- Email: [email protected]
Culture of human vascular endothelial cells on a positively charged polystyrene surface, Primaria: comparison with fibronectin-coated tissue culture grade polystyrene
Culture of ham
vasc~~ endotherm cells on a positivelycharged polystyrenesurface, Primaria: comparisonwith fibronec~,coated tissue culture grade polystyrene ClaudeKlein-Sayer,SylvieHemmendingerand Jean-Pierre Cazenave INSERM U.3 11. Laboratok de BioLgia et Pharmacologic des ikractions du Sang avac /es Vaisseaux et /es Biomatkiaux, Centre Rtigional de Transfusion Sanguine. 10 rue Spielmann, 67085 Strasbourg-Chdax, France fReeeked 2 November 198 7; revised 9 February 1988; accepted 15 February 1988)
Two culture surfaces, fibronectin-coated tissue culture grade polystyrene and a surface-modified polystyrene called Primaria (Falcon), were compared. The morphological (contact inhibition and cobblestone aspect), biological (production of von Willebrand factor and prostacyclin) and physiological (growth activity, non-thrombogenicity and regeneration after mechanical injury) properties of human endothelial cells were studied. ~hesion and growth of endothelial cells at low and clonal density were identical on both substrates and the biological properties were preserved. Regeneration of injured endothelium was less easy to study on Primaria polystyrene because the extracellular matrix was damaged during the lesion process. Nevertheless, Primaria polystyrene can easily be substituted for fibronectin coating in growth experiments. especially at very low seeding density. Keywords: Cell adhesjon, polystyrene, ~bronacti~, cell-polymer
The presence of a substratum, either adsorbed adhesive protein, such as fibronectin (FN), or deposition of an extracellular matrix (ECM) on the artificial surface of a culture dish is necessary for optimal growth of adherent cells. Depending on the cell type to be grown, several kinds of pretreatment have been proposed: proteins of the culture surface such as laminin’, gelatin’ and collagen of different isotypes? 4 or complex matrices such as the amniotic membrane5 and ECM produced by vascular or cornea1 endothelial cells (EC)“-8. These methods are costly and time-consuming, especially when ECM is used, because cells have first to be cultured for the production of ECM. It is now well established that adhesion and growth of EC is greatly improved by coating the culture surface with FN or ECM’. The thromb~enicityof vascular prostheses implanted in the cardiovascular system is a critical step that prevents development of new artificial materials, especially for surgical replacement of vessels of small diameter. The thromboresistance of these biomaterials can be improved by pharmacological manipulation, design of new surface physico-chemical properties and seeding and growth of Correspondence to Dr J.P. Cazeoave. 0 1989
interactions
ECg~‘o. The surface chemistry of polystyrene can be modified in bulk by covalently introducing nitrogen radicals conferring new characteristics of a positively charged surface, manufactured under the name of Primaria. The surface chemistry of Primaria as analysed by electron binding energy measurements closely resembles coating by polylysine, collagen types I and IV and ECM. The objectives of this paper were to compare the adhesion, growth, repair and biological properties of human vascular EC on polystyrene surfaces, either modified chemically by introducing charged groups as in Primaria, or coated with the adhesive plasma protein FN.
MATERIALS
AND
METHODS
Mato~als Culture media: M 199 (with Hank’s salts) containing HEPES, RPM1 1640, antibiotics (penicillin-streptomycin). fungizone, trypsin-EDTA solution, L-glutamine, were from Gibco, Paisley, UK. Collagenase type I CLS was from Seromed, Biochrom, Berlin, FRG. The 35 mm tissue culture grade poiystyrene (TCGP) dishes, Corning (25000 HA-20) were from Corning Glass Works, New York, NY, USA.
Butterworth 8 Co (Publlshers) Ltd. 0142-9612/89/020085-06SO3.00 Eiomaterials
1989, Vol IO March
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Culture of human vascular endothelial cells: C. Klein-Sayer
et al.
Primaria 35 mm culture dishes, Falcon 3801, were kindly provided by Beckton-Dickinson, Grenoble, France. Pooled human blood serum was prepared from 30 to 50 normal blood donors and the preparation of human brain extract (HBE), containing growth promoting activity, was described previously’ ‘.
EC culture Human EC from umbilical veins were obtained” and cultured in the following medium: M 199/RPMI v/v, IO mM HEPES, 2 mM L-glutamine, penicillin-streptomycin (100 unit/ml and 100 pg/ml respectively), fungizone 2.5 ,ug/ml, supplemented with 30% pooled human serum. Cryopreservation of EC was done in 90% culture medium (M 199/ RPM1 v/v 30% serum), 10% DMSO. Routinely, EC was seeded on TCGP dishes precoated with a therapeutic cryoprecipitate (Centre Regional de Transfusion Sanguine, Strasbourg, France) prepared from human plasma and enriched in FN (56% of total protein content); other major proteins were fibrinogen, albumin and immunoglobulins. The surface was coated with this fraction to give a surface concentration of 10 pg/cm2 FN; preliminary experiments using purified human FN gave similar results12.
Growth assay After thawing, EC (104/cm2) was seeded in 35 mm diameter culture dishes. The medium was changed 24 h later to remove non-adherent material and every second day afterwards. Cell growth measurements were performed by counting trypsinized EC under phase contrast microscopy or by directly enumerating EC on photographs”.
