MICROVASCULAR

RESEARCH,

The Isolation

10,286-297

(1975)

and Culture of Capillary from Epididymal Fat

Endothelium

ROGERC. WAGNERAND MAUREENA. MATTHEWS Department of Biological Sciences, University of Delaware, Newark, Delaware 1971I Received February II,1975 Capillaries were isolated from collagenase-dissociated epididymal fat by centrifugation to remove floating adipocytes and subsequent filtering to remove blood elements and free stromal cells. Isolated capillaries were cultured in Medium 199 and 20 % fetal calf serum. Spreading of endothelial cells from the capillary explants could be directly observed. Nonendothelial cells were removed from primary cultures by treatment with thimerosal. A small percentage of the cells spreading from the explants exhibited cusp-shaped or circular profiles, which were no longer observed after the cells had spread into monolayers. Purified primary cultures consisted of sheets of close-packed polygonal cells. Electron microscopy revealed that micropinocytic vesicles were present in abundance both at the cell’s free surfaces and enclosed within the cytoplasm. Endothelial cells were frequently joined by intracellular junctions. Weibel-Palade bodies were not evident in those cells observed by electron microscopy. Upon subculturing, the cells progressively exhibited pleomorphism.

INTRODUCTION The importance of endothelial transport mechanisms in healthy tissues and in conditions leading to vascular diseaseis becoming increasingly more evident. Isolated systemsof endothelium facilitate the study of endothelial cell biology by minimizing variables of the in viva environment. The biochemistry, pharmacology, and metabolism of endothelial cells can be studied in a more accurately defined system. Dependence and interactions between endothelium and surrounding tissues, however, are compromised by such simplication. Endothelial cells have been dissociated from the walls of large blood vessels(Lazzarini-Robertson, 1959; Maruyama, 1963; Pomerat and Slick, 1963; Fryer et al., 1966; Jaffe et al., 1972; Lewis et al., 1973; Shepro et al., 1973; Gimbrone et al., 1974). Collagenasewas first used by Maruyama (1963) to dissociate endothelial cells specifically from the walls of large blood vessels.The effectivenessof collagenasedigestion in providing relatively pure isolates of endothelial cells for subsequent subculturing has been confirmed by a number of later studies (Fryer et al., 1966; Jaffe et al., 1972, 1973; Lewis et al., 1973; Shepro et al., 1973; Gimbrone et al., 1974).Culturing of such isolates is complicated by the relatively low mitotic activity of vascular endothelium (Schoefl, 1963; Tannock and Hayashi, 1972)and by contaminating smooth muscle and connective tissue cells (Gimbrone et al., 1974). Cultures of endothelial cells have been obtained, however, by utilizing specific culture media that are selectivefor endothelium (Lewis et al., 1973)and differential susceptibility to trypsin of cells in primary culture Copyright0

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(Gimbrone et al., 1974) and by minimizing contaminating cell types in the original isolate (Shepro et al., 1973). Isolated capillaries have been prepared from epididymal fat (Wagner et al., 1972) and brain (Joo and Karnushina, 1973; Orlowski et al., 1974; Brendel et al., 1974). Capillary endothelial cells are of special interest regarding transport processessince they are the focus of most blood-tissue exchanges. Isolated capillary endothelium provides a unique system for studying the cell biology of capillary function and constitutes a source for culturing of endothelial cells as well. We describe here a technique for the isolation and culture of capillary endothelium derived from epididymal fat. METHODS AND MATERIALS Capillary Isolation

The distal two-thirds of epididymal fat pads from adult (350-400 g) rats (Flow Labs. Inc.) were aseptically removed and pinned out flat on beeswaxin the bottom of a Petri dish containing lo-ml sterile Delbecco’s PBS (pH 7.4). Portions of fat free of large blood vessels(greater than 60-pm diam) were removed from the pads under a dissection microscope (Fig. 1). This microvascularized fat was placed in 50-ml screwcap Erlenmeyer flasks (2 pads/flask) containing lo-ml Delbecco’s PBS (pH 7.4), Worthington Type II Collagenase (7.5 mglml), bovine serum albumin (5 mg/ml), and a magnetic stirring bar. The fat was incubated with gentle stirring at 37” for 45 min or until the pieces of fat were digested into a slurry. The collagenase digest was

FIG. 1. Rat epididymal fat pad pinned out flat in a Petri dish. Large blood vesselscan be distinguished from exclusively microvascularized fat. (x 1.5.)

