THE ANATOMICAL RECORD 236541-652 (1993) Lymphatic Endothelium Isolation, Characterization and Long Term Culture LEE V. LEAK AND MICHAEL JONES The Ernest E . Just Laboratory of Cellular Biology, Department of Anatomy, Howard University College of Medicine, Washington, DC 20059 (L.V.L.);Laboratory Animal Medicine and Surgery, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892 (M.J.) ABSTRACT Using a collagenase trypsin-EDTAtreatment, we have been able to successfully isolate and grow primary cultures of the lymphatic endothelium (LEC) that were subcultured, frozen for storage, subsequently thawed with good recovery and growth, and serially subcultured. The morphological features of cultured LEC were consistent with that observed for the endothelium of intact lymphatic vessels. A prominent feature of growing cultures was the appearance of large vacuoles in the perinuclear region of the cytoplasm, which became filled with fluid and cell debris engulfed from the culture medium. The basal cell surface lacked a well defined basal lamina and anchoring filaments were observed extending from the basal plasmalemmal surface into the underlying substratum. LEC in cultures were also positive for Factor VIII-related antigen. However, specific granules, characteristic of Weibel-Palade bodies were not observed in ultrathin sections of confluent cultures. F-actin was identified in LEC cultures using fluorescein phalloidin, and in confluent cultures actin filaments were located at the periphery of the cell as a continuous circumferential thin band and short filamentous bundles in the central part of the cell. By using heparin and endothelial cell growth supplement in the culture medium we have been able to grow stable cultures of lymphatic endothelial cells that could be maintained when serially subcultured for over two years. These LEC cultures provide an in vitro model for investigating the function and biochemical properties of the lymphatic endothelium. o 1993 Wiley-Liss, Inc. Key words: Lymphatic Endothelium, Cell Culture, Factor VIII-Related Antigen, Actin Filaments, Anchoring Filaments Lymphatic vessels like blood vessels are lined by a layer of simple squamous endothelial cells. For many years the lymphatics were regarded as passive tubes (Hall et al., 1965; Leak and Burke, 1966). While the biochemical properties of the lymphatic endothelium are not well understood, it is now known that the lymphatics are not merely a series of vessels which passively serve as draining tubes, but they constitute a dynamic system of vessels which play an important role in the maintenance of interstitial fluid homeostasis, the recirculation of lymphocytes and the spread of various diseases including cancer (Carr, 1983; Leak and Ferrans, 1991). Beginning as blind-ended tubes, lymphatics serve an active role in the removal of fluids, plasma proteins, and cells from the connective tissue areas for return to the blood circulatory system. The ability to culture blood vascular endothelial cells from macro and micro vessels over the past two decades has provided much information on the culture, growth requirements, and biochemical processes of the blood vascular endothelium (Gimborne, 1976; Goldsmith et al., 1984; Jaffe, 1984; Jaffe et al., 1973; Piovella et al., 1978; Schwartz, 1978; Wagner et al., 1982; Gordon et 0 1993 WILEY-LISS, INC. al., 1983). These in vitro studies have yielded a considerable body of new information regarding the function of the blood vascular endothelial cells and they also provide a valuable tool for further investigations into the role of the endothelium in normal and pathologic processes. Beginning with the studies of Johnston and Walker (19841, methods are now being developed for both primary (Gnepp and Chandler, 1985; Jones and Yong, 1987) and long-term (Leak, 1991, 1992) culture of lymphatic endothelial cells. Our long-term interest in the lymphatic vascular system in health and disease has prompted us to undertake studies aimed a t the isolation and long-term culture of the lymphatic endothelium in order to further characterize the functional and biochemical properties of the lymphatic endothelial cell. MATERIALS AND METHODS Reagents and Media Medium 199, penicillin, streptomycin, 2% gelatin, fetal bovine serum, heparin, endothelial cell growth Received January 13, 1993; accepted April 9, 1993. 