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Isolation and cryopreservation of human peripheral blood monocytes
CRYOBIOLOGY
Isolation
23, 531-536 (1986)
and Cryopreservation
TS. TSVETKOV,
of Human Peripheral
Blood Monocytes
CH. NICKOLOV, A. BUCKURESHTLIEV, L. TSONEY, R. TERZIEV, AND D. POPOV
Central Problem Laboratory
for
Cryobiology
and Freeze-Drying.
M. MINCHEFF,
Sofia, Bulgaria
A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983)) is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up IO 0.65 x lo9 cells/75 cm* surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution. 0 1986 Academc he,,. Inc.
Monocytes can be obtained from peripheral human blood by several techniques based on functional (adherence) or physical (density) properties of these cells. Methods based upon cell density arc often used in the United States (6, 7, ll), but their widespread application has been hampered elsewhere by high equipment costs (e.g., elutriators). Low yields of viable monocytes have been reported with the older methods, using adherence of cells to glass or plastic surfaces (1, 9, 10). Recently Freundlich and Avdalovic (4) reported a method for isolation of viable monocytes. The technique includes reversible attachment of monocytes on plasma-gelatin coated flasks through specific receptors on the cell membrane for Iibronectin (FN) (2). As long as the cell density of the suspension, which is layered on the plasma-gelatin coated surface, is low (2 x lo6 cells/ml), this method is suitable for obtaining monocytes for laboratory use only. A possible way to increase monocyte yield per unit area could be sought by multiplying the FN active sites on the gelatin-glass surface, provided that the bulk of the plasma fibronectin is present in the cryoprecipitate. The latter could be used instead of plasma for coating the bottom surface of standard glass flasks.
Received June 18, 1985; accepted May 21, 1986
On the other hand the current processing of donated blood often leaves the buffy coats out of use. A possible benefit could be sought through the utilization of pooled buffy coats for obtaining monocytes. The proper utilization of monocytes often requires cryoprcservation, and in the present paper we investigate the possibility to freeze cells isolated by such a technique. MATERIALS
AND METHODS
Buffy coats (BC I) are separated after centrifugation of several ACD preserved blood units, pooled together, and stored at 2-8°C for 24 hr. Leukocytes in BC I are concentrated through centrifugation and BC II is collected. Each 100 ml BC II is diluted in 25 ml ACD plasma and in 25 ml isotonic saline prior to use. Glass flasks (Usifroid type, 500 ml, 75 cm2 bottom surface) are rinsed with 5 ml gelatin solution (10% w/v in water) and incubated for 3 hr at 160°C. Cryoprecipitate is obtained from previously converted heparinized ACD plasma (5000 E heparin and 6 ml 1 M CaCl, are added consecutively to 100 ml ACD plasma) and it is dissolved in l/5 vol of the cryosupernatant plasma. Ten milliliters dissolved cryoprecipitate is added to each gelatin coated flask. After incubation at 37°C for 30 min, the dissolved cryoprecipitate is removed and the flasks are washed with 10 ml MgCl, enriched isotonic saline (0.836 g
531 001 l-2240186 $3.00 Copyright 0 1986 by Acadenuc Press. Inc. All nehts of reproductmn rn any term reserved
532
TSVETKOV
MgCl,, 6 H,O in 500 ml isotonic saline). The gelatine-cryoprecipitate coated flasks can be dried at 37°C after washing and stored at room temperature for at least 1 month. Mononuclear leukocytes are obtained from diluted BC II by centrifugation on Polysep (Pharmachim, Bulgaria) denisty gradient (d = 1077 g/cm3) (Boyum, 1968). Mononuclear cells, isolated from 100 ml BC II, are purified from platelets by centrifugation at 540g for 20 min and resuspended in 100 ml 20% human serum in MgCl,-enriched isotonic saline. (The ABO compatibility of sera is of no importance.) Erythrocyteigranulocyte sediment (EGS) is left for further fractioning. Twenty-five milliliters of the mononuclear suspension is layered on each of the bottoms of the gelatine-cryoprecipitate treated flasks and these are incubated at 37°C for 40 min. Nonadherent cells are transferred into a separate vessel and the flasks are washed gently four times with 25 ml of MgCl,enriched saline. Adherent cells in each flask are detached after the addition of 25 ml EDTA buffer (0.2 g NaEDTA in 500 ml Dulbecco’s medium, pH = 7.2), vigorous agitation, and incubation at 22°C for 15 min. followed by more vigorous agitation. The EGS is diluted up to 100 ml vol in isotonic saline and left at 22°C. The supernatant is observed after intervals of 30 mitt, 1 hr, and 2 hr, for leukocyte type content. The adherent mononuclear cells are concentrated five times and slowly diluted with
ET AL.
