, and has been questioned both by investigators and federal regulatory agencies, who feel that the inherent toxicity and clinical objections to Me+SO preclude its extensive and routine use in patients. Glycerol requires reexamination as a cryoprotective additive for preserving leukocytes for transfusion; no clinical side effects of glycerol have been observed based on extensive experience by Rowe (18, 19, 41, 42, 43) and others (22, 34, 56, 57) with transfusion of red cells preserved in glycerol. Recent studies on cryopreservation of platelets by Rowe and colleagues (lo- 13) in the Laboratory of Cryobiology at The New York Blood Center now suggest that glycerol could readily be used in place of Me,SO to achieve a high yield and good functional integrity of platelets which are also viable when transfused into patients. This symposium paper describes early studies on successful cryopreservation of rabbit peritoneal granulocytes with Me&SO which were viable based upon demonstration of phagocytic activity. It further describes more recent studies aimed at our

OF GRANULOCYTES

199

principal goal of developing a simple procedure for preserving human granulocytes at low temperatures, using glycerol instead of Me,SO as the cryoprotective additive. Studies on Phagocytic Activity of Rabbit Granulocytes Cryopreserved in Me,SO One of the earliest attempts to use a functional viability assay for cryopreserved granulocytes was reported by Rowe and colleagues (39, 40, 44) who utilized an in vitro test of phagocytosis as an in vivo test of granulocyte function. These studies were patterned after work by Karnovsky (23) and others (52, 53, 55) who, using guinea pig polymorphonuclear leukocytes, found that phagocytosis was accompanied by an increase in oxygen uptake and glucose utilization. Phagocytosis is a process requiring an expenditure of energy and this energy is obtained from glycolysis. Granulocytes were harvested from adult rabbits 18 hr after intraperitoneal injection of 12% sodium caseinate which produces an exudate rich in polymorphonuclear leukocytes. Inert polystyrene latex particles (PSP) of various sizes obtained from Dow Chemical Company were used for promoting the phagocytic event and utilization of oxygen was measured in a conventional Warburg apparatus at 37°C. Phagocytosis as measured by oxygen uptake was stimulated by addition of PSP (1.171 wrn) to fresh granulocytes as seen in Fig. 1. The PSP particles were added to the medium from the side arm of the Warburg flask. Evolved CO, was absorbed by potassium hydroxide in the center well. Undiluted particles were approximately 10% solids. Dilution of the particles resulted in lower oxygen utilization and less phagocytosis, indicating that phagocytosis is dependent in part on the number of particles. The data in Fig. 2 show that glucose utilization was increased with particles of three different sizes, indicating that phagocytosis of larger particles requires a greater expenditure of energy. Hence,

200

ROWE

AND

LENNY

0 NO PARTICLES A PARTlCLES(UNML., X PARTICLES~I:IOOIL.~

0 1

20

40

2

TIME.MIN.

60

60

TIME.

MIN.

100 120

160 0 O%DYSO-PSP

140

/

I20 I: $lOOS

: 60s 0" w 60q OWDMSO-UNFROZEN a,o%oYSO-UNFROZEN x ,OXDMSOtPSPLl ,r,UNFROZEN q IOY.DMSO-FROZEN O,OXDMSO+(PSP,-FROZEN

3

1 0

1

20

40

I

60

,

SO

TIME.MIN.

/

100 120

4

I 0

20

40

60

60

TIME,

MIN.

100 120

FIG. 1. Oxygen consumption during phagocytosis of polystyrene latex particles (1.171 pm) by fresh rabbit peritoneal granulocytes. FIG. 2. Effect of polystyrene latex particle size on oxygen consumption during phagocytosis by fresh rabbit granulocytes. FIG. 3. Effect of dimethyl sulfoxide (DMSO or Me,SO) on oxygen utilization with and without phagocytosis of polystyrene latex particles (1.31 pm) by freshrabbitgranulocytes. FIG. 4. Oxygen consumption and phagocytosis by unfrozen and frozen rabbit granulocytes in 10% DMSO (M&SO) at a rate of 1”Cimin.

energy expenditure during phagocytosis is added Me,SO must be removed from the related to both the number and size of par- cells. When Me,SO was removed by ticles being engulfed. washing in a balanced saline medium, the rate of respiration was restored to 80-90% Prior to using Me,SO as a cryoprotective agent for rabbit granulocytes, Me,SO was of the original value. Figure 3 shows that tested for its effect on the respiration of the when phagocytosis was carried out in the cells. Oxygen uptake was markedly inhib- presence of different concentrations of ited as the concentration of Me,SO was in- Me,SO (DMSO) using PSP (1.31 pm) creased. The depressant effect of Me,SO in stimulation, oxygen consumption stimuconcentrations above 2.5% indicates that lated by phagocytosis was decreased pro-

CRYOPRESERVATION

portionately as MqSO was increased. At a concentration of 5% MqSO there was no effect on phagocytosis but at 15% Me,SO complete inhibition of phagocytosis occurred. Rabbit peritoneal granulocytes were frozen using a controlled rate freezer (BF3-Union Carbide Corp.) at a rate of l”C/min to -2S”C, then at a rate of S”C/min to -8O”C, followed by immersion into LN,. The final concentration of Me,SO was 10% in the 1.2 ml ampule containing 1 x lo6 cells/ml. Following thawing, the cells were washed by gradual dilution with buffered saline to remove the Me,SO, and PSP (1.31 pm) were added to frozen and unfrozen cells treated in the same manner. The data in Fig. 4 show that there was a loss of about 50% of the frozen granulocytes but when stimulated, the granulocytes frozen in 10% Me,SO were able to utilize 02, i.e., phagocytize PSP to a greater extent than the unfrozen cells. These results first demonstrated the functionality of cryopreserved granulocytes by using an in vitro test of phagocytosis to simulate an important in vivo property of granulocytes. Encouraging results with cryopreservation of rabbit granulocytes using Me&SOgave impetus to investigations into cryopreservation of human peripheral granulocytes with glycerol. STUDIES

