- Email: [email protected]
The culture of vascular endothelial cells to confluence on microporous membranes
THE CilLTU,SECF VASCuIq9 __ EPJXYIX~LIAL CELLS TO CONFLUENCE ON MICRO?OR!XS .YEXBFA?1ES
E. McCall, J. Povey and 3. C. Dumon& , Department of Imsluno1'7'gy St Thomas' Hospital ?l&iiC21 School, London SF1 7EH, gnited Kingdom.
(Received 18.6.1981; in revised form 2.11.1931. Accepted Sy Editor J.B. Smith) ABSTRACT
We have designed a two compartment system in which upper and lower compartments are separated by a layer of vascular endothelial cells T'ne culture of pig aortic endothelial grown on a porous membrane. cells and their growth to confluence on porous ?TFE membranes is described and the principal characteristics of membrane-cultured endothelium are compared with those of similar cells cultured on solid surfaces. While growth of endothelium to visual confluence was slower on the PTFE membranes than on solid surfaces, gross morphology and ratio of 6 keto prostaglandin F1a to prostaglandin E_ A decrease in fluid flow ‘ production by the cells was similar. across the membrane-cultured cells was associated with their growth to visual confluence: from low levels, fluid flow could be increased markedly by altering the environmental temperature of the cells. The possible uses and limitations of such a system in the interpretation of the role of endothelial cells in selective transport and compartmentation of fluid and cellular components of the blood are discussed.
INTRODUCTION The importance of vascular endothelial cell function lies in many areas of human disease (1). Endothelium provides the ultimate blood: tissue barrier [2) and is thus a target in the spread of neoplastic disease (3, 4), in thrombosis and atherosclerosis (5) and in the events associated with
Key words :
Vascular endothelium,
prostaglandins,
417
membrane
culture
Ye
have
ap,rsached
this
problem
*by
culturin,-
vasc_Lar
en.Soth2liC
cells
In preliminary experiments, Lie found that thin micro3 -porous polytetrafluoroethylene (PTFE) membranes :rleremost suitabLe, ar,d accordingly :ce desi3ne" a two compartiient culture system in iihici:,upper anA lower compartments are separated by an endothelial cell layer growing on a porous membrane. In this paper we describe the clllture system, C'ne principal characteristics of membrane-caltured endothelial cells an.3 their growth to confluence. on
porous
membrmes.
MATERIALS Experimental
AND METBODS
design
The aim of the experiments was threefold. First, the growth characteristics of pig aortic endothelial cells cultured on PTFE membranes were compared with those of similar cells cultured on Nlunc plastic and on glass; second, prostaglandin (PG)EZ and prostacyclin (assayed as 6 keto PGFIJ) production by cells cultured on these three surfaces were compared, as a biochemical marker of their behaviour; third, fluid flow across the membranecultured endothelial cells was measured, both in monolayers at apparent confluence, and after experimental disturbance of these monolayers. Endothelial
cell culture
Endothelial cells were isolated from pig aortas by a modification of the procedure described by Hayes et al (9). Aortas of 6 month old pigs were rinsed with 20 ml O.OOlM phosphate buffered saline (DBS), pH 7.4, and then injected with 10 ml collagenase solution (types I, II or VI, Sigma) in PBS, containing 200 U collagenase. After incubation (15 min., 37eC), endothelial cells were rinsed free with RPMI 1640 medium (Gibco, Europe) supplemented with 2 mM glutamine, 20% foetal calf serum (Gibco, Europe), 200 U/ml penicillin and 100 U/ml streptomycin (Glaxo) ("culture medium"). The cells were then washed twice by centrifugation (200 g, 5 min.) and the cell pellet was resuspendedcin 2.5 ml culture medium and grown in T25 flasks, (Nunc, Gibco, Europe) at 37 C in a humidified incubator (95% air/54 CO2). After 24 hours, cells were rinsed in PBS and the culture medium was changed. cells were subcultured from the flasks by brief At or near confluence 6 treatment (5 min., 37 C) with 2.5 ml 0.05% trypsin (Difco)/O.Oi% disodium EDTA w/v in PBS. They were washed twice by centrifugation, resuspended in fresh medium, counted (by haemocytometer) and adjusted to tine required concentration for seeding onto Nunc plastic, glass coverslips or PTFE membranes.
Endothelial cell cultures (1.0 ml) were undertaken at seeding densities of 104-2~10~ cells/cm2 on PTFE membranes (area: 0.5 cm2), on glass coverslips (area: 2 cm2) or plastic wells (area: 2 cm2). These were examined at intervals by inverted phase-contrast microscopy, so as not to disturb the cultures, and their gross appearance and time to visual confluence :cas noted.
FIG. 1 Endothelial cell culture on microporous PTFE membranes. Fig. la shows a Plastic cylinder bearing a PTFE membrane glued to its lower end and foming an upper compartment inserted into the well of a Linbro plate which "Lg. lb is a fcms the lower compartment of the membrane culture system. lcw power SEM (x 250) of the growing surface of an endothelial culture showing the ribbed nodular structure of the underlying PTFE membrane. Fig. lc is an inverted phase-contrast photo-micrograph (x 400) of visually and functionally confluent endothelium on a PTFE membrane supported 2mm above the floor of a Linbro well containing culture medium (see Fig. la). Fig. Id is a transmission EM(x 7500) of the overlapping junctional zones between adjacent endothelial cells: tight junctions are not evident.
(the gift of- 7.. i. Gore and Associates) was gll;ed ta,ut to the laxer *ni 3: a lipped zylinder of autoclavable plastic 15 mm long and .3T"y?interr$al diameter, naking a chamber Whose loder surface (area: 2.5 cx 2) x35 f3,32ed attached to ::?e :7olders *dere dips?:? briefly Sy tie meflbrane. T:?e ne*ran@s in et:hanol, sterilized by boiiing in deionisad g:G X2 stored in PPKI lib:; supplemented with glutamine, penicillin 39.2 strept3~_jcL?.for 24 ti$_r3 SeElr_ 1. use. T:?e c‘n&ers were t:?en placed in the we>is of a Lldrc! TC 21~i~l'~e~l plate (Flow Laboratories? and 1 ml cell suspension at t:?e reTJired concentration was pipette", into each chamber (Fig 13:. cp113 xsre rssai:.**:! ‘2.i ‘i-.2 membrane, while the levels of ciilture aedi>uiiinside and outside the cha,Zber equilibrated immediately.
