21 (I 975) 259-272 (‘1Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
.4rhuws&rosis,
THE
PORCINE
D. N. SLATER Division
AND
ENDOTHELIAL
CELL
IN TISSUE
259
CULTURE
J. M. SLOAN*
of Pathology,
University
qf‘ Shefield,
ShcTjfield (Great
Buitaitl
J
(Received September 9th, 1974) (Accepted November l9th, 1974)
SUMMARY
Endothelial cells from porcine aorta and inferior vena cava have been harvested, using trypsin, EDTA or collagenase, and grown in tissue culture. Growth-behaviour. cytology, scanning and electronmicroscopy findings are reported. It is hoped this technique will prove useful in the investigation of atherosclerosis.
Key words : Atherosclerosis Pig - Pinocytotic
- Endothelial
cell - Microjlaments
- Multitubular
that
bodies -
vesicles - Tissue culture
INTRODUC’i-IOh
Despite the importance about
its metabolism
of endothelium
and the changes
as emphasised
by Oliver’, little is known
that occur in atherosclerosis.
An operational
difficulty is that the endothelial cell is intimately attached to other vascular structural components. There have been few tissue culture studies using pure cell lines of endothelial cells. Fryer et a/.2, extending the original work of Maruyama3, obtained cultures of human umbilical arterial and venous endothelium after removing the cells from the aorta using a technique with trypsin. However, cellular replication was not good and the ultrastructure was not studied. Buonassis? isolated rabbit aortic endothelium with collagenase; he achieved good cellular replication and showed that the cells had the capacity to synthesise and excrete sulphated mucopolysaccharides. Jaffe et al.” and Gimbrone et al.6 have recently obtained replicating human venous endothelium using the collagenase technique. This communication reports that replicating endo-
* Present address: Ireland.
Institute
of Pathology,
Queen’s University
of Belfast, Belfast, Northern
I>. N.
260 thelial
cells can be obtained
using trypsin,
MATERIALS
EDTA
from
both
porcine
arterial
SLATER,
and venous
J. M. SLOAN
endothelium,
or collagenase.
AND METHODS
All materials
were supplied
by Biocult
Laboratories
unless
otherwise
stated.
Harvesting endotfzelial cells Five to ten cm segments of aorta, inferior vena cava or pulmonary veins were obtained within 3 min of slaughter from young male pigs (20-30 weeks old). The segments were washed in Hanks’ balanced salt solution (BSS) and transported in Hanks’ BSS at 4°C. All minor vessels were ligated and the ends of the segments were clamped trypsin
with bull-dog
w/v in Hanks’
clips. The sacs were inflated with 5-10 ml of 0.25 x,
BSS, 1 in 5000 EDTA
in Dulbecco’s
calcium
and magnesium
free phosphate buffered saline (Flow Laboratories) or phosphate buffered saline with calcium and magnesium containing 1 mg/ml collagenase (Sigma Type III: Fraction “A”). After incubation at 37°C one bull-dog clip was removed and the contents transferred
into a sterile 30 ml universal
container
(Sterilin
Ltd.). The sac was cut
open in its longitudinal axis and the luminal surface gently scraped with a stainless steel scalpel blade (Swann-Morton No. 22). The blade must be inclined at an angle of approximately 60” to the intimal surface. The endothelial cells were washed off the blade with a jet of culture medium (see below) and the samples centrifuged at 1000 rpm for 5 min. The supernatants resuspended
were discarded
in 2 ml of culture
finally added to 4 ml aliquots
medium; of culture
and the white pellets of endothelial cells I ml suspension of endothelial cells were
medium.
