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Endothelial cell transplantation
Cell Transplantation, Vol. 4, No. 4, pp. 401-410, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0963-6897/95 $9.50 + .OO
Pergamon 0963-6897(95)00023-2
Original Contribution ENDOTHELIAL
CELL TRANSPLANTATION STUART
Section
of Surgical
Research,
K.
Department of Surgery, University of Arizona 1501 N. Campbell Ave., Tucson, AZ 85724
Biomaterials;
INTRODUCTION
Historical Perspectives While the function of blood vessels in maintaining body function are described in the earliest investigations of modern physiology and medicine, the function of the endothelial cell which lines these blood vessels has only become evident during the last 25 yr. The need to replace lost organ function has resulted in clinically applicable methods for whole organ transplantation as well as cell transplantation. Yet the need for viable endothelium on vascular transplants (e.g., coronary artery bypass grafts, saphenous vein grafts) remained overlooked during the development of other types of cellular transplantation. The development and first clinical use of synthetic vascular grafts provided initial evidence of the importance of the lumenal endotheha1 cell lining on native blood vessels. These synthetic ACCEPTED
Health
Sciences
Center,
grafts functioned satisfactorily in large diameter positions such as abdominal aorta interposition grafts. On the other hand, smaller diameter synthetic grafts, especially those used to bypass arteries to distal positions below the knee, exhibit reduced long term patency. In general, synthetic vascular grafts with internal diameters less then 6 mm exhibit patencies that are usually deemed clinically unacceptable. The lack of satisfactory long term patency of small diameter grafts has been attributed to the inherent thrombogenicity of their lumenal surface. While all synthetic grafts exhibit thrombogenicity, large diameter grafts (i.e, > 6 mm in diameter) do not exhibit significant thrombosis related failure. Smaller diameter grafts have less tolerance for lumenal thrombosis especially when there is limited flow to the peripheral tissue being revascularized. Several polymer based strategies have been proposed and evaluated including the bonding of heparin, streptokinase or other antithrombogenic molecular species on the lumenal surface (8,20). These methods do exhibit improved short term patency, however these effects are measured in terms of days and not years necessary for improved polymer function. Small diameter vessel bypass surgery has therefore been limited to autologous native blood vessels and most predominantly saphenous vein. These vessels do permit revascularization of ischemic tissue with bypass to the level of the peroneal/tibial artery in the leg and coronary artery bypass. The satisfactory function of these autologous vessels has been attributed to the presence of viable endothelial cells on the lumenal surface. The importance of maintaining the integrity of endothelium during vein harvest has been extensively stressed (2,5,23,24). Clearly the lack of viable endothe-
0 Abstract - Endotbelial cells line the lumenal surface of all elements of the vascular system. These cells exhibit numerous metabolic functions necessary for the maintenance of homeostasis. The critical role of endothelium in maintaining normal blood vessel function is exemplified by the poor clinical performance of small diameter polymeric vascular grafts which fail due, in part, to the lack of a functional endothelium on the lumenal surface. Extensive research has explored the potentiality of transplanting endothelial cells onto polymeric vascular grafts to improve graft function. Several critical issues have been explored including the source of endothelial cells for transplantation, the interaction of endothelium with polymers and the healing process of endothelial cell transplanted grafts. The future of endothelial cell transplantation will also include the use of these cells as vehicles for genetic engineering. 0 Keywords - Endothelium; Collagenase; Thrombogenicity; Gene therapy.