A two compartment system was thus formed >lith the ITFE membrane culture supported 2 mm above the floor of the Licbro +;ell (Fig la). Gross morphological and ultrastructural appearances of membrane-cultured endothelilum are shown in Figs lb-ld. PGE, and 6 keto PGF1a determinations -i Samples of culture medium from membrane-, plastic- and glass-cultured Wr endothelial cell suspensions were collected in tubes containing 10 '?+I indomethacin (Merck, Sharp and Dohme). PGE2 and 6 keto PGFlr were extracted from the samples and measured by radioimmunoassay (10). Tubes containing 0.1 ml standard PG solution or a dilution of the sample, 8000 dpm 3H-PG in 0.1 ml and 0.1 ml antiserum (diluted to give 40% binding in the absence of added PG), were incubated overnight at 4'C. PG bound to the antiserlum was separated from free PG by dextran charcoal and the antibody-bound radioactivity was counted in an LKB Rackbeta counter. 3H-PGEZ and 3H-6 keto PGF1a were obtained from the Radiochemical Centre, Amersham, standard PGs were the gift of Dr J. E. Pike, Upjohn, Kalamazoo and antisera (whose binding parameters and cross reactions have been described elsewhere) (10, 11) were the gift of Dr F. Dray (Institut Pasteur, Paris). The recovery of standard prostaglandins added immediately before extraction and assay was 70-90x (PGE*) and SO-80% (6 keto PGF1a). Inter-assay precision (5 experiments) had a coefficient of variation o f 5.3% and intra-assay precision (10 experiments) of 11,3%. 3H-thymidine
uptake by endothelial
cell cultures
In order to determine whether acquisition of visual confluence might be associated with a diminution of DNA synthesis, endothelial cells :
ENDOTHELILX
Vo1.2$, 'X0.516
Studies of fluid flow and albumin bearing endothelial cell cultures
ON POROUS ME?lBMES
compartmentation
421
across PTFE membranes
PTFE membranes attached to holders 25 mm long were prepared as described above, to form chambers similar to those used for growth and morphology studies. These chambers were suspended over sterile bijou bottles containing 4 ml culture medium and 1 ml cell suspension (adjusted to give a seeding density 'of 1 x lo5 cells/cm2) was pipetted into these upper compartments. The equilibr,ation of culture medium across the membrane, resulted in the membrane with the cells retained on its surface, being covered by culture medium to a depth of 2-3 mm. Fluid flow across the membrane supporting the endothelial At each time, cells was measured at 24, 48, 72 and 96 hrs after seeding. the chamber was removed from the bijou bottle, the medium above the membrane was carefully aspirated, the chamber wasoplaced over an empty sterile weighed hijou bottle and 1 ml warmed (37 C) culture medium was pipetted into the chamber. At 30 minute intervals the bijou bottle was replaced witha fresh empty weighed bottle. The amount of fluid accumulating in the bottles was measured by weight differences. This process was repeated for up to 180 minutes, or until no fluid remained in the upper chamber. To study compartmentation of albumin across 96-hour membrane cultures, the medium in the upper compartment was replaced by 1.0 ml medium containing 20 ug (0.02 PCi) of iodinated human serum albumin (1Z51-HSA, Amersham, 2.5 pCi/mg : 20 vg = 25,000 cpm approx.). After gentle mixing, 20 ~1 samples of fluid were withdrawn from the upper compartments for y-counting. The membrane cultures were then placed successively over empty weighed bijou bottles (see above). Fluid accumulation in the lower compartments (bijoux) from O-30 min, 30-60 min, 60-90 min, 90 -120 min and 120-180 min was estimated by weighing; and where possible, 20 ~1 volumes were then taken fox- y-counting. Cumulative values were calculated to relate accumulation of "'1-HSA and of total fluid in the lower compartments from zero time to 30, 60, 90, 120 and 180 minutes; and the results were expressed as percentages and ratios. Experimental
disturbance
of the endothelial
monolayer
Cells were cultured on membranes in 25 mm chambers for 72 or 96 hours, and fluid flow across the endothelium was measured as described above. After 150 minutes, the chambers, suspended over sterile empty weighed bijou bottles, were placed at 4OC for 5 minutes and then returned to 37 C. Fluid flow was measured 10 minutes after returning the chambers to 3J°C, and again 15 minutes later. RESULTS Growth of endothelium
on different
surfaces(Table
I)
Cells obtained from a single primary culture at or near confluence were subcultured onto PTFE membranes, Nunc plastic TC wells or glass coverslips. They were examined daily by inverted phase-contrast microscopy, and apparent confluence was judged by eye. Cells seeded onto PTFE membranes attached to the substrate as rapidly as endothelium seeded onto Nunc plastic or glass, but while the gross appearance of the cultures resembled that of cells seeded on plastic or glass surfaces, their time to confluence was different (Table I). On glass or Nunc plastic, the minizum seedinn density at which visual confluence could be attained was 1 x 10 cells/cm2: at this density, confluence was reached in 6 days. With increasing seeding density, the time
$22
ESDOTHELIb?l ON POROCS !QZfBBRXUES
Vol.24
)
so.s/i
taken to reach confluence decreased: at a seeding density of 2.5 x 13* cells/cm2, confluence was reached in 4 days, and at 5 x 10' cells/cm2, confluence was reached in 3 days. Cells cultured on PTFE membranes required a minimum seeding density of 2.5 x lo4 cells/cm2 to attain confluence: at cell densities lower than this, confluence was never reached, even after prolonged incubation. In addition, at any given seeding density, the time taken for cells cultured on PTFE membranes to reach confluence was greater than that taken by cells cultured on Nunc plastic or glass. Cells seeded on PTFE membranes at 5 x 104/cm2 took 5 days to reach confluence, while similar populations seeded on Nunc plastic or glass took only 3 days. In some 30% of the experiments, cells failed to reach confluence, despite a high initial seeding density, and apparently normal attachment and spreading in the first 24 hours of culture. 3
and 6 keto PGPla determinations
(Table I; Fig21
Levels of PGE2 and 6 keto PGFla were measured in endothelial cell supernatants 24 hours after seeding the cells on PTFE membranes, Nunc plastic and glass coverslips. Results, expressed as ng PC/lo6 cells originally seeded, and corrected for cross reactions and recovery, are shown in Table I. As can be seen, the levels of both prostaglandins varied widely from one experiment to another (see Fig 2) : in 5 experiments the amounts of PGE2 and 6 keto PGFla produced by cells cultured on PTFE membranes ranged from 30.5 to 194.8 ng/106 cells and from 46.4 to 154.8 ng/106 cells respectively. Within an experiment, an individual cell population cultured on PTFE membranes generally produced more PGE2 and 6 keto PGFla than on glass or on Nunc plastic (Fig. 2). Differences in the PGE2 levels were significant (membrane: glass, p<0.025; membrane: Nunc plastic, ~0.025; glass: Nunc plastic, p
culture
(Table
II)
As judged by 3H-thymidine up take, DNA synthesis diminished as visual confluence was acquired, whether the endothelial cells were cultured on a Comparison of thymidine uptake solid (glass) phase or on PTFE membranes. rates and seeding densities on the two surfaces (Table II) suggests that the membrane-cultured cells may exhibit a somewhat higher metabolic activity than Although DNA synthesis and PG production cells cultured on a solid phase. were not measured in the same experiments, the greater production of both PGE2 and 6 keto PGFIa by membrane-cultured endothelium in the first 24 hours (Table I) may perhaps be ascribed to a greater metabolic activity on a porous substrate and to a greater ease of cell spreading on solid surfaces.
vo1.24,
So.516
ENDOTHELIW
OX POROUS ?lEYBRXUES
:2 3
TABLE I Characteristics
of vascular endothelium
cultured on PTFE membranes
PTFE Membranes
h‘1un.c ?lastic
Glass Coverslips
Time to confluence from seeding density of: 104 cells/cm2 5~10~ cells/cm* 105 cells/cm2
5-6 days 4-5 days
6 days 3-4 days 2-3 days
6 days 3-4 days 2-3 days
Prosta landin (PG) production* z cells) in first 24 hrs: (nq/'lO PGE2
mean (range)
112..0 (30.5-194.8)
76.7 (26.7-88.4)
59.3 (30.1-i02.8)
6 keto PGFla
mean (range)
lU8.3 (46.4-154.8)
85.3 (24.6-196.4)
6.1.9 (35.5-106.3)
Ratio 6 ketoFla/E2
mean (range)
1.13 (0.64-1.52)
1.08 (0.4-1.39)
1.15 (0.73-1.41)
-*?G levels expressed as nq PG produced in the 24 hours following seeding (at 5 x lo4 cells/cm*) per lo6 cells originally seeded. Mean values and range given for 5 experiments.
TABLE II 3 Diminution in H-thymidine in relation to acquisition
uptake by endothelial of visual confluence
cell cultures
_)
Substrate* and seeding density
JH-thymidine uptake (cpm/culture)? between 12-15 hr 24-27 hr 48-51 hr 72-75 hr -
Glass coverslips (5 x 104/cm2)
Expt 1 Expt 2 Expt 3
PTFE Expt 4 membranes Expt 5 (1.6 x 105/cm2)Expt 6
122 183 110
4354 4099 4363
1472* 1078* 1915*
582 607 704
20-23 hr
42-45 hr
66-69 hr
90-93 hr
5630 10519 8890
26582 14483 20734
* Visual confluence attained on glass at 48 hrs membranes at 90 hrs (expts 4-6) Wean
values of two or three replicate
cultures
22348 12585 22706
2722* 4613* 6610*
(expts l-3) and on PTF'E
424
ESDOTHELILY
Expercment
ON POROCS YEYBRAXES
I
Experiment
So.j/S
2
1_
a0
60 . 40 -
L’o1.24,
7 rl
FIG. 2 Wo experiments illustrating total and relative amounts of 6 keto PGFla (hatched bars) and PGE2 (open bars) produced (in ng/106 seeded ce I. 1s) during the first 24 hours of endothelial cell culture on PTFE membranes 'Ml), glass coverslips ('G') and plastic wells ('P').
