Preservation of polymorphonuclear neutrophils at cryogenic temperatures (−80 °C)

CRYOBIOLOGY

17, 112- 119 (1980)

Preservation

of Polymorphonuclear Temperatures

Neutrophils (-80°C)

at Cryogenic

TATSUHISA YAMASHITA Laborarory

of Physiological

Chemistry,

School of Medicine, Tokyo, Japan 113

In a previous study (16), the effect of commonly employed cryoprotective agents such as dimethyl sulfoxide (Me,SO), glycerol, and ethylene glycol upon cell function was examined as a first step to select a cryoprotectant of choice for the freeze-preservation of polymorphonuclear neutrophils (PMNs). It was found that chemotactic migration, the inhibition of which appeared to be a more sensitive indicator among impaired leukocyte functions, hardly was affected by the exposure of PMNs to cryoprotectant and subsequent washing, provided that a low concentration was used. In this communication, therefore, we report that the cryoprotective agent at a low concentration was used alone or in combination with additives such as albumin, ATP (adenosine triphosphate), and glucose to protect guinea pig neutrophils against freeze-thaw injury. MATERIALS

AND

METHODS

Polymorphonuclear neutrophils (PMNs) obtained from 0.12% glycogen-induced peritoneal exudates of guinea pigs were washed twice with phosphate-buffered saline (PBS) without divalent cations [PH 7.4, PBS(-)] after removal of contaminating erythrocytes by a hypotonic treatment with 0.2% saline and finally suspended in phosphate-buffered saline containing 1 mM MgSO, and 0.07 mM CaCl, (PBS) as described previously Leukocytes.

(16). Received June 7, 1979; accepted December

14, 1979.

Copyright All rights

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

University,

Bunkyo-ku,

Preservation. To the freezing medium containing 0.45 ml PBS, 0.5 ml of PMN suspension (2 x lo7 cells/ml), and 0.2 ml of the additive (such as bovine albumin, ATP, and glucose) in PBS, the endocellular cryoprotectant (0.05 ml) was added slowly to a concentration of 4.2%. The final concentrations of albumin were 1,2,4, and 8%; glucose, 2.5, 5, and 7.5%; and ATP, 2.5, 7.5, 15, and 50 mM. In the case of ATP, cryoprotectants were added after preincubation of PMNs in the freezing medium containing ATP for 1 hr at room temperature. In the control PMN sample, 0.2 ml PBS was used instead of the additive. These freezing mixtures were frozen to -80°C in sealed plastic pipet tubes using a deep freezer. The period of storage varied from 1 hr to 1 week. Examination of cells was carried out after rapid thawing in a 37°C water bath and two washes (added very slowly) with a large excess of PBS (ca. 10 ml). Trypan blue exclusion, chemotaxis, and phagocytosis. These functions were as-

sayed essentially as described previously (16). Chemotaxis was assayed in a modified Boyden chamber using a Toyo membrane filter (pore size 3 pm) in the presence of a chemoattractant (the supernatant from a sonicated suspension of Escherichia coli grown for 20 hr at 37°C in a polypeptone medium). Chemotaxis is expressed as the distance from the top of the filter to the farthest two cells at the same focal plane, according to the method of Zigmond and Hirsch (18). The phagocytosis assay was modified slightly. As reported previously 112

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CRYOPRESERVATION

(16)) under our experimental conditions, the percentage of cells containing bacteria increased with increasing incubation time, but heat-killed PMNs (lOO’C, 20 min) did not show such an increase, indicating a true ingestion of bacteria by non-heat killed PMNs. In this paper, therefore, the ability of PMNs to phagocytize bacteria was examined only with PMNs incubated with heat-killed bacteria for 30 min under the same conditions as reported previously. TO the incubation mixture containing 0.5 ml PMN suspension ( lo7 cells/ml), 0.3 ml PBS, and 0.1 ml fresh homologous serum in a siliconized Pyrex tube was added 0.1 ml of saline suspension containing Sfaphylococcus aureus (lo* cells/ml). The tubes were shaken continuously at 37°C. After a 30-min incubation, the PMN suspension was smeared on a glass slide, fixed with methanol, and stained with Gram stain. Heat-killed PMNs were treated under the same conditions except that a heat-killed PMN suspension was used to check the possibility that PMNs had adsorbed bacteria outside the cells. Ingestive ability was expressed oy subtracting the percentage of cells with bacteria obtained with heat-killed PMNs from the percentage of cells with bacteria obtained with non-heat treated PMNs. In some cases, after 30 min incubation, aliquots of the PMN suspension were washed twice with PBS, suspended in PBS containing 4% autologous serum, and smeared. The result was almost the same as that obtained by the above procedure. Adhesion. After thawing and two washes with PBS, PMNs in a density of 107/mlwere suspended in PBS with bovine albumin at a concentration of 4 g/100 ml (pH 7.4, albumin-PBS). One milliliter of cell suspension was placed onto a column of a 5-ml plastic syringe containing 5 g of glass beads, equilibrated with 1 ml of albumin-PBS. One milliliter was taken out by dropping from the bottom of the column. After standing for 15 min, cells were eluted with 4 ml of albumin-PBS at the rate of about 1

