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Lymphatic endothelium isolation characterization and long term culture.

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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.
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