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lial cells on the lumenal surface of polymeric grafts is a significant cause of continued synthetic graft thrombogenicity. Development of Seeding Technologies In 1978, Dr. Malcolm Herring first reported the successful isolation of endothelial cells from segments of vein and their subsequent transplantation onto synthetic vascular grafts using a method he termed endothelial cell seeding (11). The seeding method involved the mixing of freshly isolated cells with autologous blood and the use of this cell-blood mixture to preclot the graft. This procedure resulted in a coagulum of cells intermixed in clotted blood on the surface of the graft. The expectation was that endothelium would subsequently migrate to and proliferate on the lumenal surface resulting in the establishment of a contiguous endothelial cell lining. Subsequent work from the laboratories of Graham (9), Stanley (32), and Schmidt (26) confirmed the ability to isolate large vessel endothelium from segments of vein and established in implant studies in dogs that endothelial cell linings formed on seeded grafts. With these pioneering studies the evaluation of methods for endothelial cell transplantation onto vascular grafts focussed on several critical issues including source and methods for cell isolation, methods for cell deposition and the preparation of polymer surfaces to enhance cell attachment. Numerous other questions needed to be addressed before expanded clinical use of endothelial cell transplantation could be realized. Source of Endothelium for Transplantation The first source of cells for graft seeding were venous derived and used without further culturing. The yield of cells from a viable piece of vein was considered relatively low equaling approximately 1 X lo4 cells for the entire vein segment harvested (16). This number of cells would provide only a sparse coating on the lumenal surface of a polymeric vascular graft used for distal bypass in the leg and a significant cell proliferation would be necessary to establish monolayers. Studies by Kempzinski et al. (25), determined that even if a significant number of seeded cells could be obtained, a significant number of cells would be lost from the graft during the restoration of blood flow necessitating even more cell proliferation. For this reason several investigative groups began to evaluate alternate sources of cells especially sources which could increase the number of cells present in the initial inoculum. The use of culturing techniques to expand the number of endothelial cells available for transplantation has been possible for non human endothelial cells since the 1920s (17) however, human endothelial cell prolif-
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eration in culture has been comparatively slow. Several breakthroughs in the 1980s permitted the increased proliferative capacity of human endothelial cells in culture. Maciag (18) identified an endothelial cell growth factor later classified as heparin binding growth factor which stimulated, albeit in a very limited way, growth of human umbilical vein endothelial cells. In 1983, Dr. Susan Thornton working with Dr. Elliot Levine identified heparin as a necessary cofactor with heparin binding growth factor for the stimulation of human umbilical vein endothelial cell growth (34). Jarrell and coworkers quickly reported that heparin and heparin binding growth factor would stimulate human adult endothelial cell growth in culture (13). These endothelial cells included vein derived endothelium analogous to the cells to used for graft seeding. For the first time autologous endothelial cells could be procured and expanded in culture permitting high density seeding of vascular grafts. The use of large vessel derived endothelium for transplantation posed several questions which affected clinical acceptance. First, derivation of these cells required a separate surgical procedure to derive a segment of suitable vein for cell isolation. Since patients requiring the vascular procedure were suffering from systemic vascular disease, the inclusion of an additional surgical procedure involving the removal of a segment of native vein was not universally acceptable. In addition, the yield of endothelial cells from segments of vein was limited to a maximum of 1 x lo5 cells per cm2 surface area of vein harvested. This value represents the density of endothelial cells on a vein segment and procurement of 100% of the endothelium from the lumenal surface of veins is rarely achieved. Hence, the actual seeding density of endothelial cells using vein derived cells is low requiring the seeded cells to undergo significant proliferation to create a monolayer on seeded grafts. While culturing of cells before seeding can dramatically increase cell number, culturing raises several other concerns. Among these concerns are the fact that the endothelial cells are being treated with growth factors to stimulate their growth. Since endothelial cells have one of the lowest inherent proliferation rates in normal blood vessels, stimulating their growth by addition of growth factors forces them into a condition they rarely undergo under normal conditions. For these reasons investigators have actively pursued other sources of endothelial cells for transplantation. In 1984 Jarrell and Williams (13) reported methods for the isolation of autologous microvessel endothelial cells from adipose tissue for use in cell transplantation. The first anatomic site reported as a source of adipose tissue was fat deposits associated with omentum. This