an equal volume of precooled to 4°C cryoprotective solution (20 ml Me,SO + 20 ml human serum + 60 ml isotonic saline). The diluted cell suspension is transferred into aluminium (thin wall) containers. The thickness of the frozen specimen is 2 mm. The containers are left at - 30°C for 60 min and then transferred into liquid nitrogen. The specimens are thawed in water bath at 40°C with constant agitation for 15-20 sec. The samples are diluted four times the volume with precooled to 4°C isotonic sucrose. To define the cell type and their functional activity, the following criteria are used: -Cell number is estimated by a routine method in a Burker’s chamber. -Cell type is defined on a Giemsa stained smear. -Functional activity is determined using the test for phagocytosis of formaldehyde-treated red blood cells (8). The serum is removed after the incubation when the phagocytosis is performed with cryopreserved cells. The cells are washed with isotonic saline after vigorous agitation, and resuspended in human serum before the smear is made. The percentage of phagocytizing cells is defined as the average of 1000 counted. -The determination of nonspecific esterase is done by a routine cytochemical method (5). RESULTS
Each 100 ml of BC 1 contains 1.58 * 0.06
TABLE I Total Number of Cells and Number of Mononuclear Cells ( x IO9 cells) Isolated from 100 ml BC 1 in Different Stages of Isolation Procedure (Mean Values Out of 23 Experiments) Cell fractions BC 1
BC II
1.58 k 0.06 k 0.05
1.32 2 0.07 0.90 k 0.05
Cell contents Total cell count Mononuclear cells a IL, intermediate
layer.
0.97
IL” 0.614 0.595
k 0.03 2 0.04
EGS 0.518 i- 0.0s i- 0.01
0.144
CRYOPRESERVATION
0
533
OF HUMAN BLOOD MONOCYTES
0.30
s
+
it l
c +
f
0.25
CI +
+
+
a+
:
+
+ + l
/’
+
z
0.05
-D
10
$ I
0
0.25
0.5 0.75
1.0
Numberof mononuclear cells 1x10’) layered on 75 cm2surfece FIG. I. Relationship between cell density of the layered suspension and (a) the yield of adherent phagocytizing cells; (b) the loss of nonattached phagocytizing cells; (c) the loss of nonattached monocytes (96). Mean values of 23 experiments. (+) One run value; (0-j mean values of esterase-positive cells; (0- - -) mean values of phagocytizing monocytes.
x lo9 leukocytes (0.97 2 0.05 x IO9 and at the mononuclear cell number is remononuclear cells). The preparation of BC duced only by 7.22% (Table 1). The Polysep II leads to a loss of total 16.46% leukocytes system used in these experiments yields a
FIG. 2. Phagocytosis performed with fresh monocytes (X 800).
534
TSVETKOV ET AL.