642 L.V. LEAK AND M. JONES supplement, an extract of bovine pitutitary gland containing growth promoting factors for vascular endothelial cells, antisera against Ulex europaeus I lectin, and a solution of 0.5 gfL trypsin-0.2 g/L EDTA were purchased from Sigma Chemical Co. (St. Louis, MO). Fungizone was purchased from E.R. Squibb and Sons, Inc. (Princeton, NJ). Rhodamine labeled antiserum against antihemophilic factor (AHF, Factor VIII), was purchased from Binding Site Inc. (San Diego, CA), and fluorescein phalloidin was purchased from Molecular Probes, Inc. (Junction City, OR). ISOLATION AND HARVEST OF LYMPHATIC ENDOTHELIAL CELLS (LEC) Bovine mesenteries were obtained immediately after slaughter from a local abattoir and placed in plastic containers and kept warm until brought to the laboratory. The lymphatic endothelial cell cultures described herein were obtained from the major mesenteric lymphatic vessel of one animal. Lymphatic endothelial cells were isolated from bovine mesenteric lymphatic vessels using a modification of the methods described by Johnston and Walker (1984). Sterile methods were employed throughout the isolation and culture procedures. To visualize the lymphatic vessels, 0.5% trypan blue in phosphate buffered saline (PBS) was injected into mesenteric lymph nodes, after which efferent lymphatic vessels were instantly outlined and a segment of the mesenteric lymphatic vessels (15-25 cm in length) was ligated and dissected from its tributaries and both ends were cannulated with Radiopaque Fep Teflon I.V. Catheters (14 to 20 gauge, Abbott Hospitals Inc., N. Chicago, IL). Following cannulation, each vessel was flushed with PBS to clear the lumen of trypan blue and cells. To isolate the lymphatic endothelial lining cells, each vessel was first filled with a solution of collagenase (1.5 mg/ml in PBS) for 15 minutes and second with a solution of 0.25% trypsin-0.02% EDTA for 10-15 minutes. After each treatment the vessel was flushed with PBS into separate 15 ml centrifuge tubes and centrifuged at 200g for 10 minutes. After centrifugation the supernatant was removed and the faint cell pellet was suspended in and washed twice with PBS. After the second wash, the pellet obtained from each treatment was suspended in 2 ml of Medium 199 with 20-25 mM HEPES buffer and Earle’s salts containing L-glutamine, 15% fetal bovine serum, 100 pglml endothelial cell growth supplement, 100 pg/ml heparin, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2.5 pg/ml fungizone. This complete medium is referred to as lymphatic endothelial cell (LEC) complete medium. The isolated lymphatic endothelial cells obtained from each treatment were plated into separate multiwell plates that were coated with 1%gelatin. Cultures were maintained at 37°C in a humidified incubator with 5% CO,. Examination of cultures with phase optics revealed that a large number of cells had also been isolated in the treatment with trypsin-EDTA. Therefore, in subsequent isolation procedures the cell pellets obtained from both treatments were combined prior to plating. After the first day cells were fed with fresh medium, and one half of the medium replaced with fresh medium every two days for the first week. After the first week of culture the medium was completely changed once weekly with LEC complete me- dium. The cells were maintained in LEC complete medium until the cells reached confluence. Subculture of Lymphatic Endothelial Cells In choosing cells for the continuous subculture of lymphatic endothelial cells, we selected cells from cultures that were at near confluence while still in a period of active growth. At this point, cultures were rinsed twice with PBS- (calcium and magnesium free) and then treated with 0.25% trypsin-0.02% EDTA until cells began to retract and round up (3-5 minutes). The trypsin-EDTA was removed and replaced with LEC complete medium. Cells were dislodged from the plastic surface by gentle agitation of medium using a pipette. Once free, the cells were routinely replated to freshly coated 60 mm x 15 mm petri dishes at a ratio of 1:3 and placed in the incubator for 30 minutes. After this short incubation time the floating cells were removed by pipetting off the culture medium and briefly rinsing the petri dish with PBS- . Adherent cells were refed with LEC complete medium and returned to the incubator. Subculturing With Microcarrier Beads Microcarrier beads Cytodex I11 (Jacobson and Ryan, 1982)were used as a matrix to subculture LEC without the use of proteolytic enzymes. The beads were hydrated in PBS and sterilized by autoclaving. After soaking beads in LEC complete medium for 12-24 hours, 0.5 ml to 1ml of beads in LEC complete medium were added to confluent LEC cultures. Lymphatic endothelial cells were allowed to migrate and