113

OF GRANULOCYTES

ml/min. The procedure was carried out at 37°C. Total PMN counts were made on the starting cell suspension and eluates. Cyanide-insensitive oxygen uptake durOxygen consumption ing phagocytosis.

was measured polarographically with a Clarke oxygen electrode (Rank Brother, England) with magnetic stirring at 37°C. The period chosen for the measurement of oxygen consumption was the first 7-8 min following the brief lag period. The reaction started with the addition of bacteria. The assay medium contained 2 ml PMN suspension (2 x 10’ cells/ml), 0.02 ml of 0.2 M KCN, and 0.1 ml of heat-killed Staphylococc~s aureus (lOlo cells/ml) in saline. In the respiration experiment, the freezing mixture contained 2.2 ml of PMN suspension (2 x lo7 cells/ml), 0.44 ml of the additive, and 0.11 ml of Me,SO. After freezing and thawing, PMNs were washed twice with Krebs - Ringer phosphate buffer without Ca”+, pH 7.4 (KRPB), and suspended in 2.2 ml of KRPB. Materials. Bovine albumin (Fraction V Powder) was purchased from Sigma Chemical Company. Glass Beads (Glasperlen, 0.45-0.50 mm in diameter) were obtained from B. Braun Melsungen A. G. Membrane filters (TM-300, 3.0 pm, size 25 m/m) were obtained from Toyo Roshi Company, LTD. RESULTS

Effect of cryoprotectant on postfreezing neutrophil chemotaxis and trypan blue exclusion. The inhibition of chemotactic

migration has proven to be a more sensitive indicator of impaired leukocyte function (16), therefore, the degree of integrity of postfreezing PMNs was first examined by checking the degree of inhibition of chemotaxis along with trypan blue exclusion. Freezing PMNs without cryoprotective agents caused the death of cells after even 1 hr of storage, while PMNs with cryoprotectants survived, indicating that cryoprotective agents were able to protect

114

TATSUHISA

YAMASHITA

TABLE 1 Effects of Cryoprotective Agents on Postfreezing Neutrophil Chemotaxis

Agent

Concentration (%)

Neutrophil migration (Percentage of control)” 1 hr

24 hr

72 hr

Me&30

4.2 8.3

57 + 16b 32 t 8

33 2 10 34 * 20

30 + 17 15 f 7

Ethylene glycol

4.2 8.3

18 t- 8 82-6

13 + 8 ST1

10 t 5 5k-2

Glycerol

4.2 8.3

623 421

321 221

321 321

” Neutrophil migration is expressed as a percentage of migration compared to the control cells. Control = chemotaxis of washed fresh PMNs after preparation. The migration value for washed fresh cells is 55 + 5 wrn in 60 min at 37°C. b The mean 2 standard deviation of four samples.

