Int. J. Exp. Path (1992), 73, 491-501

The seeding of human aortic endothelial cells on the extra-cellular matrix of human umbilical vein endothelial cells Denis E. Solomon Research Division, Miami Heart Institute, Miami Beach, FL 33140, USA

Received for publication 6 December 1991 Accepted for publication 20 February 1992

Summary. A post confluent layer (6th passage) of human umbilical vein endothelial cells (HUVECs) was treated with 3mM ethylene diamine tetra-acetic acid (EDTA) to expose the subendothelial extra-cellular matrix (ECM). Normal human aortic endothelial cells (HAECs) harvested by mechanical scraping were seeded onto the ECM of the HUVECs. The cells quickly attached and proliferated with normal morphology. To ensure confluency the HAECs were pooled after a brief trypsin/EDTA incubation and seeded onto the ECM of the same HUVECs (6th passage) cell line. They attached within 2 hours, and the cells grew to confluence displaying cobblestone morphology characteristic of phenotypic endothelium. HUVECs (11th passage) were seeded onto (6th passage) HUVECs ECM. The cells attached, proliferated to confluence within the normal time interval (7-8 days) and were positively characterized. A Corvita 6mm graft supplied with a gelatin/heparin matrix was densely seeded with HUVECs (6th passage). These cells also proliferated to confluence. The implications for improving the design of arterial grafts are discussed.

Keywords: human aortic endothelial cells, human umbilical vein endothelial cells, extracellular matrix, arterial grafts

The isolation and tissue culture of human vascular endothelial cells (ECs) has involved the use of enzymes, usually collagenase (Jaffe et a]. 1973a), or mechanical scraping (Ryan & White 1985), and the seeding of the isolated endothelial cells on a gelatin or fibronectin substrate. Fibronectin has been the most popular choice of substrate, since it was described as the primary protein of the extra-cellular matrix (ECM) underlying calf bovine aortic ECs (Kramer et a). 1980). Later studies (Fry et a]. 1984) showed that the pattern of pro-

teins, including type IV basement membrane procollagen, thrombospondin, and fibronectin secreted by cultures of human adult vena cava and aortic endothelial cells in vitro resembled the composite proteins described by Sage and Bornstein (1982) for human umbilical vein endothelium. Kramer et a]. (1980) also included a technique using 2mm ethylene diamine tetra-acetic acid (EDTA), a modification of earlier work (Rosen & Culp 1977), which induced the detachment of a monolayer of calf bovine aortic ECs from the underlying

Correspondence: Dr Denis E. Solomon, Research Division, Miami Heart Institute, 4701 Meridian Avenue, Miami Beach, FL 33140, USA. 49'