INVOLVING

ISOLATION

AND

OF GRANULOCYTES

201

nent laboratory were utilized as starting material for preparation of granulocytes. However, these impure leukocyte concentrates prepared from whole human blood contain not only granulocytes but also platelets, lymphocytes, and red cells. To obtain a homogeneous preparation of granulocytes for preservation studies, initial efforts were directed toward the isolation and purification of the granulocyte population from the leukocyte concentrate. The leukocyte concentrates were prepared from whole blood by centrifugation for 10 min at 1800 t-pm at 4°C in a Lourdes 30-R centrifuge. After removal of plateletrich plasma, the buffy coat containing leukocytes and contaminating red cells was removed. The leukocyte concentrates were suspended in 300 ml buffered NaCl (0.8%) containing dextrose (0.2%) and recentrifuged for 3 min at 4000 rpm in a Sorvall RC-3 centrifuge with an HG-4 rotor. The supernatant containing debris and residual platelets was expelled and the remaining buffy coat along with contaminating red cells was retained for further purification. of Leukocytes Attempts to separate the leukocytes from the major portion of contaminating red cells involved density gradient centrifugation with Ficoll and sedimentation with dextran.

Separation

1. Density gradient centrifugation Ficoll. The leukocyte suspension

with

was carefully layered on 9% (w/v) Ficoll (PharCollection macia) in a siliconized tube. After centrifuA regional center, The New York Blood gation for 40 min at 1500 rpm (approx 400g) Center, is dedicated to a program of frac- in a Sorvall GLC- 1, attempts were made to tionating whole blood into its cellular and separate granulocytes. Various concentraplasma components. This practice of pre- tions of Ficoll were subsequently tried but paring various cellular components from no definitive bands of granulocytes were whole blood results in a potentially large observed. In addition, the Ficoll was found source of granulocyte concentrates. As im- to cause aggregation of the leukocytes and proved methods of preservation are devel- thus the Ficoll method of granulocyte sepoped, the routine separation of leukocyte aration was discontinued. concentrates from normal whole blood will 2. Sedimentation with dextran. The folbecome a major source of granulocytes. lowing procedure was used for separation For this reason, leukocyte concentrates of granulocytes using solutions of dextran prepared by The Blood Center’s compo- (Pharmacia) (MW 228,000): PREPARATION

OF

HUMAN

GRANULOCYTES

202

ROWE AND LENNY

(a) One volume of leukocyte suspension was mixed with 2 vol of 3% dextran in 0.9% NaCl . (b) The suspension was allowed to stand for 1 hr at 4°C. (c) The top layer of each tube was aspirated and saved. (d) The bottom layer was resuspended (1: 1) in 2% dextran in 0.9% NaCl for 30 min at 4°C. (e) The top layer was again aspirated to extract additional leukocytes and was recombined with the other leukocytes. This extra dextran sedimentation step was found to increase the leukocyte yield by approximately twofold. The dextran-sedimented leukocytes were concentrated by centrifugation at 1000 rpm (15Og)for 10 min and the pellet resuspended in modified Hanks medium (MHM). This dextran sedimentation method is currently used for leukocyte isolation (see Fig. 5). Separation of Red Cells from Leukocytes Although efficient for separation of leukocytes, the dextran sedimentation WHOLEBLOOD + BUFFYCOAT

x DEXTRAN s EOIMENTPlTiON REMOVE RBC's

(-30%)

+

CONCENTRATE LEUKOCYTES + ADD PLASMATEIN HYPOTONICSHOCK W,TH Hz0 TO REMOVE REMAfNlNGM's +

FIG. 5. Separation procedure for human granulocytes.

method presented a problem of contaminant red cells. Two approaches were studied for removal of contaminating red cells: I. Osmotic shock with glycerolized cells. Prior experience with red cells has shown that glycerolized red cells can be hemolyzed by osmotic shock when flooded suddenly with saline. This glycerol-osmotic shock approach was explored as a potential method for removing contaminating red cells from granulocytes. Preliminary experiments with granulocytes suspended in various glycerol solutions indicated that glycerol concentrations approaching 15% did not damage them. Accordingly, leukocyte suspensions containing red cells were suspended in various concentrations of glycerol (in 0.9% NaCl) ranging from 2 to 14%. After a 5-min equilibration period, glycerolized cells were flooded with an excess of saline (0.8% NaCl containing 0.2% dextrose). Following concentration by centrifugation, the cells were resuspended in the glycerol solutions and the osmotic shock treatment repeated. Although the osmotic shock treatment caused hemolysis of the red cells, it apparently damaged the granulocytes as evidenced by extensive clumping. Therefore, this approach involving pretreatment of leukocyte suspensions with glycerol and osmotic shock to selectively hemolyse red cells was not considered to be feasible. 2. Hypotonic shock with water. Dextransedimented granulocyte suspensions were centrifuged and resuspended in titrated modified Hank’s medium (MHM): K,HPOI, 1.568 g/liter; NaCl, 8.12 g/liter: glucose, 2.0 g/liter; sodium citrate, 3.8 g/liter; pH 7.6. Six milliliters of cold distilled water were added to 2 ml of suspension for 30 set, immediately followed by addition of 2 ml of 3.5% NaCl to restore isotonicity with continuous mixing. After centrifugation the cells were again subjected to hypotonic shock treatment. Occasional clumping was noted in some preparations when hypotonic shock treat-

