[76] The isolation and cultivation of mononuclear phagocytes

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[76]

dibutyryl-cyclic AMP (DBC), iodide transport is altered in a complex manner (see Wilson et al.3), which seems to be the counterpart to the phenomenon occurring in rats treated with TSH in vivo as described by Halmi et al. 26 The results of two experiments illustrating these responses to TSH and DBC are shown in Fig. 6. Following the addition of TSH, there is a transient depression of the 1311 C:M which lasts about 2 hr; then the C : M gradually rises to stimulated levels as the incubations are continued. These and other findings have indicated that the TSH affects iodide transport in two rather distinct ways. First, the TSH rapidly increases the permeability of the cell membrane toward the outward diffusional leak of iodide so that the 1311 C: M ratio falls. Second, the TSH, operating by means of a slowly acting mechanism, gradually augments the activity of an inwardly directed iodide pump, which then leads to the increases in the 13~I C:M. Knopp et al. 2 have accumulated extensive experimental evidence that this TSH stimulation of the iodide pump proceeds through a series of intermediate reactions which include in sequence: (1) increased cAMP production, (2) increased DNA-dependent RNA synthesis, and (3) increased protein synthesis. Clearly, the next step in the elucidation of this phenomenon should be the isolation and/or identification of the role of this TSH-induced protein in the stimulation of the iodide pump. N. S. Halmi, D. K. Granner, K. J. Doughman, B. H. Peters, and G. Muller, Endocrinology 67, 70 (1960).

[76] T h e I s o l a t i o n a n d C u l t i v a t i o n of M o n o n u c l e a r Phagocytes B y ZANVIL A. COHN

The mononuclear phagocytes are a widely distributed group of cells which play important roles in a variety of physiological and pathological situations. They originate in the bone marrow, and the earliest recognizable form is the rapidly dividing p r o m o n o c y t e . These give rise to m o n o c y t e s , which after a short period of maturation in the marrow, are released into the blood stream, where they represent 3-10% of the total white blood ceils. The monocyte has an intravascular 1/2T of approximately 22 hours and under steady-state conditions randomly emigrates into the tissues from all capillary beds. Once in the extravascular compartment or when attached to walls of vascular sinuses, the monocyte matures into a larger, long-lived, more functionally active cell, the macrophage. Cer-

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tain organs, such as the liver (Kupffer cell), spleen, lymph nodes and lung (alveolar macrophage), contain large numbers of macrophages which are involved in the endocytosis and intracellular degradation of autochthonous and foreign molecules. Sources and Isolation of Monocytes and Macrophages The major work on this cell series has been performed on the circulating monocyte and tissue macrophage. As yet, the bone marrow promonocyte has not been obtained in sufficient numbers or purity for more exacting analysis.

The Separation of Monocytes from Blood Blood represents the only source of bona fide monocytes. Two approaches are feasible for obtaining relatively homogeneous populations which may be subsequently maintained in vitro. The first employs buffy coat cells and depends upon the preferential adherence and longevity of monocytes in culture. An example for the cultivation of chicken blood monocytes1 will be detailed although modifications of the method are applicable to all species. Ten milliliters of heparinized blood is obtained from the wing vein. Five milliliters of blood is layered over 5 ml of an albumin solution (8.4 ml of 35% albumin plus 2.6 ml of Hanks' balanced salt solution) contained in 15-ml conical centrifuge tubes. The tubes are centrifuged at 2000 rpm (International No. 1) for 15 minutes, resulting in a leukocyte layer at the plasma-albumin interface. The leukocytes are withdrawn with a Pasteur pipette, pooled, and washed twice with 10 ml of 4% albumin-Hanks' solution. Final resuspension is made in 10 ml of 30% chicken serum, 30% Fischer's medium, 40% Hanks' solution, and 0.002% Phenol Red. One-milliliter aliquots are then dispensed to Leighton tubes with flying cover slips. The initial cell population is heterogeneous and contains all white cell elements. After incubation for 24 hours at 37 °, the monocytes are firmly adherent and other leukocytes are removed by vigorous washes. This leaves a relatively homogeneous monolayer of monocytes, many of which have been involved in extensive phagocytosis of other cellular elements. With species in which large numbers of granulocytes are present, the culture should be washed at both 6 and 24 hours after incubation since these cells produce large amounts of lactic acid and pH control is difficult. A second method depends upon the separation of monocytes from other formed elements of the blood, avoids extensive phagocytosis and L. Weiss and D. W. Fawcett, J. Histochem. Cytochem. 1, 47 (1953).