Time
(min)
FIG. 3 Fluid flow, measured at 30 minute intervals for up to 180 minutes, across endothelial cells cultured on PTFE membranes for 24 (n = 81, 48 (n = 15), 72 (n = 12) or 96 (n = 5) hours. Vertical bars indicate standard errors.
vol.i~S, ~0.516
EXDOTHELILW
Fluid flow measurements(Figs
$25
ON POROUS XEXBRAWMES
3,Sa)
Endothelial cells were seeded onto PTFE membranes at a cell density of 1 x 105 cells/cm', and after 24, 43, 72 and 96 hours of culture, fluid flow across the cells and membrane was measured at 30-minute intervals for up to 180 nin_:tes. Total (ie cumulative) fluid flow (in ul) across membrane cultl.:resat 34-96hrs is shown in Fig. 3 and flow rates are shown in Fig. 5a. In the absence of added cells, fluid flow across the membrane was 972 * _.6t:L (m 5 s.e.m; n = 5) in the first 5 minutes of the experiment. Fluid flow across cells cultured on PTFE membranes was first m,easured 24 hours after seeding (n = 8). glow across the cells and membrane in the first 30 minutes of the experiment was 789 2 7 pl(m i s.e.m.) and after 90 minutes, virtually all the fluid originally pipetted into the chambers above the membrane had been collected in,bijou bottles. After 48 hours of culture (n = 16), when there had been considerable growth and spreading of the cells, flow in the first 30 minutes of the experiment was 126 2 16 ul (m ? s.e.m.1 and after Fluid flow was further 150 minutes, total flow was 440 i: 30 ul (m * s.e.m.1. reduced with increasing time of culture: after 72 hours culture (n = 12), flow in the first 30 minutes of the experiment was 82 + 16 ul(n + s.e.m.), and after 96 hours of culture (n = 51, when the cells appeared confluent (as judged by microscopic examination), flow in the first 30 minutes of the experiment was 11 ?: 4 ~1 (m C s.e.m.) and total flow after 180 minutes was only 102 < 12 (m + s.e.m.). Compartnentation
of albumin across membrane
cultures
(TABLE III)
These results on fluid flow measurements led us to examine the barrier function of PTFE-cultured endothelium for albumin, using a similar experimental design to the system designed for fluid flow. A series of 96-hour membrane cultures was studied in which fluid flow in the first 180 min ranged from 9.03to 41.0% and which in the first 30 mins ranged from zero to 9.8% (Table III). As judged by passage of 1251-HSA from the upper to the lower compartment, the proportional accumulation of albumin was related to the extent of fluid transfer, though consistently lower at each time of measurement from 30 to 180 minutes of test. Table III shows that on average there was about a 30% restriction of albumin compartmentation below the restriction of fluid flow across the membrane cultures exposed to a hydrostatic pressure of 2 cm 'of tissue culture fluid. Experimental
alteration of the endothelial
cell layer(Figs
4, 5b)
The effect of temperature change on fluid flow across 72 hour and 96 hour endothelial cell cultures was measured in 6 experiments. IFol.Lowingincubation at 4OC for 5 minutes, there was a marked increase in flow across the membrane cultures (Fig 4). The increased flow was measurIn 3 experiments, able within 15 minutes of the temperature alteration. marked changes were observed during this period, while in the other 3 experiments, the increase in the flow had a slower onset. In all 6 experiments, some 95% of the fluid originally present in the upper compartment had appeared in the lower compartment 30 minutes afteg the temperature alteration (ie 25 minutes after replacing the cultures at 37 C). The effect of temperature 'shock' on fluid flow rates is shown in Fig. 5b.
426
ELDOTHELIL.
IOCC
L
a00
-
600
-
400
-
200
-
OS POROUS XEXSRQES
i’ol.LG,
?;0.5;5
‘;
“E Z :
E
0 ” L
; 3 c ._ -0 .3
Z i 0 30
60
90
Time
120
IS0
(min)
I80
+
FIG. 4 Effect of temperature change on fluid flow across endothelial cells cultured on PTFE membranes (6 experiments). After 72 hrs (upper 4 graphs) or 96 hrs (lower 2 graphs) of culture, fluid flow measurements at 37 C were made at 30-min intervals for 150 mins. At this time (arrowed) the cultures were placed at 4OC for 5 min and then returned immediately to 37OC. Fluid flow measurements were then made after a further 10 min (at 165 min) and after a further 25 min (at 180 min).
(al
(b)
i 40
hrs
in
culture
Pb
72
at
37O
effect
of
5 men at
4'
FIG.5 Fig Sa shows fluid flow rates (measured during 5 min) across the PTFE membrane alone ('M alone') and (from O-30 min) across membrane cultured endothelium 24, 48, 72 and 96 hrs after seeding (n = 4). Fig 5b shows fluid flow rates across 72 hr (n = 4) and 96 hr (n = 2) membrane cultures from 120-159 min (open columns) and in the 25 min following cold shock at 4OC (hatched columns). Vertical bars indicate standard errors.
L’0 1 . .I -, ’
?;0.5/6
EKDOTHELIUX
$27
ON POROUS XEVXRtiES
TABLE III Relation between passage of albumin and of fluid across membrane-cultured endothelium into lower compartment Duration of test period
Total flow of albumin Experiment or fluid (and ratio) r 2
lISI_HSA
0 0
Mean ratio
no (chamber assembly)
(alb/fluid)
3
4
5
2.7% 4.7% 0.57
6.2% 9.2% 0.67
6.7% 9.5% 0.68
0.64
4.3% 5.2% 0.83
2.7% 6.0% 0.45
11.3% 14.4% 0.78
11.3% 15.7% 0.72
0.70
0 0
30 min 1
Fluid Ratio
60 min 1
1251-HSA Fluid Ratio
9C min c
1251-HSA Fluid Ratio
5.2% 5.4% 0.96
4.3% 5.2% 0.83
2.7% 6.0% 0.45
16.0% 21.7% 0.74
16.3% 21.4% 0.74
0.74
L
'*'I-HSA Fluid Ratio
5.2% 6.8% 0.76
11.4% 13.6% 0.84
7.7% 15.6% 0.49
19.6% 25.8% 0.76
22.2% 29.3% 0.76
0.72
-I
1251-HSA Fluid Ratio
7.4% 9.0% 0.82
17.6% 20.3% 0.87
20.1% 31.3% 0.64
28.8% 36.5% 0.79
31.03 41.0% 0.76
0.79
l-
120 min-
180
min
0 0
Average ratio of accumulation of '*'I-HSA to total fluid in lower compartment:
0.72
DISCUSSION In this paper we desaribe the culture of vascular endothelium on PTFE membranes. We find that cells subcultured on this surface retain the gross appearance and biochemical characteristics of cells cultured on solid surfaces, and we demonstrate that their growth to visual confluence is associated with an increasing capacity to retain fluid. PTFE membrane is a microporous structure in which a matrix of nodules is interconnected by fibrils. It is inert, is rendered transparent with alcohol and may be heat sterilized. These properties make it particularly useful in the design of a translucent two-compartment culture system where the gross appearance and integrity of cell cultures can be observed, without disturbance, under sterile conditions during growth. In experiments in which unmodified dacron and nucleopore filters were tested as substrates for endothelial culture, endothelium failed to reach confluence. One difficulty experienced in using PTFE membranes was in the preparation of samples for transmission electron microqraphy. The membrane could not be snapped off from the cell layer: the section had to be cut with the PTFE in place, which resulted in smeared electron micrographs. However, the cell layer could be readiLy visualised by scanning electron microscopy.