Regeneration
of injured endothelium in vitro
A mechanical lesion of the endothelium was produced in ~itro’~. EC from a confluent monolayer was specifically detached by application of a calibrated disk of cellulose polyacetate paper (Sepraphore III Gelman n* 51003, Ann Arbor, Ml, USA) under sterile conditions. The regeneration of the denuded area was measured as a function of time on fixed and stained samples by two methods. In the first, the stained culture dishes were transluminated on a negatoscope. The remaining lesions were drawn on cardboard, cut and the area obtained by weighing12. In the second, the image of the Petri dish was directly projected by means of a photographic enlarger on to a digitized tablet and the area of the lesion measured by means of a pen stylu~‘~. Both methods gave similar results.
an antibody Paris)15.
provided by J. Maclouf
(INSERM
U 150
Quantification of platelet adhesion on confluent endothelium and subendothelium “‘lndium-oxine labelled human washed platelets’6 (1 ml, 3 x lo5 platelets/mm3 in Tyrode albumin buffer) were incubated for 30 min at 37°C without agitation, in the presence of an EC monolayer previously injured (two lesions per dish, 6 mm diameter each). The platelet suspension was discarded and the cell monolayer was rinsed carefully five times with PBS after 30 min. Then one lesion was made on each sample on a preformed lesion and one on the intact cell monolayer. The detached platelets or EC plus platelets stuck to the polyacetate paper were counted in a y counter (LKB Instruments, Turku, Finland) in order to determine platelet density on endothelium and subendothelium. Further staining of the dishes with MGG allowed a visual control of the interactions of platelets with the surfaces.
RESULTS Comparison of EC growth on TCGP, FN-coated TCGP and Primaria EC was seeded in Corning polystyrene (tissue culture grade), FN-coated Corning TCGP and Primaria positively charged polystyrene surface 35 mm diameter culture dishes. Cell adhesion was measured at day 1 and cell growth was checked at days 2,4 and 7 by two different methods. One series of samples was trypsinized and cells counted in a haemocytometer. A second series of samples was fixed and stained with MGG and cell density was determined on photographs from random areas. The results correlated, although the cell density determined on photographs was always higher (ca. IO%, data not shown). The number of adherent EC obtained per dish as a function of time for the three different types of culture surface is represented on Figure 1. The highest cell density was obtained on Primaria. The aspect of the different EC layers at day 7 varied according to the surface of culture (Figure 2a, b and c). The EC layer looked patchy and irregular when cells were grown on TCGP not previously coated with adhesive proteins (Figure 2a). A uniform monolayer of confluent EC was obtained at day 7 when cells were grown on TCGP precoated
Secretion of von Willebrand factor and prostacyclin after stimulation with human thrombin Primary confluent EC monolayers were rinsed three times with PBS to eliminate traces of serum. Two millilitres of medium containing 1% human serum albumin and 5 or 1 NIH units of human purified a-thrombin per ml were added to the EC14. After 24 h incubation, the supernatants were collected, centrifuged at 12 OOOg for 1 min to eliminate cellular debris and frozen at 30°C until tested. The cell layers were stained with May-Grunwald-Giemsa (MGG) and cell density was determined on photographs of the stained layers. The endothelial cell von Willebrand factor (vWF) was measured by radioimmunoassay (Immunotech, Marseille, France) or by ELISA assay (Asserachrom, Diagnostica Stago, Asnieres, France). The stable metabolite of prostacyclin 6-keto-PGFla, was measured by radioimmunoassay using
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CULTURETIME (OAYS) Figure 1 Number of adherent cells par dish as a function of time. Average curves obtained by two methods: cell count after trypsinization and determination of cell density on photographs. P (0). tissue culture grade polystyrene; Pr (0). Primaria; FN (A). fibronectin.
Cuhure of human vascular endothelial cek
C. Klein-Sayer
et al.
at low (860 and 86 EC/cm*) and at clonal (8.6 EC/cm*) density. HBE was added at optimal concentration, 5OO&ml, every second day to the culture medium containing 30% serum. The culture dishes were observed macroscopically, after fixing and staining, at days 8 and 12. The aspect of the different cultures is presented in Figure 3. Cell proliferation was always enhanced when HBE was present. The cell growth on Primaria was comparable to cell growth on FN-coated polystyrene at each of the three cell concentrations tested.
Regeneration
of an endothelial
lesion
A mechanical lesion (6 mm diameter) was made on confluent EC grown on FN-coated TCGP or Primaria. The regeneration of the lesion was studied as a function of time on MGGstained samples by measuring the uncovered area. The percentage of uncovered area after 96 h zf regeneration and the time necessary to recover 50% of the lesion was measured for cells grown on FN or Primaria polystyrene (Table 7). The regeneration time (T50) was slightly longer when the lesion was made on EC cultured on Primaria. The slopes of the regeneration curves were compared using a Student’s ‘t’ test17 and found to be significantly different (P < 0.05). An explanation for this difference may be given by observing figure 4. At time zero, the detachment of EC grown on Primaria was not easy; the cell layer detached in patches and the underlying ECM was damaged. The repair process of FN-coated TCGP had already begun 24 h after injury. On Primaria, migrating cells were seen at the borderof the lesion, but the matrix was markedly damaged at the time of injury. This indicates that the matrix produced by EC grown on Primaria did not stick tightly to the positively charged surface.
Production of vWF and 6-keto PGFla stimulated with th~mbin
by EC
After stimulation with purified human ~-thrombin (Table 2) vWF and 6-keto PGF 1a were measured in supernatant from confluent EC grown on FN-coated TCGP or Primaria. In both circumstances, EC was able to produce vWF and PGI, after stimulation with thrombin. Figure 2 Aspect of the d~ere~t cellular tayers at day 7 when EC was grown on tissue cufture grade: la) polystyrene, (b,l fibronectin or fc) Primaria. Original magnification X40.
with FN or on Primaria not pre-exposed to adhesive proteins. EC grown on FN-coated TCGP or Primaria, exhibited the characteristic contact inhibited cobblestone pattern upon reaching confluence. EC grown on Primaria started to overlap and to form areas of high cell density (Figure 2) although without forming multilayers at post-confluence. When EC was passaged on Primaria it did not overlap until reaching post confluence. When Primaria polystyrene was pretreated with FN, no difference from TCGPcoated with FN could be demonstrated (data not shown).
