A simple method of obtaining monocytes in suspension

Journal oflmmunologicaIMethods, 69 (1984) 71-77 Elsevier

71

JIM03043

A Simple Method of Obtaining Monocytes in Suspension H e l i o s R. R e c a l d e 1 Centro Trasfusionale, Ospedale Prooinciale di Magenta, Magenta, Italy

(Received 24 August 1983, accepted 20 December 1983)

Relatively pure monocyte suspensions may be obtained by density flotation after simple conditioning of leukocytes. This consists of incubating blood or buffy coat cells in plasma containing NaC1 at a higher than physiological concentration. As a result the density of granulocytes and lymphocytes increases greatly, but the density of monocytes changes very little. It is possible with one centrifugation on a slightly modified density gradient to obtain 90% pure monocyte suspensions with 55% yield. More than 95% purity but lower yield is obtained simply by increasing the NaC1 concentration. The membrane characteristics of monocytes and their capacity to function normally remain unaltered by this treatment and cells so obtained develop in culture into macrophages normally. This method is much cheaper and simpler than others described, and is applicable equally to either small or large numbers of leukocytes. Key words: monocyte separation - macrophages - density gradient cell separation

Introduction

There is growing interest in studying the immunological and non-immunological functions of the monocyte-macrophage system in man. Such studies require large numbers of high-purity monocytes. Published methods are, generally speaking, expensive and complicated (Berthold, 1981; Feige et al., 1982; Gadeberg et al., 1979; Norris et al., 1979; Pertoft et al., 1980; Zanella et al., 1981; Zembala et al., 1982). This paper describes an alternative and simple method which makes it possible very cheaply to obtain monocytes that are relatively pure from small or large quantities of blood. The method is based on changes in mononuclear cells after brief incubation in plasma made hypertonic with NaCI. Within limits, this treatment causes a marked increase in the density of lymphocytes as a result of H 20 loss, whereas the density of monocytes changes very little because of their intense pinocytosis. This paper outlines the simplest method of obtaining monocytes in suspension, making use of this phenomenon together with separation on a density gradient by a slightly modified BOyum's technique. 1 Correspondence to: Helios R. Recalde, Via Langosco 11, 27100 Pavia, Italy. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

72 Materials and Methods

Source of leukocytes (1) Buffy coats and plasma were obtained by centrifugation of blood bags taken with CPD as anticoagulant. Buffy coat preparations contained 10-20% (v/v) erythrocytes, 10-20 x 10 6 leukocytes/ml, and 200-400 x 10 6 platelets/ml (leukocyte-rich plasma). (2) For the study of membrane antigens, and when it was not necessary to obtain a high number of monocytes, small samples of 2 ml of anticoagulated blood with 0.2 ml of Na citrate at 3.5% concentration were used. In these samples, platelets were eliminated by one of the following procedures: (a) 20 ~1 of bovine thrombin (Behringwerke) containing 30 U / m l were added to samples previously incubated at 37°C and agitated continuously on a rotor for 15 min. In this way platelet aggregation and partial defibrination were obtained. (b) After centrifugation at 600 x g for 10 min the buffy coats were collected and washed twice with 6-8 vols. of PBS containing 0.4 m g / m l of E D T A N a 2 centrifuging each time at 200 x g for 5 rain. The sediment was then resuspended in 4 vols. of autologous plasma without platelets.

Conditioning of cells Different volumes of 9% NaCI solution were added to leukocyte-rich plasma as indicated in Table I and the cells incubated at 37°C for either 10, 30, or 60 rain. The additions were made in 3 portions during incubation. On the basis of the results obtained the procedure chosen as most convenient was the gradual addition of 25/~1 of 9% NaCI solution during 30 min incubation, for every ml of leukocyte-rich plasma or blood. Five microliters of 9% NaCI were first added and the cells incubated in a water bath at 37°C for 10 min, then 10/~1 with further incubation for 10 rain, and then another 10/~1 for a final 10 min incubation. The blood was then diluted with 2 vols. of saline solution of similar osmolality (made by adding 27 ~tl of 9% NaC1 to every ml of normal saline). Immediately afterwards, separation was performed by centrifugation on a density gradient.

Preparation of the density gradient A standard solution of Ficoll-Hypaque was prepared according to B6yum (1968). For every ml of this solution volumes of NaCI were added so as to obtain the final osmolalities indicated in Table I. The mixture selected was that obtained by adding 2.8 mg of crystalline NaC1 to every ml of Ficoll-Hypaque, so as to give a final osmolality of 385 mosM/kg, as determined by the freeze-etching point method. The final density was 1080. The same final osmolality and density were obtained after identical additions to each ml of Lymphoflot (Biotest). Separation of monocytes was obtained by layering 1 vol. of the hyperosmotic separation medium under 3 vols. of previously conditioned and diluted blood or leukocyte-rich plasma and centrifuging at 600 x g for 15 min at ambient temperature. The layer of cells at the interface between the Ficoll-Hypaque cushion and the plasma was collected, washed twice with cold PBS and resuspended in RPMI 1640 containing 25 mM Hepes (Gibco), 30% autologous serum and 100/~g/ml gentamicin.