Endothelial cell transplantation
fat is highly vascularized with an endothelial cell density capable of theoretically providing > 1 x lo6 endothelial cells/gm fat (14). The methods described by Jarrell and Williams (14) represented modifications of the methods originally described by Wagner in the earlier 1970s reported for the isolation of endothelial cells from rat epididymal fat (36). These rat derived microvascular endothelial cells have undergone extensive characterization and evaluation of cellular function (19,41). The isolation methods for endothelial cells from human omental fat were similar to the earlier methods reported by Wagner in that the fat is treated with crude Clostridial collagenase to digest extracellular matrix and release cells. The separation of endotheha1 cells from adipocytes is relatively easy based upon the density differences in these two cell types. During centrifugation at relatively low speeds (100 x g force) adipocytes rise to the top of an aqueous buffer and dense endothelial cells are pelleted. The pelleted endothelial cells are subsequently washed by centrifugation to remove collagenase. Methods for the isolation of endothelial cells from autologous human fat have undergone significant refinement since these earliest reports as well as extensive characterization of the cells derived. The major methodological improvement in these methods has been the use of liposuction derived SC fat as a source of tissue (38). A patient undergoing a vascular procedure therefore does not need to undergo a laparotomy to remove omental or perirenal fat, but rather removal of a small amount (50 cc) of fat can be achieved using a hand held syringe/cannulae device. Removal of fat is achieved in less then 5 min requiring a small (~1 cm)
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skin incision. Liposuction derived fat is also advantageous since the tissue is essentially minced into small pieces during the liposuction process accelerating the digestion process. Figure 1 illustrates the morphology of liposuction derived fat. At this lower magnification scanning electron micrograph the individual adipocytes with average diameters of approximately 100 microns are easily identified. Much higher magnification (Fig. 2) illustrates the presence of microvascular elements in association with the surface of adipocytes. Identity of Cells in Human Fat A significant controversy was established in the field of endothelial cell derivation from fat when Visser, et al. reported that omental tissue derived from humans contained predominantly mesothelial cells and not endothelium (35). This report cast significant doubt on the types of cells derived from adipose tissue and whether mesothelial cells and not endothelium were actually being used for transplantation. Since the original report by Visser, et al. additional reports have clarified this controversy (22,40). First, investigators have erroneously used the terms omentum and omental fat as interchangeable terms to describe tissue harvested. Omentum and omental fat are histologically and anatomically distinct tissues. Omentum represents a vascularized tissue with a thick, multilayered serosal surface composed predominantly of mesothelial cells (35). Visser, et al. were correct in stating that this tissue exhibits a predominance of mesothelial cells. Adipose tissue derived from sites in association with the omentum exhibits a very different histology and is composed of adipocytes and microvascular endothe-
SIZE BARFig. 1. Scanning electron micrograph of human liposuction-derived low magnification (x78; bar = 100 pm).).
SC fat illustrating
the predominance
of adipocytes
at this
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SIZE BARFig. 2. Higher magnification dothelial tube on the surface bar = 10 pm).
scanning electron micrograph of human liposuction-derived fat illustrating a microvascular enof an adipocyte. This microvessel is surrounded by a dense layer of extracellular matrix (x600;
lial cells. This adipose tissue is covered by a serosal layer rich in mesothelial cells, however, careful dissection of the thick adipose deposit will significantly reduce the contribution of mesothelial cells to the final isolate. The use of adipose tissue derived from SC fat using liposuction has provided an easily obtainable source of tissue devoid of mesothelial cells. Recently a complete characterization of cells found in SC fat was performed and reported (40). The conclusion of this work was that SC fat contains predominantly adipocytes and endothelial cells. In fact, prior to any tissue digestion human SC fat contains in excess of 85% endothelium based on the total cells present per unit volume of fat. Following tissue digestion and separation of adipocytes by centrifugation, the final cell isolate is highly enriched in pure endothelium.