428
E?;I)OTHELILJM ON POROUS XEYBRQES
Morphologically, endothelium cultured on PTFE membranes resembled that grown on solid surfaces. Early growt‘n on membranes often followed the lines of the nodules, but at confluence, the cells grew as a uniform layer over the membrane. In those electron micrographs of endothelial cells cultured on PTFE membranes which could be adequately examined, structure and junction formation of cells cultured on membranes resembled those of cells c,ultured on solid plastic and glass surfaces. Attainment of visual confluence on both solid and porous surfaces was accompanied by diminution of cellular DNA synthesis (as judged by uptake of 3H-thymidine). This suggests that endothelial cells exhibit contact inhibition of growth on both porous and non-porous substrates. Endothelial cells grown on PTFE membranes required a higher seeding density and took longer to attain confluence than those grown on Nunc plastic or glass; this may be related to the structure of the membranes. Membranes were sterilized in deionised water and soaked in serum-free medium for 2.4 hours before seeding. Soaking the membranes in culture medium containing 20% foetal calf serum or in a 2% gelatin solution (12), did not further improve attachment of the endothelial cells, nor indeed did it increase the proportion of cultures reaching confluence. Shortly after seeding, cells were frequently observed growing first along the lines of nodules rather than across the pores. It may be that it is difficult for the cells to bridge the pores, and that growth on solid parts of the membrane is preferred. Even the surface of the solid nodules is not smooth, and these two difficulties may contribute to the slower acquisition of confluence observed. In addition, the uneven surface of the membrane will increase the actual growth area available to the cells. Another factor which may contribute to the slower acquisiton of confluence is the composition of the membrane. It is possible that PTFE is a substrate on which endothelial cells do not spread rapidly. To investigate this endothelial cell growth on solid PTFE surfaces could be compared with growth on other solid surfaces used for cell culture; and plating efficiences should be compared on solid and microporous PTFE surfaces. Since the discovery of prostacyclin (13), there have been many reports of PGI2 synthesis, as well as PGE2 synthesis, both by vascular tissue (14) and cultured endothelial cells (15-19). In particular, it has been reported recently (18) that the ratio of PGI2 and PGE2 produced by endothelial cells in culture may vary with environmental variables, such as pH, and also with It seemed possible subculture, time in culture and the age of the cells. that the growth of cells on different substrates might also constitute a In the variable which would result in altered ratios of PG production. experiments described here, cells from a single population were seeded onto the different substrates, and subsequently cultured under identical conditions. Amounts of PGE2 and 6 keto PGFla, the major stable metabolite of PGI2, measured in the supernatants of cell cultures, varied widely from one cell However, within each separate experiment, the ratio population to another. of 6 keto PGFla/PGEz production was close to 1.1, whether the endothelial cells were cultured on PTFE membrane, glass or Nunc plastic, irrespective of the total amounts of prostaglandin-'producedSince this ratio is subject to alteration depending upon culture conditions(l8) it seems probable that growth on PTFE membranes does not constitute a trauma for the endothelial cells. Total PG production was somewhat greater in membrane cultures than in solid-phase culture (Table 1). It is known that PG release occurs only from living, metabolically active cells (J L Gordon, pers. comm. 1981) but
Vol.Zli, No.516
EN~OTHELILJM ON ?OROCS ?fE?lBkmES
-29
does not occur during cell lysis(20). Accordingly these differences in PG production may reflect differences in metabolic activity as well as plating efficiency of endothelial cells on the three surfaces. Fluid flow across the membrane-cultured endothelium was measured during The growth of the cultures and also after their experimental disturbance. progressive decrease in fluid flow measured over the first four days of culture was associated with growth of the cells to apparent confluence, as judgea by eye. When fluid flow measurements were continued beyond 96 hr (4 days) no further decrease in flow was observed, and on some occasions there This was associated with a roundwas an increase with continuing culture. ing up of the cells in the centre of the culture. Mean fluid flow across the endothelial cell layers after 96 hrs of culture was 66 ~l/cm~/hour. Other workers, measuring fluid flux across cell monolayers cultured on L)orous membranes have obtained figures for the net flux of between 7-26 l_ljcm2/hr (21.-22)Jn both of these studies however, the cells cultured were an epithelial In the cell line (MDCK cells) and flux was measured using an Ussing chamber. experiments described in this paper, flow was measured in a system in which there was a 2 cm head of pressure (ie 1 ml fluid on an area of 0.5 cm'). Under these conditions, apparently confluent endothelial monolayers did not markedly restrict the passage of albumin to a greater degree than the passage 'of fluid; on average, the lower compartment contained 72% of the albumin concentration expected of a complete transudate. Endothelial cells in vivo possess both tight and gap junctions: it appears that these junctions -transmit water, solutes and small proteins, and that the tight junctions are the site of molecular sieving (1). In addition, an exaggeration of the small changes in the tight junctions which occur under physiological conditions may be responsible for the inter-endothelial gaps observed in inflammatory sites -in vivo (23). In the present experiments tight junctions could not be seen by transmission electron microscopy. It is of interest that J Ryan and U Ryan (pers. comm., 1980) found that endothelial cells possessing both tight gap junctions, once cultured -in vitro and passaged with trypsin, formed only gap junctions. It is not clear whether it is the use of trypsin, or the conditions of cell culture that account for the ready passage of albumin; in our experiments we used cultured cells which had been passaged with EDTA/trypsin. In conclusion, we have demonstrated that endothelial cells can be grown to confluence on porous membranes, and that the cultures resemble, by several criteria, those grown on solid surfaces. Using the two-compartment system described, we can measure fluid flow across monolayers at apparent confluence, and its alteration after experimental disturbance of the cells. The system that we have described may prove a useful adjunct to techniques currently available for studying the functions of endothelium in transport and compartmentation. ACKNOWLEDGEMENTS We should like to thank the Wellcome Trust and W. L. Gore and Associates for financial support. Preliminary results were presented at 'The Biology of the Endothelial Cell', Cold Spring Harbor, May 5 - 10, 1980.
?EFE?.E?:CES In: Gszarcrl ?k?okg~, H.;J. Florev 1. FLOPEY, H.W. Inflammation. London: Lloyd-Luke (Medical Books) Ltd., 1970, pp. ;I-123.
(Ed.)
2. JAFFE, E-A., NACHMAN, R.L., BECKER, C.G. and MINICK, C.R. Culture of human endothelial cells derived from umblical veins. Indentification by morphologic and immunologic criteria. J. Clir..I~vBs;., 52, 2745-2756, 1973. 3. AUSPRUNK, D.H. and FOLKMAN, J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumour angiogenesis. k%croVascz~Zar Research, 14, 53-65, 1977. 4. KRAMER, R.H. and NICOLSON, G.L. Interaction of tumour cells with vascular endothelial cell monolayers: A model for metastatic invasion. Proc. Nat. Acad. Sci. U.S.A., 76, 5704-5708, 1979. 5. WOOLF, N. Thrombosis 1978.
and Atherosclerosis.
Br. Med. ~AZL., 34, 137-142,
6. CONN, D.L., MCDUFFIE, F.C., KAZMIER, F-J., SCHROETER, A.L. and SUN, N.C.J. Arth. Rhewn., 19, Coagulation Abnormalities in Rheumatoid Disease. 1237-1243, 1976. Quantitative investigations of the adhesive 7. ATHERTON, A. and BORN, G.V.R. ness of circulating polymorphonuclear leucocytes to blood vessel walls. J. Physiol. (Land.), 222, 447-474, 1972. M.A. Culture of vascular endothelium. 8. GIMBRONE, Thrombosis, 3, i-28, 1976.
.?rog. Hemostasis &
9. HAYES, L.W., GOGUEN, C.A., CHING, S. F. and SLAKEY, L.L. Angiotensinconverting enzyme: accumulation in medium from cultured endothelial cells. Biochem. biophys. Res. comm., 82, 1147-1153, 1978. 10. DRAY, F., CBARBONNEL, B. and MACLOUF, J. glandins F1a, E and E2 in human plasma. 5, 311-318, 1975.
Radioimmuoassay
of prosta-
Eur. J. Clin. Invest.,
11. GEROZISSIS, K. and DRAY, F. Prostaglandins in the isolated testicular capsule of immature and young adult rats. Pros taglaxdins, 13, 777-783. 1977. 12. FOLKMAN, J., HAUDENSCHILD, C.C. and ZETTER, B.R. Long-term culture of Proc. Nat. Acad. Sci, U.S.A., 76, capillary endothelial cells. 5217-5221, 1979. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an Nature (LOVE.), unstable substance that inhibits platelet aggregation. 263, 663-665, 1976.
13. MONCADA, S., GRYGLEWSKI, R., BUNTING, S. and VANE, J.R.
14. VANE, J.R. and MONCADA, S. Prostacyclin, blood cells and vessel walls: In: Ciba Foundation Symposifan,71, 79-97. functional interactions. Amsterdam, Oxford and Princeton: Excerpta Medica, 1980.
VO
..24,
5’0.516
EXDOTHELIL?i ON POROUS ?fE?BRANES
431
15. GIMBRONE, M.A. and ALEXANDER, R.W. Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium. So?snos, 189, 219-220, 1975. 16. DUMONDE, D.C., JOSE, P-J., PAGE, D.A. and WILLIAMS, T.J. Production of prostaglandins by porcine endothelial cells in culture. 3~. ~7. ?harmacoz.., 61, 5134-505P, 1977. 17. WEKSLER, B.B., MARCUS, A.J. and JAFFE, E.A. Synthesis of prostaglandin 12 (prostacyclin) by cultured human and bovine endothelial cells. ,"roc. PIat. Acacl.Sci. U.S.A., 74, 3922-3926, 1977. 18. AGHR, A., PEARSON, J.D., and GORDON, J.L. Radioimmunoassay of 6-oxoprostaglandin Flu and prostaglandin E2 produced by pig aortic endothelium in culture. Biochem. Sot. Transactions,7, 1065-1066, 1979. 19. JOSE. P.J., PAGE, D.A., WOLSTENHOLME, B-E., WILLIAMS, T.J. and DUMONDE, I2.C Brandykinin-stimulated prostaglandin E2 production by endothelial cells and its modulation by anti-inflammatory compounds. InfZa7ruzation, 5 (4) 375-390 (1981).
20. MCCALL, E.M. inflammation.
Prostaglandins
and polymorphonuclear
.?hD.thesis, &rive&i&
leucocytes _
in
of London, 1980.
21. MISFELDT, D.S., HAMAMOTO, S.T. and PITELKA, D.R. Transepithel ia transport in cell culture. Proc. Nat. Acad. Sci. U.S.A., 73, 1212-1216, 1976. 22. CEREIJIDO, M., ROBBINS, E.S., DOLAN, W-J., ROTUNNO, C.A. and SABATINI, D.D. Polarized monolayers formed by epithelial cells on a perme‘able and translucent support. J. Cell. BioZ., 77, 053-800, 1978.
