4’7, 173-177 (1989)
Effects of in Vitro Aging on Human Endothelial Cell Adherence Dacron Vascular Graft Material JOHN
of Surgery, Jefferson Medical College, Philadelphia, Submitted
July 18, 1988
surface. In this study, we have examined the use of cultured human EC and their potential for graft endothelialization. Although tissue culture allows rapid expansion of EC to generate a large number of cells over a brief period of time, culture also introduces other variables such as the effects of culture conditions and in vitro cellular aging [6, 71. The effects of these variables could be important to EC-graft interactions and might be important in the selection of a method of cell incubation.
The adherence and growth characteristics of cultured human adult large vessel endothelial cells (EC) maintained on substrate-coated polyethylene terephthalate in the form of woven dacron vascular graft were examined. Two different populations of EC, a low passage (EC (low)) and a high passage (EC (high)) population, were incubated at cell densities from 10’ to lo6 EC/cm2 for 24 hr. Cell counts were performed at 24 hr and after 14 days in tissue culture. At 24 hr on collagen I/III-coated Dacron, EC adherence was independent of the number of passages or the incubation density. When examined after 14 days in culture only EC (low) incubated at 10’ EC/cm2 maintained initial cell numbers. Human plasma precipitated upon Dacron was necessary before significant cell growth occurred. We conclude that increasing in vitro EC age is associated with decreasing attachment and growth on Dacron. Growth on this important vascular replacement surface requires low passage EC incubated at a high density and the presence of plasma proteins in the substrate coating. 0 1989 Academic Press, Inc.
METHODS Isolation and Culture of Large Vessel Human Adult Endothelial Cells Human adult EC were isolated from vessels obtained from brain-dead, heart-beating cadaver organ donors and cultured according to our previously published reports [ 79]. EC were obtained by treating the luminal vessel surface with crude collagenase (Worthington Type I; Worthington Diagnostic Systems, Inc., Freehold, NJ) and grown in 25-cm* tissue culture flasks of polystyrene coated with 1% gelatin. Culture medium contained medium 199,20% heat-inactivated fetal calf serum, 90 pg/ml heparin (porcine), and 20 kg/ml endothelial cell growth factor (ECGF). The EC identity was established by typical cobblestone morphology in culture and by positive staining for Factor VIII-related antigen.
INTRODUCTION Vascular graft endothelialization has been studied extensively using the technique of seeding freshly isolated venous EC onto Dacron and expanded polytetrafluoroethylene (ePTFE) surfaces [l-4]. Although successful in dogs, this method requires 6 to 8 weeks before a significant percentage of the luminal surface is covered by endothelial cells (EC). An alternate approach to endothelializing a surface more rapidly is by placing cultured EC onto the graft surface . These could be placed onto the surface at a high density, resulting potentially in a “near-confluent” surface at implantation or shortly thereafter. Two variables that might critically affect the rate of endothelialization are (1) the number of EC that remain adherent to the surface following incubation and (2) the ability of attached EC to grow and cover the remaining exposed
Selection of EC As a preliminary requirement of this study, we selected several cultured EC lines that demonstrated rapid growth in culture upon tissue-culture polystyrene. Cellular senescence of human EC has been reported with increasing in vitro age, but both the low and high passage EC demonstrated rapid proliferation on polystyrene and therefore could be classified as nonsenescent cells [lo]. EC for this study were selected from two separate categories based upon their in vitro age. Early or low passage EC, designated EC (low), were defined as EC that were isolated from a vessel and placed into primary culture. After growth to confluence, the cells underwent one subcultivation at a 2 to 3 split ratio before use in the present
’ To whom correspondence and reprint requests should be addressed at Jefferson Medical College, Department of Surgery, 1025 Walnut Street, Philadelphia, PA 19107. 173
0022.4804/89 $1.50 Copyright 0 1989 by Academic Press, Inc. rights of reproduction in any form reserved.