For scanning
electron
microscopy
the cells were grown in 5 cm glass petri dishes containing 13 mm diameter cover slips (Chance Ltd.). For electron microscopy they were grown in 30 ml tissue culture flasks (Falcon Plastics). Culture media At first we used Medium
199 with Earle’s
BSS containing
2.00 g/l NaHCOs
(Flow Laboratories) gassed in 5 “/oCOa/air or Medium 199 with Hanks’ BSScontaining 0.35 g/l NaHCOs (Flow Laboratories). In later experiments we used Medium 199 with Hanks’ BSS containing Hepes buffer. 100 ml were all supplemented by 20 ml of foetal bovine serum, 0.5 ml of 200 mM glutamine and 100 units of penicillin and 100 pg of streptomycin/ml. Subculture and quantitation Subculture was achieved using 0.15% trypsin w/v Hanks’ BSS. Single cell suspensions were obtained by ultrasonification for 5 set in an ultrasonic bath (Kerry K5200). Cells were counted in a haemocytometer and viability was assessed with I “/;; trypan blue.
PORCINE
tNDOTHELlAL
CELL IN I’ISSUE CULTURL
261
Microscopy
Cells were studied For light microscopy I5 min in methanol.
during
growth
using a Leitz Diavert
inverted
microscope.
cells were either fixed for 30 min in IO”,, formal-saline or for For scanning electron microscopy the cells were fixed for I ht
in 3 % phosphate-buffered
glutaraldehyde,
washed in phosphate-buffered
distilled water and finally slowly dried in air. The coverslips were mounted stubs with DAG915 conductivity paint and coated with gold-palladium. viewed on a Cambridge S4 Stereoscan at IO kV. Cells were prepared for transmission electron ley’s techniquei.
Cells were fixed in 37; phosphate
microscopy buffered
saline, glass on scanning They were
by modifying
glutaraldehyde
Brink-
and post-
fixed in I :‘:, osmium tetroxide for 30 min. They were stained with 2 y,, aqueous uranyl acetate for 30 min and dehydrated in ethanol. Subsequently they were cleared with upgraded strengths of hydroxypropylmethacrylate to Epon. The Epon was peeled off the flasks and selected areas bored out. Sections were cut on a Porter-Blum MT2B ultramicrotome and viewed on an AEI Corinth SO0 electron microscope at 60 kV. RESULTS
Light microscopy and electron microscopy showed that the endothelial ccli lining of the aortic and venous sacs no longer remains after harvesting. There appear5 to have been minimal
intimal
disruption
with the internal
elastic
lamina
remaining
intact. Incubation
for 20 min appeared
cells using 0.25 ‘/” trypsin, the incubation venous
EDTA
satisfactory
for harvesting
(I in 5000) or collagenase
times had to be approximately
doubled
aortic
(I mg/ml).
to obtain
similar
endothelial However. numbers
of
cells. Cell viability
of both aortic and venous cells after incubation was approximatelc 95 ‘:,, and effluent cell numbers were between 2 and 5 Y IO”. Cellular attachment varied between 20 and 60’%, of the initial inocula; this proportion depended more on the number of cells in an aggregate rather than on the number of cells per flask. Aggregates over 20 cells appeared to establish themselves less readily. Cellular attachment was present in 3-6 hr and confluent monolayers of 2-3 IO” cells per flask were achieved in 6-9 days. To achieve confluent monolayers the initial inoculum had to be greater than 5-7 x 10” cells per flask; with sm2ller numbers individual cell colonies were obtained. With the exception of the initial incubation time no significant differences were found between aortic and venous cells. Although all experiments involved primary cultures, subcultures at the end of every week were possible and one cell-line has been maintained for 3 months.
D.
262
Fig.