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centrifuged at 100g for 3 min, and the floating cake of adipocytes and the supernatant were decanted. The vascular pellet was washed with 5 ml of Delbecco’s PBS and recentrifuged at 100 g for 3 min. The washed pellet (0.2 ml packed pellet/fat pad) was resuspendedin 5 ml of Medium 199 (Earle’s salts plus bicarbonate), fetal calf serum (20x), gentamycin (50 pg/ml), and amphotercin B (2.5 ,ug/ml). The vascular suspension was then filtered through a stainless steel screen(200~,umholes) attached to a suction flask. Tufts of capillary networks and smaller fragments were retained by the screen (Fig. 2), and free cells in suspensionwere filtered through the holes. The capillaries were washed from the screen with a jet of culture medium (15 ml) from a 30-ml syringe

FIG. 2. Isolated capillary network. Single red blood cells can be seen in the vessel’s luminae. (x300.) Scale marker = 33 pm.

(20-gaugeneedle)into a plastic Petri dish, transferred to centrifuge tubes, and repelleted at 100 g for 3 min. The entire isolation procedure was performed in a laminar flow hood, and sterile apparatus and solutions were used in all operations. Culture Conditions The tinal capillary pellet (0. l-ml packed capillaries/fat pad) was resuspendedin 5 ml of Medium 199 (Earle’s salts plus bicarbonate), fetal calf serum (20 %), gentamycin (50 pg/ml), and amphotercin B (2.5 fig/ml). The suspension was transferred to either 25 cm2 Falcon flasks (5 ml/flask) or round or square glass cover slips (1 ml/cover slip) and incubated at 37” in a 5 % CO, atmosphere. After 72 hr of incubation, the medium was removed and replaced with fresh medium containing 0.46-2.30 pg/ml sodium ethyl mercurithiosalicylate (thimerosal) (Aldrich Chemical Co., Inc.). Thereafter the medium was replaced daily with fresh medium con-

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taining thimerosal until monolayers of cells were observed. At this point the cells were cultured in thimerosal-free medium. Upon reaching confluence, the cells were dissociated (0.25 % trypsin in Ca, Mg-free Earle’s BSS) for 7 min, pelleted at 100g for 3 min, resuspendedin fresh medium, and placed into fresh culture flasks. Light Microscopy

Round glass coverslips containing capillary explants or cell monolayers were attached to a thin plexiglass slide chamber. Medium was added (1 ml), and the chamber was sealed with another coverslip. The preparation was then observed with phase contrast microscopy. Electron Microscopy