Growth of EC on FN-coated polystyrene Primaria, at low and clonal density
Platelet adhesion to endothelium and subendothelium of EC grown on FN-coated TCGP or Primaria Platelet adhesion to confluent EC or to subendothelium was expressed as platelets/mm* (Table 3). The adhesion of platelets to the monolayer was the same when EC was cultured on FN-coated TCGP or Primaria. The number of platelets adhering to subendothelium exposed by the lesion (6 mm diameter) was higher when EC was grown on FN rather than on Primaria. Observation of the stained samples under the microscope showed a uniform matrix in the case of FN, but a damaged matrix in the case of Primaria. Thus in this experiment, we confirm that the matrix formed by the EC grown on Primaria plastic was easily detached from the positively charged TCGP surface. This was not the case when TCGP was precoated with FN, collagen or ECM’*.
or on
The effect of HBE on primary human endothelial cell proliferation is described elsewhere”. HBE enhances the growth of EC seeded at low or clonal density on FN or ECM. In the present assay, we compared the growth of EC on Primaria with that on FN-coated polystyrene. EC was seeded
DISCUSSION Endothelial cells, especially when seeded at low or clonat density, are difficult to establish in culture unless ECM or at least one of its components (FN, collagen or laminin) is adsorbed on the surface of the culture dish7. We compared
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Cuifure of human vascular endorhelial cells: C. Klein-Sayer
et a/.
FIBRONECTIN
PRIMARIA
860
DAY 8 cells seeded
+HBE
/ art2
+HBE
DAY12 86 ccl IS seeded +HBE
/ cm*
+HBE
DAY 12 8.6 cells +HBE
seeded
/cm*
+HBE
Figure 3 Macroscopic morphological appearance of vascular EC seeded ar low and clonal dens& and grown on FN or Primaria in rha presence of 30% serum wirh or wirhour HBE. MGG staining.
some of the properties of human EC grown on FN-coated standard tissue culture polystyrene and on Primaria, a modified polystyrene introducing positively charged groups at the surface. Primaria did not need to be precoated with FN. Cultures could be easily established on both surfaces when EC was seeded at low and clonal density and both responded
Table 1 Comparison of the ~egene~ari~ of confluent EC grown on FN or Primana polysryremz. Thawed EC was grown to conflrrence on FNor Primeda polystyrene in rha presence of 30% serum. Size of the lesion at rime zero: 6 mm diameter. Regenerarion process in rhe presence of 10% serum Fibronectin
Primaria
Time in hours necessary to repair 50% of the lesion
T50 = 41.5
T50 = 46.7
% of remaining lesion at 96 h of regeneration
6
12.6
to a crude extract of human brain containing growth promoting activity, probably FGF. in proliferation experiments, EC grew faster and the cell density was higher at confluence on Primaria than on FN-coated polystyrene. After confluence, EC grown on Primaria partially lost its contact-
Tattle 2 Stimulation of confluent EC with human purified a-Thrombin (6 unir/m& exp 7: 1 unitlml. exp 2) in medium M 1SS/RPMf v/v. 1% HSA for 24 h. Pmdocrion of von WiJ/eb~and facror (vWF~ and 6-kero-PGFfa in colrom supemaranrs
Cells/cm*
n=2
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1989, Vol 10 March
f SD (Exp 1) (EXP 2)
vWF m unit/l O6 cells (Exp 1) n = 2 fSD (Exp 2) n = 4 6-keto-PGFl a q/l
O6 cells (Exp l)n=2
Fibronectin
Primaria
59166+_3000 70 166 rt 6106
100375+8481 89291 f 7591
130 + 22.6
97.5 239 + 17.3
183.5
70
44.6
Cuflure of human vascular endathefial cells: C. Klein-Sayer
FIBRONECTIN
et al.
PRIMARIA ?t 24 hours
t,96
Figure 4 Aspect of the lesion of EC wnw magnification ~40. MGG staining.
on FN or Primaria durino the process of regenerarion at 24 h (lesion margin) and 96 h (remaining ksionj
inhibited morphol~y like EC grown on FN and began to overlap. We suggest that EC does not spread as well on Primaria as on FN-coated TCGP, therefore the cell density on Primaria can be much higher and displays an overlapping pattern upon post-confluence. Nevertheless, after passaging EC on Primaria, a uniform monolayer of non-overlapping EC could be obtained in preconfluent and confluent stages. The r~eneration process of injured confluent endothelium and the thrombogenicity of subendothelium were more difficult Table3 Comparison of the adhesion of human washed platelets fo endothelium and subendothelium when EC was 9rown to confluence on FM or Primaria Number of adherent platelets/mm2
Endathelium Subendothelium n=2
hours
Fibronectin
Primaria
18 329 64 263
19493 12 641
Original
to study when EC had grown on Primaria. It is possible that the matrix synthesized by EC itself did not have the same properties as when EC was grown on FN or that the matrix did not adhere well to the Primaria surface. The biological properties of EC were generally maintained when cultured on Primaria, as far as PGI, and vWF synthesis and release after thrombin stimuiation were concerned. The role of positively charged surface coatings like poly-o-lysine or polyethylenimide, has been successfully demonstrated, in the first case to isolate plasma membrane from eukaryotic cells’* and in the second case to grow EC and HeLa cells”. Furthermore, Swiss mouse 3T3 fibroblasts and MM 14 mouse myoblast cell lines were shown to be able to grow well on Primaria2’ and the authors claim a possible role for some nitr~en-containing surface group in cell growth. We have shown that Primaria culture dishes which present nitrogen groups at the surface, can advantageously replace FN-coated TCGP to grow EC. It is especially of interest when establishing EC cultures at low or clonal density in the absence of surface precoating with FN and
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Culture of human vascular endotheliel cells: C. Klein-Soyer et al.
without supplementary growth factor. However, it has to be noted that at post-confluence, the adherence of the EC to the culture dish surface when EC was grown on Primaria was not as good as on FN-coated TCGP. The resistance of the EC monolayer to flow rate in these conditions might be less efficient and remains to be investigated.