73

Identification of cells by membrane receptors and cytochemistry Monocytes with receptors for IgG were identified by rosette formation with sheep erythrocytes coated with rabbit IgG anti-sheep erythrocytes at a subagglutinating dose (EA). One hundred microliters of EA at a concentration of 2 x 108 E A / m l in R P M I were mixed with 100/~1 of mononuclear cells at a concentration of a 2 x 10 6 cells/ml in R P M I without serum, centrifuged at 100 x g for 5 min and incubated at 4°C for 60 min. The percentage of monocytes forming EA rosettes were assessed by microscopic examination of resuspended cells adhered to slides by cytocentrifugation, and processed to reveal esterase (Yam et al., 1971). Cells with intense, diffuse esterase activity were identified as monocytes and among these, those with 3 or more adherent EA were classified as monocytes with receptors for IgG. Monocyte phagocytic capacity was assessed by the same procedure except that after centrifugation the cell sediment was incubated for 60 min at 37°C. The percentage of phagocytic monocytes was assessed by counting esterase positive cells with one or more intracytoplasmatic erythrocytes. Percentages were calculated from counts of at least 200 cells.

Monocyte bactericidal activity A modified procedure of Territo and Martin (1977) was used. Staphylococcus aureus was added to 100 btl of a suspension of mononuclear cells in R P M I 1640 containing 5% inactivated AB serum to obtain a ratio bacteria : cells of 50 : 1. As a control, bacteria were added to 100 ~1 of the same suspension of mononuclear cells which had been treated for 5 min with 0.01% glutaraldehyde (Fluka) in PBS and washed twice. The mononuclear cell suspension contained 1 x 10 6 cells/ml and the percentage of monocytes had been previously determined. Bacteria and cells were centrifuged at 200 x g for 5 rain and incubated for 120 rain at 37°C to promote ingestion and killing of the bacteria. After incubation 2 ml of sterile distilled water was added and the cells were agitated vigorously for 10 min. Serial dilutions were prepared and staphylococcal colonies were counted by the pour plate method with trypticase soy agar. The percentage of bacteria killed by the mononuclear cells was calculated as [(A - B)/B] x 100, where A was the number of colonies counted after incubation with fixed mononuclear cells and B the number after incubation with living mononuclear cells. Trial experiments demonstrated that purified lymphocytes were not capable of killing bacteria. The index of monocyte bactericidal activity was expressed as: percentage of bacteria killed by mononuclear cells/percentage of monocytes present in mononuclear cells.

Results

Variation in density of mononuclear cells following incubation in hypertonic plasma The results shown in Table I were obtained by using the buffy coats of bags from 5 blood donors. After incubation in medium containing physiological quantities of NaC1 the percentage of interface monocytes was as reported by other workers and did not change significantly even after 60 min incubation at 37°C. However, when

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the NaC1 concentration in the incubation medium and in the gradient was increased a much greater percentage of monocytes was found at the interface, even though the yield was smaller. Incubation time acted synergistically, within limits, on the effect caused by the hypertonic medium. Beyond 60 min incubation, the purity and yield of monocytes obtainable differed greatly from one donor to another, and monocyte survival and capacity to function was compromised (data not shown). The optimum conditions, as described above, were gradual addition of 25 t~l of a solution of NaC1 at 9% for every ml of blood to be conditioned over a period of 30 min at 37°C, and centrifuging on Lymphoflot containing 2.8 mg of NaCI per ml. Monocytes used to assess functional capacity (Table II) were obtained by this method. Buffy coats stored at 4°C for 24 h before conditioning gave preparations of similar purity but with slightly lower yield (data not shown). When small samples of blood anticoagulated with CPD or with citrate and treated with thrombin were used in the same manner, monocyte suspensions of purity comparable with that shown in Table I were obtained, but with 15-30% lower yield, since the thrombin added to eliminate platelets produced small aggregates of both leukocytes and platelets. Buffy coats twice washed with PBS-EDTA and resuspended in autologous plasma gave results comparable with those shown in Table I. Similar results were obtained with small samples anticoagulated with EDTA. Samples anticoagulated with heparin did not give good separation. Lymphocytes sedimented with other leukocytes could be recovered by removing the layer of Lymphoflot and adding to the cell sediment 3 vols. of autologous plasma at physiological osmolality. After incubation for 60 rain at 37°C the lymphocytes regained their original density and could be processed by the usual method for separation of mononuclear cells (BSyum, 1968). Subsets of lymphocytes obtained in this way were not studied.