Endothelial cells were cultured on square glass coverslips that had been sprayed lightly with Teflon. After various intervals of incubation, the coverslips with attached cells were placed in glutaraldehyde (2% in Tyrodes cacodylate buffer, pH 7.4) for 30 min and in osmium tetroxide (1% in buffer) for 30 min. Then they were dehydrated in a graded seriesof ethanol. They were infiltrated in 100% propylene oxide and finally in Epon. The coverslips were then inverted on square Epon-filled molds (Polysciences, Inc.) and polymerized at 60” for 48 hr. The glass coverslips were dissociated from the plastic containing the embeddedcells by immersion in liquid nitrogen according to the method of Chang (1971). Areas or single cells were selected with a phase contrast microscope, demarcated with a glass stylus, and punched out with a metal punch (No. 5 Junior Hand Punch, Robert Whitney, Inc.). The punched disk was then inverted (cell side up) and made to adhere with Epon to the face of a blank Epon block. The cells were sectioned tangentially with a diamond knife at 400-500 A. The sections were then picked up on 300-mesh grids, counterstained with uranyl acetate (60 min) and lead citrate (10 min), and observed with a Phillips 201 electrqn microscope. RESULTS The final pellet in the isolation procedure contained tufts of capillary beds and fragments of capillaries (vesseldiameter : 1O-40 pm) in which single erythrocytes could be observed (Fig. 2). Some free cells were observed in the medium surrounding the capillaries as well as a few erythrocytes. Electron microscopy revealed that the vessel walls were composed of a single layer of endothelial cells joined by intercellular junctions (Fig. 3). Occasional pericytes were observed closely adhering to the periphery of the capillary walls. Smooth muscle cells were not observed in those sections taken, nor was there any evidence of other stromal cell types. After incubation for 5-6 hr, the capillary explants had attached, and cells were observedspreading out upon the substratum (Fig. 4). Someof the spreading cells (IO-1 5 %) exhibited cusp-shapedor ring-shaped profiles (Fig. 5). It appearedas though extensions of thesecells had curved around to join upon themselves.Electron microscopy of a ring cell (Fig. 6) revealed micropinocytic vesicles characteristic of capillary endothelial cells. No junctions were seenjoining the cellular extensions, however, and the cyto-

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FIG. 3. EM :ron micrcIgraph of a cross section of an isolated capillary. The ve:se1 Ivalls are composed of two enldoth elialI cel!s joined by intercellular junctions (arrows). The cytoplal sm i!J filled w6th micropinocy rtic vesicAes. Note ) red blood cell in the capillary lumen. (~22,500.) Scale mar.ker = 0.7 firn.

FIG. 4. A capillary explant after 2 hr of incubation. Cells can be seen spreading from the capi llary onto the sut Bstrate.(x290.) Scale marker = 34 pm.

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FIG. 5. An endothelial cell exhibiting a circular profile in which cellular ex:tensions amrear tcI have joined upon themselves. Square denotes area of cell included in Fig. 6. (x600. .) Scale nlark .er ==l 6 pm.

FIG. 6. Electron micrograph of a transverse section through the circular ccClII in Fig ;. 5. Mic:r( lpinocytic vesicles are present, free in the cytoplasm, and attached to the plasm a -ane. (x 32!,ooo.) Scale Inarker = 0.3 pm.

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plasm appeared to form a continuous ring. As the cells spread into sheets,these anomalous cell shapeswereno longer detectable. Capillary explants were situated at the center of slowly spreading islands of epithelial cells. However, cells exhibiting pleomorphic characteristics ranging from fusiform to very large, flat polynucleate cells, were observed infrequently (less than 10%) within and between the endothelial monolayers. Upon treatment with thimerosal (0.46-l .07 pg/ml), the pleomorphic-cell types began to round up, detach from the substratum, and finally fragment into debris. This effect was evident 12-14 hr after addition of thimerosal to the culture medium and continued for periods of 12-14 days without apparent alteration in the epithelial cells. Higher concentrations (1.68-2.50 w/ml) resulted in destruction of endothelial cells as well. After treatment with thimerosal for 12-14 days, during which the medium was replenished daily to remove cell debris, the pleomorphic-cell types were no longer evident. At this time, the culture was comprised of close-packedmonolayers of flat epithelial cells (Fig. 7). These cells exhibited granular perinuclear cytoplasm and minimal cytoplasmic vacuolation (Fig. 7). Electron micrographs of transverse sections through monolayers showed numerous micropinocytic vesicles attached to the plasma membrane and free in the cytoplasm (Fig. 8). It appeared as though micropinocytosis was occurring at the top free surface of the cells as well as at the cell periphery. Small bundles of filaments (100-A diam) were sparsely scattered throughout the cytoplasm, and intercellular junctions between opposing plasma membraneswere encountered frequently (Fig. 8). Structures characteristic of Weibel-Palade bodies were not observed.

FIG. 7. Primarycultureof capillary endothelium. The cells have spread out to form continuous sheets in a close-packed arrangement. (x290.) Scale marker = 34 pm.