ACKNOWLEDGEMENTS The authors are grateful to Mrs C. Helbourg and Mrs M.O. Bernhard for excellent secretarial assistance. C. Klein-Soyer and J.P. Cazenave are research investigators, respectively Charge and Directeur de Recherche at the lnstitut National de la Sante et de la Recherche Medicale.
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REFERENCES Timpl, R., Rohde, H., Gehran-Robey, P., Rennard, S.I., Foidard, J.M. and Martin, G.R., Laminin a glycoprotein from basement membranes, J. Biol. Chem. 1979, 254, 9333-9937 Jaffe, E.A.,Cultureof humanendothelialcells.Transp/anr. Proc. 1980, 12,49-53 Kleinman, ILK., Klebe, R.J. and Martin, G.R., Role of collagenous matrices in the adhesion and growth of cells, J. Ce//. Biol. 198 1,88, 473-485 Chazou, E.I., Alexeev, A.V., Antonov, A.S., Koteliansky, V.E.. Leytin, V.L., Lyubimova. E.V., Repin, VS., Sviridov, D.D., Torchilin, V.P. and Smirnov, V.N., Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intracellular collagen matrix, Proc. Nat/. Acad. Sci. 198 1, 78. 5603-5607 Madri, J.A., Endothelial cell-matrix interactions in hemostasis, in Progress in Hemostasis and Thrombosis (Vol. 6) (Ed. TH.H. Spaet), Grune and Stratton, New York, 1982, pp l-24 Gospodarowicz, D., Vlodavsky, I., Greenburg, G. and Johnson, L.K., Cellular shape is determined by the extracellular matrix and is responsible for the control of cellular growth and function, in Hormones and Cell Culture (Vol. 6A) (Eds G.H. Sato and R. Ross), Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor Laboratory, USA 1979, pp 561-592 Gospodarowicz. D., Cheng, J., Hirabayashi, K. and Tauber, J.P., The extracellular matrix and the control ofvascular endothelial and smooth muscle cell proliferation, in Celllnferactions (Eds J.T. Dingle and J.L. Gordon), Elsevier North Holland Biomedical Press, 198 1, pp 1 35-l 65
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Macarak, E.J. and Howard, P.S., Adhesion of endothelial cells to extracellular matrix proteins, J. Ce//. Physiol 1983, 116, 76-86 Van Wachem, P.8.. Interactions of Cultured Human Endothelial Cells with Polymeric Surfaces, Proefschrih Thesis, University of Twente, The Netherlands, 1987 Pusineri, C. and Cazenave, J.P. Techniques for modifying artificial surfaces, in Blood-Surface Interactions. Biological Principles Underlying Haemocompatibility with Artificial Materials (Eds J.P. Cazenave, J.A. Davies, M.D. Kazatchkineand W.G. van Aken) Elsevier,Amsterdam, 1982, pp 209-235 Klein-Soyer, C., Stierle, A., Bouderbala, 8. and Cazenave, J.P., Effect of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture, Biol. Ceil. 1984, 52. 9-20 Klein-Soyer, C., Beretz, A., Millon-Collard, R., Abecassis. J. and Cazenave, J.P., A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process, Thrombos. Haemost. 1986, 56, 232-235 Wittendorp-Rechenmann, E., Vonesch. J.L., Rechenmann, R.V., Klein-Soyer, C. and Cazenave, J.P., Development of a computerassisted methology combined with a specific autoradiographic procedure. Application to the study of human endothelial cell regeneration, lnnov. Tech. Biol. Med. 1988, 9, 16-28 Freyssinet, J.M., Gauchy, J. and Cazenave, J.P., The effect of phospholipids on the activation of protein C by the human thrombinthrombomodulin complex, Biochem. J. 1986, 238, 151-l 57 Maclouf, J., Radioimmunoassayfor 6 keto PGF,,, Merhods fnzymol. 1982,86,273-286 Eber, M., Cazenave, J.P., Grob, J.C., Abecassis. J. and Methlin, G., lndium labelling of human washed platelets; kinetics and in viva sequestration sites, in Blood Cells in Nuclear Medicine, Part /, Ceil Kinetics end Bio-distribution (Eds M.R. Hardeman and Y. Najean) Martinus Nijhoff Publishers, Boston, 1984, pp 29-42 Tallarida, R.J. and Murray, R.8.. Manualof Pharmacologic Calculations with Computer Programs Springer-Verlag. New York, 1981, ppll-12 Jacobson, B.S., Isolation of plasma membrane from eukaryotic cells on polylysine coated polyacrylamide beads, Biochem. Biophys. Acfa. 1977,471,331-335 Jacobson, B.S. and Ryan, U.S., Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive, negative or collagen coated, Tissue CL?//.1982, 14, 69-83 Chinn, J.A., Horbett, T.A., Ratner, B.D., Schway, M.B., Haque, Y. and Hauschka, S.D., Enhancement of serum fibronectin adsorption and the cloned plating efficiencies of Swiss mouse 3T3 fibroblast and MM 14 mouse myoblast cells on polymer substrates modified by radio frequency plasma deposition, J. Colloid. Interface Science 1988 (in press)
vasc~~ endotherm cells on a positivelycharged polystyrenesurface, Primaria: comparisonwith fibronec~,coated tissue culture grade polystyrene ClaudeKlein-Sayer,SylvieHemmendingerand Jean-Pierre Cazenave INSERM U.3 11. Laboratok de BioLgia et Pharmacologic des ikractions du Sang avac /es Vaisseaux et /es Biomatkiaux, Centre Rtigional de Transfusion Sanguine. 10 rue Spielmann, 67085 Strasbourg-Chdax, France fReeeked 2 November 198 7; revised 9 February 1988; accepted 15 February 1988)