23. MAJNO, G., PALADE, G.E. and SCHOEFL, G.I.
Studies in inflammation. II. The site of action of histamine and serotonin along the vascular tree. A topographic study. J. Biophys. biochem. cytol., 11, 607-626, 1961.
E. McCall, J. Povey and 3. C. Dumon& , Department of Imsluno1'7'gy St Thomas' Hospital ?l&iiC21 School, London SF1 7EH, gnited Kingdom.
(Received 18.6.1981; in revised form 2.11.1931. Accepted Sy Editor J.B. Smith) ABSTRACT
We have designed a two compartment system in which upper and lower compartments are separated by a layer of vascular endothelial cells T'ne culture of pig aortic endothelial grown on a porous membrane. cells and their growth to confluence on porous ?TFE membranes is described and the principal characteristics of membrane-cultured endothelium are compared with those of similar cells cultured on solid surfaces. While growth of endothelium to visual confluence was slower on the PTFE membranes than on solid surfaces, gross morphology and ratio of 6 keto prostaglandin F1a to prostaglandin E_ A decrease in fluid flow ‘ production by the cells was similar. across the membrane-cultured cells was associated with their growth to visual confluence: from low levels, fluid flow could be increased markedly by altering the environmental temperature of the cells. The possible uses and limitations of such a system in the interpretation of the role of endothelial cells in selective transport and compartmentation of fluid and cellular components of the blood are discussed.
INTRODUCTION The importance of vascular endothelial cell function lies in many areas of human disease (1). Endothelium provides the ultimate blood: tissue barrier [2) and is thus a target in the spread of neoplastic disease (3, 4), in thrombosis and atherosclerosis (5) and in the events associated with
Key words :
Vascular endothelium,
prostaglandins,
417
membrane
culture
Ye
have
ap,rsached
this
problem
*by
culturin,-
vasc_Lar
en.Soth2liC
cells
In preliminary experiments, Lie found that thin micro3 -porous polytetrafluoroethylene (PTFE) membranes :rleremost suitabLe, ar,d accordingly :ce desi3ne" a two compartiient culture system in iihici:,upper anA lower compartments are separated by an endothelial cell layer growing on a porous membrane. In this paper we describe the clllture system, C'ne principal characteristics of membrane-caltured endothelial cells an.3 their growth to confluence. on
porous
membrmes.
MATERIALS Experimental
AND METBODS
design
The aim of the experiments was threefold. First, the growth characteristics of pig aortic endothelial cells cultured on PTFE membranes were compared with those of similar cells cultured on Nlunc plastic and on glass; second, prostaglandin (PG)EZ and prostacyclin (assayed as 6 keto PGFIJ) production by cells cultured on these three surfaces were compared, as a biochemical marker of their behaviour; third, fluid flow across the membranecultured endothelial cells was measured, both in monolayers at apparent confluence, and after experimental disturbance of these monolayers. Endothelial
cell culture
Endothelial cells were isolated from pig aortas by a modification of the procedure described by Hayes et al (9). Aortas of 6 month old pigs were rinsed with 20 ml O.OOlM phosphate buffered saline (DBS), pH 7.4, and then injected with 10 ml collagenase solution (types I, II or VI, Sigma) in PBS, containing 200 U collagenase. After incubation (15 min., 37eC), endothelial cells were rinsed free with RPMI 1640 medium (Gibco, Europe) supplemented with 2 mM glutamine, 20% foetal calf serum (Gibco, Europe), 200 U/ml penicillin and 100 U/ml streptomycin (Glaxo) ("culture medium"). The cells were then washed twice by centrifugation (200 g, 5 min.) and the cell pellet was resuspendedcin 2.5 ml culture medium and grown in T25 flasks, (Nunc, Gibco, Europe) at 37 C in a humidified incubator (95% air/54 CO2). After 24 hours, cells were rinsed in PBS and the culture medium was changed. cells were subcultured from the flasks by brief At or near confluence 6 treatment (5 min., 37 C) with 2.5 ml 0.05% trypsin (Difco)/O.Oi% disodium EDTA w/v in PBS. They were washed twice by centrifugation, resuspended in fresh medium, counted (by haemocytometer) and adjusted to tine required concentration for seeding onto Nunc plastic, glass coverslips or PTFE membranes.
Endothelial cell cultures (1.0 ml) were undertaken at seeding densities of 104-2~10~ cells/cm2 on PTFE membranes (area: 0.5 cm2), on glass coverslips (area: 2 cm2) or plastic wells (area: 2 cm2). These were examined at intervals by inverted phase-contrast microscopy, so as not to disturb the cultures, and their gross appearance and time to visual confluence :cas noted.
FIG. 1 Endothelial cell culture on microporous PTFE membranes. Fig. la shows a Plastic cylinder bearing a PTFE membrane glued to its lower end and foming an upper compartment inserted into the well of a Linbro plate which "Lg. lb is a fcms the lower compartment of the membrane culture system. lcw power SEM (x 250) of the growing surface of an endothelial culture showing the ribbed nodular structure of the underlying PTFE membrane. Fig. lc is an inverted phase-contrast photo-micrograph (x 400) of visually and functionally confluent endothelium on a PTFE membrane supported 2mm above the floor of a Linbro well containing culture medium (see Fig. la). Fig. Id is a transmission EM(x 7500) of the overlapping junctional zones between adjacent endothelial cells: tight junctions are not evident.
(the gift of- 7.. i. Gore and Associates) was gll;ed ta,ut to the laxer *ni 3: a lipped zylinder of autoclavable plastic 15 mm long and .3T"y?interr$al diameter, naking a chamber Whose loder surface (area: 2.5 cx 2) x35 f3,32ed attached to ::?e :7olders *dere dips?:? briefly Sy tie meflbrane. T:?e ne*ran@s in et:hanol, sterilized by boiiing in deionisad g:G X2 stored in PPKI lib:; supplemented with glutamine, penicillin 39.2 strept3~_jcL?.for 24 ti$_r3 SeElr_ 1. use. T:?e c‘n&ers were t:?en placed in the we>is of a Lldrc! TC 21~i~l'~e~l plate (Flow Laboratories? and 1 ml cell suspension at t:?e reTJired concentration was pipette", into each chamber (Fig 13:. cp113 xsre rssai:.**:! ‘2.i ‘i-.2 membrane, while the levels of ciilture aedi>uiiinside and outside the cha,Zber equilibrated immediately.