Freeze-Thaw-Wash
TABLE 2 Recovery of Monocytes (Percentage)
1 Month storage
2 Months storage
N
After thawing
After washing
After thawing
After washing
1 2 3 4 5 Mean
89.70 96.16 76.28 74.34 72.00 81.69 2 4.75
88.23 100.00 76.28 75.47 86.66 85.33 ir 4.50
88.23 100.00 82.06 67.92 72.00 82.04 t 5.75
91.17 100.00 80.90 67.92 76.66 83.33 k 5.60
fraction consisting of 97.65% mononuclear cells, 24.1% of which are phagocytizing. The average yield in the intermediate layer is 0.614 -+ 0.03 x lo9 cells (0.595 +- 0.04 X IO9 are mononuclear cells). The EGS contains 0.518 + 0.05 x lo9 leukocytes and 27.8% of them are mononuclear. The percentage of phagocytizing cells in all the 15 experiments carried out coincides with the percentage of polymorphonuclear cells in the sediment. The centrifugation of the suspension on a Polysep gradient and the following removal of platelets results in 14% loss of the original leukocyte number. The unrecovered cells are mononuclear (Table 1). The relation between the number of mononuclear cells layered on a certain gelatin-cryoprecipitate surface and the yield of adherent cells is shown in Figs. la, b, and c. The number of adherent cells in-
creases proportionally to the number of mononuclear cells layered, up to a certain extent (0.188 X lo9 monocytes/75 cm2 surface), then remains constant (Fig. la). After that point the number of phagocytizing nonattached cells in the suspension above the gelatin-cryoprecipitate surface increases sharply (Figs. lb and c). When x lo9 the optimum cell density-O.65 cells/75 cm2 surface-is used, the yield of monocytes is about 70% of their original content in the BC I. All adherent and eluated cells are esterase positive, but only 71.35% of them are phagocytizing (Fig. 2). Freeze-preservation results in about 17% loss of the original cell content. The cell recovery ranges from 68 to lOO%, mean 83%. No cells are damaged in the washing procedure and a prolonged storage of frozen cells up to 2 months does not lead to additional injury of cells (Table 2). Cryopreser-
TABLE 3 Changes in Phagocytic Activity of Monocytes after Cryopreservation Percentage of monocytes which phagocytize during storage period N
Fresh monocytes
48 hr
1 month
2 months
1 2 3 4 5 Mean
65 57.2 62.2 75 65 64.9 k 5.6
52 61 41 50 47.4 50.3 k 3.5
42 54 48 51 48.2 48.6 + 2.8
44 48.2 45.1 40 48 45 f 1.5
Phagocytic activity of cryopreserved monocytes as percentage of the phagocytic activity of fresh monocytes 100 77.5
75
69.5
CRYOPRESERVATION
OF HUMAN BLOOD MONOCYTES
535
FIG. 3. Phagocytosis. performed with cryopreserved monocytes ( x 800)
All the adherent and eluated cells are esterase positive, but only 70% of them are phagocytizing. A possible reason could be the preliminary leukocyte storage as BC I DISCUSSION at 2 to 8°C for 24 hr. Most probably no Our results show that monocytes can be mononuclear phagocytizing cells are left in isolated by this method with utilization of EGS because the percentage of phagocycryoprecipitate instead of plasma. Cryo- tizing cells in this sediment coincides with precipitate contains more of the fibronectin the percentage of polymorphonuclear cells of the bulk plasma and it can be used for present. As only monocytes are esterase positive coating the glass-gelatin surface. In such a case an optimum cell density at certain (among mononuclears), the eluated adconditions could be reached, Most prob- herent cells are, therfore, monocytes. The ably the employment of such cell density latter could be successfully cryopreserved saturates all the active FN sites. Otherwise by routine methods. the use of diluted suspensions will result in REFERENCES lower yields (Fig. la) while the utilization of more concentrated suspensions leaves a 1. Absolm, D. R., van Oss, C. .I., and Neuman, A. V. Elution of human granulocytes from significant number of phagocytizing mononylon filters by means of van der Waals repulnuclear cells nonattached in the suspension sion forces. Transfusion, 21, 663 (1981). (Figs. lb and c). 2. Bevilaqua, M. P., Ambrani, D., Mosesson, M. W., et al. Receptors for cold-insoluble globThese results strongly support the obserulin (plasma fibronectin) on human monocytes. vation that only 30% of the monocytes are J. Exp. Med. 153, 42 (1981). lost when optimum cell density of the sus- 3. Boyum, A. Separation of leukocytes from blood pension, which is layered on the active surand bone marrow. &and. J. Clin. Lab. Invest. face, is used. (Suppl.) 21, 97 (1968). vation of the cells causes about 25-30% loss of phagocytizing activity regardless of the storage period (Table 3, Fig. 3).