grow over the surface of the microcarrier beads. After 2-3 days the beads with attached lymphatic cells were detached from the petri dish by pipetting, or by scraping with a rubber policeman, and plated onto gelatin coated petri dishes. Additional culture medium was added and the dishes placed in the CO, incubator for continued culture. After the cells migrated off the beads onto the coated surface of the petri dish the beads that were free of cells were removed by changing the culture medium. Subsequent subculture and cell transfers could be continued with this nonproteolytic procedure. Freezing LEC For Storage and Thawing for Subculture To freeze lymphatic endothelial cell, cultures were used in the log phase of growth. Culture medium was removed and cells were rinsed two times with PBS-, and then covered with trypsin-EDTA for 6-8 minutes or until cells rounded up and began to dislodge, the trypsin EDTA solution was removed and LEC complete medium was added and cells were dislodged from the plastic surface by gentle pipetting. The cell suspension was added to a 15 ml centrifuge tube and centrifuged at 200g for 10 minutes to pellet cells. The supernatant was discarded and the cell pellet resuspended in freezing medium consisting of freshly made cold LEC Complete medium with 10% DMSO a t a cell concentration of 5 x lo6 to 2 x lo7 cells per ml. A 1.5 ml of cell suspension was added to 2 ml freezer vials and kept on ice until placed in the gaseous phase of a liquid nitrogen storage tank and allowed to freeze and then left for storage. Alternatively, vials with cell suspensions were place at -70°C and allowed to freeze slowly for 2-12 hours. This was done either by placing ice cooled vials LYMPHATIC END07'HELIUM IN VITRO in a -70°C freezer, or by slow freezing in a dry ice acetone mixture and then placing vials in a -70°C freezer for 8-12 hours. Vials were then rapidly placed in liquid nitrogen and transferred to a liquid nitrogen freezer for cold storage. For the recovery of frozen cells, vials were removed from the liquid nitrogen storage cabinet and thawed at 37°C. The contents of the thawed vial were slowly pipetted into 10 ml of LEC complete medium and the cells were plated on gelatin coated dishes and processed as described for the subculture of LEC. lmmunofluorescence Studies of LEC Factor Vlll 643 Light microscope and cells were photographed with panatomic x film. Scanning Electron Microscopy Lymphatic endothelial cells grown to confluence on glass cover slips were fixed in 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 2 hours at room temperature and postfixed with 1% osmium tetroxide in 0.1 M cacodylate (pH 72)for 1 hour at 5"C, rinsed in buffer and dehydrated in an ethanol series to 100%and then infiltrated with amyl acetate and critical point dried using COz in a Balzers Critical Point Dryer (Leak, 1983). The critical-point-dried cover slips were coated with Gold-Palladium and examined in an ETEC Autoscan. To detect Factor VIII related antigens (Jaffe, 1973) cells were grown on gelatin coated glass cover slips. As cells reached near confluence, cultures were washed in Transmission Electron Microscopy PBS and fixed for 5 minutes in -20°C acetone and rinsed twice in PBS. Cells were incubated with rhodaConfluent lymphatic endothelial cells grown on mine conjugated sheep antiserum t o human Factor glass cover slips and on coated culture dishes were VIII diluted 1:lO in PBS for 45 minutes at room tem- fixed and postfixed as described above for SEM. The perature. The cover slips were washed for 5 minutes in fixed cultures were then washed in buffer and treated PBS and then mounted with glycerol and PBS (l:l), with 1% tannic acid in 0.1 M cacodylate buffer for 2 and the edges were sealed with clear nail polish and hours a t 5°C and then processed for epon embedding the slides were examined and photographed with a and sectioning for electron microscopic observation Zeiss Axiovert 10 inverted microscope. Negative con- (Leak, 1983). Ultrathin sections were obtained with a trols for Factor VIII related antigens consisted of cul- diamond knife, double stained with uranyl and lead tures of fibroblast. citrate and examined in a Phillips EM 410 electron microscope. Ulex europaeus I Lectin LEC cultures on cover slips were processed as above and incubated in FITC-conjugated anti-Ulex europaeus I lectin diluted 1:lO in PBS for 30 minutes (Holthofer et al., 19821, washed in PBS, cover-slipped, and examined by fluorescence microscopy. Negative controls consisted of cultures of fibroblast as above. RESULTS LEC