A two compartment system was thus formed >lith the ITFE membrane culture supported 2 mm above the floor of the Licbro +;ell (Fig la). Gross morphological and ultrastructural appearances of membrane-cultured endothelilum are shown in Figs lb-ld. PGE, and 6 keto PGF1a determinations -i Samples of culture medium from membrane-, plastic- and glass-cultured Wr endothelial cell suspensions were collected in tubes containing 10 '?+I indomethacin (Merck, Sharp and Dohme). PGE2 and 6 keto PGFlr were extracted from the samples and measured by radioimmunoassay (10). Tubes containing 0.1 ml standard PG solution or a dilution of the sample, 8000 dpm 3H-PG in 0.1 ml and 0.1 ml antiserum (diluted to give 40% binding in the absence of added PG), were incubated overnight at 4'C. PG bound to the antiserlum was separated from free PG by dextran charcoal and the antibody-bound radioactivity was counted in an LKB Rackbeta counter. 3H-PGEZ and 3H-6 keto PGF1a were obtained from the Radiochemical Centre, Amersham, standard PGs were the gift of Dr J. E. Pike, Upjohn, Kalamazoo and antisera (whose binding parameters and cross reactions have been described elsewhere) (10, 11) were the gift of Dr F. Dray (Institut Pasteur, Paris). The recovery of standard prostaglandins added immediately before extraction and assay was 70-90x (PGE*) and SO-80% (6 keto PGF1a). Inter-assay precision (5 experiments) had a coefficient of variation o f 5.3% and intra-assay precision (10 experiments) of 11,3%. 3H-thymidine
uptake by endothelial
cell cultures
In order to determine whether acquisition of visual confluence might be associated with a diminution of DNA synthesis, endothelial cells :
ENDOTHELILX
Vo1.2$, 'X0.516
Studies of fluid flow and albumin bearing endothelial cell cultures
ON POROUS ME?lBMES
compartmentation
421
across PTFE membranes
PTFE membranes attached to holders 25 mm long were prepared as described above, to form chambers similar to those used for growth and morphology studies. These chambers were suspended over sterile bijou bottles containing 4 ml culture medium and 1 ml cell suspension (adjusted to give a seeding density 'of 1 x lo5 cells/cm2) was pipetted into these upper compartments. The equilibr,ation of culture medium across the membrane, resulted in the membrane with the cells retained on its surface, being covered by culture medium to a depth of 2-3 mm. Fluid flow across the membrane supporting the endothelial At each time, cells was measured at 24, 48, 72 and 96 hrs after seeding. the chamber was removed from the bijou bottle, the medium above the membrane was carefully aspirated, the chamber wasoplaced over an empty sterile weighed hijou bottle and 1 ml warmed (37 C) culture medium was pipetted into the chamber. At 30 minute intervals the bijou bottle was replaced witha fresh empty weighed bottle. The amount of fluid accumulating in the bottles was measured by weight differences. This process was repeated for up to 180 minutes, or until no fluid remained in the upper chamber. To study compartmentation of albumin across 96-hour membrane cultures, the medium in the upper compartment was replaced by 1.0 ml medium containing 20 ug (0.02 PCi) of iodinated human serum albumin (1Z51-HSA, Amersham, 2.5 pCi/mg : 20 vg = 25,000 cpm approx.). After gentle mixing, 20 ~1 samples of fluid were withdrawn from the upper compartments for y-counting. The membrane cultures were then placed successively over empty weighed bijou bottles (see above). Fluid accumulation in the lower compartments (bijoux) from O-30 min, 30-60 min, 60-90 min, 90 -120 min and 120-180 min was estimated by weighing; and where possible, 20 ~1 volumes were then taken fox- y-counting. Cumulative values were calculated to relate accumulation of "'1-HSA and of total fluid in the lower compartments from zero time to 30, 60, 90, 120 and 180 minutes; and the results were expressed as percentages and ratios. Experimental
disturbance
of the endothelial
monolayer
Cells were cultured on membranes in 25 mm chambers for 72 or 96 hours, and fluid flow across the endothelium was measured as described above. After 150 minutes, the chambers, suspended over sterile empty weighed bijou bottles, were placed at 4OC for 5 minutes and then returned to 37 C. Fluid flow was measured 10 minutes after returning the chambers to 3J°C, and again 15 minutes later. RESULTS Growth of endothelium
on different
surfaces(Table
I)
Cells obtained from a single primary culture at or near confluence were subcultured onto PTFE membranes, Nunc plastic TC wells or glass coverslips. They were examined daily by inverted phase-contrast microscopy, and apparent confluence was judged by eye. Cells seeded onto PTFE membranes attached to the substrate as rapidly as endothelium seeded onto Nunc plastic or glass, but while the gross appearance of the cultures resembled that of cells seeded on plastic or glass surfaces, their time to confluence was different (Table I). On glass or Nunc plastic, the minizum seedinn density at which visual confluence could be attained was 1 x 10 cells/cm2: at this density, confluence was reached in 6 days. With increasing seeding density, the time
$22
ESDOTHELIb?l ON POROCS !QZfBBRXUES
Vol.24
)
so.s/i
taken to reach confluence decreased: at a seeding density of 2.5 x 13* cells/cm2, confluence was reached in 4 days, and at 5 x 10' cells/cm2, confluence was reached in 3 days. Cells cultured on PTFE membranes required a minimum seeding density of 2.5 x lo4 cells/cm2 to attain confluence: at cell densities lower than this, confluence was never reached, even after prolonged incubation. In addition, at any given seeding density, the time taken for cells cultured on PTFE membranes to reach confluence was greater than that taken by cells cultured on Nunc plastic or glass. Cells seeded on PTFE membranes at 5 x 104/cm2 took 5 days to reach confluence, while similar populations seeded on Nunc plastic or glass took only 3 days. In some 30% of the experiments, cells failed to reach confluence, despite a high initial seeding density, and apparently normal attachment and spreading in the first 24 hours of culture. 3
and 6 keto PGPla determinations
(Table I; Fig21
Levels of PGE2 and 6 keto PGFla were measured in endothelial cell supernatants 24 hours after seeding the cells on PTFE membranes, Nunc plastic and glass coverslips. Results, expressed as ng PC/lo6 cells originally seeded, and corrected for cross reactions and recovery, are shown in Table I. As can be seen, the levels of both prostaglandins varied widely from one experiment to another (see Fig 2) : in 5 experiments the amounts of PGE2 and 6 keto PGFla produced by cells cultured on PTFE membranes ranged from 30.5 to 194.8 ng/106 cells and from 46.4 to 154.8 ng/106 cells respectively. Within an experiment, an individual cell population cultured on PTFE membranes generally produced more PGE2 and 6 keto PGFla than on glass or on Nunc plastic (Fig. 2). Differences in the PGE2 levels were significant (membrane: glass, p<0.025; membrane: Nunc plastic, ~0.025; glass: Nunc plastic, p
culture
(Table
II)
As judged by 3H-thymidine up take, DNA synthesis diminished as visual confluence was acquired, whether the endothelial cells were cultured on a Comparison of thymidine uptake solid (glass) phase or on PTFE membranes. rates and seeding densities on the two surfaces (Table II) suggests that the membrane-cultured cells may exhibit a somewhat higher metabolic activity than Although DNA synthesis and PG production cells cultured on a solid phase. were not measured in the same experiments, the greater production of both PGE2 and 6 keto PGFIa by membrane-cultured endothelium in the first 24 hours (Table I) may perhaps be ascribed to a greater metabolic activity on a porous substrate and to a greater ease of cell spreading on solid surfaces.
vo1.24,
So.516
ENDOTHELIW
OX POROUS ?lEYBRXUES
:2 3
TABLE I Characteristics
of vascular endothelium
cultured on PTFE membranes
PTFE Membranes
h‘1un.c ?lastic
Glass Coverslips
Time to confluence from seeding density of: 104 cells/cm2 5~10~ cells/cm* 105 cells/cm2
5-6 days 4-5 days
6 days 3-4 days 2-3 days
6 days 3-4 days 2-3 days
Prosta landin (PG) production* z cells) in first 24 hrs: (nq/'lO PGE2
mean (range)
112..0 (30.5-194.8)
76.7 (26.7-88.4)
59.3 (30.1-i02.8)
6 keto PGFla
mean (range)
lU8.3 (46.4-154.8)
85.3 (24.6-196.4)
6.1.9 (35.5-106.3)
Ratio 6 ketoFla/E2
mean (range)
1.13 (0.64-1.52)
1.08 (0.4-1.39)
1.15 (0.73-1.41)
-*?G levels expressed as nq PG produced in the 24 hours following seeding (at 5 x lo4 cells/cm*) per lo6 cells originally seeded. Mean values and range given for 5 experiments.
TABLE II 3 Diminution in H-thymidine in relation to acquisition
uptake by endothelial of visual confluence
cell cultures
_)
Substrate* and seeding density
JH-thymidine uptake (cpm/culture)? between 12-15 hr 24-27 hr 48-51 hr 72-75 hr -
Glass coverslips (5 x 104/cm2)
Expt 1 Expt 2 Expt 3
PTFE Expt 4 membranes Expt 5 (1.6 x 105/cm2)Expt 6
122 183 110
4354 4099 4363
1472* 1078* 1915*
582 607 704
20-23 hr
42-45 hr
66-69 hr
90-93 hr
5630 10519 8890
26582 14483 20734
* Visual confluence attained on glass at 48 hrs membranes at 90 hrs (expts 4-6) Wean
values of two or three replicate
cultures
22348 12585 22706
2722* 4613* 6610*
(expts l-3) and on PTF'E
424
ESDOTHELILY
Expercment
ON POROCS YEYBRAXES
I
Experiment
So.j/S
2
1_
a0
60 . 40 -
L’o1.24,
7 rl
FIG. 2 Wo experiments illustrating total and relative amounts of 6 keto PGFla (hatched bars) and PGE2 (open bars) produced (in ng/106 seeded ce I. 1s) during the first 24 hours of endothelial cell culture on PTFE membranes 'Ml), glass coverslips ('G') and plastic wells ('P').
