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THE CULTURE OF MONONUCLEAR PHAGOCYTES: A BRIEF OVERVIEW
D. O. Adams P. J. Edel s on
Cell culture has become a fundamental tool for studying the mononuclear phagocytes, and the basic requirements for culture of these cells have been described (1, 2) . Many of the technical details for doing this successfully are des- cribed at length in this volume. Techniques for obtaining specific types of mononuclear phagocytes and for separating, counting, and identifying mononuclear phagocytes are presented below. This overview will outline briefly some of the general problems, in culturing these cells.
The basic conditions useful for culturing most mononuclear phagocytes are not complex (1 - 3). A variety of basal media including medium 199, Dulbecco1s minimum essential medium, and Eagle's minimum essential medium have been successfully em- ployed. In some situations, basal media may be supplemented with nutrients, such as pyruvate, nonessential amino acids, vitamins, and ascorbic acid, or may be replaced by richer me- dia such as MEM-alpha. The pH is usually controlled by the C02 - bicarbonate buffer system. Organic buffers such as HEPES may be quite useful for work in room air, but can be toxic. In
METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.
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2 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES
general, cultures of mononuclear phagocytes do best at a den- sity where the cells are neither crowded nor sparse
(2 - 3 x 10 adherent macrophages/cm ) . The size and shape of the culture vessel are usually not critical, but the diffi- culties in removing adherent macrophages generally make it ad- visable to establish the primary culture in the type of vessel that will be ultimately employed for each experiment. In the end, the culture conditions for mononuclear phagocytes that will prove optimal for a given experiment cannot be completely determined a priori and must be established empirically for each function.
Cultures of mononuclear phagocytes are generally supple- mented with serum (1, 2) . Fetal bovine serum, neonatal bovine serum, or equine serum at 10 - 40% (v/v) are commonly used.
The serum can prove to be a major variable in altering function of the cultures. The content of endotoxin, of pesticides or other agricultural chemicals, of hormones, of antimacrophage antibodies, and perhaps of other as yet unidentified substances such as trace elements may stimulate or inhibit the function of macrophages (4, 5 ) . Consequently, it is best to screen each lot of serum for its suitability in each assay of macrophage function in which the investigator is interested. Furthermore, the apparent lability of certain serum constituents may make special handling of the serum necessary to obtain the desired results (1). On occasion (e.g., quantification of secreted pro- teases in the absence of serum inhibitors of proteases such as a2-macroglobulin), establishment of cultures of mononuclear pha- gocytes in the absence of serum is desirable (6). The serumless medium of Neumann - Tytell is useful for this purpose, as it will support vigorous cultures of macrophages for several days.
The health and viability of macrophages being cultured should be established each day by screening the culture plates with an inverted phase microscope. In healthy cultures, the macrophages form monolayers of large, plump, stellate, phase- dense cells (Fig. 1 ) . By contrast, unhealthy cultures contain sparse accumulations of round, phaselucent, adherent macro- phages overlaid by numerous refractile, detached cells.
A major pitfall in study of macrophages in vitro is their physiologic plasticity. Various functions of mononuclear pha- gocytes, even under apparently optimal culture conditions, may be reduced or lost after only a few hours of cultivation (7).
Furthermore, subtle alterations in the culture environment can Fig. 1. Phase photomicrographs taken with an inverted phase microscope of cultures of murine peritoneal macrophages elicited bv thioglucollate broth. *600. (Ά) Four hours after explantation. (B) One day after exvlantat ion. Note the num- erous, prominent vacuoles in the wellspaced macrophages.
I. OBTAINING AND CULTURING MONONUCLEAR PHAGOCYTES W** V Î ^
Fig. 1Ά
4 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES strikingly modify certain functions of macrophages. Heparin, a frequent constituent of washout medium, is one of a class of polyanionic substances that are known to stimulate pinocytosis
(8). Trace concentrations of endotoxin can strikingly-stimu- late other functions of macrophages (4). Control of these variables requires, at minimum, meticulous replication of the details of each experimental protocol.
The goal of in vitro studies of macrophage function has been to better understand the mechanisms of their function in vivo. As we develop better ways of assessing and regulating their behavior in culture, we shall hopefully come closer to understanding their physiology in situ.
REFERENCES
1. D. 0. Adams. Macrophages. In "Methods of Enzymology,"
Vol. LVIII, Cell Culture (W. Jakoby and I. Pastan, eds.), pp. 495-505. Academic Press, New York, 1979.
2. P. J. Edelson and Z. A. Cohn. In "In Vitro Methods of Cell-mediated and Tumor Immunity" (B. R. Bloom and J. R.
David, eds.), p. 333. Academic Press, New York, 1976.
3. G. D. Wasley and R. John. The cultivation of mammalian macrophages in vitro. In "Animal Tissue Culture" (G. D.
Wasley, ed.), p. 101. Butterworth, London, 1972.
4. J. B. Weinberg, H. A. Chapman, Jr., and J. B. Hibbs, Jr.
Characterization of the effects of endotoxin on macrophage tumor cell killing. J. Immunol. 121: 72, 1978.
5. Z. A. Cohn and B. Benson. The in vitro differentiation of mononuclear phagocytes to the influence of serum on granular formation, hydrolase production, and pinocytosis.
J. Exp. Med. 121: 836, 1966.
6. D. O. Adams. Effector mechanisms of cytolytically-
activated macrophages. I. Secretion of neutral protease as an effect of protease inhibitors. J. Immunol. 124: 286, 1980.
7. D. 0. Adams and P. A. Marino. Evidence for a multistep mechanism of cytolysis: The interrelationship between
capacity for cytolysis, target binding, and secretion of cytolytic factor. J. Immunol. 126: 981, 1981.
8. Z. A. Cohn and E. Parks. The regulation of pinocytosis in mouse macrophages. II. Factors inducing vesicle formation.
J. Exp. Med. 125-. 213, 1967.