Isolation and Subculture Once bovine mesenteric lymphatic vessels were outlined with trypan blue, large segments of the vessels were easily visualized and could be dissected from the mesentery and processed for isolating the lymphatic Actin fibers in LEC endothelial lining cells. Cell pellets obtained by the For detection of f-actin, cells were grown on gelatin isolation procedures described herein were found to coated glass cover slips to confluence, washed twice yield single lymphatic endothelial cells and small with PBS, and fixed in 3.7% formaldehyde in PBS for clumps consisting of 3 to 6 cells. During overnight cul10 minutes a t room temperature. Following fixation, ture the isolated cells attached to the gelatin coated cells were washed twice with PBS and the cover slips multiwell plates (Fig. 1). Growth was apparent within were placed in a solution of -20°C acetone for 5 min- 2 to 3 days as individual cells and small clusters of cells utes and then air dried. Cover slips were incubated in began to form distinct colonies. The cells grew closely a 1:20 solution of fluorescein phallotoxin (Wulf et al., apposed to each other and remained as a monolayer. 1979) a t room temperature for 30 minutes. Cover slips After about 2 to 3 weeks in culture several wells had were then washed twice in PBS and mounted on slides grown to near confluence with most of the cells demwith 1:l solution of PBS glycerol. Slides were examined onstrating a uniform cobblestone appearance, characby fluorescence microscopy as above. teristic of the endothelium (Fig. 2). Confluent cultures were treated with trypsin-EDTA and the isolated cells Silver nitrate staining of LEC subcultured. Lymphatic endothelium like the blood Mesenteric LEC cultures grown to confluence were vascular endothelium becomes adherent within 30 stained with silver nitrate using the method of Poole et minutes to 1 hour after being plated on gelatin coated al. (1958) with modifications. LEC grown on gelatin culture dishes. Therefore, contaminating cells such as coated cover slips to confluence were treated with 5% fibroblast or smooth muscle cells which appear as elonglucose for 1-2 minutes, 0.4% AgNO, for 1 minute, gated spindle shaped cells that are reported to grow in rinsed with 5%glucose for 1 minute, then treated with whorls or in parallel arrays could be eliminated during 1% NH,Br and 3% cobalt bromide for 3 minutes and subculture by plating cells in gelatin coated multiwell rinsed with 5%glucose. The cover slips were exposed to plates and allowing lymphatic endothelial cells to adUV light for 1-2 minutes, fixed in 4%formalin for 2 here for only 30 minutes, after which the non adherent hours or overnight, rinsed in PBS, and then stained in cells were withdrawn and the dishes rinsed briefly Diff-Quik, rinsed, dehydrated in ethyl aclohol and in- with PBS- to remove the remaining non-adherent filtrated with xylene before mounting slides with per- cells. With this procedure cultures were obtained that mount. Slides were examined with a Zeiss Universal were free of contaminating smooth muscle cells. 644 L.V. LEAK AND M. JONES Fig, 1. Phase contrast light micrographs of lymphatic endothelial cells adhering to gelatin coated culture dishes as single cells (a, x 900) and in a cluster of four cells (b,x 800). Fig. 2. Light photomicrograph of LEC culture growing closely apposed to each other with dividing cells shown at arrow at 2 weeks following isolation. x 1,000. LYMPHATXC ENDOTHELIUM IN VITRO Fig. 3. Light photomicrograph showing LEC attached to cytodex microcarrier beads. Lymphatic endothelial cells have migrated onto this microcarrier bead to form a monolayer of cells over its surface as observed in phase contrast optics (a, x 1,4001and after fixation and staining with Diff Quik (b,x 1,300). Fig. 4. Light photomicrograph of LEC showing numerous vacuoles (v) throughout the cytoplasm. In some cells the vacuoles completely surround the nucleus (arrow). Note multinucleated cell in upper top region of micrograph (double arrows). x 800. 645 Figs. 5-6. 