536
TSVETKOV ET AL.
4. Freundlich, B., and Avdalovic, N. Use of gelatinplasma coated flasks for isolating human peripheral blood monocytes. J. Immunol. Methods 62, 31(1983).
5. Hayhoe, F. G. J., and Quaglino, D. “Haematological Cytochemistry.” Churchill Livingstone, EdinburglLondoniNew York, 1980. 6. Hunt, S. M., Lionetti, F., and Valeri, C. R. Isolation and cryopreservation of monocytes from plateletpheresis cellular residue. Transfusion 23, 387 (1983). 7. Lionetti, F. J., Hunt, S. M., Lin, P. S., et al. Pres-
ervation of human granulocytes. II. Characteristics of granulocytes, obtained by counterflow centrifugation. Trunsfusion 17, 465 (1977). 8. Nickolov, Ch. Study of some properties of ABO
test sera by using the methods of phagocytosis of erythrocytes, treated with formaldehyde in vitro. Folin Huematol. 108, 838 (1981). 9. Rabinovitz, I. Separation of monocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observation. BIood23,
811 (1964).
10. Senn, A. S., et al. Fitration leukapheresis: leukocyte production. In “Cell Separation and Cryobiology” (H. Rainer, Ed.) p. 163. Schattauer Verlag, Stuttgart/New York, 1978. 11. Stevenson, H. C., et al. System for obtaining large numbers of cryopreserved human monocytes, purified by leukapheresis and counter current centrifugation elutriation (CCE). J. Immunol. Methods 62, 353 (1983).
Isolation
23, 531-536 (1986)
and Cryopreservation
TS. TSVETKOV,
of Human Peripheral
Blood Monocytes
CH. NICKOLOV, A. BUCKURESHTLIEV, L. TSONEY, R. TERZIEV, AND D. POPOV
Central Problem Laboratory
for
Cryobiology
and Freeze-Drying.
M. MINCHEFF,
Sofia, Bulgaria
A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983)) is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up IO 0.65 x lo9 cells/75 cm* surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution. 0 1986 Academc he,,. Inc.
Monocytes can be obtained from peripheral human blood by several techniques based on functional (adherence) or physical (density) properties of these cells. Methods based upon cell density arc often used in the United States (6, 7, ll), but their widespread application has been hampered elsewhere by high equipment costs (e.g., elutriators). Low yields of viable monocytes have been reported with the older methods, using adherence of cells to glass or plastic surfaces (1, 9, 10). Recently Freundlich and Avdalovic (4) reported a method for isolation of viable monocytes. The technique includes reversible attachment of monocytes on plasma-gelatin coated flasks through specific receptors on the cell membrane for Iibronectin (FN) (2). As long as the cell density of the suspension, which is layered on the plasma-gelatin coated surface, is low (2 x lo6 cells/ml), this method is suitable for obtaining monocytes for laboratory use only. A possible way to increase monocyte yield per unit area could be sought by multiplying the FN active sites on the gelatin-glass surface, provided that the bulk of the plasma fibronectin is present in the cryoprecipitate. The latter could be used instead of plasma for coating the bottom surface of standard glass flasks.