study as low passage EC cells. EC (low) were obtained from both an iliac artery segment and an iliac vein segment. Late or high passage EC, designated EC (high), were isolated from a vessel and then maintained in culture medium until at least 10 subcultivations had taken place. These EC were not senescent in that they maintained rapid proliferative growth to confluence when cultured upon tissue culture polystyrene (See Table 1). EC (high) were obtained from two separate donor iliac arteries and one iliac vein. Graft Surface Preparation Surfaces of Cooley Graft Woven Low Porosity Dacron (supplied by Meadox Medicals, Oakland, NJ) were immobilized in a plastic ring (Beem Capsule, Polysciences, Fort Washington, PA) providing a surface area of 0.5 cm2 . All subsequent surface treatments, seeding, culture, and staining were performed on this immobilized graft surface. Collagen Preparation Interstitial collagen types I and III were prepared from human placenta according to the procedures of Madri and Williams [ 111. Minced and freeze-dried placenta was solubilized with 0.5 M acetic acid. Types I/III collagen were precipitated with 1.4 M NaCl, and the precipitate was collected and dialyzed against buffer. The flocculent collagen was freeze-dried and stored at -20°C until use. Collagen was added to grafts following its solubilization in 0.0174 M acetic acid and dilution to 0.32% collagen with ice-cold medium 199 and NaHC03. The collagen solution was then layered onto the graft surfaces and incubated at 37’C for 30 min. The surface was washed with culture medium prior to EC seeding. Platelet Rich Plasma Preparation Plasma was prepared from fresh human blood drawn into tubes with buffered sodium citrate. Platelet-rich
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plasma (PRP) was prepared by centrifuging the blood sample at 300g for 4 min and collecting the supernatant. Immediately prior to coating the graft, 0.02 M CaC12 was added. After the plasma was added, the graft surface was incubated at 37°C for 30 min prior to EC incubation. Endothelial
Confluent T-25 flasks of EC (low) and EC (high) were briefly treated with a trypsin solution (0.25% trypsin, 0.04% EDTA). The cells were washed with culture medium and incubated upon the graft surfaces at varying concentrations. The grafts were either untreated or pretreated with collagen I/III, PRP, or a combination of collagen I/III and PRP as described above. The seeded grafts were placed in culture medium and incubated in a 5% COP atmosphere at 37’C. The graft surfaces were evaluated by light microscopy on Days 1 and 14 for quantitative cell counts. Light Microscopy EC seeded grafts were washed with Dulbecco’s phosphate-buffered saline (pH 7.4 containing 0.1% serum albumin) and then fixed with 95% ethanol for 15 min at 22°C. Grafts were then rinsed twice with distilled water and stained for 2 min with Gill’s hematoxylin (Fisher Scientific Co., Fairlawn, NJ). After two more rinses with water, the grafts were placed in Scott’s Tap Water Substitute for 1 min. The grafts were then rinsed twice with water and 95% ethanol, mounted, and viewed under a Nikon diaphot microscope. Statistical
All experiments were performed using triplicate surfaces. At the completion of the experiment, the surfaces were stained and cells were counted in 10 representative high power fields (400X). The mean cell count and the standard error of the mean were determined and compared using the Student t test. Significant changes in cell number were chosen at P < 0.05. RESULTS
Rates of Human
Days to reach confluence polystyrene Initial
10s cells/cm* 5 X lo3 cells/cm’ lo4 cells/cm’
8 6 4
a 6 4
Note. Cultured human EC were seeded onto polystyrene tissue culture wells and observed until confluence was reached. No significant difference was noted between EC (low) and EC (high) or between cells of arterial or venous origin.
Adherence Studies on Collagen-Coated Dacron
Cultured EC were incubated onto collagen-coated woven Dacron graft material at different initial cell densities. After a 24-hr incubation period, the surfaces were washed and the remaining attached EC were counted (Fig. 1). EC-graft adherence was independent of cell age in culture (EC (low) vs EC (high)) and the initial cell density. The percentage adherent cells at 24 hr ranged from 26 to 58%. Growth Studies on Collagen-Coated Dacron Cultured EC were incubated at 103, 5 X 103, 104, and lo6 EC/cm’ and placed into tissue culture for 14 days.