CRYOPRESERVATION

ment was used to reduce red cell contamination. Several approaches were used to refine the method and eliminate clumping: (a) DNase treatment. Shohet and Mohler (48) have pointed out that DNase treatment of granulocyte preparations will eliminate clumping. DNase I (beef pancreas, Sigma) and DNase II (hog spleen, Sigma) were added to the system at various concentrations. The oligonucleotidohydrolase DNase I was more efficient in eliminating clumping at concentrations of 1.0 mg/ml and higher, while DNase II was not effective in eliminating clumping. (b) Colloidal protein treatment. Because of their colloidal properties, gelatin and a 5% plasma protein fraction (Plasmatein, Abbott) were added to granulocyte suspensions to minimize physical contact of the leukocytes and to prevent clumping. Addition of gelatin to the granulocytes was not effective, but addition of 5% Plasmatein not only prevented clumping effectively but also facilitated the resuspension of the granulocytes.

203

OF GRANULOCYTES

preparations demonstrated that some cells were phagocytizing dye particles and particulate matter. STUDIES

INVOLVING

INDICATORS

LYSOSOMES

AS

OF CRYOINJURY

As part of our working hypothesis on freezing injury in granulocytes, we believe that lysosomes appear to be implicated in cryoinjury. The loss of granulocyte function through cryoinjury may be due to lysosomal damage that results in the release of hydrolytic enzymes into the cytosol. Uncontrolled digestion by lysosomal hydrolases would then lead to loss of cell function and ultimate lysis of the granulocyte (49). Based on previous work in our laboratory involving lysosomes and cryoinjury in platelets (lo- 13) we have concentrated our initial studies on the use of the lysosomal enzyme @glucuronidase as a key indicator of cryoinjury primarily because this enzyme is not membrane-bound as are such lysosomal enzymes as acid phosphatase and cathepsin. Since this enzyme was to be used as an indicator of cryoinjury, it was Purification of Granulocytes necessary to characterize fully the enzyme Contaminating leukocytes along with in the granulocyte system under various hypotonically shocked red cells were re- conditions. I. Characterization of /3-glucuronidase, moved from the impure granulocyte preparation by centrifugation in a Sorvall GLC- 1. The enzymatic assay for p-glucuronidase Centrifugation for 1 min at 500 rpm was ef- was performed using the homogenate and fective in yielding a granulocyte pellet free clear supernatant of both frozen and nonof contaminating lymphocytes and red frozen granulocytes. The nonionic detercells. The complete procedure developed gent Triton X-100 was added to the homogfor preparing granulocyte concentrates is enate for determination of total activity; summarized in Fig. 5. free activity was determined from the clear The final granulocyte suspension pre- supernatant. The percent of total activity pared by dextran sedimentation, hypotonic found in the supernatant was then calcushock, and the Plasmatein procedure was lated and taken as a measure of lysosomal subjected to microscopic examination damage. The optimal conditions for the enunder ordinary light and phase contrast zymatic assay of P-glucuronidase as modconditions. The granulocytes were ob- ified after Talalay et al. (54) involved the served to be intact, viable and with homo- use of phenolphthalein glucuronide as subgeneity in excess of 90%. Based on the strate. Data showing the effect of increasing ability of the granulocytes to exclude the concentrations of phenolphthalein glucdye trypan blue, viability of the cells was uronide on the activity of granulocyte pdemonstrated to be in excess of 90%. Wet glucuronidase are presented in Fig. 6. A

ROWE

AND

LENNY .YJo

l -.

.

.240

\

.

.lao

.120

\

.

,060

0

0.5

6

1.0

2.0

1.5

!,l#tll

0

2.5

4.2

3.8

PHENOLPHTHALEIN GLUCURONIDE CONCENTRATION, MM

0 0.1 0.2

0.4

0.8

4.6

5.4

5.0

PH

.lL!--1.3

2.6

3.9

5.2

.

,100

,300

. .

.0&o *MO

L

5 dI z 4 P Y a

.// ,100

R 8

,020

/ 0

9

15

30

0

120 TIME, MIN.

10

0.9

1.8

2.7

3.6

GRANULOCYTECONCENTRATION, MG/ML

FIG. 6. Effect of substrate concentration on p-glucuronidase activity.

FIG. 7. Effect of pH on P-glucuronidase activity. FIG. 8. Effect of Triton X-100 on release of /3-glucuronidase. FIG. 9. Effect of time on p-glucuronidase activity at constant granulocyte concentration. FIG. 10. Effect of granulocyte concentration on P-glucuronidase activity.