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results in a more homogeneous population of cells in the same state of maturity. Horse blood is a useful source 2 since the red cells form rouleaux spontaneously. The techniques to be described can also be applied to human blood. Five hundred milliliters of horse blood is collected from the jugular vein into flasks containing 0.1 volume of 0.1 M citrate-saline; 40-ml aliquots are dispensed to tubes, and the erythrocytes are allowed to sediment for 45 minutes. The leukocyte-rich plasma is sedimented at 100 g per 12 minutes, leaving platelets in the supernatant. After decanting the plasma layer and brief drainage the packed white cells are resuspended in 35% albumin (sterile, preservative free, Pentex, Inc., Kankakee, Illinois) and diluted with phosphate-saline pH 7.4 to a final albumin concentration of 27%. After a white cell count, 27% albumin is added to give a final leukocyte concentration of 75 × 106/ml; 5-ml aliquots were placed in lusteroid tubes and centrifuged in the swinging-bucket rotor (Model SCR, Lourdes Instrument Corp.) at 2400 g for 36 minutes at 12 °. This yielded a compact pellet containing erythrocytes and granulocytes. The less dense monocytes were found in a surface pellicle along with varying numbers of lymphoid cells. The pellicle was harvested by means of a curved Pasteur pipette and the cells pooled in phosphatesaline containing 0.05 mg/ml heparin. The average yield from 450 ml of blood containing 4% of its white cells as monocytes was 100 × 106 monocytes or a recovery of approximately 70%. To obtain pure populations of monocytes, their adhesiveness to glass surfaces was exploited. The washed cells were resuspended in tissue culture medium (TC No. 199, 20% newborn calf serum) to a monocyte density of 1-1.5 × 106/ml and dispensed to glass culture vessels. After incubation at 37 ° for 2 hours in an air-5 % CO2 atmosphere, the monolayer was washed 3-4 times with TC No. 199. This removed all lymphocytes, leaving a uniform, adherent monolayer of monocytes. The technique of BSyum3 employing Ficoll-Isopaque density gradients is quite useful. This results in a band of lymphocytes, monocytes, and platelets. The platelets are removed by differential centrifugation, and the monocytes are recovered by their adherence to a glass surface. This method is rapid, well adapted for small quantities of human blood, and results in good yields of pure monocytes. Peritoneal M a c r o p h a g e s Unstimulated M o u s e Cells. The mouse peritoneal cavity contains reasonable numbers of resident macrophages which may be isolated rapidly

2W. E. Bennett and Z. A. Cohn, J. Exp. Med. 123, 145 (1966). 'A. B/3yum,Scand. 1. Clin. Lab. Invest. 21, 77 (1968).