Two culture surfaces, fibronectin-coated tissue culture grade polystyrene and a surface-modified polystyrene called Primaria (Falcon), were compared. The morphological (contact inhibition and cobblestone aspect), biological (production of von Willebrand factor and prostacyclin) and physiological (growth activity, non-thrombogenicity and regeneration after mechanical injury) properties of human endothelial cells were studied. ~hesion and growth of endothelial cells at low and clonal density were identical on both substrates and the biological properties were preserved. Regeneration of injured endothelium was less easy to study on Primaria polystyrene because the extracellular matrix was damaged during the lesion process. Nevertheless, Primaria polystyrene can easily be substituted for fibronectin coating in growth experiments. especially at very low seeding density. Keywords: Cell adhesjon, polystyrene, ~bronacti~, cell-polymer
The presence of a substratum, either adsorbed adhesive protein, such as fibronectin (FN), or deposition of an extracellular matrix (ECM) on the artificial surface of a culture dish is necessary for optimal growth of adherent cells. Depending on the cell type to be grown, several kinds of pretreatment have been proposed: proteins of the culture surface such as laminin’, gelatin’ and collagen of different isotypes? 4 or complex matrices such as the amniotic membrane5 and ECM produced by vascular or cornea1 endothelial cells (EC)“-8. These methods are costly and time-consuming, especially when ECM is used, because cells have first to be cultured for the production of ECM. It is now well established that adhesion and growth of EC is greatly improved by coating the culture surface with FN or ECM’. The thromb~enicityof vascular prostheses implanted in the cardiovascular system is a critical step that prevents development of new artificial materials, especially for surgical replacement of vessels of small diameter. The thromboresistance of these biomaterials can be improved by pharmacological manipulation, design of new surface physico-chemical properties and seeding and growth of Correspondence to Dr J.P. Cazeoave. 0 1989
interactions
ECg~‘o. The surface chemistry of polystyrene can be modified in bulk by covalently introducing nitrogen radicals conferring new characteristics of a positively charged surface, manufactured under the name of Primaria. The surface chemistry of Primaria as analysed by electron binding energy measurements closely resembles coating by polylysine, collagen types I and IV and ECM. The objectives of this paper were to compare the adhesion, growth, repair and biological properties of human vascular EC on polystyrene surfaces, either modified chemically by introducing charged groups as in Primaria, or coated with the adhesive plasma protein FN.
MATERIALS
AND
METHODS
Mato~als Culture media: M 199 (with Hank’s salts) containing HEPES, RPM1 1640, antibiotics (penicillin-streptomycin). fungizone, trypsin-EDTA solution, L-glutamine, were from Gibco, Paisley, UK. Collagenase type I CLS was from Seromed, Biochrom, Berlin, FRG. The 35 mm tissue culture grade poiystyrene (TCGP) dishes, Corning (25000 HA-20) were from Corning Glass Works, New York, NY, USA.
Butterworth 8 Co (Publlshers) Ltd. 0142-9612/89/020085-06SO3.00 Eiomaterials
1989, Vol IO March
85
Culture of human vascular endothelial cells: C. Klein-Sayer
et al.
Primaria 35 mm culture dishes, Falcon 3801, were kindly provided by Beckton-Dickinson, Grenoble, France. Pooled human blood serum was prepared from 30 to 50 normal blood donors and the preparation of human brain extract (HBE), containing growth promoting activity, was described previously’ ‘.
EC culture Human EC from umbilical veins were obtained” and cultured in the following medium: M 199/RPMI v/v, IO mM HEPES, 2 mM L-glutamine, penicillin-streptomycin (100 unit/ml and 100 pg/ml respectively), fungizone 2.5 ,ug/ml, supplemented with 30% pooled human serum. Cryopreservation of EC was done in 90% culture medium (M 199/ RPM1 v/v 30% serum), 10% DMSO. Routinely, EC was seeded on TCGP dishes precoated with a therapeutic cryoprecipitate (Centre Regional de Transfusion Sanguine, Strasbourg, France) prepared from human plasma and enriched in FN (56% of total protein content); other major proteins were fibrinogen, albumin and immunoglobulins. The surface was coated with this fraction to give a surface concentration of 10 pg/cm2 FN; preliminary experiments using purified human FN gave similar results12.
Growth assay After thawing, EC (104/cm2) was seeded in 35 mm diameter culture dishes. The medium was changed 24 h later to remove non-adherent material and every second day afterwards. Cell growth measurements were performed by counting trypsinized EC under phase contrast microscopy or by directly enumerating EC on photographs”.