hr cryostorage when frozen in Me,SO. granulocytes from cold damage during cryostorage. However, a considerable dif- These findings suggest that Me,SO would ference among the cryoprotective agents be superior to ethylene glycol and glycerol was observed in protective effectiveness as a cryoprotective agent (cf. (16)). Thus against freeze-thaw injury of PMNs. As Me,SO was used as the cryoprotective seen in Table 1, the protective effect of the agent in subsequent experiments. Combined effects of cryoprotectant and cryoprotectant on chemotactic inhibition on postfreezing neutrophil depended upon the type and concentration additive of the cryoprotectant and the cryostorage chemotaxis and dye exclusion. As can be seen in Table 3, chemotaxis of PMNs froperiod. Cryoprotectants in a concentration zen in Me,SO alone was considerably supof 4.2% protected PMNs more effectively from chemotactic inhibition than did these pressed with increase in preservation time, agents at a concentration of 8.3% where resulting in approximately 70% inhibition of only Me,SO exhibited a protective effect. chemotaxis in 3 days when most PMNs could exclude the dye (Table 4). However, When the agent was used in a concentration of 4.2%, PMNs frozen in Me,SO retained l-week preservation brought about the approximately 60% of their chemotaxis as death of 50% of the cells with about 80% compared to control PMNs in 1 hr, and about 30% of the control’s migration in 24 TABLE 2 and 72 hr. However, the chemotaxis of Effects of Cryoprotective Agents on Postfreezing PMNs frozen in glycerol was completely Neutrophil Trypan Blue Exclusion suppressed after even 1 hr of storage. Trypan blue exclusion (%) Ethylene glycol, too, showed little effecConcentration tiveness. On the other hand, trypan blue Agent 1 hr 24 hr 72 hr W) exclusion by PMNs was affected by the 4.2 89 + 5” 86 k 7 84 c 5 type of cryoprotectant and the concentra- lb&SO 8.3 81 k5 6ak12 78*9 tion of the agent, and the storage time had Ethylene 4.2 82 r 6 76 + 3 66 + 10 a.3 65 2 12 71 k 8 51 + 10 little effect (Table 2). Greater than 60% of glYCO1 4.2% glycerolized frozen PMNs could not Glycerol 4.2 34 k 16 28r20 2429 8.3 38 k 9 33 r 12 32 -c 12 exclude the dye after even 1 hr, while at 0 The mean k standard deviation of four samples. least 80% PMNs were viable even after 72

CRYOPRESERVATION

OF GRANULOCYTES

115

TABLE 3 Chemotaxis of Polymorphonuclear Neutrophils Frozen in Me,SO with and without Additives Neutrophil migration (Percentage of control)” Additive

Concentration

1 hr

24 hr

72 hr

168 hr

None

-

59 2 15h

37 + 15

28 k 14

16 t 8

Glucose

2.5% 5% 7.5%

52 t 20 49 k 19 33 t 12

34 + 14 23 + 14 5-+2

30 -c 18 26 k 18

20 + 6 10 lr 6 -

35 + 15 40 2 20 34 + 21 aggregated

34 + 17 36 + 18 29 + 18 -

24 k 22 21 k 17 17 5 18 -

46 + 51 r 49 f 38 k

18 k 5 27 + 10 22 % 8 22 2 14

ATP

Albumin

2.5 mM 7.5 mM 15 mh4 50 mM 1% 2% 4% 8%

71 5 78 t 77 t 56r

13 9 5 16

81 t 85 ? 84 t 75 k

18 17 17 14

55 k 53 k 49 t 44 +

21 16 18 17

18 12 16 10

a Neutrophil migration is expressed as a percentage of migration of control cells. Control = chemotaxis of washed fresh PMNs without Me,SO or additives after preparation. The migration value for washed fresh cells is 69 ? 20 pm in 60 min at 37°C. h The mean rt standard deviation of four to nine samples.

chemotactic inhibition. Therefore, the combined effects of Me&SO and albumin (a type of exocellular cryoprotectant), or energy-supplying agents such as glucose and ATP (widely used in freezepreservation of red blood cells) were studied. The combination of Me,SO and glucose did not cause improvement in

chemotactic inhibition, as compared to Me&SO alone, but rather completely inhibited chemotaxis in the 24-hr samples at a high concentration of glucose. Viability of PMNs at that time also had decreased considerably. Addition of ATP to the preservation medium did not bring about a very remarkable improvement in chemotaxis

TABLE 4 Trypan Blue Exclusion of Polymorphonuclear Neutrophils Frozen in Me,SO with and without Additives Trypan blue exclusion (%) Additive None Glucose

ATP

Albumin

Concentration

1 hr

24 hr

72 hr

168 hr

-

90 k 5”