I,
A confluent
endothelial
Fig. 2. Three-day
cells appear
culture
cells. HE,
of h-day arterial
showing
its monolayer
J. M. SLOAN
nature
with gian
40.
venous endothelitm
indistinct
endothelium
N. SLATER,
and perinuclear
showing
nuclei in various
halos can be seen. HE,
stages of mitosis. The edges
” 100.
of the
PORCINE
ENDOTHELIAL
CELL
IN TISSUE
The cells had a uniform
‘6.3
C‘ULTURL
epithelioid
polygonal
appearance,
occasionally
being
slightly elongated. Measurements in the longitudinal axis varied between 40 and 60~. Occasionally giant cells were present, being approximately 150,u in diameter, but the nuclei were of a similar size (Fig. I). Mitotic especially proached.
figures were numerous
in young colonies
around the periphery. Mitotic figures became less as confluency was apThe nuclei contained one to three nucleoli (Figs. 2 and 3). Although edges
were indistinct,
staining
with silver nitrate
showed a patchy distribution
of “cement”
lines; thisevidentlydependedon howclosethecontact was betweencells. Phasecontrast microscopy showed a granular cytoplasm that, because of greater cytoplasmic depth, was more marked in the perinuclear region. In some cells there was a less granular perinuclear
area which appeared
ence between
as a semicircular
arterial
and venous
electron
microscope
endothelium
halo. There was no apparent in both young
differ-
(3 day) and confluent
cultures.
Fig. 3. Scanning The nucleoli
appear
(S.E.M.)
as white dots in darkened
appearance
of 3-day colonies
areas representing
of arterial
the nuclei. S.E.M..
endothelium
180.
D. N. SLA’TER,
264
J. M. SLOA N
Fig. 4. Numerous pits are seen on the surface of the cell representing pinocytotic vesicles. S.E.M., 2200.
hj
electron microscopic The outstanding feature was the presence of numerous pits in the cell surface, which were interpreted as possibly representing pinocytotic vesciles (Fig. 4). Most varied in size between 100 nm and 2500 nm. The number of vesicles of this size on the Scunnillg
upper surface varied between I50 and 250 per cell. They were more numerous in the perinuclear region and occasionally giant vesicles were present up to I x I04 nm. However, these were rare in normal endothelial cell cultures and when present were limited to not more than two per cell. The nuclei assumed a flattened hollow appearance with nucleoli standing out prominently. At high magnification occasional gaps were evident between adjacent cell membranes (Fig. 5). No surface spikes were seen comparable to those seen in transmission electron microscopy. The surface appearance of arterial and venous endothelium appeared similar at all ages. Transmission
electron microscopy
Each cell is banded
by a cell membrane
which has the usual
unit membrane
PORCINE
EkDOTHtLIAL
Fig. 5. Small layer.
S.E.M.,
CELI.
gaps are apparent
IN TISSUE
between
CULTURL
adjacent
arterial
265
endothelial
cells of a &day
confluent
mono-
7200.
(Fig. 6). Microvilli
were seen projecting
from the upper cell surface:
1000 nm in height and 100 nm in diameter (Fig. 7). The intercellular junctions had three main configurations.
these were 300--
The cells “butted”
onto each other, slightly overlapped or formed a “mortice” type ofjunction. Although cell membranes became closely apposed, they did not fuse. However, the cytoplasm was always
dense
near the regions
of apposition
(Fig. 8). Areas of cytoplasm
also
occasionally seemed dense where the cell membrane happened fortuitously to rest on the base of the flask. The pinocytotic vesicles (Fig. 7) were either smooth or “coated”x. The smooth vesicles measured from 70 nm to 150 nm and sometimes two or three appeared partially fused. Their membrane was of the same thickness as the cell membrane, from which they seem to be derived by evagination. They were distributed reasonably uniformly on both cell surfaces in the cytoplasm. The coated vesicles were seen less frequently; they measured between 80 nm and 120 nm. The coating appeared to possess radiating projections about I5 nm in length and 15 nm apart.
266
D. N. SLATER,
Fig. 6. Transmission cell sectioned
electron
vertically.
Fig. 7. An arterial and a microvillus.
microscope
Its flattened
6-day endothelial T.E.M..