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and simply.' Pathogen-free mice, 25-30 g, are killed rapidly with chloroform or cervical dislocation. The abdomen is flushed with 70% ethanol and the skin reflected leaving an intact peritoneal membrane. Two milliliters of heparinized phosphate-saline is injected intraperitoneally. After gentle massage, the fluid is aspirated into a Pasteur pipette yielding 3-5 × 106 cells, of which 50% are macrophages and the remainder are lymphocytes. The fluids are pooled and sedimented at 250 g for 5 minutes; the cell pellet resuspended in TC No. 199-20% newborn calf serum to yield a final cell density of 2-2.5 × l0 Gcells. The suspension is dispensed to glass or plastic culture vessels and allowed to incubate at 37 ° for 30 or more minutes in a 5% CO2-air atmosphere. When plastic culture dishes are employed, the incubation should be at least 60 minutes. At this time the monolayer is rinsed vigorously 3-4 times with warm tissue culture medium to remove nonadherent lymphoid cells. This yields a homogeneous population of macrophages and a rare ( < 1 % ) mesothelial and mast cell. Granulocytes are not present in the unstimulated cavity, nor are erythrocytes. Stimulated Mouse Cells. A variety of agents have been employed to increase the number of macrophages that may be harvested from the mouse peritoneal cavity. 5,6 Three to four days after injection, as many as 1-2 × 107 macrophages may be obtained, along with varying numbers of lymphocytes and granulocytes. With many of these inflammatory agents a brisk, early, polymorphonuclear leukocyte response occurs, and remnants of white cells and other debris are phagocytized by the macrophages. These procedures also produce a greater contamination with replicating mesothelial cells and yield a more heterogeneous macrophage population. Oil-lnduced Rabbit Peritoneal Macrophages. Rabbits weighing 3-4 kg, are injected intraperitoneally with 50 ml of sterile mineral oil (Nujol, extra heavy, Plough, Inc.) by means of a No. 18 n e e d l e / F o u r days later, the animals are sacrificed and 100 ml of pyrogen-free saline is injected into the cavity. The abdomen is then opened and the oily, exudate fluid is removed with a broad-tipped 25-ml pipette and dispensed to centrifuge tubes. The fluid is then centrifuged at 250 g for 5 minutes at room temperature, and the upper oil layer and intermediate supernatant fluid are removed. These fractions contain macrophages with many oil droplets and are discarded. The cell pellet is then resuspended, washed, and diluted to the appropriate cell density. This procedure yields 1-2 X 10 s cells, ' Z . A. Cohn and B. Benson, J. Exp. Med. 121, 153 (1965). rE. R. Unanue and J. C. Cerottini, J. Exp. Med. 131, 711 (1970). B. F. Argyris, 1. Exp. Med. 128, 459 (1968). 7Z. A. Cohn and E. Wiener, J. Exp. Med. 118, 991 (1963).

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more than 90% of which are exudate macrophages and 20-30% contain occasional, small oil droplets. Normal Rabbit Alveolar Macrophages. Alveolar macrophages may be obtained from many species by means of tracheal lavage. The technique for the rabbit will be described in detail. 7,8 Rabbits weighing 3-4 kg are sacrificed by injecting 50 ml of air into the marginal ear vein. The upper end of the trachea is clamped, the chest is opened, and the lungs, heart, and trachea are dissected free and removed in toto. The heart, lymph nodes, and connective tissue are then carefully removed, blood is expelled from the pulmonary vessels, and the remaining trachea and lungs are carefully washed in a beaker of saline. The latter steps are of importance in obtaining erythrocyte-free preparations. A plastic catheter is inserted through the cut end of the trachea, and 25 ml of saline is injected into each main stem bronchus. The distended lung is then wrapped in moist gauze, inverted over a beaker, and gently kneaded to expel the washings. The procedure is repeated once more; the harvest is pooled and centrifuged, and the cell pellet is washed. An average number of 2 × 107 cells are obtained, of which over 95% are large, uniformly sized macrophages. BCG-lnduced Alveolar Macrophages. Larger numbers of cells may be obtained for biochemical analysis by evoking a macrophage response in the lungs. The procedure is a modification of that" described by Myrvik et al. 7,9 Bacillus Calmette-Gu6rin (BCG) are obtained as a dead, lyophilized powder and suspended in 0.01% Tween 80-saline to a concentration of 15 mg/ml. Two injections, each 1.0 ml, are given to rabbits via the marginal ear vein on successive days. Lungs are obtained from animals 3.5-4.0 weeks after inoculation and harvested by the procedure described for normal alveolar macrophages, except that three washes of the lung are carried out and the cells are pooled. A total of 1-3 × l0 s cells is obtained, of which 85 % are typical, large macrophages, some of which are binucleate or multinucleated giant cells. This represents as much as 4-6 ml of packed cells. The Maintenance and Culture of Macrophages in Vitro Certain of the mononuclear phagocytes may be maintained in suspension culture for short periods of time or on glass or plastic surfaces for prolonged cultivation. In part, the choice depends upon the particular parameter of cell function under study. Short-Term Suspension Cultures. This method is particularly useful for studies of phagocytosis in which biochemical and cell fractionation SQ. N. Myrvik, E. S. Leake, and B. Fariss, J. lmmunol. 86, 128 (1961). 9Q. N. Myrvik, E. S. Leake, and S. Oshima, I. lmmunol. 89~ 745 (1962).