83 2 13

87 + 4

53 2 5

79 _f 14 88 + 12 83 r 13

74 _t 10 71 iz 10 44 + 10

78 -c 8 67 k 12 35 ? 9

48 f 18 55 ? 12

77 + 15 77k 11 73 2 10 aggregated

79 s 19 82 2 6 78 5 22 -

44 k 20 44 e 23 39 f 21

88 + 89 iz 85 2 79 +

57 ? 59 + 54 2 65 k

2.5% 5% 7.5% 2.5 mM 7.5 mM 15 mA4 50 mM 1% 2% 4% 8%

88 2 88 t 87 + 88 r

7 7 9 7

92 2 92 r 93 + 94 +

6 1 4 5

0 The mean f standard deviation of four to nine samples.

77 2 80? 85k 83 2

17 12 11 9

4 3 13 11

23 25 24 21

116

TATSUHISA

YAMASHITA

TABLE 5 Effects of Additives on Chemotaxis and Trypan Blue Exclusion of Neutrophils Frozen in Ethylene Glycol or Glycerol Neutrophil migration” (% of control)

Trypan blue exclusion (%)

Additive

1 hr

24 hr

72 hr

1 hr

Ethylene glycol None Glucose ATP Albumin

13 k 5” 7+1 13 + 5 12 lr 6

11 -1-4 12 2 7 10 k 4 14 2 8

8+2 10 k 6 7*1 422

83 -+ 9 71 2 2 72 + 3 84 k 8

75 k 53 k 49 f 81 +

1 6 8 14

67 2 56 + 64 + 83 +

Glycerol None Glucose ATP Albumin

7t7 725 4?2 5kl

4kl 4kl 522 4+1

27 + 41 r 29 + 56 +

17 + 15 + 15 + 53 2

2 2 4 13

19 f 4 27 + 7 25 YL16 52 4 14

421 3*1 3+2 2+1

2 24 10 15

24 hr

72 hr 8 12 9 3

(1Neutrophil migration is expressed as a percentage of migration of control cells. Control = chemotaxis of washed fresh PMNs without cryoprotectant or additives after preparation. The migration value for washed fresh cells is 53 ? 4 pm in 60 min at 37°C. h The mean of two samples. The concentration of additives was as follows: glucose, 2.5%; ATP, 7.5mM; albumin, 4%.

and dye exclusion, although it did exert to less inhibited than was chemotaxis. For some degree a protective effect against example, when forzen in Me,SO alone, freeze injury in 1-hr preservation. However, only a 20-30% inhibition of phagocytosis at ATP concentrations of 50 n&f, all PMNs and adhesion was observed as compared were dead with aggregation in 24 hr- with the control, although chemotaxis was preserved samples. Among the additives inhibited by 70% (cf. Table 3). A combinaused here, albumin gave the most potent tion of Me,SO and glucose did not improve the protective effect of Me,SO, nor did alprotection against chemotactic inhibition regardless of concentration used, whereas bumin, which displayed protection against chemotactic impairment. On the other no distinct effect of albumin on viability was observed when compared to Me,SO hand, the combination of Me,SO and ATP alone. Table 5 shows the combined effects markedly protected phagocytosis and adheoxygen conof ethylene glycol or glycerol with these sion. Cyanide-insensitive additives on chemotaxis and dye exclusion. sumption of PMNs during phagocytosis Neither chemotactic inhibition nor the abil- was inhibited considerably with all PMNs ity to exclude dye was improved by the after 3-day freeze preservation regardless combination of these agents with additives of the presence of additive. except in the case of albumin where a DISCUSSION cumulative effect on protection against dye exclusion injury was observed. Dye exclusion, chemotaxis (motility), phagocytosis, oxygen consumption, inhibiRecovery of functions of postfreezing neutrophils other than chemotaxis. The de- tion of bacterial growth, myeloperoxidase gree of the recovery of functions other than (l), etc., commonly are employed as the in chemotaxis was studied with PMNs after vitro methods in assessment of granulocyte freeze-preservation for 3 days when most integrity. These functions, however, were cells still were viable. As can be seen in impaired to different degrees by freezing Table 6, phagocytosis and adhesion were and thawing, and chemotaxis and oxygen

CRYOPRESERVATION

117

OF GRANULOCYTES

TABLE 6 Phagocytosis, Adhesion, and Oxygen Consumption of Neutrophils Frozen in Me&SO for 3 Days with and without Additives Additive

Phagocytosis’