50.000.
nature
appearance is apparent.
cell showing
(T.E.M.)
of a 3-day-old
T.E.M..
pinocytotic
J. M. SLOAN
arterial
endothelial
/’ 5000.
vesicles, myofilaments,
a microtubule
PORCINE
ENDOTHELIAL
Fig. 8. Cells from bowing
cytoplasmic
CELL
confluent density.
h-day
IN TISSUE
venous
CULTURF
endothelium
267
showing
As seen here. a microvillus
close apposition
commonly
occurs
and increased
in this position.
neighT.E.M..
50.000.
Long and short fragments
of microtubules
(Fig. 7) were present
measuring
25
nm in diameter. No attachment to cellular structures was seen except to centrioles during mitosis. Cytoplasmic filaments (Fig. 7) were present varying in diameter between 4 and 7 nm. The fine filaments tended to form aggregates, especially near the cell membrane, but again no direct attachment to the cell membrane was seen. The thicker filaments tended to form apparently haphazard arrangements within the cytoplasm. The Golgi apparatus contained the classical stacked smooth-surfaced cislernae and vesicles. Varying amounts of free ribosomes and rough-surfaced endoplasmic reticulum were present, but smooth-surfaced endoplasmic reticulum was less frequent. The mitochondria were of two types. Some were of the classical type with linear cristae, while others were of the tubular-vesicular type that is associated with steroidproducing cells. The latter type were up to 4,~ in length and of varying shape including branched and horse-shoe forms (Fig. 9). one to two Microtubular dense bodies (Fig. 10) were also seen, approximately
268
D. N. SLATER. J. M. SLOAN
Fig. 9. Tubular-vesicular
mitochondria
in a 3-day-old
venous
endothelial
cell. T.E.M.,
,’ 50,000.
per ultra-thin section. Usually these were round structures, derived from unit membrane, of diameter 100-700 nm and with a dense granular cytoplasm containing up to 200 microtubules of diameter 15-20 nm. Some had an irregular outline and others a more elongated appearance. Rarely a second type of microtubular body possessed a double
membrane
with microtubules
of diameter
25 nm.
Osmiophilic droplets (Fig. I I) within the cytoplasm. were limited to one or two per cell. Free electron-dense granules were consistent with small amounts of glycogen. No significant differences were noted between arterial and venous endothelium at 7 days. However, in younger cells the cytoplasmic free ribosomes were more numerous than the attached ribosomes on the rough endoplasmic reticulum (Fig. 9). DISCUSSION
After overcoming the technical difficulties of separating a cell line, believed to be endothelial in origin, the next problem was that of finding a satisfactory marker for
PORCINE
Fig.
ENDOTHELIAL
10. A 6-day-old
appear
to be related
CELL
IN TISSUE
venous endothelial to the formation
CULTURE
cell showing
of a pinocytotic
269
a multibubular vesicle. T.E.M.,
dense body and the microvilli j
50,000.
the identification of endothelial cells. Although unit membrane multitubular dense bodies are usually accredited to Weibel and Palades, similar bodies were probably first described by Pease and Paule lo. To our knowledge they have not been described in fibroblasts and smooth muscle cells - the major source of cellular contamination in endothelial cell cultures - but they have been described in hamster plateletsll and human capillary
pericytests.
However,
they provide
a useful ultrastructural
marker
for
vascular endothelium. Burri and Weiberts considered they were concerned with blood coagulation but their origin and function remain an enigma. Imai and Thomasl” consider that, in swine, the endothelial cells in the proliferative atheromatous lesions develop an increased number of multibubular dense bodies. Although ultrastructural differences have been reported between human and swine arterial and venous endothelium, after 4 days no major morphological difference was apparent between cultured arterial and venous endothelium. These results seem to suggest that ultrastructural differences tend to disappear when the cells are exposed to a common environment. However, functional differences - such as the time required to harvest the
D. N. SLATER, J. M. SLOAN
270
Fig. 11. A 6-day arterial endothelial cell showing an osmophilic droplet within the cytoplasm. T.E.M., s 125,000.