647 LYMPHATIC ENDOTHELIUM IN VITRO Subculture With Microcarrier Beads Freezing for Storage and Subculturing Within a few minutes (5-10) after inoculating microcarrier beads to cultures of lymphatic endothelial cells many of the beads became attached t o the cells and after 30 minutes to one hour the cells began to spread onto the surface of the beads. By two days many of the beads were covered and the cells presented a cobble stone morphology similar to cells growing on the surface of multiwell plates or culture dishes (Fig. 3). For transfer and subculture, cell covered microcarrier beads were dislodged from the culture dishes as described under methods. The lymphatic endothelial cells were forzen and thawed and subcultured with good recovery of many viable cells that were able to sustain continued growth. The cells maintained their morphology and function to secrete coagulation factor (Factor VIII) as determined by immunofluorescent studies (see below). Growth Rates of LEC Lymphatic endothelial cells plated on gelatin and grown in LEC complete medium grew rapidly with a doubling time of 24-30 hours. This growth rate could be maintained over long periods of continued passages with the use of heparin and growth factor in the medium. MORPHOLOGICAL STUDIES Light Microscopy Silver Nitrate Staining To confirm their endothelial nature as continuous sheets of flattened cells we utilized the silver nitrate method to outline the intercellular borders of confluent LEC cultures (Poole et al., 1958). In cultures exposed to silver nitrate prior to fixation a precipitate from the silver nitrate reaction was localized along the intercellular borders of adjacent cells (Fig. 5 ) . By staining the cells with Diff-Quik the nuclei of lymphatic endothelial cells were brightly stained and appeared as oval structures containing several prominent nucleoli. Cells in the process of undergoing mitosis were also observed. For the most part the cells were extremely flattened with their margins closely apposed to form intercellular junctions. Examination of culture dishes a t one hour after subULTRASTRUCTURAL STUDIES OF LEC culturing with an inverted phase microscope showed Scanning Electron Microscopy (SEM) lymphatic endothelial cells firmly attached to the surface of the coated petri dish. At 3 to 5 days after subConfluent LEC cultures observed in the scanning culturing, small colonies were apparent which prolif- electron microscope showed a continuous sheet of cells erated and spread to form a continuous sheet of cells. growing in a monolayer (Fig. 6). The lymphatic endoAs LEC cultures grew to near confluence a prominent thelial cells were extremely flattened with the nuclei feature observed in many cells was the appearance of showing only a slight elevation above the surface of the numerous vacuoles which surrounded the nucleus (Fig. surrounding cytoplasm. The regions of contact between 4). In time these vacuoles became filled with dense adjacent cells were seen as irregular ridges which ingranules (cell debris) engulfed from the culture me- dicated sites of overlapping cell margins (Fig. 6). The dium forming a dense corona around the nucleus. As apical cell surface was usually smooth with only an growth of cultures continued there was a decrease in occasional bleb or projection extending from its surface. the vacuoles that contained dense granules. This Transmission Electron Microscopy (TEM) of LEC marked reduction of vacuoles with dense granules would suggest the breakdown of vacuolar content by Ultrathin sections of confluent cultures examined lysosomal enzymes for utilization by the cell. In high with the transmission electron microscope showed the density cultures that were undergoing rapid growth Golgi complex and the usual cytoplasmic organelles in rates the cells also showed an elongated shape while the perinuclear region. In addition, numerous vacuoles still maintaining a monolayer. However as cultures of varying sizes were also observed (Fig. 7). The adjareached confluence the cells resumed the typical polyg- cent cell margins were observed in ultrathin sections to onal shape which indicated a cessation of growth. To be closely apposed to each other with interdigitating, maintain the LEC in long term cultures the cells were overlapping and specialized adhering sites a t regions of disassociated at near confluence while many cells were peripheral cell to cell contact (Fig. 8a,b). There were still in an active growth phase. Following trypsin- also areas where overlapping cell margins were sepaEDTA treatment cells were plated in 60 mm petri rated by wide gaps, reminiscent of patent intercellular dishes and then to larger dishes for subsequent passage junctions of lymphatics in vivo. The basal surface for freezing and for storage. lacked a well defined basal lamina, a trait that is also characteristic of the lymphatics in vivo. Prominent along the basal surface were filamentous structures very similar t o the anchoring filaments described for the intact lymphatic capillary vessel (Leak and Burke, Fig. 5. The close apposition of adjacent LEC is demonstrated in 1968). The filaments were attached or anchored to the cultures treated with the silver nitrate method is illustrated in this photomicrograph. The silver nitrate reaction forms a dense precipi- plasmalemmal surface and extended into the underlytate which is localized along the intercellular junctions of adjacent ing substratum of the culture surface (Figs. 7, 8b). cells (*I. Vacuoles appear as clear circular areas in the cytoplasm. The Plasmalemmal vesicles were observed along both basal nuclei contain several prominent nucleoli (arrows). x 2,200. and apical surfaces and vesicles also occurred throughFig. 