cell isolation has resulted in a general acceptance of crude collagenase for cell isolation, including methods resulting in human cell transplantation. However, concerns with the numerous components present in crude collagenase have prompted investigators to evaluate methods to purify those components present in crude collagenase necessary for cell isolation. Purification of collagenase for use with endothelial cell isolation has been achieved (4,28). Investigators have reported that two major components of crude collagenase, specifically trypsin and pure collagenase, are necessary for successful endothelial cell isolation from both large blood vessels and microvascularized fat tissue. In conclusion, numerous sources of endothelial cells for subsequent cell transplantation have been described including large vessel and microvascularized fat. Cell culturing techniques have been described to increase the number of endothelial cells available.
Collagenase- Critical Enzyme in Endothelial Cell Isolation A method common to all processes described for endothelial cell isolation has been the use of collagenase to digest tissue prior to cell isolation. Use of the term collagenase suggests a degree of enzyme purity, yet this is not the case. The collagenase used in most endothelial cell transplantation studies reported to date exists as the crude lyophilized powder that results from the culture of Clostridium histolyticum (4,28). This powder contains numerous other components in addition to specific collagenases. Among the other components are a variety of proteases, proteins, metals, ions and toxins as well as residual components of C. histolyticum cell walls. The wide spread use of collagenase for
Preclinical Animal Trials of Endothelial Cell Transplantation With the availability of methods for the isolation of endothelial cells, their deposition onto grafts and an understanding of the conditions necessary to establish shear resistant endothelial association with graft surfaces, numerous investigators have performed animal trials to assess the efficacy of endothelial cell transplantation toward improved function of small diameter (<6 mm) prosthetic grafts (1,3,7,15,27,29-31,33,39). A majority of these studies have been performed in canine models due to the general acceptance of this model as being most similar to humans with respect
Endothelial
cell transplantation
to graft healing characteristics. The precise method of graft placement (carotid, aortofemoral or femoral), use of antiplatelet drugs, type and use of control grafts (paired carotid or femoral grafts as compared to unpaired aortic grafts) varies significantly among investigators. The rationale for different methods of graft preparation and placement are equally justified. For example, authors argue that while the use of paired grafts (e.g., carotid or femoral artery) permits comparison of cell transplanted grafts with control grafts on the contralateral side, the failure of one of the paired grafts could result in altered function of the graft on the contralateral side. This effect cannot occur in unpaired grafts. On the other hand a significant disadvantage of unpaired models is the known variability of thrombotic state of animals especially related to platelet reactivity, coagulation and fibrinolytic capacity. No universally acceptable model of small diameter graft evaluation has been established. Table 1 illustrates a compilation of the results of published trials using 4 mm inner diameter polymer grafts in animal models of endothelial cell seeding. The analysis presented in this table has been performed using nonpaired statistical analysis within each study. The significance of endothelial cell transplantation can be evaluated first by assessing fractional increase in patency between seeded and nonseeded grafts. In this column a positive fractional increase indicates a benefit of seeding, a zero increase indicates no benefit and a negative fractional increase indicates seeding has a negative influence on graft patency. The 95 % confidence limits have been provided for each study to indicate variability. The major conclusions from this table are the consistently observed positive benefit of endothelial cell seeding with respect to graft patency. While the benefit of seeding is not always statistically significant, seeding is the most common predictor of improved patency. While all of these reports claim a positive patency effect of endothelial cell transplantation, only two reports provide evidence of a statistically significant improvement in patency. Another common feature of most previously reported animal trials of endothelial cell transplantation is the duration of the implant phase for no more then 3 mo. Recent reports have established the durability of endothelial cell linings on cell transplanted grafts for periods extending to one year (39). The long term behavior of endothelialized grafts with respect to improved patency has not been definitively established. A definitive conclusion of nearly all endothelial cell transplantation studies in animals has been the observed ability to accelerate the formation of an endothelial cell lining on the lumenal surface of polymeric grafts (Fig. 3). This consistent finding has prompted
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SIZE BAR (4
SIZE BAR @I Fig. crk
3. Scanning electron micrographs (Meadox;
x60;
bar = 100 $j.
of (a) ePTFE
.