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FIG. 8. Electron micrograph of a transverse section through endothelial cells in monolayer. Numerous micropinocytic vesicles are present, both free in the cytoplasm and attached to the plasma membrane. Intercellular junctions are present between adjacent cells. Cytoplasmic filaments are sparsely scattered throughout the cytoplasm (arrows). (~112,500.) Scale marker = 0.09 pm.

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After placement in thimerosal-free medium, the cells reached confluence within 2-3 weeks, depending upon the density of the original capillary isolate. Doubling times between the first and second passageaveraged between 12 and 17 days. Subcultured endothelial cells progressively developed more pleomorphic characteristics. After the third passage,all of the cells exhibited a large flattened profile with many processes overlapping those of neighboring cells. Some of the pleomorphic cells were also polynucleate. After the fourth passage,the cells were cultured for 5 weeks without reaching confluence. DISCUSSION The epididymal fat pad has been used as a source for the isolation of adipocytes (Rodbell, 1964; McKee1 and Jarett, 1970) as well as vascular endothelium (Wagner et al., 1972). Its advantages as a source of capillaries are twofold: since it functions simply as an insulator for the epididymus and as a fat depot, large adipocytes comprise the bulk of the tissue, limiting the amount of stromal elements; also, the bouyancy of the adipocytes renders their separation from the stroma a simple matter of flotation. Selection of microvascularized fat results in minimal contamination by larger blood vessels.Further removal of blood elements and free stromal cells by filtration yields an enriched preparation of capillaries. The viability of this capillary isolate is evidenced by the ability of the capillary cells to ingest ferritin by micropinocytosis as well as the activity of various enzymes(Wagner et al., 1972). Maruyama (1963) first used 20% fetal calf serum in culturing endothelium and was followed by Fryer et al. (1966) and Jaffe et al. (1972). A standard culture medium (Medium 199) plus 20 % fetal calf serum supports the growth of large blood-vessel derived endothelium (Gimbrone et al., 1974)and maintains capillary endothelial cell in culture as well. Isolated capillaries provide an important advantage in establishing the source from which cultured cells arise since the spreading of cells from seededcapillary explants can be directly observed. Contamination with vascular smooth muscle cells has been a problem in obtaining pure endothelial cultures from the walls of large blood vessels (Gimbrone et al., 1974).Sincecapillaries lack external muscle layers and are essentially naked with the exception of occasional pericytes, this problem is minimized. The lack of cells with ultrastructural features characteristic of smooth muscle in capillaryderived cultures substantiates this claim. The rapid proliferation and overgrowth of fibroblasts has impeded progress in obtaining primary cultures of specific cell types. It is probable that the pliomorphic contaminants observed in primary cultures of capillary endothelium are fibroblastic in nature, and some may also have originated from pericytes on the surface of the isolated capillaries. The mercurial compound sodium ethylmercurithiosalicylate (thimerosal) has been used successfully for the specific removal of fibroblastoid cells from primary cultures of pancreas islets with no apparent effect on the endocrine functions of the cultured islet cells (Braaten et al., 1974). Thimerosal reversibly inhibits sulfhydryl enzymes (Esplin, 1965), but the mechanism of its toxic effect on fibroblasts is unknown. It may be theorized that thimerosal acts on mitosing cells, in which a rounded conformation renders attachment to the substratum very tenuous. Over an extended period of time, rapidly proliferating cells such as fibroblasts would be select-