4.5

CRYOPRESERVATION

concentration of 1.25 mM phenolphthalein glucuronide yielded maximum velocity in assays of P-glucuronidase when the reaction was performed at a final pH of 4.2 with 50 rnM acetate buffer (Fig. 7). The concentration of Triton X-100 required to maximally release P-glucuronidase from the granulocyte was found to be 0.4%/mg cell protein (Fig. 8). The reaction was terminated by the addition of 7% perchloric acid (HClO,) instead of 10% trichloroacetic acid (TCA) because of turbidometric interference by TCA. The samples were titrated with developing solution (0.1 M glycine + NaOH to a pH - 13) to insure optimal OD and stability (final pH, 10.2- 10.4). The parameters described above were specifically designed to insure an assay reaction with zero order kinetics, i.e., determination of enzyme activity independent of substrate concentration and incubation time. The data in Fig. 9 show that pglucuronidase release is linear with time indicating that the reaction rate is zero order throughout a maximum of 2 hr. A plot of the effect of limiting enzyme concentration for zero order kinetics (Fig. 10) shows linearity with enzyme concentration, indicating that 2.0 mg protein/2 ml incubation medium is optimal (final concn, 1 mg/ml).

granulocytes with a ground glass hand homogenizer was the preferred method for release of lysosomes from the cell because this treatment per se did not damage the lysosomes. The extent of freezing damage to the lysosomes was determined by the fraction of total enzyme released into the medium after homogenization of the granulocytes. Figure 11 shows a direct linear relationship between damage to the granulocytes by freezing and percent lysosomal enzyme released. Fractions of granulocytes in the suspensions were prepared from whole and damaged preparations lysed by repeated freezing and thawing. Complete lysis resulted in total release of @glucuronidase which indicated that /I-glucuronidase was a very sensitive indicator of lysosomal damage. These results also demonstrated the absence of autolysis (by liberated hydrolases) of remaining intact lysosomes. pglucuronidase release therefore was used in subsequent experiments as the key indicator of lysosomal membrane injury in the granulocyte. 3. Effect of glycerol on lysosomes. Development of freezing procedures to preserve granulocytes involved the use of

.

100 -

2. @-Glucuronidase as an indicator of damage to the granulocyte lysosome. Since

an intact outer membrane could prevent the escape of lysosomai enzymes into the extracellular milieu, the granulocyte preparation was treated with sucrose or was homogenized. This treatment was designed to prevent the masking of damage to the granulocyte lysosomes which might go undetected if the plasma membrane remained intact. Sucrose (0.34 M) has been shown to lyse selectively the outer membrane of cells but with granulocytes some damage occurs to the lysosomes. This type of sucrose treatment was found to be unreliable and time-consuming. Homogenization of the

205

OF GRANULOCYTES

80 -

H

60 -

1 0

L

20

40

60

80

100

LYSIS OF GRANULOCYTFS, X

FIG. 11. Releaseof granulocytes.

P-glucuronidasefrom lysed

206

ROWE

AND

glycerol as the cryoprotective additive. To function as a cryoprotective additive, glycerol must be able to permeate the granulocyte membrane without causing damage to the cell. Experiments therefore were undertaken to determine if glycerol per se exhibited any detrimental osmotic or toxic effect on the granulocyte as evidenced by damage to lysosomes. Granulocytes were suspended in various concentrations of glycerol ranging from 0 to 30% and incubated for 30 min at room temperature. The samples were then homogenized and assayed for p-glucuronidase activity. The data in Fig. 12 indicate that glycerol in the concentration range of O-15% was not damaging to the granulocyte lysosomes as evidenced by lack of release of p-glucuronidase above the background level. When granulocytes were treated with sucrose to lyse the outer cell membrane for liberation of intact lysosomes, a high background activity (40%) of available P-glucuronidase activity was observed due to damage of the lysosomes by sucrose (Fig. 12). Figure 12 also shows that glycerol concentrations in excess of 10% induced

1W I

GLYCEROL CONCENTRATION. X

12. Effect of glycerol concentration on release of /3-glucuronidase from fresh granulocytes with ( 0) and without (A) s&rose treatment. FIG.

LENNY

further lysosomal damage as evidenced by increased P-glucuronidase activity. The lack of damage by glycerol per se on fresh granulocytes is of particular significance since it suggests that granulocytes, unlike platelets, are not overtly damaged by exposure to high concentrations of glycerol and supports our hypothesis that glycerol is a potentially effective cryoprotectant for preserving viable granulocytes. STUDIES

ON

PERMEATION

KINETICS

OFGLYCEROL

Glycerol is considered to be an intracellular cryoprotective additive because, in the case of human red cells, it is freely permeable through the membrane. In platelets, however, the permeation kinetics of glycerol and Me,SO differ (11, 12) in that a partial membrane barrier exists to the free permeation of glycerol that results in cell volume changes, while DMSO is freely permeable. To determine that glycerol is an effective cryoprotectant for preservation of granulocytes, it was necessary to know whether or not glycerol is capable of permeating the membrane to the extent necessary to achieve the intracellular concentration sufficient to confer optimal cryoprotection (10, 12, 17, 32). The uptake and intracellular distribution of glycerol in isolated granulocytes was determined using a triple isotope method under equilibrium conditions (11). The procedure involved measuring the difference between the amount of 14C-labeled glycerol trapped in the extracellular space of the granulocyte and the amount of glycerol in the granulocyte pellet. Extracellular space was measured using [14C]inulin (which is nonpermeable) and total granulocyte pellet space with 3H,0 (which is freely permeable). This method for determining uptake also provided a means for detecting changes in cell volume which might accompany uptake. Cell volume (intracellular space) was calculated on the basis of the difference between the measured granulo-