Received June 18, 1985; accepted May 21, 1986
On the other hand the current processing of donated blood often leaves the buffy coats out of use. A possible benefit could be sought through the utilization of pooled buffy coats for obtaining monocytes. The proper utilization of monocytes often requires cryoprcservation, and in the present paper we investigate the possibility to freeze cells isolated by such a technique. MATERIALS
AND METHODS
Buffy coats (BC I) are separated after centrifugation of several ACD preserved blood units, pooled together, and stored at 2-8°C for 24 hr. Leukocytes in BC I are concentrated through centrifugation and BC II is collected. Each 100 ml BC II is diluted in 25 ml ACD plasma and in 25 ml isotonic saline prior to use. Glass flasks (Usifroid type, 500 ml, 75 cm2 bottom surface) are rinsed with 5 ml gelatin solution (10% w/v in water) and incubated for 3 hr at 160°C. Cryoprecipitate is obtained from previously converted heparinized ACD plasma (5000 E heparin and 6 ml 1 M CaCl, are added consecutively to 100 ml ACD plasma) and it is dissolved in l/5 vol of the cryosupernatant plasma. Ten milliliters dissolved cryoprecipitate is added to each gelatin coated flask. After incubation at 37°C for 30 min, the dissolved cryoprecipitate is removed and the flasks are washed with 10 ml MgCl, enriched isotonic saline (0.836 g
531 001 l-2240186 $3.00 Copyright 0 1986 by Acadenuc Press. Inc. All nehts of reproductmn rn any term reserved
532
TSVETKOV
MgCl,, 6 H,O in 500 ml isotonic saline). The gelatine-cryoprecipitate coated flasks can be dried at 37°C after washing and stored at room temperature for at least 1 month. Mononuclear leukocytes are obtained from diluted BC II by centrifugation on Polysep (Pharmachim, Bulgaria) denisty gradient (d = 1077 g/cm3) (Boyum, 1968). Mononuclear cells, isolated from 100 ml BC II, are purified from platelets by centrifugation at 540g for 20 min and resuspended in 100 ml 20% human serum in MgCl,-enriched isotonic saline. (The ABO compatibility of sera is of no importance.) Erythrocyteigranulocyte sediment (EGS) is left for further fractioning. Twenty-five milliliters of the mononuclear suspension is layered on each of the bottoms of the gelatine-cryoprecipitate treated flasks and these are incubated at 37°C for 40 min. Nonadherent cells are transferred into a separate vessel and the flasks are washed gently four times with 25 ml of MgCl,enriched saline. Adherent cells in each flask are detached after the addition of 25 ml EDTA buffer (0.2 g NaEDTA in 500 ml Dulbecco’s medium, pH = 7.2), vigorous agitation, and incubation at 22°C for 15 min. followed by more vigorous agitation. The EGS is diluted up to 100 ml vol in isotonic saline and left at 22°C. The supernatant is observed after intervals of 30 mitt, 1 hr, and 2 hr, for leukocyte type content. The adherent mononuclear cells are concentrated five times and slowly diluted with
ET AL.
an equal volume of precooled to 4°C cryoprotective solution (20 ml Me,SO + 20 ml human serum + 60 ml isotonic saline). The diluted cell suspension is transferred into aluminium (thin wall) containers. The thickness of the frozen specimen is 2 mm. The containers are left at - 30°C for 60 min and then transferred into liquid nitrogen. The specimens are thawed in water bath at 40°C with constant agitation for 15-20 sec. The samples are diluted four times the volume with precooled to 4°C isotonic sucrose. To define the cell type and their functional activity, the following criteria are used: -Cell number is estimated by a routine method in a Burker’s chamber. -Cell type is defined on a Giemsa stained smear. -Functional activity is determined using the test for phagocytosis of formaldehyde-treated red blood cells (8). The serum is removed after the incubation when the phagocytosis is performed with cryopreserved cells. The cells are washed with isotonic saline after vigorous agitation, and resuspended in human serum before the smear is made. The percentage of phagocytizing cells is defined as the average of 1000 counted. -The determination of nonspecific esterase is done by a routine cytochemical method (5). RESULTS
Each 100 ml of BC 1 contains 1.58 * 0.06
TABLE I Total Number of Cells and Number of Mononuclear Cells ( x IO9 cells) Isolated from 100 ml BC 1 in Different Stages of Isolation Procedure (Mean Values Out of 23 Experiments) Cell fractions BC 1
BC II
1.58 k 0.06 k 0.05
1.32 2 0.07 0.90 k 0.05
Cell contents Total cell count Mononuclear cells a IL, intermediate
layer.