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analyses are involved. 1°,1x It may be employed with any of the cell populations mentioned in the preceding sections, but is less useful when limited numbers of cells are available or when populations have a tendency to aggregate. Studies have been carried out with mouse, TM rabbit, 1~ and guinea pig 14 macrophages. Rabbit alveolar macrophages, which have a large cell volume, are maintained at 1 × 10T/ml whereas the normal or induced peritoneal cell can be incubated at 3 × 107/ml. The medium will vary in terms of the particular experiment. In most cases the cells will do well for many hours in H a n k s ' solution, Gey's, minimal Eagle's or a more enriched medium, such as TC No. 199. The presence of autologous serum or fetal calf serum at 1 0 - 2 0 % final concentration maintains cell viability and serves as an additional buffer system. The culture vessel can be either plastic or glass and agitation by slow rolling or in a rotary shaker is necessary to keep cells in suspension and to facilitate contact in phagocytosis experiments. L o n g - T e r m Cultures. Both monocytes and macrophages can be maintained for days or weeks and are particularly useful for immunological and biochemical studies. In the majority of cases the cells will increase in size and complexity but do not divide or synthesize DNA. The monocytes from most species are easily cultivated and mature into macrophages upon prolonged cultivation. 1~ Not all populations of macrophages may be maintained. Particular difficulty has been encountered with rabbit and human alveolar macrophages and the induced peritoneal ceils of all small rodents other than the mouse. This difficulty is reflected in the continuous loss of cells from the culture dish and the inability to maintain a constant population for longer than 24 hours. Further studies are necessary on the medium and serum requirements for rabbit and guinea pig macrophages. Unstimulated M o u s e Peritoneal Macrophage. T h e mouse macrophage is the cell of choice for long-term experiments and has been employed for studies on phagocytosis, pinocytosis, TM lysosome formation, ~7 intracellular digestion, 18 plasma membrane synthesis, 19 and cholesterol metabolism. 2° 10Z. A. Cohn, J. Exp. Med. 120, 869 (1964). laZ. A. Cohn and E. Wiener, J. Exp. Med. 118, 1009 (1963). a2E. Unanue and B. A. Askonas, Immunology 15, 287 (1968). 13G. B. Mackaness, J. Exp. Med. 112, 35 (1960). 14R. Oren, A. Farnham, A. Saito, K. Milofsky, and M. L. Karnovsky, J. Cell Biol. 17, 487 (1963). 15Z. A. Cohn, Advan. lmmunol. 9, 163 (1968). ~Z. A. Cohn, J. Exp. Med. 124, 557 (1966). ~7Z. A. Cohn, M. E. Fedorko, and J. G. Hirsch, J. Exp. Med. 123, 757 (1966). ~8B. A. Ehrenreich and Z. A. Cohn, J. Exp. Med. 129, 227 (1969). 19Z. Werb and Z. A. Cohn, J. Biol. Chem. 247, 2439 (1972). ~Z. Werb and Z. A. Cohn, 1. Exp. Med. 134, 1545 (1971).