Regeneration
of injured endothelium in vitro
A mechanical lesion of the endothelium was produced in ~itro’~. EC from a confluent monolayer was specifically detached by application of a calibrated disk of cellulose polyacetate paper (Sepraphore III Gelman n* 51003, Ann Arbor, Ml, USA) under sterile conditions. The regeneration of the denuded area was measured as a function of time on fixed and stained samples by two methods. In the first, the stained culture dishes were transluminated on a negatoscope. The remaining lesions were drawn on cardboard, cut and the area obtained by weighing12. In the second, the image of the Petri dish was directly projected by means of a photographic enlarger on to a digitized tablet and the area of the lesion measured by means of a pen stylu~‘~. Both methods gave similar results.
an antibody Paris)15.
provided by J. Maclouf
(INSERM
U 150
Quantification of platelet adhesion on confluent endothelium and subendothelium “‘lndium-oxine labelled human washed platelets’6 (1 ml, 3 x lo5 platelets/mm3 in Tyrode albumin buffer) were incubated for 30 min at 37°C without agitation, in the presence of an EC monolayer previously injured (two lesions per dish, 6 mm diameter each). The platelet suspension was discarded and the cell monolayer was rinsed carefully five times with PBS after 30 min. Then one lesion was made on each sample on a preformed lesion and one on the intact cell monolayer. The detached platelets or EC plus platelets stuck to the polyacetate paper were counted in a y counter (LKB Instruments, Turku, Finland) in order to determine platelet density on endothelium and subendothelium. Further staining of the dishes with MGG allowed a visual control of the interactions of platelets with the surfaces.
RESULTS Comparison of EC growth on TCGP, FN-coated TCGP and Primaria EC was seeded in Corning polystyrene (tissue culture grade), FN-coated Corning TCGP and Primaria positively charged polystyrene surface 35 mm diameter culture dishes. Cell adhesion was measured at day 1 and cell growth was checked at days 2,4 and 7 by two different methods. One series of samples was trypsinized and cells counted in a haemocytometer. A second series of samples was fixed and stained with MGG and cell density was determined on photographs from random areas. The results correlated, although the cell density determined on photographs was always higher (ca. IO%, data not shown). The number of adherent EC obtained per dish as a function of time for the three different types of culture surface is represented on Figure 1. The highest cell density was obtained on Primaria. The aspect of the different EC layers at day 7 varied according to the surface of culture (Figure 2a, b and c). The EC layer looked patchy and irregular when cells were grown on TCGP not previously coated with adhesive proteins (Figure 2a). A uniform monolayer of confluent EC was obtained at day 7 when cells were grown on TCGP precoated
Secretion of von Willebrand factor and prostacyclin after stimulation with human thrombin Primary confluent EC monolayers were rinsed three times with PBS to eliminate traces of serum. Two millilitres of medium containing 1% human serum albumin and 5 or 1 NIH units of human purified a-thrombin per ml were added to the EC14. After 24 h incubation, the supernatants were collected, centrifuged at 12 OOOg for 1 min to eliminate cellular debris and frozen at 30°C until tested. The cell layers were stained with May-Grunwald-Giemsa (MGG) and cell density was determined on photographs of the stained layers. The endothelial cell von Willebrand factor (vWF) was measured by radioimmunoassay (Immunotech, Marseille, France) or by ELISA assay (Asserachrom, Diagnostica Stago, Asnieres, France). The stable metabolite of prostacyclin 6-keto-PGFla, was measured by radioimmunoassay using
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CULTURETIME (OAYS) Figure 1 Number of adherent cells par dish as a function of time. Average curves obtained by two methods: cell count after trypsinization and determination of cell density on photographs. P (0). tissue culture grade polystyrene; Pr (0). Primaria; FN (A). fibronectin.
Cuhure of human vascular endothelial cek
C. Klein-Sayer
et al.
at low (860 and 86 EC/cm*) and at clonal (8.6 EC/cm*) density. HBE was added at optimal concentration, 5OO&ml, every second day to the culture medium containing 30% serum. The culture dishes were observed macroscopically, after fixing and staining, at days 8 and 12. The aspect of the different cultures is presented in Figure 3. Cell proliferation was always enhanced when HBE was present. The cell growth on Primaria was comparable to cell growth on FN-coated polystyrene at each of the three cell concentrations tested.
Regeneration
of an endothelial
lesion
A mechanical lesion (6 mm diameter) was made on confluent EC grown on FN-coated TCGP or Primaria. The regeneration of the lesion was studied as a function of time on MGGstained samples by measuring the uncovered area. The percentage of uncovered area after 96 h zf regeneration and the time necessary to recover 50% of the lesion was measured for cells grown on FN or Primaria polystyrene (Table 7). The regeneration time (T50) was slightly longer when the lesion was made on EC cultured on Primaria. The slopes of the regeneration curves were compared using a Student’s ‘t’ test17 and found to be significantly different (P < 0.05). An explanation for this difference may be given by observing figure 4. At time zero, the detachment of EC grown on Primaria was not easy; the cell layer detached in patches and the underlying ECM was damaged. The repair process of FN-coated TCGP had already begun 24 h after injury. On Primaria, migrating cells were seen at the borderof the lesion, but the matrix was markedly damaged at the time of injury. This indicates that the matrix produced by EC grown on Primaria did not stick tightly to the positively charged surface.
Production of vWF and 6-keto PGFla stimulated with th~mbin
by EC
After stimulation with purified human ~-thrombin (Table 2) vWF and 6-keto PGF 1a were measured in supernatant from confluent EC grown on FN-coated TCGP or Primaria. In both circumstances, EC was able to produce vWF and PGI, after stimulation with thrombin. Figure 2 Aspect of the d~ere~t cellular tayers at day 7 when EC was grown on tissue cufture grade: la) polystyrene, (b,l fibronectin or fc) Primaria. Original magnification X40.
with FN or on Primaria not pre-exposed to adhesive proteins. EC grown on FN-coated TCGP or Primaria, exhibited the characteristic contact inhibited cobblestone pattern upon reaching confluence. EC grown on Primaria started to overlap and to form areas of high cell density (Figure 2) although without forming multilayers at post-confluence. When EC was passaged on Primaria it did not overlap until reaching post confluence. When Primaria polystyrene was pretreated with FN, no difference from TCGPcoated with FN could be demonstrated (data not shown).