CRYOPRESERVATION

cyte pellet and the extracellular space. Based on the cell volume, the intracellular molar concentration was determined and compared to the exogenous molar concentration. The uptake and distribution of glycerol in granulocytes is shown in Table 1. The granulocyte suspensions were equilibrated with various concentrations of glycerol ranging from 0 to 15%. Using [14C]inulin and 3H,0, the extracellular and total spaces were measured and from the difference of these, the intracellular space was calculated. The data show that the intracellular space, i.e., cell volume, remained constant and was independent of glycerol concentration. Using [14C]glycerol, the ratio of intracellular to extracellular distribution of glycerol was found to be near unity (average 1.03) over the range of concentrations studied. These results were very encouraging since they indicated that glycerol is freely permeable to the granulocyte plasma membrane and that glycerol does not cause overt changes in cell volume that might be potentially damaging to the granulocyte. STUDIES INVOLVING FREEZING OF GRANULOCYTES IN GLYCEROL

The enzyme P-glucuronidase has been characterized as a useful indicator of cryoinjury to the lysosomes of granulocytes. A direct relationship has been found to exist between the release of this hydrolytic lysosomal enzyme and freeze-thaw injury to granulocytes. The cryoprotective additive glycerol did not appear to have any

207

OF GRANULOCYTES

adverse effect on the granulocyte lysosomes and also appeared to permeate granulocytes with facility. Studies therefore were undertaken to determine the range of freezing rates necessary to prevent cryoinjury to granulocytes in the presence of various concentrations of glycerol. Granulocyte preparations were suspended in various glycerol solutions to achieve final concentrations of 5, 10, 15, and 20%. The glycerolized suspensions were placed in tubes with uniform crosssectional thickness of 4 mm and frozen at various rates. Freezing rates were monitored with a copper-constantan thermocouple and a Honeywell strip chart recorder. To determine the most effective cooling rates three ranges of freezing rates were studied-slow, moderate, and rapid. Slow-rate freezing was controlled by cooling at approximately YClmin throughout the critical cryoinjury range (between the heat of fusion of the cell specimen and -35°C). Moderate-rate freezing was achieved by controlled cooling at lOO”C/min utilizing cold vapor from liquid nitrogen, while rapid-rate freezing involved the use of liquid nitrogen to obtain uncontrolled rates of approximately 100OWminute. All samples were stored in liquid nitrogen (- 196°C) prior to thawing. Figure 13 shows the effect of three ranges of freezing rates on human granulocytes suspended in various concentrations of glycerol. The release of P-glucuronidase was used as the indicator of freeze-thaw injury to the granulocyte lysosomes. These studies demonstrated that cooling at rapid

TABLE 1 Uptake and Distribution of Glycerol in Granulocytes Exogenous glycerol VfJ)

Total space 6.4

3 6 9 12 15

20.6 20.2 20.5 20.5 20.5

Extracellular space (4)

Intracellular space (PI)

Intracellular/ extracellular glycerol ratio

1.5 7.5

13.1 12.7 13.0 12.5 12.7

0.95 1.13 1.06 1.02 1.08

7.5 8.0 7.8

208

ROWE

AND

LENNY

mine recovery and yield based on numerical count and dye exclusion using trypan blue. Table 2 shows three slow cooling rates at different glycerol concentrations. A cooling rate of l”C/min and a glycerol concentration of H-20% resulted in the highest cell recovery and yield of viable cells based on trypan blue exclusion. At a rate of S”C/min, the best recovery and yield occurred at a glycerol concentration of 20%. The lowest recoveries and yields at all glycerol concentrations were obtained at a L slightly faster cooling rate of 1OWmin. lo a, 5 15 0 These freezing studies indicated that GLYCEROL CONCENTRATION,% glycerol is an effective cryoadditive that FIG. 13. Effect of cooling rate on preservation of protects granulocyte lysosomes from granulocyte lysosomes in the presence of glycerol. freezing damage. Further studies now are necessary to define more precisely both the (1000Wmin) and moderate rates (lOO”C/ optimal glycerol concentration and the min) resulted in considerable injury to the most effective cooling rate using additional granulocyte lysosomes. Slow-rate freezing, viability criteria including phagocytosis, on the other hand, afforded greater protec- chemotaxis, and other commonly accepted tion to the granulocytes as evidenced by the assay systems (3, 18, 45, 51). least amount of P-glucuronidase release DISCUSSION and, hence, the best preservation of granulocyte integrity. A concentration of Research in our Laboratory of Cryobiol15% glycerol afforded the greatest protec- ogy has been aimed at understanding the tion to the granulocytes frozen at this slow mechanisms involved in achieving succooling rate. cessful cryopreservation of the cellular Based on the data in Fig. 13 which components of blood. Studies on cryoshowed that slow cooling rates at all preservation of peritoneal granulocytes from glycerol concentrations resulted in least rabbits using Me,SO as the cryoprotective damage to lysosomes and best viability, ad- additive indicated that it was feasible to efditional studies were carried out to deter- fect preservation of viable cells. Viability COOLING RATE:

TABLE 2 Effect of Slow Cooling Rates on Granulocyte

Viability

and Yield

Freezing rate l”C/min Trypan

blue

Glycerol (%I

Recovery (%)

exclusion (%)

Nonfrozen 5

100 57 74 82 71

94 47 42 58 73

10 15 20

10Wmin

5”Clmin Yield

Yield viable

(%)

Recovery (%)

exclusion (%)

viable cells (%)

94 29 33 51 53

100 37 47 64 81

94 36 59 65 69

94 14 30 45 59

CdlS

Trypan blue

Yield

viable

Recovery (%)