(Impra;
30 micron internodal
distance;
x240; bar = 10 pm) and (b) Da-
_
a number of investigators to evaluate endothelial cell transplantation in human trials. Human Studies of Endothelial Ceil Transplantation As previously described, Herring was the first to use enzymatically harvested endothelial cells from veins to treat vascular grafts in humans. These studies were continued from 1978 until 1982 and used Dacron grafts in the lower extremity (10). The most definitive finding in these studies was the deleterious effect of smoking on patency. The results did suggest an improved patency in seeded grafts as compared to nonseeded controls. Herring subsequently began a randomized
trial of endothelial cell transplantation using 6 mm i.d. ePTFE grafts in bypasses between the proximal and distal popliteal artery segments. Most impressive was the statistically significant improvement in patency of seeded vs. control grafts after both one and two year follow-up. Again patency was better in nonsmoking patients. Since these pioneering studies several other groups have initiated, albeit small in number, clinical trials of endothelial cell seeding (12,37,42). Significant variation exists in the performance of these trials making comparison of results extremely difficult. Variables such as the size (4 mm to 10 mm internal diameter), graft type (ePTFE vs. Dacron), placement (above knee vs. below knee arterial vs. aorto bifemoral bypasses),
Endothelial cell transplantation
use of cell culture (immediate transplantation vs. culture), type of endothelial cells (venous derived vs. SC fat vs. omental fat vs. omenturn), method of deposition (seeding in plasma or blood vs. gravity deposition vs. pressure deposition-sodding) and use of anticoagulants are controlled within each study but permit very little comparison of data and the inability to conclude definitively on the benefit of endothelial cell transplantation toward improved prosthetic graft patency. Figure 4 illustrates the structure of ePTFE (Fig. 4a) and Dacron (Fig. 4b) grafts and demonstrates the immense difference in their structure. These differences make comparisons between the healing characteristics of these materials difficult. Standardized Method of Endothelial Cell Transplantation for Prosthetic Grafts As just stated there is no standard method universally accepted between investigators for endothelial cell isolation and transplantation for use with polymeric bypass grafts. Our cell transplantation team has spent the past decade optimizing conditions for the use of fat derived microvascular endothelial cells for endothelial cell transplantation onto vascular grafts. The key elements of this process include the following critical steps. First, we have selected liposuction using a handheld liposuction syringe-cannulae system as the most reproducible method to procure SC fat from the abdominal wall of patients (38). Fat digestion is subsequently performed using a partially purified Clostridium histolyticum derived collagenase which has been selected for optimal cell isolation and cell function characteristics (28). The most critical function of the
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cells assessed has been dissociation of fat into a single cell suspension and the ability of cells to rapidly (~20 min) adhere and spread on polymeric surface. During the process of endothelial cell isolation we have found it critical to treat grafts (ePTFE) with autologous serum in an aqueous medium (media 199E). This graft treatment is critical to wet the polymeric surface and thus remove air from the surface, and to permit serum borne proteins, especially fibronectin and vitronectin, to adsorb to the surface of the graft. Endotheha1 cells are resuspended in media 199E containing autologous serum in a volume sufficient to fill the inner volume of the graft. The graft is then connected to a syringe containing excess serum containing medium and the graft pressurized to force fluid through and cells into the interstices of the graft. The pressure gradient is maintained until 4 graft volumes pass through the interstices or until 5 min has passed. Our studies have established a homogeneous deposition of cells results on the lumenal surface of grafts treated this way. Grafts are subsequently implanted while maintaining the inner surface of the graft moist with serum containing medium. Our clinical experience using this method of microvessel endothelial cell isolation and deposition using the process we have termed sodding is ongoing. Morphological evaluations of grafts retrieved from patients has been extremely limited but of great interest since they establish our ability to accelerate the formation of endothelial cell linings on human grafts (21) (Fig. 5). The evaluation of the efficacy of endothelial cell transplantation toward improved graft patency are ongoing in controlled clinical trials.