ET AL.: ENDOTHELIAL
were incubated onto plasma-coated Dacron, a statistically significant rise in cell number was observed. The actual final cell count at 14 days was 9.6 X lo* EC/cm2, indicating a near confluent coverage of the surface. DISCUSSION
DENSlTY CM ’ )
FIG. 1. Initial adherence of cultured EC to collagen-coated Dacron. Human EC were incubated at different densities upon collagen-coated woven Dacron and placed in tissue culture. (IS) EC (low) or low passage EC. (B) EC (high) or high passage EC. After 24 hr, the surfaces were washed and the remaining cells were counted. Each experiment was performed in triplicate surfaces and each surface was evaluated by six different cell field counts. No significant differences between EC (low) and EC (high) were observed as measured by the Student t test. Values are expressed as means + standard errors of the means. Values on the y-axis were determined by dividing the number of EC present at 24 hr by the number of EC initially incubated upon the surface.
The surfaces were washed and the remaining EC were counted (Fig. 2). No growth occurred and statistically significant drops in cell number when compared to the cell number on Day 1 were observed for all experiments except when EC (low) were incubated upon Dacron at lo5 EC/ cm’. For EC (low) incubated at lo5 EC/cm2, the number of attached cells remained constant over the 14-day period. To determine if the lack of growth using EC (high) was related to EC source, high passage EC from venous and arterial sources were compared. The long-term adherence for venous EC (high) = 12 f 9% compared to arterial EC (high) = 24 ? 5.2%. There was no statistical difference between the values. Effect of Substrate Treatment
One major direction in blood-contacting surface research remains the examination of vascular endothelium as a lining on prosthetic surfaces to lessen or eliminate thrombogenicity. Historically vascular graft “seeding” using freshly derived EC has resulted in a surface that is initially thrombogenic but that becomes less thrombogenie as it becomes partially endothelialized by 6 to 8 weeks. More recently it has been demonstrated that an EC monolayer can form rapidly (within 1 hr) from the time of initial cell contact with certain surfaces [lo]. These rapidly formed monolayers require a large number of cells and rely on rapid attachment of the cells to the surface rather than small cell numbers and subsequent growth to confluence. A major problem with the rapid monolayer technique has been the procurement of enough EC to allow 10’ cells/cm2 to incubate with the graft. Freshly isolated large vessel EC would rarely be obtainable in the human unless very small graft segments were used. Freshly isolated microvessel EC are an alternate cell type that may supply large numbers of EC for high density incubation [ 121. A second alternative solution is the use
on EC Growth on Dacron
Cultured EC were incubated at lo5 cells/cm2 upon plain woven Dacron or Dacron-coated with plasma, collagen I/ III, or plasma-collagen I/III combined. Data from arterialderived EC at low and high passages are presented in Figs. 3A and 3B, but equivalent results were observed using venous derived EC. In Fig. 3A, the initial adherence of EC to the substrate-coated surface after 24 hr is presented. Although EC (low) always demonstrated higher initial adherence when compared to EC (high), only EC (low) adherence to plasma-coated dacron was statistically superior. When subsequent adherence and growth over a 14-day period were examined (Fig. 3B), all surfaces demonstrated a drop in cell count when EC (high) were studied. Use of EC (low) resulted in maintenance of cell number when plain, collagen I/III-coated Dacron and plasmacollagen I/III-coated Dacron were studied, When EC (low)
DENSITY CM ’ 1
FIG. 2. Long-term adherence of cultured EC to collagen-coated Dacron. Human EC were incubated at different densities upon collagencoated woven Dacron and placed into tissue culture. @) EC (low) or low passage EC. (m) EC (high) or high passage EC. Cell number was evaluated at 24 hr and at 14 days and the change in cell number was determined. Experiments were performed on triplicate surfaces and each surface was evaluated by six different cell field counts. Values on theyaxis were determined by dividing the number of EC present at 14 days by the number of EC present at 24 hr. $ denotes a significant drop in cell number over the 13-day period as determined by the Student t test (P < 0.05). t denotes a significant difference between EC flow) and EC (high).