492 D.E. Solomon 100 Mug/ml heparin and 50 Mg/ml of a crude subendothelial ECM, leaving the 'native' ECM intact. Other workers have used 20mM extract of endothelial cell growth suppleethylene glycol-bis-(,B-aminoethyl ether)- ment (ECGS) (Maciag et al. 1979). For N,N,N,N'l-tetra-acetic acid (EGTA) or 0.5% HAECs, the concentration of ECGS was sodium deoxycholate (Herman & Castellot doubled. 198 7); or 100mM NH4 OH (Sakariassen et al. 1983). Cell culture of HUVECs In this report, it is demonstrated that the ECM of human umbilical vein endothelial Human umbilical vein endothelial cells cells (HUVECs) may be used as a tissue- (HUVECs) were isolated by perfusion of a culture substratum for human aortic endo- single umbilical cord with 0.2% collagenase thelial cells. The potential implications for (Jaffe et al. 1973a). The cannulae were the future design of arterial grafts are dis- fastened to the umbilical cord by preautoclaved disposable plastic twist ties. cussed. Haemostats maintained the position of the cannulae. Isolated ECs were seeded onto Materials and methods T-2 5 gelatin-coated flasks, and incubated at 37°C in a 5% C02/95% air mixture. Tissue culture Feeding was thrice weekly and confluence Tissue culture flasks were obtained from was reached in 7-8 days. Confluent monoCorning, New York. Primaria coated 24-well layers of HUVECs were subcultured (1:2 split) dishes were obtained from Falcon (Becton by mechanical means via a rubber policeDickinson), Lincoln Park, New Jersey. Anti- man. Confluence was routinely reached in biotics and materials for the preparation of 7-8 days. cell culture media were obtained from Gibco, Grand Island, New York. Foetal bovine Preparation of HUVECs ECM serum (FBS) was purchased from Hyclone, A 2-day-old post-confluent monolayer (6th Logan, Utah. Heparin (from porcine intesti- passage) of HUVECs (Fig. 1) was washed nal mucosa) and bovine fetuin were obtained three times with Ca2+/Mg2+-free (CMF) from Sigma, St Louis, MO. Type 1 collage- (Dulbecco's phosphate-buffered saline) DPBS nase was obtained from Worthington, Free(pH 7.4). Aliquots of 3 mm EDTA/(CMF) hold, NJ and calf bovine hypothalamus, from DPBS, a modification of the method of Pel-Freez, Rogers, Arizona. Dil-Ac-LDL and Kramer et al. (1980), were added to the Dil-LDL were purchased from BioMedical incubated flask (T-25) until a cell-free ECM Technologies Inc., Stoughton, MA. Goat was obtained. Flasks containing prepared antiserum to Human Factor VIII and FITC cell-free ECM overlaid with (CMF) DPBS were Antigoat IgG were purchased from Miles stored in the incubator and were viable up to Scientific, Naperville, IL. a week thereafter.

Culture medium The culture medium for both the HUVECs and human aortic endothelial cells (HAECs) was Medium 199 with Earle's salts, supplemented with 2 mM L-glutamine, 20% heatinactivated FBS, 100 U/ml penicillin, 100 ,ug/ml streptomycin, 10 Mg/ml gentamicin, and 10 mm HEPES (pH 7.4). Additional supplements were 100 Ig/ml bovine fetuin,

The seeding of HUVECs on a HUVECs ECM The ECM from identical HUVECs (6th passage) cell line was prepared as described above. A T-2 5 flask containing a postconfluent (11th passage) HUVECs monolayer was subjected to a brief 0.05% trypsin/ 0.02% EDTA incubation. After pelleting, the cells were resuspended in culture medium, and seeded onto the 6th passage ECM. After

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Seeding of human aortic ECs on extra-cellular matrix of umbilical vein cells 495 2 hours, the majority of HUVECs had attached (Fig. 2), grew to confluence within 18 hours and were positively characterized. The seeding of HUVECs onto a Corvita 6 mm graft A T-25 of identical HUVECs (6th passage) was enzymatically harvested and resuspended in culture medium as described above. Under sterile conditions a 4-inch (10-cm) section of a Corvita 6-mm graft supplied with a gelatin/ heparin matrix (Graft no. B002212B) was clamped at one end and placed into a sterile glass Petri dish. HUVECs were seeded via a 2-ml sterile plastic disposable pipette, and the open end clamped. The graft was manually rolled. At 10-minute intervals the cell seeding was repeated, and the covered Petri dish was incubated. After 2 days, the graft was slit open lengthwise and pinned at its edges onto a sterile section of dental wax plate sitting in the glass Petri dish. Examination on a Leitz diavert microscope was followed by the addition of fresh culture medium plus ECGS to the Petri dish. Two days later it was observed that the HUVECs had reached confluence. Owing to a lack of facilities, this could not be photographed but was witnessed.

Cell culture of HAECs A 6-inch (15-cm) segment of human aorta was transported to the laboratory in a sterile container filled with cold serum-free culture medium. The donor was a 3 6-year-old male. On cutting open the aortic segment, under sterile conditions, no clinical manifestations of arterial disease were observed. The luminal surface, which was smooth and shiny with an off-white coloration, was scraped with a scalpel exactly as described (Ryan & White 1985). The cells were shaken off the blade into culture medium, pelleted, and resuspended with culture medium. Since this was a pilot experiment, the volume of HAECs suspension was subdivided into four aliquots

which were seeded onto four T-25 flasks containing HUVECs ECM. Accurate cell counts could not be determined since small sheets of endothelium were observed floating in the culture medium in each of the four flasks.