6. A scanning electron micrograph of LEC confluent culture. out the cytoplasm. The oval nucleus occupied the cenThe intercellular junction between adjacent cells are also observed tral region of the cell and its perinuclear region con(J).The cytoplasm is extremely flattened except in areas occupied by the nucleus (*I, which appears as a slight elevation or domed shaped tained an extensive Golgi complex with numerous vesicles associated with its periphery (Figs. 7,9). While areas. X 15,000. 648 L.V. LEAK AND M. JONES a small number of dense granules were observed in the perinuclear region, we did not observe distinct granules that were characteristic of Weibel-Palade bodies. Mitochondria and rough endoplasmic reticulum were observed in the perinuclear as well as the attenuated regions of the cytoplasm (Figs. 7, 9). Numerous vacuoles of varying sizes were prominent throughout the perinuclear region and corresponded to the large vacuoles observed with phase optics (cf. Figs. 4, 7). In ultrathin sections many of these were translucent while others contained an electron dense material (Figs. 7,9). Microtubules and cytoplasmic filaments were observed throughout the cytoplasm with many areas containing bundles of cytoplasmic filaments which could be identified as f-actin according to their size and distribution pattern. lrnrnunofluorescence Studies Immunof luorescence studies to demonstrate the presence of Factor VIII related antigens were carried out on non-confluent as well as confluent cultures. Examination on non-conf luent cells with immunof luorescence microscopy showed only a small number of cells exhibiting a weak staining reaction for factor VIII related antigens. However, examination of confluent cultures of both early and late passage cells showed an intense staining reaction in the perinuclear region (Fig. 10). On the other hand, immunofluorescence studies for the demonstration of Ulex europaeus I agglutinin a lectin specific for some a-L-fucose-containing glycocompoundsfailed to show cell surface staining for this lectin, which has been shown to be a marker for blood vascular endothelial cells (Holthofer et al., 1982). Lymphatic endothelial cells were also stained with fluoresein phallotoxin which is specific for f-actin (Wulf et al., 1979). Confluent LEC cultures exhibited a very intense staining reaction for f-actin, which appeared as dense bundles in the periphery of the cytoplasm and dense bundles extending across the central cytoplasm (Fig. 11). Long-Term Cultures With repeated passages (12-15) LEC cultures contained a small number of very large flattened cells which often contained several nuclei. These cells had a large cytoplasm to nuclear ratio and were similar to those described by Gospodarowicz et al. (1976) for the blood vascular endothelium after continued cell passage. As cultures reached near confluence we have continued to subculture, freeze, thaw, and reculture them for over 2 years with their morphology and growth pattern remaining stable. DISCUSSION Fig. 7. Ultrathin sections of lymphatic endothelium from a confluent culture showing a portion of the nucleus (n),G l g i apparatus (G), mitochondria (m), and numerous vacuoles (v) of various sizes. Filamentous structures (arrows) extend from the basal surface which resemble anchoring filaments of lymphatics in vivo. x 16,200. The colorless nature of lymphatic vessels makes it difficult to visualize the lymphatic system in vivo. However, the elusive nature of lymphatics can be overcome by the injection of vital dyes to outline the walls of these delicate vessels (Hudack and McMaster, 1932; Leak and Burke, 1966). The Of this method for labeling mesenteric lymphatic vessels has also facilitated the visualization of lymphatic vessels for isolating lymphatic endothelial cells for in vitro growth (Johnston and Walker, 1984). In the present study we have used a modification of the procedure described by LYMPHATIC ENDOTHELIUM