0.97
IL” 0.614 0.595
k 0.03 2 0.04
EGS 0.518 i- 0.0s i- 0.01
0.144
CRYOPRESERVATION
0
533
OF HUMAN BLOOD MONOCYTES
0.30
s
+
it l
c +
f
0.25
CI +
+
+
a+
:
+
+ + l
/’
+
z
0.05
-D
10
$ I
0
0.25
0.5 0.75
1.0
Numberof mononuclear cells 1x10’) layered on 75 cm2surfece FIG. I. Relationship between cell density of the layered suspension and (a) the yield of adherent phagocytizing cells; (b) the loss of nonattached phagocytizing cells; (c) the loss of nonattached monocytes (96). Mean values of 23 experiments. (+) One run value; (0-j mean values of esterase-positive cells; (0- - -) mean values of phagocytizing monocytes.
x lo9 leukocytes (0.97 2 0.05 x IO9 and at the mononuclear cell number is remononuclear cells). The preparation of BC duced only by 7.22% (Table 1). The Polysep II leads to a loss of total 16.46% leukocytes system used in these experiments yields a
FIG. 2. Phagocytosis performed with fresh monocytes (X 800).
534
TSVETKOV ET AL.
Freeze-Thaw-Wash
TABLE 2 Recovery of Monocytes (Percentage)
1 Month storage
2 Months storage
N
After thawing
After washing
After thawing
After washing
1 2 3 4 5 Mean
89.70 96.16 76.28 74.34 72.00 81.69 2 4.75
88.23 100.00 76.28 75.47 86.66 85.33 ir 4.50
88.23 100.00 82.06 67.92 72.00 82.04 t 5.75
91.17 100.00 80.90 67.92 76.66 83.33 k 5.60
fraction consisting of 97.65% mononuclear cells, 24.1% of which are phagocytizing. The average yield in the intermediate layer is 0.614 -+ 0.03 x lo9 cells (0.595 +- 0.04 X IO9 are mononuclear cells). The EGS contains 0.518 + 0.05 x lo9 leukocytes and 27.8% of them are mononuclear. The percentage of phagocytizing cells in all the 15 experiments carried out coincides with the percentage of polymorphonuclear cells in the sediment. The centrifugation of the suspension on a Polysep gradient and the following removal of platelets results in 14% loss of the original leukocyte number. The unrecovered cells are mononuclear (Table 1). The relation between the number of mononuclear cells layered on a certain gelatin-cryoprecipitate surface and the yield of adherent cells is shown in Figs. la, b, and c. The number of adherent cells in-
creases proportionally to the number of mononuclear cells layered, up to a certain extent (0.188 X lo9 monocytes/75 cm2 surface), then remains constant (Fig. la). After that point the number of phagocytizing nonattached cells in the suspension above the gelatin-cryoprecipitate surface increases sharply (Figs. lb and c). When x lo9 the optimum cell density-O.65 cells/75 cm2 surface-is used, the yield of monocytes is about 70% of their original content in the BC I. All adherent and eluated cells are esterase positive, but only 71.35% of them are phagocytizing (Fig. 2). Freeze-preservation results in about 17% loss of the original cell content. The cell recovery ranges from 68 to lOO%, mean 83%. No cells are damaged in the washing procedure and a prolonged storage of frozen cells up to 2 months does not lead to additional injury of cells (Table 2). Cryopreser-
TABLE 3 Changes in Phagocytic Activity of Monocytes after Cryopreservation Percentage of monocytes which phagocytize during storage period N
Fresh monocytes
48 hr
1 month
2 months
1 2 3 4 5 Mean
65 57.2 62.2 75 65 64.9 k 5.6
52 61 41 50 47.4 50.3 k 3.5
42 54 48 51 48.2 48.6 + 2.8
44 48.2 45.1 40 48 45 f 1.5
Phagocytic activity of cryopreserved monocytes as percentage of the phagocytic activity of fresh monocytes 100 77.5
75
69.5
CRYOPRESERVATION