Identification HUVECs were seeded onto 35-mm gelatin coated Petri dishes, whereas HAECs were seeded into a number of wells (pre-coated with HUVECs ECM) of a 24-well Primaria plate. Both HUVECs and HAECs displayed cobblestone morphology at confluence and were positively characterized as endothelial cells by their ability to synthesize Factor VIII: von Willebrand complex as determined by indirect immunofluorescence (Jaffe et al. 1973b), and by their uptake of acetylated low density lipoprotein (Voyta et al. 1984). A Nikon Diaphot phase-contrast microscope, equipped with a reflected-light fluorescence system and Nikon 3 5-mm camera, was used throughout this study.

Results HAECs started to attach to the cell-free HUVECs ECM within an hour after seeding and incubating the flasks at 3 7°C in a 5% C02/9 5% air mixture. Within 16 hours, even the small sheets of endothelium had attached (Fig. 3), and there was progressive outgrowth of endothelial cells with normal morphology. The culture medium was aspirated off, the cell layer was washed with DPBS (pH 7.4), and fresh culture medium plus ECGS were added. Small primary colonies of cells did not remain rounded but spread laterally though maintaining cell-cell contact. Cells growing away from an adhered endothelial sheet formed an enlarging concentric circle (Fig. 4). No cells or sheets of cells were observed lifting off once they had bedded down and no multi-layers were observed. The culture medium was changed thrice weekly and the

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contents of the flasks were examined three times a day. Based only on observations, it was concluded that cell growth proceeded in its usual fashion as was noted previously on a 0.2% gelatin/DPBS substrate (D.E. Solomon and U.S. Ryan, unpublished). Seven days after seeding it was realized, given the slow proliferative rate of HAECs, that each flask did not contain a sufficient population of cells to ensure confluency. A rubber policeman could not be used for pooling the contents of the individual flasks. The uprooted ECs became trapped in the admixed gelatin/ECM substrate and failed to attach to a fresh ECM substratum (of the same HUVECs 6th passage cell line) even after 48 hours. Hence, the contents of each flask was briefly incubated with 0.05% trypsin/0.02% EDTA, pooled, and seeded into a single flask containing HUVECs ECM (of the same HUVECs 6th passage cell line). Aliquots were seeded as described previously for identity tests. The cells started to attach within 20 minutes and within 2 hours the vast

majority of cells had bedded down and had secreted a cellular matrix. There was no evidence of cell or ECM debris. The cells proliferated to confluence displaying cobblestone morphology characteristic of phenotypic endothelium (Fig. 5), took up Dil-Ac LDL (Fig. 6) and produced Factor VIII: von Willebrand complex. This confluent monolayer was subcultured (1: 2 split) by enzymatic means and seeded onto ECMs prepared from HUVECs (now at 8th passage). Attachment, cell division and cell proliferation were observed. Discussion A HUVECs ECM with superimposed HAECs provided the latter cell population with a substrate they seemed to 'recognize' and to which they preferentially attached, since they produce a very similar ECM. Using a 0.2% gelatin/DPBS substrate previously (D.E. Solomon and U.S. Ryan, unpublished), small endothelial sheets took 7-12 days for