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Phase Contrast Microscopy. Cells are harvested from the peritoneal cavity as described previously. For morphological studies, 1 ml of washed cells (2 × 106) are allowed to adhere to flying cover slips in Leighton tubes in a medium containing 20% NBCS and TC No. 199 for 30-45 minutes before vigorous rinsing. The medium is then replaced and the ceils cultivated in an atmosphere of 5% CO2-air at 37 °. At the time of harvest, the medium is removed and the cells fixed with 1.0 ml of ice cold, buffered, 1.25% glutaraldehyde for 10 minutes. The cover slip is rinsed with distilled water, inverted over a drop of water on a slide, sealed, and examined. This procedure is also useful for a variety of cytochemical and autoradiographic proceduresY 1 Mass Cultures. Large numbers of cells are often required for biochemical determinations and may be cultured on either glass or plastic surfaces. Glass T-flasks (15 or 30 cm 2) are often useful ~° and do not require the use of a COs incubator. When employed, the initial cell suspension is allowed to adhere for 60 minutes before the removal of lymphocytes. The flasks are gassed with 5 % CO2-air and tightly stoppered. In the case of plastic petri dishes, cells are allowed to adhere for 1-2 hours before rinsing and are then maintained in a CO2 incubator. In general, the adherence and spreading of macrophages is slower on plastic surfaces. Once in culture, the medium may be changed every third day for a period of many weeks. Macrophages are not removed from glass or plastic surfaces with either trypsin, E D T A or a mixture of the two and require physical methods. A rubber or plastic policeman is used to scrape the cells from the dish surface into an appropriate volume of diluent, i.e., water, salt solutions. For the purposes of electron microscopy, the cells may be fixed on the dish surface, then scraped and pelleted for further processing. 17 M e d i u m . A variety of the usual tissue culture media are adequate for the maintenance of macrophage cultures. In this laboratory, TC No. 199 or MEM supplemented with glutamine have been employed. Penicillin at 1000 units/ml is the only antibiotic employed and contamination of cultures is a minor problem with macrophages. The most important variable in the culture of mouse cells is the selection of an appropriate serum. Mouse serum, in addition to being difficult to obtain in sufficient quantities, contains high levels of fatty acids, and results in the rapid accumulation of large numbers of triglyceride containing lipid droplets. For many purposes fetal calf, newborn calf and horse serum have proved adequate for long-term cultures. Newborn calf and adult bovine sera contain natural antibodies directed against the ~IZ. A. Cohn and B. Benson, J. Exp. Med. 121, 279 (1965).

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plasma membranes of both mouse macrophages and erythrocytes. 22 In the presence of complement, these sera lyse mouse cells whereas in its absence they stimulate pinocytosis and lysosome formation in a dose dependent relationship. Since bovine hemolytic complement is only partially susceptible to 56 ° at 30 minutes, all sera are aged at 4 ° for 2 weeks before use. Fetal calf serum lacks the antimouse antibody and is a less effective inducer of pinocytosis. For routine cultures all sera are employed at a concentration of 20% v/v, but may be increased to as high as 50% for the more rapid formation of lysosomes. Properties of Cultured Mouse Macrophages In the presence of 2 0 - 5 0 % aged calf serum, macrophages do not divide but increase in size and protein content and demonstrate progressive increments in their content of lysosomes 23 and lysosomal hydrolases. Functional properties, such as pinocytosis and phagocytosis, are enhanced, and large amounts of plasma membrane are interiorized to form endocytic vacuoles. The cells become highly oriented on the glass surface, demonstrating a perinuclear area filled with secondary lysosomes or dense granules surrounding a multicentric Golgi apparatus. The peripheral cytoplasm contains long mitochondria extending to the tips of pseudopods and lucent pinocytic vesicles which can be seen moving centripetally toward the perinuclear area. Moderate amounts of rough-surfaced endoplasmic reticulum and occasional polysomes are present. A more detailed account of their properties can be found in review articles. 1~ ~z. A. Cohn and E. Parks, J. Exp. Med. 126, 941 (1967). 2~Z. A. Cohn and M. E. Fedorko, in "Lysosomes in Biology and Pathology" (J. Dingle and H. Fell, eds.), Vol. 1, p. 43. North-Holland Publ., Amsterdam, 1969.

[77]

Cultured

Cell Systems and Methods

for Neurobiology

B y BRUCE K. SCHRIER, SAMUEL n . WILSON, and MARSHALL NIRENBERG

Methods' are described for the culture of cells from the nervous system of the mouse or rat, and for determining the activities of enzymes required for communication between neurons, such as choline acetyltransferase (EC 2.3.1.6); tyrosine hydroxylase (EC 1.14.3a); glutamate decarboxylase (EC 4.1.1.15 ) ; acetylcholinesterase (EC 3.1.1.7), and catechol O-methyltransferase (EC 2.1.1.1). The methods are sufficiently 1S. H. Wilson, B. K. Schrier, J. L. Farber, E. J. Thompson, R. Rosenberg, A. Blume, and M. Nirenberg, J. Biol. Chem. 247, 3159 (1972).