IN VITRO 649 Fig. 8. The intercellularjunctions between adjacent LEC in confluent cultures are illustrated in these electron micrographs.a: Adjacent cells are extensively overlapping (J-1) and also form interdigitations J-2), x 10,800. b Adjacent cells are closely apposed by short overlapping cell margins (J), and filaments (arrows) extend from the basal cytoplasmic surface. x 29,000. Johnston and Walker (1984) for the isolation, growth, characterization, and long-term culture of LEC from bovine mesenteric lymphatic vessels. When plated on gelatin coated dishes using culture medium containing a growth factor, (Gospodarowicz and Bialecki, 1978) lymphatic endothelial cells grew as a monolayer of closely apposed cells. As the cultures reached confluence they exhibited a cobblestone appearance characteristic of lymphatic endothelial cells in vitro (Gnepp and Chandler, 1985; Johnston and Walker, 1984;Jones and Yong, 1987). LEC cultures monitored with the phase microscope showed good growth and a retention of the lymphatic endothelial cell morphology as primary cultures were subcultured, frozen, thawed, and then serially subcultured for over 2 years. In high density cultures that were undergoing rapid growth rates the cells also showed an elongated shape while still maintaining a monolayer. In plating cells for subcultures, cells were allowed to adhere to gelatin coated dishes for 30 min, and the nonadherent and floating cells were removed. This short adhesion time permitted us to obtain cultures of LEC that were free of contaminating smooth muscle cells. The examination of these cells using immunofluorescence methods to detect Factor VIII related antigens (Jaffe et al., 1973; Rosen et al., 1981) showed that all of the cells were positive for Factor VIII with most of the cells exhibiting an intense reaction for the antigen by immunofluorescence. The presence of this characteristic pattern for Factor VIII in all cells provides a further indication that contamination free cultures of lymphatic endothelial cells had been achieved. This observation also correlates with the finding of others for lymphatic endothelial cells in vitro (Johnston and Walker, 1984; Gnepp and Chandler, 1985; Jones and Yong, 1987), including a lymphatic endothelial cell line (CH3) derived from a massive recurrent chyle-containing retropreitoneal lymphangioma by Way et al. (1987). Weibel-Palade bodies (Weibel and Palade, 1964) have been demonstrated to represent intracellular sites for the concentration of Factor VIII (Von Willebrand protein) in the vascular endothelium (Wagner et al., 1982; Warhol and Sweet, 1984). Although we were unable to detect specific granules that were characteristic of Weibel-Palade bodies in bovine LEC in ultrathin sections analyzed in the transmission electron microscope, there was an intense staining exhibited in immunofluorescence studies for Factor VIII antigens. This positive reaction for Factor VIII, may be reflective of the active synthetic state of these cells in culture and may represent a state where the coagulation factor is being actively secreted via the constitutive pathway, in contrast to conditions where the cells have reached a stable non-growth state where the pathway for synthesized Factor VIII then becomes regulated and the protein is directed to secretory granules until receiving a 650 L.V. LEAK AND M. JONES Fig. 9.Electron micrograph of portion of lymphatic endothelium showing many cytoplasmic organelles in the perinuclear region. Rough endoplasmic reticulum (er) and vacuoles (v) of various sizes with some containing a n electron dense substance (arrow) are also found throughout the cytoplasm. X 20,000. Fig. 10. LEC from confluent culture showing intense reaction for Factor VIII antigen. x 2,500. signal for exocytosis (Farquhar, 1985;Pfeffer and Rothman, 1987). The failure to demonstrate these structures in some cultures of both blood vascular and lymphatic endothelial cells have been attributed to fewer Weibel-Palade bodies in the endothelium of various size vessels (Gimborne, 1978; Haudenschild, 1984; Jones and Yong, 1987; Zetter, 1984). The lymphatic endothelium in vivo is characterized as an extremely flattened layer of endothelial cells which is much flatter than the blood vascular endothelium (Leak, 1970,1984; Leak and Burke, 1966).The attenuated nature of the lymphatic endothelium was also exhibited in confluent cultures when examined in both scanning and transmission electron microscopes. Although the lymphatic endothelial cells in the present study were derived from the larger collecting