Time
(min)
FIG. 3 Fluid flow, measured at 30 minute intervals for up to 180 minutes, across endothelial cells cultured on PTFE membranes for 24 (n = 81, 48 (n = 15), 72 (n = 12) or 96 (n = 5) hours. Vertical bars indicate standard errors.
vol.i~S, ~0.516
EXDOTHELILW
Fluid flow measurements(Figs
$25
ON POROUS XEXBRAWMES
3,Sa)
Endothelial cells were seeded onto PTFE membranes at a cell density of 1 x 105 cells/cm', and after 24, 43, 72 and 96 hours of culture, fluid flow across the cells and membrane was measured at 30-minute intervals for up to 180 nin_:tes. Total (ie cumulative) fluid flow (in ul) across membrane cultl.:resat 34-96hrs is shown in Fig. 3 and flow rates are shown in Fig. 5a. In the absence of added cells, fluid flow across the membrane was 972 * _.6t:L (m 5 s.e.m; n = 5) in the first 5 minutes of the experiment. Fluid flow across cells cultured on PTFE membranes was first m,easured 24 hours after seeding (n = 8). glow across the cells and membrane in the first 30 minutes of the experiment was 789 2 7 pl(m i s.e.m.) and after 90 minutes, virtually all the fluid originally pipetted into the chambers above the membrane had been collected in,bijou bottles. After 48 hours of culture (n = 16), when there had been considerable growth and spreading of the cells, flow in the first 30 minutes of the experiment was 126 2 16 ul (m ? s.e.m.1 and after Fluid flow was further 150 minutes, total flow was 440 i: 30 ul (m * s.e.m.1. reduced with increasing time of culture: after 72 hours culture (n = 12), flow in the first 30 minutes of the experiment was 82 + 16 ul(n + s.e.m.), and after 96 hours of culture (n = 51, when the cells appeared confluent (as judged by microscopic examination), flow in the first 30 minutes of the experiment was 11 ?: 4 ~1 (m C s.e.m.) and total flow after 180 minutes was only 102 < 12 (m + s.e.m.). Compartnentation
of albumin across membrane
cultures
(TABLE III)
These results on fluid flow measurements led us to examine the barrier function of PTFE-cultured endothelium for albumin, using a similar experimental design to the system designed for fluid flow. A series of 96-hour membrane cultures was studied in which fluid flow in the first 180 min ranged from 9.03to 41.0% and which in the first 30 mins ranged from zero to 9.8% (Table III). As judged by passage of 1251-HSA from the upper to the lower compartment, the proportional accumulation of albumin was related to the extent of fluid transfer, though consistently lower at each time of measurement from 30 to 180 minutes of test. Table III shows that on average there was about a 30% restriction of albumin compartmentation below the restriction of fluid flow across the membrane cultures exposed to a hydrostatic pressure of 2 cm 'of tissue culture fluid. Experimental
alteration of the endothelial
cell layer(Figs
4, 5b)
The effect of temperature change on fluid flow across 72 hour and 96 hour endothelial cell cultures was measured in 6 experiments. IFol.Lowingincubation at 4OC for 5 minutes, there was a marked increase in flow across the membrane cultures (Fig 4). The increased flow was measurIn 3 experiments, able within 15 minutes of the temperature alteration. marked changes were observed during this period, while in the other 3 experiments, the increase in the flow had a slower onset. In all 6 experiments, some 95% of the fluid originally present in the upper compartment had appeared in the lower compartment 30 minutes afteg the temperature alteration (ie 25 minutes after replacing the cultures at 37 C). The effect of temperature 'shock' on fluid flow rates is shown in Fig. 5b.
426
ELDOTHELIL.
IOCC
L
a00
-
600
-
400
-
200
-
OS POROUS XEXSRQES
i’ol.LG,
?;0.5;5
‘;
“E Z :
E
0 ” L
; 3 c ._ -0 .3
Z i 0 30
60
90
Time
120
IS0
(min)
I80
+
FIG. 4 Effect of temperature change on fluid flow across endothelial cells cultured on PTFE membranes (6 experiments). After 72 hrs (upper 4 graphs) or 96 hrs (lower 2 graphs) of culture, fluid flow measurements at 37 C were made at 30-min intervals for 150 mins. At this time (arrowed) the cultures were placed at 4OC for 5 min and then returned immediately to 37OC. Fluid flow measurements were then made after a further 10 min (at 165 min) and after a further 25 min (at 180 min).
(al
(b)
i 40
hrs
in
culture
Pb
72
at
37O
effect
of
5 men at
4'
FIG.5 Fig Sa shows fluid flow rates (measured during 5 min) across the PTFE membrane alone ('M alone') and (from O-30 min) across membrane cultured endothelium 24, 48, 72 and 96 hrs after seeding (n = 4). Fig 5b shows fluid flow rates across 72 hr (n = 4) and 96 hr (n = 2) membrane cultures from 120-159 min (open columns) and in the 25 min following cold shock at 4OC (hatched columns). Vertical bars indicate standard errors.
L’0 1 . .I -, ’
?;0.5/6
EKDOTHELIUX
$27
ON POROUS XEVXRtiES
TABLE III Relation between passage of albumin and of fluid across membrane-cultured endothelium into lower compartment Duration of test period
Total flow of albumin Experiment or fluid (and ratio) r 2
lISI_HSA
0 0
Mean ratio
no (chamber assembly)
(alb/fluid)
3
4
5
2.7% 4.7% 0.57
6.2% 9.2% 0.67
6.7% 9.5% 0.68
0.64
4.3% 5.2% 0.83
2.7% 6.0% 0.45
11.3% 14.4% 0.78
11.3% 15.7% 0.72
0.70
0 0
30 min 1
Fluid Ratio
60 min 1
1251-HSA Fluid Ratio
9C min c
1251-HSA Fluid Ratio
5.2% 5.4% 0.96
4.3% 5.2% 0.83
2.7% 6.0% 0.45
16.0% 21.7% 0.74
16.3% 21.4% 0.74
0.74
L
'*'I-HSA Fluid Ratio
5.2% 6.8% 0.76
11.4% 13.6% 0.84
7.7% 15.6% 0.49
19.6% 25.8% 0.76
22.2% 29.3% 0.76
0.72
-I
1251-HSA Fluid Ratio
7.4% 9.0% 0.82
17.6% 20.3% 0.87
20.1% 31.3% 0.64
28.8% 36.5% 0.79
31.03 41.0% 0.76
0.79
l-
120 min-
180
min
0 0
Average ratio of accumulation of '*'I-HSA to total fluid in lower compartment:
0.72
DISCUSSION In this paper we desaribe the culture of vascular endothelium on PTFE membranes. We find that cells subcultured on this surface retain the gross appearance and biochemical characteristics of cells cultured on solid surfaces, and we demonstrate that their growth to visual confluence is associated with an increasing capacity to retain fluid. PTFE membrane is a microporous structure in which a matrix of nodules is interconnected by fibrils. It is inert, is rendered transparent with alcohol and may be heat sterilized. These properties make it particularly useful in the design of a translucent two-compartment culture system where the gross appearance and integrity of cell cultures can be observed, without disturbance, under sterile conditions during growth. In experiments in which unmodified dacron and nucleopore filters were tested as substrates for endothelial culture, endothelium failed to reach confluence. One difficulty experienced in using PTFE membranes was in the preparation of samples for transmission electron microqraphy. The membrane could not be snapped off from the cell layer: the section had to be cut with the PTFE in place, which resulted in smeared electron micrographs. However, the cell layer could be readiLy visualised by scanning electron microscopy.