Growth of EC on FN-coated polystyrene Primaria, at low and clonal density
Platelet adhesion to endothelium and subendothelium of EC grown on FN-coated TCGP or Primaria Platelet adhesion to confluent EC or to subendothelium was expressed as platelets/mm* (Table 3). The adhesion of platelets to the monolayer was the same when EC was cultured on FN-coated TCGP or Primaria. The number of platelets adhering to subendothelium exposed by the lesion (6 mm diameter) was higher when EC was grown on FN rather than on Primaria. Observation of the stained samples under the microscope showed a uniform matrix in the case of FN, but a damaged matrix in the case of Primaria. Thus in this experiment, we confirm that the matrix formed by the EC grown on Primaria plastic was easily detached from the positively charged TCGP surface. This was not the case when TCGP was precoated with FN, collagen or ECM’*.
or on
The effect of HBE on primary human endothelial cell proliferation is described elsewhere”. HBE enhances the growth of EC seeded at low or clonal density on FN or ECM. In the present assay, we compared the growth of EC on Primaria with that on FN-coated polystyrene. EC was seeded
DISCUSSION Endothelial cells, especially when seeded at low or clonat density, are difficult to establish in culture unless ECM or at least one of its components (FN, collagen or laminin) is adsorbed on the surface of the culture dish7. We compared
Eiomaierials
1989. Vol 10 March
87
Cuifure of human vascular endorhelial cells: C. Klein-Sayer
et a/.
FIBRONECTIN
PRIMARIA
860
DAY 8 cells seeded
+HBE
/ art2
+HBE
DAY12 86 ccl IS seeded +HBE
/ cm*
+HBE
DAY 12 8.6 cells +HBE
seeded
/cm*
+HBE
Figure 3 Macroscopic morphological appearance of vascular EC seeded ar low and clonal dens& and grown on FN or Primaria in rha presence of 30% serum wirh or wirhour HBE. MGG staining.
some of the properties of human EC grown on FN-coated standard tissue culture polystyrene and on Primaria, a modified polystyrene introducing positively charged groups at the surface. Primaria did not need to be precoated with FN. Cultures could be easily established on both surfaces when EC was seeded at low and clonal density and both responded
Table 1 Comparison of the ~egene~ari~ of confluent EC grown on FN or Primana polysryremz. Thawed EC was grown to conflrrence on FNor Primeda polystyrene in rha presence of 30% serum. Size of the lesion at rime zero: 6 mm diameter. Regenerarion process in rhe presence of 10% serum Fibronectin
Primaria
Time in hours necessary to repair 50% of the lesion
T50 = 41.5
T50 = 46.7
% of remaining lesion at 96 h of regeneration
6
12.6
to a crude extract of human brain containing growth promoting activity, probably FGF. in proliferation experiments, EC grew faster and the cell density was higher at confluence on Primaria than on FN-coated polystyrene. After confluence, EC grown on Primaria partially lost its contact-
Tattle 2 Stimulation of confluent EC with human purified a-Thrombin (6 unir/m& exp 7: 1 unitlml. exp 2) in medium M 1SS/RPMf v/v. 1% HSA for 24 h. Pmdocrion of von WiJ/eb~and facror (vWF~ and 6-kero-PGFfa in colrom supemaranrs
Cells/cm*
n=2
88
Eiomarerials
1989, Vol 10 March
f SD (Exp 1) (EXP 2)
vWF m unit/l O6 cells (Exp 1) n = 2 fSD (Exp 2) n = 4 6-keto-PGFl a q/l
O6 cells (Exp l)n=2
Fibronectin
Primaria
59166+_3000 70 166 rt 6106
100375+8481 89291 f 7591
130 + 22.6
97.5 239 + 17.3
183.5
70
44.6
Cuflure of human vascular endathefial cells: C. Klein-Sayer
FIBRONECTIN
et al.
PRIMARIA ?t 24 hours
t,96
Figure 4 Aspect of the lesion of EC wnw magnification ~40. MGG staining.
on FN or Primaria durino the process of regenerarion at 24 h (lesion margin) and 96 h (remaining ksionj
inhibited morphol~y like EC grown on FN and began to overlap. We suggest that EC does not spread as well on Primaria as on FN-coated TCGP, therefore the cell density on Primaria can be much higher and displays an overlapping pattern upon post-confluence. Nevertheless, after passaging EC on Primaria, a uniform monolayer of non-overlapping EC could be obtained in preconfluent and confluent stages. The r~eneration process of injured confluent endothelium and the thrombogenicity of subendothelium were more difficult Table3 Comparison of the adhesion of human washed platelets fo endothelium and subendothelium when EC was 9rown to confluence on FM or Primaria Number of adherent platelets/mm2
Endathelium Subendothelium n=2
hours
Fibronectin
Primaria
18 329 64 263
19493 12 641
Original
to study when EC had grown on Primaria. It is possible that the matrix synthesized by EC itself did not have the same properties as when EC was grown on FN or that the matrix did not adhere well to the Primaria surface. The biological properties of EC were generally maintained when cultured on Primaria, as far as PGI, and vWF synthesis and release after thrombin stimuiation were concerned. The role of positively charged surface coatings like poly-o-lysine or polyethylenimide, has been successfully demonstrated, in the first case to isolate plasma membrane from eukaryotic cells’* and in the second case to grow EC and HeLa cells”. Furthermore, Swiss mouse 3T3 fibroblasts and MM 14 mouse myoblast cell lines were shown to be able to grow well on Primaria2’ and the authors claim a possible role for some nitr~en-containing surface group in cell growth. We have shown that Primaria culture dishes which present nitrogen groups at the surface, can advantageously replace FN-coated TCGP to grow EC. It is especially of interest when establishing EC cultures at low or clonal density in the absence of surface precoating with FN and
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1989, Vol 10 March
83
Culture of human vascular endotheliel cells: C. Klein-Soyer et al.
without supplementary growth factor. However, it has to be noted that at post-confluence, the adherence of the EC to the culture dish surface when EC was grown on Primaria was not as good as on FN-coated TCGP. The resistance of the EC monolayer to flow rate in these conditions might be less efficient and remains to be investigated.