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FIG. 3. Adherence of Cultured EC to Substrate-Coated Dacron. Human arterial EC were incubated at 10’ EC/cm2 upon plain Dacron (D), Dacron precoated with plasma (D + P), collagen I/III (D + C), or plasma-collagen I/III combination (D + P + C) and placed in tissue culture. (El) EC (low) or low passage EC. (a) EC (high) or high passage EC. Cell number was evaluated at 24 hr and at 14 days and the change in cell number was determined. Experiments were performed on triplicate surfaces and each surface was evaluated by six different cell field counts. In A, the initial adherence over a 24-hr period was determined by dividing the number of EC present at 24 hr by the number of EC initially seeded upon the surface. In B, the long-term adherence over a I3-day period was determined by dividing the number of EC present at 14 days by the number of EC present at 24 hr. Statistically significant better initial adherence to plasma-coated Dacron was observed for EC (low) compared to EC (high). EC (low) gave statistically better long-term adherence to all surfaces compared to EC (high). All EC (high) demonstrated a significant fall in cell number between Day 1 and Day 14. EC (low) seeded onto plasma-coated Dacron demonstrated a significant rise in cell number, reaching a final density of 9.6 X 10’ EC/cm*. Statistical significance was determined by the Student t test (I’ < 0.05). A significant rise in cell number is denoted by *, a fall in cell number by (I), and a significant difference between EC (low) and EC (high) by (7).
of cultured EC as a source of large numbers of cells . The use of this source has been examined in uiuo using canine EC and is able to produce a monolayer in the usual canine-seeding experiment . We have examined more closely the variables that might affect the success of using cultured EC. As a preliminary requirement of this study, we selected several cultured EC lines that demonstrated rapid growth in culture upon tissue-culture polystyrene. Cellular senescence of human EC has been demonstrated and should presumably be avoided in any EC-seeding experiments . Both the low and high passage EC demonstrated rapid proliferation on polystyrene and therefore could be classified as nonsenescent cells. This is an important distinction for the later studies. Initial adherence to collagen-coated Dacron demonstrated no significant differences as a function of EC age in culture. This suggests that culture does not affect or has a minor effect on cell mechanisms involved in shortterm adherence. These include but are not limited to such things as fibronectin receptor sites and other surface binding mechanisms involved in initial cell-surface contact . Initial EC incubation density also had no significant effect on initial adherence, suggesting that cell competition for surface binding sites is not an important variable at cell densities at or below lo6 cells/cm’. Long-term adherence to collagen-coated Dacron demonstrated a significant drop in cell number over a 14-day period except when EC (low) were incubated at lo5 cells/
cm2. These data suggest that specific conditions may be necessary before continued adherence is possible. The specific conditions may include early passage cells and a high density of cells. This could in part explain the sporadic graft coverage that occurs in EC-seeded grafts. If EC were not uniformly seeded on the graft, areas of high and low density could occur. The areas of high density might proceed to growth and endothelial monolayer formation, whereas the low density areas would diminish in cell count and fail to endothelialize. After determining that a high density incubation was necessary in vitro to maintain long-term adherence, we then sought to determine whether substrate-coating of the Dacron would affect continued adherence. Substrate could not correct the drop in adherence seen with high passage EC. Substrate had a significant effect on adherence with low passage EC. The addition of plasma to the surface allowed the EC to remain attached to the surface and increased the cell number up to the level of a confluent surface (10’ cells/cm2). This same trend was noted when plasma was combined with collagen I/III, suggesting that plasma may contain a factor(s) that supplements cell attachment to Dacron. Plasma is known to contain fibronectin, a glycoprotein that aids in cell adhesion . Plasma also contains albumin and many other components. Normally EC are maintained in culture in the presence of fetal calf serum, which should supply these components. It is possible, however, that some factor(s) could become deficient during culture and is not replaced