428
E?;I)OTHELILJM ON POROUS XEYBRQES
Morphologically, endothelium cultured on PTFE membranes resembled that grown on solid surfaces. Early growt‘n on membranes often followed the lines of the nodules, but at confluence, the cells grew as a uniform layer over the membrane. In those electron micrographs of endothelial cells cultured on PTFE membranes which could be adequately examined, structure and junction formation of cells cultured on membranes resembled those of cells c,ultured on solid plastic and glass surfaces. Attainment of visual confluence on both solid and porous surfaces was accompanied by diminution of cellular DNA synthesis (as judged by uptake of 3H-thymidine). This suggests that endothelial cells exhibit contact inhibition of growth on both porous and non-porous substrates. Endothelial cells grown on PTFE membranes required a higher seeding density and took longer to attain confluence than those grown on Nunc plastic or glass; this may be related to the structure of the membranes. Membranes were sterilized in deionised water and soaked in serum-free medium for 2.4 hours before seeding. Soaking the membranes in culture medium containing 20% foetal calf serum or in a 2% gelatin solution (12), did not further improve attachment of the endothelial cells, nor indeed did it increase the proportion of cultures reaching confluence. Shortly after seeding, cells were frequently observed growing first along the lines of nodules rather than across the pores. It may be that it is difficult for the cells to bridge the pores, and that growth on solid parts of the membrane is preferred. Even the surface of the solid nodules is not smooth, and these two difficulties may contribute to the slower acquisition of confluence observed. In addition, the uneven surface of the membrane will increase the actual growth area available to the cells. Another factor which may contribute to the slower acquisiton of confluence is the composition of the membrane. It is possible that PTFE is a substrate on which endothelial cells do not spread rapidly. To investigate this endothelial cell growth on solid PTFE surfaces could be compared with growth on other solid surfaces used for cell culture; and plating efficiences should be compared on solid and microporous PTFE surfaces. Since the discovery of prostacyclin (13), there have been many reports of PGI2 synthesis, as well as PGE2 synthesis, both by vascular tissue (14) and cultured endothelial cells (15-19). In particular, it has been reported recently (18) that the ratio of PGI2 and PGE2 produced by endothelial cells in culture may vary with environmental variables, such as pH, and also with It seemed possible subculture, time in culture and the age of the cells. that the growth of cells on different substrates might also constitute a In the variable which would result in altered ratios of PG production. experiments described here, cells from a single population were seeded onto the different substrates, and subsequently cultured under identical conditions. Amounts of PGE2 and 6 keto PGFla, the major stable metabolite of PGI2, measured in the supernatants of cell cultures, varied widely from one cell However, within each separate experiment, the ratio population to another. of 6 keto PGFla/PGEz production was close to 1.1, whether the endothelial cells were cultured on PTFE membrane, glass or Nunc plastic, irrespective of the total amounts of prostaglandin-'producedSince this ratio is subject to alteration depending upon culture conditions(l8) it seems probable that growth on PTFE membranes does not constitute a trauma for the endothelial cells. Total PG production was somewhat greater in membrane cultures than in solid-phase culture (Table 1). It is known that PG release occurs only from living, metabolically active cells (J L Gordon, pers. comm. 1981) but
Vol.Zli, No.516
EN~OTHELILJM ON ?OROCS ?fE?lBkmES
-29
does not occur during cell lysis(20). Accordingly these differences in PG production may reflect differences in metabolic activity as well as plating efficiency of endothelial cells on the three surfaces. Fluid flow across the membrane-cultured endothelium was measured during The growth of the cultures and also after their experimental disturbance. progressive decrease in fluid flow measured over the first four days of culture was associated with growth of the cells to apparent confluence, as judgea by eye. When fluid flow measurements were continued beyond 96 hr (4 days) no further decrease in flow was observed, and on some occasions there This was associated with a roundwas an increase with continuing culture. ing up of the cells in the centre of the culture. Mean fluid flow across the endothelial cell layers after 96 hrs of culture was 66 ~l/cm~/hour. Other workers, measuring fluid flux across cell monolayers cultured on L)orous membranes have obtained figures for the net flux of between 7-26 l_ljcm2/hr (21.-22)Jn both of these studies however, the cells cultured were an epithelial In the cell line (MDCK cells) and flux was measured using an Ussing chamber. experiments described in this paper, flow was measured in a system in which there was a 2 cm head of pressure (ie 1 ml fluid on an area of 0.5 cm'). Under these conditions, apparently confluent endothelial monolayers did not markedly restrict the passage of albumin to a greater degree than the passage 'of fluid; on average, the lower compartment contained 72% of the albumin concentration expected of a complete transudate. Endothelial cells in vivo possess both tight and gap junctions: it appears that these junctions -transmit water, solutes and small proteins, and that the tight junctions are the site of molecular sieving (1). In addition, an exaggeration of the small changes in the tight junctions which occur under physiological conditions may be responsible for the inter-endothelial gaps observed in inflammatory sites -in vivo (23). In the present experiments tight junctions could not be seen by transmission electron microscopy. It is of interest that J Ryan and U Ryan (pers. comm., 1980) found that endothelial cells possessing both tight gap junctions, once cultured -in vitro and passaged with trypsin, formed only gap junctions. It is not clear whether it is the use of trypsin, or the conditions of cell culture that account for the ready passage of albumin; in our experiments we used cultured cells which had been passaged with EDTA/trypsin. In conclusion, we have demonstrated that endothelial cells can be grown to confluence on porous membranes, and that the cultures resemble, by several criteria, those grown on solid surfaces. Using the two-compartment system described, we can measure fluid flow across monolayers at apparent confluence, and its alteration after experimental disturbance of the cells. The system that we have described may prove a useful adjunct to techniques currently available for studying the functions of endothelium in transport and compartmentation. ACKNOWLEDGEMENTS We should like to thank the Wellcome Trust and W. L. Gore and Associates for financial support. Preliminary results were presented at 'The Biology of the Endothelial Cell', Cold Spring Harbor, May 5 - 10, 1980.
?EFE?.E?:CES In: Gszarcrl ?k?okg~, H.;J. Florev 1. FLOPEY, H.W. Inflammation. London: Lloyd-Luke (Medical Books) Ltd., 1970, pp. ;I-123.
(Ed.)
2. JAFFE, E-A., NACHMAN, R.L., BECKER, C.G. and MINICK, C.R. Culture of human endothelial cells derived from umblical veins. Indentification by morphologic and immunologic criteria. J. Clir..I~vBs;., 52, 2745-2756, 1973. 3. AUSPRUNK, D.H. and FOLKMAN, J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumour angiogenesis. k%croVascz~Zar Research, 14, 53-65, 1977. 4. KRAMER, R.H. and NICOLSON, G.L. Interaction of tumour cells with vascular endothelial cell monolayers: A model for metastatic invasion. Proc. Nat. Acad. Sci. U.S.A., 76, 5704-5708, 1979. 5. WOOLF, N. Thrombosis 1978.
and Atherosclerosis.
Br. Med. ~AZL., 34, 137-142,
6. CONN, D.L., MCDUFFIE, F.C., KAZMIER, F-J., SCHROETER, A.L. and SUN, N.C.J. Arth. Rhewn., 19, Coagulation Abnormalities in Rheumatoid Disease. 1237-1243, 1976. Quantitative investigations of the adhesive 7. ATHERTON, A. and BORN, G.V.R. ness of circulating polymorphonuclear leucocytes to blood vessel walls. J. Physiol. (Land.), 222, 447-474, 1972. M.A. Culture of vascular endothelium. 8. GIMBRONE, Thrombosis, 3, i-28, 1976.
.?rog. Hemostasis &
9. HAYES, L.W., GOGUEN, C.A., CHING, S. F. and SLAKEY, L.L. Angiotensinconverting enzyme: accumulation in medium from cultured endothelial cells. Biochem. biophys. Res. comm., 82, 1147-1153, 1978. 10. DRAY, F., CBARBONNEL, B. and MACLOUF, J. glandins F1a, E and E2 in human plasma. 5, 311-318, 1975.
Radioimmuoassay
of prosta-
Eur. J. Clin. Invest.,
11. GEROZISSIS, K. and DRAY, F. Prostaglandins in the isolated testicular capsule of immature and young adult rats. Pros taglaxdins, 13, 777-783. 1977. 12. FOLKMAN, J., HAUDENSCHILD, C.C. and ZETTER, B.R. Long-term culture of Proc. Nat. Acad. Sci, U.S.A., 76, capillary endothelial cells. 5217-5221, 1979. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an Nature (LOVE.), unstable substance that inhibits platelet aggregation. 263, 663-665, 1976.
13. MONCADA, S., GRYGLEWSKI, R., BUNTING, S. and VANE, J.R.
14. VANE, J.R. and MONCADA, S. Prostacyclin, blood cells and vessel walls: In: Ciba Foundation Symposifan,71, 79-97. functional interactions. Amsterdam, Oxford and Princeton: Excerpta Medica, 1980.
VO
..24,
5’0.516
EXDOTHELIL?i ON POROUS ?fE?BRANES
431
15. GIMBRONE, M.A. and ALEXANDER, R.W. Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium. So?snos, 189, 219-220, 1975. 16. DUMONDE, D.C., JOSE, P-J., PAGE, D.A. and WILLIAMS, T.J. Production of prostaglandins by porcine endothelial cells in culture. 3~. ~7. ?harmacoz.., 61, 5134-505P, 1977. 17. WEKSLER, B.B., MARCUS, A.J. and JAFFE, E.A. Synthesis of prostaglandin 12 (prostacyclin) by cultured human and bovine endothelial cells. ,"roc. PIat. Acacl.Sci. U.S.A., 74, 3922-3926, 1977. 18. AGHR, A., PEARSON, J.D., and GORDON, J.L. Radioimmunoassay of 6-oxoprostaglandin Flu and prostaglandin E2 produced by pig aortic endothelium in culture. Biochem. Sot. Transactions,7, 1065-1066, 1979. 19. JOSE. P.J., PAGE, D.A., WOLSTENHOLME, B-E., WILLIAMS, T.J. and DUMONDE, I2.C Brandykinin-stimulated prostaglandin E2 production by endothelial cells and its modulation by anti-inflammatory compounds. InfZa7ruzation, 5 (4) 375-390 (1981).
20. MCCALL, E.M. inflammation.
Prostaglandins
and polymorphonuclear
.?hD.thesis, &rive&i&
leucocytes _
in
of London, 1980.
21. MISFELDT, D.S., HAMAMOTO, S.T. and PITELKA, D.R. Transepithel ia transport in cell culture. Proc. Nat. Acad. Sci. U.S.A., 73, 1212-1216, 1976. 22. CEREIJIDO, M., ROBBINS, E.S., DOLAN, W-J., ROTUNNO, C.A. and SABATINI, D.D. Polarized monolayers formed by epithelial cells on a perme‘able and translucent support. J. Cell. BioZ., 77, 053-800, 1978.
23. MAJNO, G., PALADE, G.E. and SCHOEFL, G.I.
Studies in inflammation. II. The site of action of histamine and serotonin along the vascular tree. A topographic study. J. Biophys. biochem. cytol., 11, 607-626, 1961.