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D.E. Solomon 500 attachment, in contrast to the 16 hours retention on grafts was cited as a major being reported here. The HAECs should be problem (Herring & LeGrand 1989; Herring recognized as normal healthy cells and do 1991). Reports to date speak of pre-coating not constitute a selective population (Anto- the graft with a single constituent of the nov et al. 1986) because mechanical scrap- ECM, for example, either with collagen IV or ing of the aortic segment would yield a truly fibronectin or a pre-clot matrix (Vohra et al. representative harvest of ECs. Multinuc1991). leated 'variant' ECs were scarce. The age of Some commercially available 6-mm grafts the donor, 36 years, is below that required (Corvita) are sold impregnated with a gelafor major age related endothelial change in tin/heparin matrix, which is perfectly suithuman arteries (Cotton & Wartman 1961; able for the seeding of HUVECs. It should be Tokunaga et al. 1989). recalled that the tissue culture of HUVECs Primary isolates of HUVECs are not necess- employed a 0.2% gelatin/ DPBS matrix and ary for ECM substrate preparation, because heparin was a constituent of the culture any post-confluent monolayer of cells medium. A preliminary experiment has obtained from a single umbilical cord should determined that HUVECs would adhere to be underlaid with an identical non-thrombo- such a graft, and that cell proliferation would genic ECM, regardless of passage number, proceed to confluence. Removal of the as long as the cell line is pure and healthy. HUVECs monolayer at post-confluence deIt is noteworthy that the prepared ECM scribed herein would leave a substratum used here was derived from 6th passage which would support the growth of endotheHUVECs. lium. Of interest was that HUVECs (11th 3 mM EDTA/(CMF) DPBS was utilized to passage) harvested by trypsin digestion retract the HUVECs monolayer because 2 attached to the ECM (Fig. 2) of the same mM EDTA (Kramer et al. 1980), was found to HUVECs cell line (6th passage) as was used remove only 98% of the monolayer. The ECs for the growth of the aortic endothelium. rounded and detached individually or in Although clinical details of both mothers small clusters, in contrast to the tissue-like whose umbilical cords were used could not sheet of intact cells (whose viability was not be obtained, it is presumptive at present that determined) observed by Herman and ECMs of HUVECs share a common phenoCastellot (1987), using 20 mm EGTA. type. This principle may also be applicable to The HUVECs were minimally affected by the tissue culture of microvascular ECs. the 3mM EDTA/(CMF) DPBS treatment. Gelatin-coated microcarrier beads, seeded Detached cells, collected in serum-free cul- with HUVECs, could probably be used to ture medium, pelleted and resuspended in provide a very useful substrate for long-term culture medium, attached to a 0.2% gelatin/ tissue culture of HAECs. DPBS substrate, grew to confluence in the Thus far, HAECs from two additional normal interval (7-8 days) and were posiaortic segments (one of which was atherotively characterized. This viability suggested sclerotic) have been mechanically scraped that the HUVECs were detached in a manner and in both cases attachment to ECMs which did not disrupt their ECM. The cell free (derived from different HUVECs cell lines) ECM would not be capable of exciting an and growth of phenotypic endothelium were immunological reaction because the latter achieved. are cell-mediated. Shear stress factors need to be determined The results of these tissue culture studies to ascertain the strength of the HAECs suggest an improvement in the design of adhesion to the HUVECs ECM. Further invesarterial graft surfaces. Published reports of tigative work will encompass studies on the the EC seeding of vascular prostheses have so haemostatic functions of the HAECs and will far yielded poor results and endothelial cell form the basis of the next report.

Seeding of human aortic ECs on extra-cellular matrix of umbilical vein cells 50I Acknowledgements Dr David Crutchley is thanked for his helpful discussions and I thank Dr Parry Larsen for a tutorial on his clinical experiences with vascular prostheses. Mr Andy Toledo is also thanked for his help with the maintenance of human umbilical vein endothelial cell lines. The umbilical cords were gifts from the Nursing Staff, the Obstetrics Department, Mount Sinai Medical Center, Miami Beach, FL. The segments of human aorta were supplied by the Department of Transplantation Surgery, University of Miami, School of Medicine. The author is grateful for the ongoing cooperation of Les Olson and his staff. Dr Leonard Pinchuk of Corvita Corporation (Miami, Florida) generously donated the section of 6-mm graft. Miss Andrea Roth did an admirable job in typing and collating this paper. Mr Paul Cooper of the Photography Section, Nuffield Orthopaedic Centre, University of Oxford, is thanked for his excellent technical assistance.

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