Trypan blue exclusion (%I

100 40 52 69 84

% 51 36 26 22

96 21 19 18 19

CdlS

(%I

CRYOPRESERVATION

was determined by a phagocytosis assay developed to simulate an important in viva function of granulocytes. Our phagocytosis assay involved measurement of energy utilization and oxygen consumption during phagocytosis of polystyrene latex particles. As an extension of our studies, Skeel et al. (50) have subsequently reported on a more sensitive assay of phagocytosis involving measurement of hexose monophosphate shunt activity using labeled [ 14CJ glucose. From our investigations on other cell systems we have formulated a working hypothesis on freezing injury to explain in part the inability to freeze-preserve such fragile cells as platelets and granulocytes. Pertinent aspects of our working hypothesis are: 1. Lysosomes appear to be implicated in freezing injury. A direct relationship exists between freeze-thaw injury and release of lysosomal enzymes, e.g., p-glucuronidase, cathepsin, and acid phosphatase (10, 13). Studies with platelets show that labilization and release of these hydrolytic enzymes from lysosomes prior to and after freezing result in damage to subcellular organelles which in turn causes concomitant loss of cell function (10, 13). Studies on lysosomes as targets of freezing injury and approaches used to prevent this lysosomal damage have resulted in the development of a new method for cryopreservation of platelets with glycerol and controlled-rate freezing (12). Our studies on granulocyte preservation indicate that lysosomes are key indicators of cryoinjury, but additional research is necessary to extend these studies and to determine the concentration of glycerol and freezing conditions which will prevent cryoinjury to granulocyte lysosomes. 2. The membrane permeability characteristics of granulocytes must allow cryoprotective additives such as glycerol to penetrate readily and exhibit intracellular cryoprotective action. Membrane permeability also creates a problem involving permeability-related size and shape

OF GRANULOCYTES

209

changes to the cell that are potentially damaging. Studies reported here indicate that glycerol is freely permeable in granulocytes under equilibrium conditions although it is not yet known whether glycerol permeation is rate dependent, temperature dependent, and concentration dependent. The membrane permeability characteristics of granulocytes are not completely understood, and as such, improperly controlled addition of cryoprotective agents will adversely affect the physiological and biochemical integrity of these cells through osmotic stress resulting from different degrees of permeability. Additional studies are necessary concerning membrane permeability characteristics of granulocytes as these properties relate to the cryoprotective additives glycerol and Mt;SO. Tlie limits of tolerance of the granulocyte to additives can be defined, and the osmotic damage resulting from addition and removal of these additives may then be minimized. 3. Cryopreservation is a function of both rate of cooling and concentration of cryoprotective additive. This relationship has been observed in red cells and platelets, i.e., the lower the additive (glycerol) concentration, the more rapid the rate of freezing required to achieve preservation of viability (10, 12, 43). Studies reported here on various freezing rates indicate that it is possible to achieve successful preservation of granulocyte lysosomes with glycerol but additional work on granulocyte viability is necessary to establish a direct relationship between freezing rate and glycerol concentration. Further research is required to define more precisely the relationship of cryoprotectant concentration to various freezing regimens to achieve a combination that will provide optimal preservation with full functional integrity and viability of the granulocyte. In this way the goal of developing a simple procedure for cryopreserving granulocytes for transfusion, using glycerol rather than Me,SO as the cryo-

210 protective reached.

ROWE AND LENNY

additive

of choice,

SUMMARY

will

be

cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of &glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of YC/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me,SO) rather than glycerol as the cryoprotective additive of choice. Although Me,SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me,SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine ACKNOWLEDGMENTS use in patients. Therefore, glycerol, with The authors gratefully acknowledge the technical its important advantage of nontoxicity, expertise of S. Chin, G. Dayian, C. Kaczmarek, and has been investigated for its potential use- N. Strick, as well as the secretarial assistance of Mary fulness as a cryoprotective additive for pre- Flynn. serving human granulocytes for transfuREFERENCES sion. 1. Absolom, D. R., and Van Oss, C. J. CryopreserGranulocyte preparations were isolated vation of Ficoll-Hypaque isolated human from impure leukocyte concentrates obgranulocytes. Cryobiology 17, 287-296 (1980). tained from the buffy coats of human whole 2. Bank, H. L. Viability of frozen granulocytes and blood. Studies on the isolation and purifigranulocyte precursors. Cryobioiogy 17, cation of the granulocytes involved separa262-272 (1980). tion by sedimentation with dextran, re- 3. Bannatyne, R. M., and Umamaheswaran, B. Bactericidal function of cryopreserved neutromoval of red cells by hypotonic shock with phils. Cryobiology 10, 338 (1973). water, resuspension with Plasmatein and 4. Bouroncle, B. A. Preservation of human normal further purification by centrifugation. Inand leukemia cells with dimethyl sulfoxide at tact viable granulocytes were obtained with -80°C. Cryobiology 3, 445 (1967). 5. Bouroncle, B. A., Ashenbrand, J. F., and Todd, a purity in excess of 90%. R. F. Comparative study of the effectiveness of Lysosomes were studied as indicators of dimethyl sulfoxide and polyvinylpyrrolidone in cryoinjury in granulocytes using /3the preservation of human leukemic blood cells glucuronidase as the key marker enzyme. at -80°C. Cryobiology 6, 409-415 (1970). This enzyme has been characterized as a 6. Cavins, J. A., Djerassi, I., Aghai, E., and Roy, A. J. Current methods for the cryopreservation sensitive indicator of damage to lysosomes of human leukocytes (granulocytes). Cryobioland a direct linear relationship has been ogy 5, 60 (1968). established between damage to granulo7. Cavins, J. A., Djerassi, I., Roy, A. J., and Klein, cytes by freezing and amount of lysosomal E. Preservation of viable human granulocytes at enzyme released. Addition or presence of low temperatures in dimethyl sulfoxide. Cryobiology 2, 129 (196.5). the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of 8. Crowley, J. P., Rene, A., and Valeri, C. R. The recovery and function of human blood leukointact granulocytes. cytes after freeze-preservation. Cryobiology 11, Studies on the permeation kinetics of 395 (1974). glycerol in granulocytes indicated that the 9. Dankberg, F. L., Persidskv, M. D., Sprung, R. J., and Olson, L. S. Effect of serum on ctyoadditive was freely permeable and did not