Monocyte functional capacity In Table II properties of monocytes obtained by this method and by the B~Syum

TABLE II CHARACTERISTICS OF ESTERASE POSITIVE CELLS OBTAINED AFTER C O N D I T I O N I N G A N D DENSITY FLOTATION

Elements with receptors for IgG c Elements with EA phagocytated d Index of bactericidal activity e

Hyperosmotic medium a

Isosmotic medium b

75

(64-88)

71

(66-82)

70

(62-81)

68

(58-79)

0.84 (0.64-0.98)

0.88 (0.62-1.10)

a Suspensions contained 85-95% of positive esterase elements. t b Suspensions contained 15-28% of positive esterase elements. Percentage average obtained from 5 different donors, extreme values in brackets. d Obtained from 3 different donors. e Average and extreme values of indices of monocytes bactericidal capacities found in 3 donors.

76 flotation method in isosmotic medium (20% monocytes and 80% lymphocytes) were compared. Cell viability in all cases exceeded 95% (trypan blue exclusion method). Both methods yield monocytes with equivalent characteristics as regards IgG receptors, capacity to phagocyte sensitized erythrocytes and capacity to kill staphylococci.

Discussion

Traditional methods of obtaining monocytes have the initial disadvantage of requiring preliminary separation of mononuclear cells. In addition, with methods based on the capacity of monocytes to adhere to plastic or glass there are changes in cell membrane properties following subsequent detachment (Gadeberg et al., 1979; Zanella et al., 1981). The elutriation method requires expensive equipment and is not suitable for use with small quantities of blood. Methods which use polyvinylpyrolidone and silica (Percoll) require careful standardization and are unsuitable for small quantities of blood, while residual silica particles may cause problems. The method described here makes it possible to obtain a layer of monocytes from buffy coats or whole blood as starting material with only one centrifugation on a gradient. The Ficoll-Hypaque mixture (BOyum, 1968) used for the separation of all mononuclear cells provides a suitable gradient for density separation of monocytes when modified by addition of NaC1. Similar results were obtained by adding the same amount of NaC1 to a commercial ready-to-use preparation. Attention to the following points is essential: (1) absence of Ca 2+ during incubation in the hyperosmotic plasma; (2) centrifugation on a gradient medium with an ionic concentration of Na + slightly higher than the conditioning medium. The presence of platelets does not interfere with the separation of monocytes but occasionally during subsequent washing, many platelets adhere to these cells. This is undesirable when the aim is to study membrane antigens and in such cases it is necessary to eliminate the platelets with thrombin or use buffy coats double-washed. The treatment described produced no change in the monocyte functions shown in Table II as compared with those of monocytes obtained by the classical method of flotation in an isosmotic gradient. Monocytes washed and resuspended in isosmotic culture medium maintained their vital functions for many days. Even after 10 days culture in autologous serum 30-50% of the monocytes initially placed in culture survived in the form of macrophages. The procedure described made it possible to obtain 90% pure monocytes even from small samples (1 ml) from newborn babies or serially obtained from adults with myeloproliferative disorders. With this method it proved possible to obtain a suspension more than 90% pure of hairy cells from 2 patients with hairy cell leukemia in whom these cells represented only 2-5% of the peripheral leukocytes. In conclusion, by the method described monocytes may be obtained in suspension very simply and much less expensively than by other methods. Judged by the product purity x yield of monocytes in the final suspension, the above results are similar or only slightly inferior to those obtained by elutriation, are similar or superior to those obtained by the use of Percoll and are decidedly superior to those

77

obtained by adherence and detachment of mononuclear cells. The method is equally suitable for use with small samples of blood or large amounts of leukocyte concentrates.

References Berthold, F., 1981, Blut 43, 367. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. Feige, U., B. Overwien and C. Sorg, 1982, J. lmmunol. Methods 54, 309. Gadeberg, O.V., J.M. Rhodes and S.O. Larsen, 1979, J. Immunol. Methods 31, 1. Norris, D.A., R.M. Morris, R.J. Sanderson and P.F. K6hler, 1979, J. Immunol. 123, 166. Pertoft, H., A. Johnsson, B. Warmegard and R. Seljelid, 1980, J. Immunol. Methods 33, 221. Territo, C.M. and J.C. Martin, 1977, J. lmmunol. 118, 187. Yam, L.T., C.Y. Li and W.H. Crosby, 1971, Am. J. Clin. Pathol. 55, 159. Zanella, A., A. Mantovani, M. Mariani, G. Silvani, G. Peri and F. Tedesco, 1981, J. Irnmunol. Methods 41,279. Zembala, M., W. Uracz, I. Ruggiero and E.M. Lemmel, 1982, Clin. Exp. Immunol. 49, 225.