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ively removed from a culture of slower growing cells. The observation that rounding up precedesdetachment of thimerosal-treated cells supports this hypothesis. The cusp-shaped and circular cells observed early in endothelial cultures, although few in number, are a constant feature of each primary culture. Their ultrastructural similarity to in vivo capillary endothelial cells attests to their origin. The circular forms are reminiscent of the curvature and joining of endothelial cells comprising the walls of the smallest capillaries. The similarity is superficial, however, since the cultured cells form flat circles, not tubes. It may be conjectured that some innate property of endothelial cells contributing to their curvature in small capillaries is expressedfor a period of time following explanation. This contention is supported by the observation that similar circular forms are induced in cultured Schwannoma cells by cyclic AMP analogues (Sheppard et al., 1975)and the similar in vivo curvature of Schwann cells and capillary endothelial cells. Endothelial cultures after 12-14 days of thimerosal treatment are characterized by flat, closely packed cells with little intercellular space.This is characteristic of epithelialderived cell lines. The presenceof micropinocytic vesiclesin the cytoplasm and attached to the plasma membrane is characteristic of capillary endothelial cells in vivo and is further evidence for the endothelial origin of these cells. Cytoplasmic filaments similar to those observed in arterial endothelium suggest a capacity for contraction (Giacome11et al., 1970). These filaments may be smooth muscle actomycin, which has been demonstrated immunologically in endothelial cells in vivo (Becker and Murphy, 1969; Becker and Nachman, 1973). Intercellular junctions joining endothelial cells in culture have also beenobservedby Lewis et al. (1973), Shepro et al. (1973) Gimbrone et al. (1974), and Haudenschild et al. (1975). Weibel-Palade bodies (Weibel and Palade, 1964), the presence of which has supported identification of endothelial cells in cultures derived from large blood vessels (Jaffe et al., 1972; Lewis et al., 1973; Gimbrone et al., 1974),were conspicuously absent in cultured capillary endothelial cells. Weibel and Palade (1964) observed these structures only occasionally in alveolar capillaries and found that they appear in capillary endothelium much less frequently than they do in arterial endothelium. It is possible that due to their paucity in capillaries, the presenceof Weibel-Palade bodies in capillary endothelial cultures was overlooked during random monitoring by electron microscopy. It is also possible that Weibel-Palade bodies do not constitute a common structure in all blood vessel endothelium and are absent in capillary endothelium. Blouse and Chacko (1974) have shown that endothelial cells lining arterioles and veinules vary considerably with regard to both functional and morphological characteristics. The intima of blood vesselsin general may be characterized by such heterogeneity of form. Progressive pleomorphism of endothelial cells in culture has been observed by Gimbrone et al. (1974) and Haudenschild et al. (1975). Haudenschild et al. (1975) began observing morphological changes in large blood-vessel-derived endothelial cultures after the second passage,although in subsequent passagessome monolayers of small epithelioid cells were retained. In capillary endothelium-derived cultures, characteristic epithelioid morphology was not evident at all after the third passage and could not be cultured beyond the fourth passage.It is possible that such pleomorphism represents a degree of dedifferentiation of endothelium in culture, although ultra-