OF HUMAN BLOOD MONOCYTES
535
FIG. 3. Phagocytosis. performed with cryopreserved monocytes ( x 800)
All the adherent and eluated cells are esterase positive, but only 70% of them are phagocytizing. A possible reason could be the preliminary leukocyte storage as BC I DISCUSSION at 2 to 8°C for 24 hr. Most probably no Our results show that monocytes can be mononuclear phagocytizing cells are left in isolated by this method with utilization of EGS because the percentage of phagocycryoprecipitate instead of plasma. Cryo- tizing cells in this sediment coincides with precipitate contains more of the fibronectin the percentage of polymorphonuclear cells of the bulk plasma and it can be used for present. As only monocytes are esterase positive coating the glass-gelatin surface. In such a case an optimum cell density at certain (among mononuclears), the eluated adconditions could be reached, Most prob- herent cells are, therfore, monocytes. The ably the employment of such cell density latter could be successfully cryopreserved saturates all the active FN sites. Otherwise by routine methods. the use of diluted suspensions will result in REFERENCES lower yields (Fig. la) while the utilization of more concentrated suspensions leaves a 1. Absolm, D. R., van Oss, C. .I., and Neuman, A. V. Elution of human granulocytes from significant number of phagocytizing mononylon filters by means of van der Waals repulnuclear cells nonattached in the suspension sion forces. Transfusion, 21, 663 (1981). (Figs. lb and c). 2. Bevilaqua, M. P., Ambrani, D., Mosesson, M. W., et al. Receptors for cold-insoluble globThese results strongly support the obserulin (plasma fibronectin) on human monocytes. vation that only 30% of the monocytes are J. Exp. Med. 153, 42 (1981). lost when optimum cell density of the sus- 3. Boyum, A. Separation of leukocytes from blood pension, which is layered on the active surand bone marrow. &and. J. Clin. Lab. Invest. face, is used. (Suppl.) 21, 97 (1968). vation of the cells causes about 25-30% loss of phagocytizing activity regardless of the storage period (Table 3, Fig. 3).
536
TSVETKOV ET AL.
4. Freundlich, B., and Avdalovic, N. Use of gelatinplasma coated flasks for isolating human peripheral blood monocytes. J. Immunol. Methods 62, 31(1983).
5. Hayhoe, F. G. J., and Quaglino, D. “Haematological Cytochemistry.” Churchill Livingstone, EdinburglLondoniNew York, 1980. 6. Hunt, S. M., Lionetti, F., and Valeri, C. R. Isolation and cryopreservation of monocytes from plateletpheresis cellular residue. Transfusion 23, 387 (1983). 7. Lionetti, F. J., Hunt, S. M., Lin, P. S., et al. Pres-
ervation of human granulocytes. II. Characteristics of granulocytes, obtained by counterflow centrifugation. Trunsfusion 17, 465 (1977). 8. Nickolov, Ch. Study of some properties of ABO
test sera by using the methods of phagocytosis of erythrocytes, treated with formaldehyde in vitro. Folin Huematol. 108, 838 (1981). 9. Rabinovitz, I. Separation of monocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observation. BIood23,
811 (1964).
10. Senn, A. S., et al. Fitration leukapheresis: leukocyte production. In “Cell Separation and Cryobiology” (H. Rainer, Ed.) p. 163. Schattauer Verlag, Stuttgart/New York, 1978. 11. Stevenson, H. C., et al. System for obtaining large numbers of cryopreserved human monocytes, purified by leukapheresis and counter current centrifugation elutriation (CCE). J. Immunol. Methods 62, 353 (1983).