CRYOPRESERVATION preservation of granulocytes. Cryobiology 15, 484-487 (1978). 10. Dayian, Cl., Chin, S. N., and Rowe, A. W. Cryoprotection of human platelets: The influence of cooling rate and glycerol concentration on activating the lysosomal enzyme P-glucuronidase. Cryobiology 8, 393-394 (1971). 11. Dayian, G., Chin, S. N., and Rowe, A. W. Differences in human platelet permeability to glycerol and DMSO. Cryobiology 8, 376 (1971). 12. Dayian, G., and Rowe, A. W. Cryopreservation of human platelets for transfusion. A glycerolglucose, moderate rate cooling procedure. Cryobiok~gy 13, l-8 (1976). 13. Dayian, Cl., and Rowe, A. W. Activation of lysosomal enzymes in human platelets during cryopreservation. Cryobiology 6, 579 (1970). 14. Freireich, E. J., Levin, R. H., Whang, J., Carbone, P. P., Bronson, W., and Morse, E. E. The function and fate of transfused leukocytes from donors with chronic myelocytic leukemia in leukopenic recipients. Ann. N. Y. Acud. Sci. 113, 1081-1089 (1964). 15. French, J. E., Jemionek, J. F., and Contreras, T. J. Liquid and cryopreservation effects on in vitro function of dog granulocyte concentrates prepared for transfusion. Cryobiology 17, 252-261 (1980). 16. French, J. E., Flor, W. J., Grisson, M. P., Parker, J. L., Sajko, C., and Ewald, W. G. Recovery, structure and function of dog granulocytes after freeze-preservation with dimethyl sulfoxide. Cryobiology 14, l- 14 (1977). 17. Frim, J., and Mazur, P. Approaches to the preservation of human granulocytes by freezing. Cryobiology 17, 282-286 (1980). 18. Glasser, L. Functional considerations of granulocyte concentrates used for clinical transfusion. Transfusion 19, l-6 (1979). 19. Graham-Pole, J., Davie, M., and Willoughby, M. L. N. Cryopreservation of human granulocytes in liquid nitrogen. J. C/in. Puthol. 30, 758 (1977). 20. Graw, R. G., Herzig, G., Perry, S., and Henderson, E. S. Normal granulocyte transfusion therapy. Treatment of septicemia due to gramnegative bacteria. N. Eng/. J. Med. 287, 367-371 (1972). 21. Hill, R. S., Mackinder, C., Challinor, S., and Postlewaight, B. Recovery of functional capacity of human granulocytes after freeze preservation with dimethyl sulfoxide. Cryobiology 16, 105-111 (1979). 22. Huggins, C. Reversible agglomeration-A practical method for removal of glycerol from frozen blood. In “Modern Problems of Blood Preservation” (W. Spielmann and S. Seidl, Eds.), pp. 138- 155. Fischer Verlag, Stuttgart, 1970.

211

OF GRANULOCYTES

23. Karnovsky, M. L. Metabolic basis of phagocytic activity. Physiol. Rev. 42, 143- 168 (1962). 24. Kessel, D., Blakely, S., Cavins, J. A., Botterill, V., and Hall, T. C. Stability of human leukocyte processes to freeze-thawing. Cryobiology 4, 209 (1968). 25. Knight, S. C., O’Brien, J. A. and Farrant, J. Injury to human granulocytes at low temperatures. Cryobiology 17, 273-281 (1980). 26. Knight, S. C., O’Brien, J. and Farrant, J. Cryopreservation of granulocytes. Cryoimmunoll Cryoimmunol.

Inserm.

62, 139 (1976).

27. Lane, T. A., and Windle, B. Granulocyte concentration functions during preservation: Effect of temperature. Blood 54, 216-225 (1979). 28. Lionetti, F. J., Hunt, S. M., Gore, J. M., and Curby, W. A. Cryopreservation of human granulocytes. Crybiology 12, 181- 191 (1975). 29. Lionetti, F. J., Hunt, S. M., Mattaliano, R. J., and Valeri, C. R. In vitro studies of cryopreserved baboon granulocytes. Transfusion 18, 685-692 (1978). 30. Lionetti, F. J., Luscinskas, F. W., Hunt, S. M., Valeri, C. R., and Callahan, A. B. Factors affecting the stability of cryogenically preserved granulocytes. Cryobiology 17, 297-310 (1980). 31. Malinin, T. L. Injury of human polymorphonuclear granulocytes frozen in the presence of cryoprotective agents. Cryobiology 9, 123-130 (1972). 32. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939 (1970). 33. McCullough, J., Weiblen, B. J., Peterson, P. K., and Quie, P. G. Effects of temperature on granulocyte preservation. Blood 52, 301-3 10 (1978). 34. Meryman, H. T., and Hornblower, M. A method for freezing and washing red blood cells using a 12, high glycerol concentration. Transfusion 145-146 (1972). 35. Meryman, H. T., and Howard, J. Cryopreservation of granulocytes. In “The Granulocyte: Functions and Clinical Utilization” (T. J. Greenwalt and G. A. Jamieson, Eds.), pp. 193-201. Alan R. Liss, New York, 1977. 36. Perry, S. Preservation of leukocytes. In “Hematology” (W. J. Williams, E. Beutler, A. J. Erslev, and R. W. Rundles, Eds.), pp. 1303-1305. McGraw-Hill, New York, 1972. 37. Perry, V. P., Kerby, C. C., and Gresham, R. B. Further observations on the collection, storage and transfusion of peripheral blood leukocytes. Ann. N. Y. Acad. Sci. 114, 651 (1964). 38. Perry, V. P., Malinin, T. I., Kerby, C. C., and Dolan, M. F. Protection of lethally irradiated guinea pigs with fresh and frozen homologous peripheral blood leukocytes. Cryobiology 1,233 (1965).