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structurally the cells retain many characteristics of the original culture (Haudenschild et al., 1975). The availability of capillary endothelial cells in culture permits more controlled investigation of factors influencing cell division, metabolism, and surface transport mechanisms. Of particular interest, in this regard, are the factors in control of micropinocytosis, which are poorly understood. ACKNOWLEDGMENT This work was supported by United States Public Health Service Grant 1 Rol HL16666-01 from the National Heart and Lung Institute. REFERENCES BECKER,C. G., AND MURPHY, G. E. (1969). Demonstration of contractile protein in endothelium and cells of the heart valves, endocardium, intima, arteriosclerotic plaques and Aschoff bodies of rheumatic heart disease. Amer. J. Puthol. 55, 1. BECKER,C.G., AND NACHMAN, R. L. (1973). Contractile proteins of endothelial cells, platelets and smooth muscle. Amer. J. Puthol. 71, 1. BLOUSE,S. H., AND CHACKO, S. (1974). Vascular endothelium: In vitro differences between artery and vein. J. Cell Biol. 63, 302. BRAATEN,J. T., MICHAEL, J. L., SCHENK, A., AND MINTZ, D. H. (1974). Removal of fibroblastoid cells from primary monolayer cultures of rat neonatal endocrine pancreas by sodium ethylmercurithiosalicylate. Biochem. Biophys. Res. Commun. 61(2), 426. BRENDEL, K., MEEZAN,E., AND CARLSON, E. C. (1974). Isolated brain microvessels: A purified, metabolically active preparation from bovine cerebral cortex. Science (Washington, D.C.) 185,953. CHANG, J. P. (1971). A new technique for separation of coverglass substrate from epoxy embedded specimens for electron microscopy. J. Ultrastruct. Res. 37,370, FRYER, D. G., BIRNBAUM, G., AND LUTTRELL, C. N. (1966). Human endothelium in cell culture. J. Atheroscler. Res. 6, 151. GIACOMELLI, F., WIENER, J., AND SHEPRO,D. (1970). Cross striated arrays of filaments in endothelium. J. Cell Biol. 45, 188. GIMBRONE, M. A., COTRAN, R. S., AND FOLKMAN, J. (1974). Humanvascular endothelial cells in culture. J. Cell Biol. 60,673. HAUDENSCHILD, C. C., COTRAN, R. S., GIMBRONE, M. A., AND FOLKMAN, J. (1975). Fine structure of vascular endothelium in culture. J. Ultrastruct. Res. 50 (l), 22. JAFFE, E. A., NACHMAN, R. L., BECKER, C. G., AND MINICK, R. S. (1972). Culture of human endothelial cells derived from human umbilical cord veins. Circulation 46,253. JAFFE, E. A., HOYER, L. W., AND NACHMAN, R. L. (1973). Synthesis of antihemophilic factor (AHF) by cultured human endothelial cells. J. Clin. Invest. 52,432. Joo, F., AND KARNUSHINA, I. (1973). A procedure for the isolation of capillaries from rat brain. Cytobios 8,41. LAZZARINI-ROBERTSON, A. A. (1959). The effects of lipoid emulsions on arterial intimal cells in tissue

culture in relation to atherosclerosis. Ph.D. Thesis, Cornell University Medical College, New York. LEWIS, L., HOAK, J. C., MACA, R. D., AND FRY, G. L. (1973). Replication of human endothelial cells in culture. Science (Washington, D.C.) 181,454. MARUYAMA, Y. (1963). The human endothelial cell in tissue culture. Z. ZeNforsch. Mikrosk. Anut. 60,69. MCKELL, D. W., AND JARETT, L. (1970). Preparation and characterization of a plasma membrane

fraction from isolated fat cells. J. Cell Biol. 44,417. POMERAT, C. M., AND SLICK, W. C. (1963). Isolation and growth of endothelial cells in culture. Nature

(London) 198, 859. RODBELL, M. (1964). Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism

and lipolysis. J. Biol. Chem. 239,375.

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SCHOEFL, G. I. (1963). Studies on inflammation. III. Growing capillaries; their structure and perme-

ability. Virchows Arch. Pathol. Anat. 331,97. SHEPPARD, J. R., HUDSON, T. H., AND LARSON, J. R. (1975). Adenosine 3’,5’-monophosphate analogs

promote a circular morphology of cultured Schwannoma cells. Science 187, 179. SHEPRO, D., ROSENTHAL, M., ROBBLEE, L. S., AND BELAMARICH, F. A. (1973). Characterization of

cultured endothelial cells. In “Abstract Volume of IV Congress of the International Society on Thrombosis and Heamostasis,” p. 232. Vienna. TANNOCK, I. F., AND HAYASHI, S. (1972). The proliferation of capillary endothelial cells. Cancer Res. 32,77. WAGNER, R. C., KRIENER, P., BARRNETT, R. J., AND BITENSKY, M. W. (1972). Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc. Nat. Acad. Sci. U.S.A. 69 (ll), 3175. WEIBEL, E. R., AND PALADE, G. E. (1964). New cytoplasmic components in arterial endothelia. J. Cell Biol. 23, 101.

The isolation and culture of capillary endothelium from epididymal fat.

MICROVASCULAR RESEARCH, The Isolation 10,286-297 (1975) and Culture of Capillary from Epididymal Fat Endothelium ROGERC. WAGNERAND MAUREENA. MA...
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