Control None Glucose ATP Albumin

64 + 3 49 k 8 48 -t 10 57 IT 6 45 + 10

Adhesiveness* 69 k 48 k 39 t 63 f 43 2

7 14 19 15 13

0, consumption’ 124 + 29 k 27 t 36 f 28 +

23 8 12 12 20

o Percentage of PMNs containing bacteria. * Percentage of PMNs which are adherent. c Atoms O/lW cetls/hr. The concentration of additives was as follows: glucose, 2.5%; ATP, 7.5mM; albumin, 4%. ” The mean f standard deviation of four to six samples. Control = washed fresh PMNs after preparation.

consumption appeared to be the functions most impaired (3, 5, 11, 12). In a previous paper (16), we also found that chemotactic migration was more sensitive to exposure to cryoprotectants than were other functions that we had examined. Therefore, the protective effect of the cryopreservatives against freeze injury to neutrophils was first examined on the basis of recovery of chemotaxis and trypan blue exclusion ability, dependent on the intactness of the plasma membrane structure after freezing and thawing. When frozen in ethylene glycol and glycerol, PMNs almost completely lost chemotactic migration, and dye exclusion ability decreased particularly in glycerolized frozen PMNs to about 40% after even 1 hr of storage at concentrations of 4.2% (Table l), suggesting that damage to the plasma membrane structure had occurred due to short-term cryopreservation. On the other hand, of the cryoprotective agents used, Me,SO exhibited the greatest protective effect and its optimal concentration was approximately 4.2% (Table 1). These findings are coincident with the previous results that Me,SO, especially at a low concentration, scarcely impaired neutrophil function, which led us to expect the superiority of Me,SO over glycerol and ethylene glycol as a cryoprotectant (16). Among the functions assessed other than chemotactic migration, increased oxygen

consumption during phagocytosis that resulted from stimulated NADPH oxidase, which produces highly reactive microbicida1 agents by the reduction of oxygen (2, 13, 14) was considerably suppressed (Table 6). Results have indicated that chemotaxis and oxygen consumption, the respective trigger sites which are presumed to be localized in the plasma membrane (4, 7, 13, 17), were very sensitive to freeze injury where dye exclusion ability remained less inhibited. These findings suggest that cryopreservation would produce the relatively specific damage of these plasma membrane-associated sites without detectable alterations in the cell membrane structure. The recovery of the PMNs’ ability to exclude dye particularly was improved by the presence of albumin in the freezing medium. Since albumin does not penetrate the cell, the albumin-induced protection seems to occur at the outer surface of the plasma membrane by protecting the membrane structure from freeze-damage through shielding the cell. The mechanism of the protective effect of ATP on the recovery of adhesion, phagocytosis, and chemotaxis (1 hr storage) is not clear. However, since these functions are thought to be associated with the superficial membrane-associated contractile mechanism of cytoplasm (6,8- 10, 15), ATP might

118

TATSUHISA

affect the stabilization of the contractile system directly or indirectly. SUMMARY

The effects of cryoprotectant alone and cryoprotectant plus additives on the preservation of polymorphonuclear neutrophils (PMNs) at cryogenic temperatures (- 80°C) were studied. A considerable difference among cryoprotective agents was observed in their protective effect against freeze injury of neutrophils. Based upon degree of chemotactic inhibition and impairment of trypan blue exclusion, Me,SO proved to be superior to ethylene glycol and glycerol as a cryoprotectant and to exhibit the best protective effect at a concentration of 4.2%. When PMNs were frozen in Me,SO alone, the ability of PMNs to exclude dye was retained after 3 days of cryopreservation, while chemotaxis was inhibited markedly. One-week preservation produced the death of 50% of the cells. To improve the protective effect of Me,SO against chemotactic inhibition by cryopreservation, additives such as glucose, ATP, and albumin were included in the freezing medium. Addition of albumin displayed the most distinct improvement in the recovery of chemotaxis, although ATP also exhibited a protective effect, especially during short-term storage. Studies on the combined effect of these additives with ethylene glycol or glycerol showed that only albumin had a considerably better protective effect against dye exclusion injury but not against chemotactic inhibition. Phagocytosis and adhesion were less inhibited by freezing than was chemotaxis. A combination of Me,SO and ATP markedly protected phagocytosis and adhesion from freeze injury. However, cyanide-insensitive oxygen uptake during phagocytosis, as well as chemotaxis, were considerably inhibited. ACKNOWLEDGMENT

The author thanks Miss E. Fujikawa for her technical assistance.

YAMASHITA

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