212

ROWE

AND

39. Rowe, A. W., and Cohen, E. Low temperature preservation of leukocytes: Freezing technique and in vitro viability criteria. (Proc. XI Congr. Int. Sot. Blood Transfusion, Sydney, 1966.) Biblio. Haemarol. 29, 779-787 (1968). 40. Rowe, A. W., and Cohen, E. Phagocytic activity and antigenic integrity of leukocytes preserved in DMSO at a cryogenic temperature (- 196°C). VOX Sang. 10, 382-385 (1%5). 41. Rowe; A. W., Derrick, J. B., Miles, W., Allen, F. H., Jr., and Kellner, A. Transfusion of blood frozen by the low glycerol-rapid freeze process. Biblio. Haemarol. 38, 320-334 (1971). 42. Rowe, A. W., Derrick, J. B., Miles, W., Allen, F. H., Jr., and Kellner, A. The biochemistry and clinical use of red cells frozen by the low glycerol-rapid freeze technique. In “Modern Problems of Blood Preservation” (W. Spielman, and S. Seidl, Eds.), pp. 184- 198. Fischer Verlag, Stuttgart, 1970. 43. Rowe, A. W., Eyster, E., and Kellner, A. Liquid nitrogen preservation of red blood cells for transfusion: A low glycerol-rapid freeze procedure. Cryobiology 5, 119- 128 (1968). 44. Rowe, A. W., Kaczmarek, C. S., and Cohen, E. Low temperature preservation of leukocytes in dimethyl sulfoxide. Fed. Proc. 22, 170 (1963). 45. Roy, A. J. Methods for assaying viability of frozen-thawed granulocytes. Cryobiolugy 15, 232(1978). 46. Schwarzenberg,

L., Mathe, G., Amiel, J. S., Cattan, A., Schneider, M., and Shlumberger, J. R. Study of factors determining the usefulness and complications of leukocyte transfusions. Amer. J. Med. 43, 206 (1967). 47. Shohet, S. B. Morphologic evidence for the in viva activity of transfused chronic myelogenous leukemia cells in a case of massive staphylococcal septicemia. Blood 32, 111 (1968). 48. Shohet, S. B., and Mohler, W. C. Low temperature preservation of chronic myelogenous leukemia cells. Cryobiology 4, 47 (1967). 49. Showell, H. J., Naccache, P. H., Sha’ati, R. I., and Becker, E. L. The effect of extracellular K+, Na+ and Ca++ on lysosomal enzyme secre-

LENNY

tion from polymorphonuclear leukocytes. J. 119, 804-811 (1977). 50. Skeel, R. T., Yankee, R. A., Spivak, W. A., Novikovs, L., and Henderson, E. S. Leukocyte preservation. I. Phagocytic stimulation of the hexose monophosphate shunt as a measure of cell viability. J. Lab. Clin. Med. 73, 327-337 (1969). 51. Stossel, T. P. Neutrophil function tests and neutrophil transfusion. Exp. Hematol. (Suppl) 5, 9 Immunol.

(1977). 52. Stossel, T. P. Phagocytosis. In “The Granulocyte:

Functions and Clinical Utilization” (T. J. Greenwalt and G. A. Jamieson, Eds.), pp. 87-102. Alan R. Liss, New York, 1977. 53. Stossel, T. P. Phagocytosis, I-III. N. Engl. J. Med. 290,717-723,

774-780,

833-839

(1974).

54. Talalay, P., Fishman, W. H., and Huggins, C.

Chromogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. J. Biol. Chem. 166, 757 (1946). 55. Talstad, I. The relationship between phagocytosis of polystyrene latex particles by polymorphonuclear leukocytes (PML) and aggregation ofPML. Stand. J. Hemarol. 9,515-523 (1972). 56. Tullis, J. L., Gibson, J. G., II, Sproul, M. T., Tinch, R. J., and Baudanza, P. Advantages of the high glycerol mechanical systems for red cell preservation; a IO-year study of stability and yield. In “Modern Problems of Blood Preservation” (W. Spielmann and S. Seidl, Eds.), pp. 161-167. Fischer Verlag, Stuttgart, 1910. 57. Valeri, C. R. “Blood Banking and the Use of Fro-

zen Blood Products.” CRC Press, Cleveland, Ohio, 1976. 58. Yankee, R. A., Freireich, E. J., Carbone, P. P., and Frei, E., III. Replacement therapy using normal and CML leukocytes. BIood 24, 844 (1964). 59. Zaroulis, C. G., and Leiderman, I. Z. Successful freeze-preservation of human granulocytes. Cryobiology 17, 311-317 (1980).