ET AL.: ENDOTHELIAL
rapidly enough for continued adherence once the cells have been placed on Dacron. Lastly, we were unable to detect differences in EC adherence as a function of EC source. It is possible that arterial EC have stronger adherence properties compared to venous EC because of their relatively high shear environment encountered in the arterial circulation. We were unable to detect a difference. These studies have been performed using the technique of incubating EC upon the surface rather than the more widely used technique of mixing the EC with clot and placing or seeding the EC-clot mixture upon the graft surface. One must use caution therefore in directly applying these data to seeding data. If, however, immediate monolayers offer earlier anti-thrombogenicity than the EC-clot seeded surface, then the techniques used in this study have direct relevance. They suggest that EC may only briefly be maintained in culture prior to use on vascular grafts. The concept of EC storage in tissue banks for later use is less attractive because of the increased number of passages necessary. Lastly, further refinements in cell culture technique or identification of different graft surfaces that do not require specialized cell and substrate conditions should be sought. CONCLUSIONS
Herring, M., Glover, A., and Glover, J. A single stage technique for seeding vascular grafts with autogenous endothelium. Surgery 84: 498,1978. Burkel, W. E., Ford, J. W., Vinter, D. W., et al. Fate of knitted dacron velour vascular grafts seeded with enzymatically derived autologous canine endothelium. Trans. Amer. Sot. Artif Intern. Organs 28: 178,1982. Graham, L., Burkel, W., Ford, W., et al. Immediate seeding of enzymatically derived autologous canine endothelium in dacron velour vascular grafts. Arch. Surg. 116: 1294, 1980. Belder, T. A., Schmidt, S. P., Falkow, L. J., and Sharp, W. L. Endothelial cell seeding of small-diameter vascular grafts. Trans. Amer. Sot. Arfif. Intern. Organs 28: 173, 1982. Graham, L. M., Burkel, W. E., Ford, J. W., Vinter, D. W., Kahn, R. H., and Stanley, J. C. Expanded polytetrafluorethylene vascular prostheses seeded with enzymatically derived and culture canine endothelial cells. Surgery 91: 550, 1982. Levine, E. M., Mueller, S. N., Grinspan, J. B., et al. Endothelial cell senescence and the aetiology of age-relateddisease. In A. Cryer (Ed.), Biochemical Z&era&ion at the Endothdium. Amsterdam: Elsevier, 1983. Jarrell, B. E., Shapiro, S., Williams, S., et al. Human adult endothelial cell growth in culture. J. Vast. Surg. 1: 757, 1984. Williams, S. K., Jarrell, B. E., Friend, L., Radomski, J. S., Carabasi, R. A., Koolpe, E., Mueller, S. N., Marinucci, T., and Levine, E. Adult human endothelial cell compatibility with prosthetic graft material J. Surg. Res. 38: 618, 629, 1985. Thornton, S. C., Mueller, S. N., and Levine, E. M. Human endothelial cells: Cloning and long-term serial cultivation employing heparin. Science 222: 623, 1983. Jarrell, B. E., Williams, S. K., Carabasi, R. A., et al. Use of an endothelial monolayer on a vascular graft prior to implantation. Temporal dynamics and compatibility with operating room. Ann. Surg. 203: 671,1986. Madri, J. A., and Williams, S. K. Capillary endothelial cell cultures: Phenotype modulation by matrix components. J. Cell Biol. 97: 153,1983. Jarrell, B. E., Williams, S. K., Stokes, G., et al. Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery. Surgery 100: 392, 1986. Radomski, J. S., Jarrell, B. E., Williams, S. K., et al. Initial adherence of human capillary endothelial cells to dacron. J. Surg. Res. 42: 133, 1987.
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ACKNOWLEDGMENTS This work was supported by a grant from the Ralph and Marian Triad Trust to B.E.J. and S.K.W. and NIH Grant HL33906.
An in vitro method of evaluating EC-graft interactions has been used to determine variables that affect EC adherence and growth when placed upon the surface of Dacron graft material. These studies have revealed that EC age in culture may affect the ability of EC to remain attached to a Dacron surface and subsequently proliferate. Initial 24-hr adherence studies may not reflect this EC age effect and therefore longer term studies are necessary to completely evaluate adherence. EC proliferation on graft material may require specific conditions in order to occur. These conditions include a low passage cultured EC, a high density EC seed, and plasma-containing components upon the graft prior to EC incubation.