ACKNOWLEDGEMENTS The authors are grateful to Mrs C. Helbourg and Mrs M.O. Bernhard for excellent secretarial assistance. C. Klein-Soyer and J.P. Cazenave are research investigators, respectively Charge and Directeur de Recherche at the lnstitut National de la Sante et de la Recherche Medicale.
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11
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REFERENCES Timpl, R., Rohde, H., Gehran-Robey, P., Rennard, S.I., Foidard, J.M. and Martin, G.R., Laminin a glycoprotein from basement membranes, J. Biol. Chem. 1979, 254, 9333-9937 Jaffe, E.A.,Cultureof humanendothelialcells.Transp/anr. Proc. 1980, 12,49-53 Kleinman, ILK., Klebe, R.J. and Martin, G.R., Role of collagenous matrices in the adhesion and growth of cells, J. Ce//. Biol. 198 1,88, 473-485 Chazou, E.I., Alexeev, A.V., Antonov, A.S., Koteliansky, V.E.. Leytin, V.L., Lyubimova. E.V., Repin, VS., Sviridov, D.D., Torchilin, V.P. and Smirnov, V.N., Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intracellular collagen matrix, Proc. Nat/. Acad. Sci. 198 1, 78. 5603-5607 Madri, J.A., Endothelial cell-matrix interactions in hemostasis, in Progress in Hemostasis and Thrombosis (Vol. 6) (Ed. TH.H. Spaet), Grune and Stratton, New York, 1982, pp l-24 Gospodarowicz, D., Vlodavsky, I., Greenburg, G. and Johnson, L.K., Cellular shape is determined by the extracellular matrix and is responsible for the control of cellular growth and function, in Hormones and Cell Culture (Vol. 6A) (Eds G.H. Sato and R. Ross), Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor Laboratory, USA 1979, pp 561-592 Gospodarowicz. D., Cheng, J., Hirabayashi, K. and Tauber, J.P., The extracellular matrix and the control ofvascular endothelial and smooth muscle cell proliferation, in Celllnferactions (Eds J.T. Dingle and J.L. Gordon), Elsevier North Holland Biomedical Press, 198 1, pp 1 35-l 65
90
Biomaterials
1989, Vol 10 March
14
15 16
17
18
19
20
Macarak, E.J. and Howard, P.S., Adhesion of endothelial cells to extracellular matrix proteins, J. Ce//. Physiol 1983, 116, 76-86 Van Wachem, P.8.. Interactions of Cultured Human Endothelial Cells with Polymeric Surfaces, Proefschrih Thesis, University of Twente, The Netherlands, 1987 Pusineri, C. and Cazenave, J.P. Techniques for modifying artificial surfaces, in Blood-Surface Interactions. Biological Principles Underlying Haemocompatibility with Artificial Materials (Eds J.P. Cazenave, J.A. Davies, M.D. Kazatchkineand W.G. van Aken) Elsevier,Amsterdam, 1982, pp 209-235 Klein-Soyer, C., Stierle, A., Bouderbala, 8. and Cazenave, J.P., Effect of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture, Biol. Ceil. 1984, 52. 9-20 Klein-Soyer, C., Beretz, A., Millon-Collard, R., Abecassis. J. and Cazenave, J.P., A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process, Thrombos. Haemost. 1986, 56, 232-235 Wittendorp-Rechenmann, E., Vonesch. J.L., Rechenmann, R.V., Klein-Soyer, C. and Cazenave, J.P., Development of a computerassisted methology combined with a specific autoradiographic procedure. Application to the study of human endothelial cell regeneration, lnnov. Tech. Biol. Med. 1988, 9, 16-28 Freyssinet, J.M., Gauchy, J. and Cazenave, J.P., The effect of phospholipids on the activation of protein C by the human thrombinthrombomodulin complex, Biochem. J. 1986, 238, 151-l 57 Maclouf, J., Radioimmunoassayfor 6 keto PGF,,, Merhods fnzymol. 1982,86,273-286 Eber, M., Cazenave, J.P., Grob, J.C., Abecassis. J. and Methlin, G., lndium labelling of human washed platelets; kinetics and in viva sequestration sites, in Blood Cells in Nuclear Medicine, Part /, Ceil Kinetics end Bio-distribution (Eds M.R. Hardeman and Y. Najean) Martinus Nijhoff Publishers, Boston, 1984, pp 29-42 Tallarida, R.J. and Murray, R.8.. Manualof Pharmacologic Calculations with Computer Programs Springer-Verlag. New York, 1981, ppll-12 Jacobson, B.S., Isolation of plasma membrane from eukaryotic cells on polylysine coated polyacrylamide beads, Biochem. Biophys. Acfa. 1977,471,331-335 Jacobson, B.S. and Ryan, U.S., Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive, negative or collagen coated, Tissue CL?//.1982, 14, 69-83 Chinn, J.A., Horbett, T.A., Ratner, B.D., Schway, M.B., Haque, Y. and Hauschka, S.D., Enhancement of serum fibronectin adsorption and the cloned plating efficiencies of Swiss mouse 3T3 fibroblast and MM 14 mouse myoblast cells on polymer substrates modified by radio frequency plasma deposition, J. Colloid. Interface Science 1988 (in press)