cells and differences terial endothelium
in fibrinolytic adapted
activity l5 - persist for at least 7-10 days. Why arto high pressure is easier to detach than the more “ad-
hesive” venous endothelium is a problem that we are investigating and which could be relevant to the vascular distribution and pathogenesis of atherosclerosis. The exact function of pinocytotic vesicles in metabolic transport and permeability remains speculative. Moreover, many scanning electron micrographs of endothelium show in our opinion artefacts such as deposited fibrin on the cell surface. Buck16 illustrates vesicles in dog aorta using the palladium-shadowed carbon-replica technique, that are similar to those in our cells. Maruffo and Portman found increased pinocytotic vesicles in experimental coronary atherosclerosis in monkeys, whereas Imai and ThomasI reported no change in porcine cerebral atheroma. The three types of intercellular junctions observed are similar to those found by Schwartz and Benditt in the ratl*. However, the gap apparatus - although it can be of several types’s - is limited to a narrowing of the space between cells or increased
271
PORCINE ENDOTHELIAL CELL IN TISSUE CULTURE
density
of the neighbouring
cytoplasm
were not seen. The absence of microvilli suit to explain but a similar inconsistency The presence
of microtubules
or both.
Areas of fusion
(“tight
on the scanning electron microscope is diffihas been reported by Albarracin and Bain20.
and microfilaments
has been widely reported
endothelium; Becker and Murphy d1 showed that the filaments contain and this laid a firm foundation for the view that endothelial cells contract. We think that an extension investigation the technique
of these studies could provide
and understanding of atherosclerosis to the human vasculature.
We have already labelled
studied
the incorporation
acetate, glucose, palmitic
into mucopolysaccharides
into lipids
the incorporation
and studied
the production
for the
we have extended
of [32P]PO4 and I%-
acid, cholesterol
and chol-
of [35S]S04 and [l%]glucose of proactivator
All these aspects
of endothelial
concentration of insulin and oestrogens. Similarly, significant metahave been found in endothelial cells isolated from patients with atherosclerotic peripheral vascular disease and in rabbits fed atherowill be subsequently
are significantly
and activator.
and by increasing bolic alterations diabetes mellitus,
genie diets. All these findings
cell metabolism
in
actomycin
new avenues
and recently
acid, oleic acid, linoleic
esterol esters. We have also studied
junctions”)
altered
by anoxia
reported.
ACKNOWL.EDGMENTS
The authors are grateful to Mr. Cox and the staff of the Sheffield Corporation Abattoir for providing porcine tissue; to Miss S. Geary for assistance with tissue culture: electron
to Mr. T. Durrant, Miss E. A. Parry and Miss V. Graham for assistance with microscopy; to Mrs. M. Row for photographic assistance and Mrs. J.
Jacques
for preparing
the manuscript.
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D. N. SLATER, I. M. SLOAN
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I1 HAYDON,G. B., AND TAYLOR,D., Microtubules in hamster platelets, J. Cell Biol., 26 (1965) 673. 12 ZELICKSON,A. S., A tubular structure in the endothelial cells and pericytes of human endothelium, J. invest. Derm., 46 (1966) 167. 13 BURRY, P. H. AND WEIBER,E. R., Beeinflussung einer spezifischen cytoplasmatischen Organelle und Endothelzellen durch Adrenalin, 2. Zelfforsch. mikrosk. Anat., 88 (1968) 426. 14 IMAI, H. AND THOMAS,W. A., Cerebral arteriosclerosis in swine, Expt. Mol. Path., 8 (1968) 330. 15 SLATER, D. N., Unpublished observations. 16 BUCK, R. C., The fine structure of endothelium and large arteries, J. Biophys. Biochem. Cytol.,
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of actinomycin in cells of heart valve, endoplaque and endocardial and myocardial Aschoff bodies,