lymphatic vessels, the morphological features exhibited by these cells in culture displayed many features that were characteristic of the lymphatic capillary endothelium in vivo (Leak, 1970,1972; Leak and Burke, 1968). Plated on gelatin coated dishes (type I collagen), the lymphatic endothelial cells grew to confluence with the basal (connective tissue or abluminal) surface lacking a well defined basal lamina. In addition, anchoring filaments were attached to the basal surface of the en- dothelium and extended into the underlying substrate. In vivo these filamentous structures serve to anchor the lymphatic capillary wall to the adjoining connective tissue and play a pivotal role in regulating the dynamics of the lymphatic wall for the permeability of interstitial fluids and particulate materials (Leak, 1971, 1972). A salient feature of the lymphatic endothelium is its ability to rapidly phagocytose particulate components from both the luminal and connective tissue surfaces, culminating in the formation of vacuoles of various sizes (Leak, 1971). Our finding of a large number of vacuoles that became filled with fluid and particulate materials engulfed from the culture medium and the subsequent breakdown of this material by lysosomal enzymes, is consistent with previous studies which demonstrated that ferritin particles and other proteins that were engulfed by lymphatic endothelial cells in uiuo were digested by lysosomal enzymes (Leak, 1971, 1972). This phagocytic property was retained by both primary and long-term cultures and further indicates the stability of the lymphatic endothelium in long-term cultures. The presence of f-actin microfilament bundles in the form of peripheral bands and short microfilament bundles in the central region of confluent lymphatic endo- 651 LYMPHATIC ENDOTHELIUM IN VITRO Fig. 11. LEC from confluent culture that was incubated with fluoresein labeled phallotoxin. Actin filament bundles appear as dense bundles in the peripheral cytoplasm (arrow)and also extending across the central cytoplasm (double arrows). x 2,500. thelial cells in culture is similar to the distribution of f-actin in confluent cultures of the blood vascular endothelium (Wong and Gotlieb, 1986). While a number of workers have demonstrated the presence of actin in lymphatic endothelial cells in vivo (Lauweryns et al., 1976; Leak, 19841, the role of actin microfilaments in lymphatic endothelial cell-cell adhesion and in the propulsion of lymph is still not understood and will require further study. The data presented here demonstrate the growth of the lymphatic endothelium in long-term culture when plated on gelatin coated surfaces in culture medium supplemented with heparin and exogenous growth factors. Basic fibroblast growth factor has been shown to be a potent mitogen for mesoderm-derived cells (Gospodarowicz et al., 1976 Terranova et al., 1985) and is capable of triggering the proliferation of vascular endothelial cells a t very low concentrations (Gospodarowicz et al., 1977). It also promotes the growth of low density blood vascular endothelial cells and extends their life span in long-term cultures (Gospodarowicz et al., 1976, 1987). The ability to isolate and grow lymphatic endothelial cells in long-term cultures provides an opportunity for studying their function and bio- chemistry. Moreover, the in vitro growth of the lymphatic endothelium offers the opportunity to examine the cell biologic behavior of these cells to better understand the role of the lymphatic vascular system under normal physiological conditions and also to delineate the events leading to pathologic states. ACKNOWLEDGMENTS This work was supported in part by NSF DCB89166625, and AHA-Holmes Award, American Heart Assoc. and a Howard University Faculty Research Support Grant. The authors thank Ms. Darlene Banks for excellent technical assistance. Appreciation is expressed to Ms. Robbin Shuff and Mr. Robert Bullock for providing the bovine mesenteries for this study. LITERATURE CITED Cam, I. 1983 Experimental lymphatic metastasis. J. Microsc., 131: 211-220. Farquhar, M.G. 1985 Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol., 1:447-488. Gimborne, M.A. 1976 Culture of vascular endothelium. In: Progress in Hemostasis and Thrombosis. T. Spaet, ed. Grune and Straton, New York, 3:l-28. Gnepp, D.R., and W. Chandler 1985 Tissue culture of human and 652 L.V. LEAK AND M. JONES canine thoracic duct endothelium. In Vitro Cell. Dev. Biol., 21: 200-206. Goldsmith, J.C., J.J. McCormick, and A. 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