SIZE BAR Fig. 4. Endothelial cell monolayer established on an ePTFE graft using autologous microvascular endothelial canine carotid artery model. This graft was explanted following 5 weeks of implantation (x480; bar = 20 pm).
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SIZE BAR Fig. 5. Scanning electron micrograph of an endothelial cell monolayer on a human polymeric with autologous liposuction-fat derived microvascular endothelial cells (x 132; bar = 20 pm).
Other Applications of Endothelial Cell Transplantation The origins of the needs and methods for endothelial cell transplantation are attributable to the search for improved small diameter prosthetic vascular grafts. This search has resulted in the development of numerous methods for endothelial cell isolation and culture as well as an understanding of endothelial cell function following transplantation. The use of endothelial cell transplantation to improve the function of small diameter prosthetic grafts has reached the phase of clinical evaluation yet the uses of endothelial cell transplantation are just beginning to be realized. With the advent of methods to transfer genetic material into cells and observe functional protein products from this genetic material, investigators began to explore cells available to be used as a cellular vehicle for gene therapy. While numerous mammalian cells are susceptible to genetic manipulation, the endothelial cell has several characteristics making its choice as a vehicle attractive. Endothelium provides the natural lining of all blood vessels in the body and is thus in constant contact with blood. For this reason a gene product would be directly accessible to the blood stream if endothelial cells were used as a vehicle. Moreover, and most likely to the surprise of many molecular biologists, the methods for endothelial cell isolation and subsequent transplantation back into the body have already been undergoing refinement for years. Methods of gene therapy using endothelium suggested by molecular biologists could thus rely on already established endothelial cell transplantation methods. Molecular
graft.
This graft was sodded
biologists have taken advantage of this extensive foundation in endothelial cell transplantation methodology previously established by others. Numerous reports of successful genetic manipulation of endothelial cells have now been reported with great promise toward replacement of defective genes in patients. The use of endothelial cell transplantation has also begun to expand beyond use in arterial bypass grafts. Again based upon established methods for cell isolation and deposition, investigators have started to explore the use of endothelial cells to improve the function of vein grafts, prosthetic arterio-venous grafts, stents, shunts, patches, ventricular assist devices and the total artificial heart. Interestingly, molecular biologists have suggested that endothelium must be genetically manipulated to improve their anticoagulant activity before their use as cellular coatings on these vascular implants (6). This tenant is not universally accepted since many investigators with long standing interest in endothelial cell transplantation believe transplanted endothelium are not genetically deficient in anticoagulant activity. Moreover, the balance between procoagulant and anticoagulant activity maintained by endothelial cells is delicate and should not be manipulated by genetic modification. The field of endothelial ceil transplantation derived from initial studies of endothelial cell-polymer interaction toward the establishment of antithrombogenic linings on small diameter vascular grafts. With the development of operating room compatible methods to isolate autologous endothelium a wide variety of polymer and native surface implants are being considered.
Endothelial cell transplantation The development
of genetic
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of novel
gene
manipulation
cells as a cellular
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REFERENCES 1. Allen, B.T.; Long, J.A.; Clark, R.E.; Sicard, G.A.; Hopkins, K.Y.; Welch, M.J. Influence of endothelial cell seeding on platelet deposition and patency in smalldiameter Dacron arterial grafts. J. Vast. Surg. 1:224232; 1984. 2. Angelini, G.D.; Breckenridge, I.A.; Psaila, J.V.; Williams, H.M.; Henderson, A.H.; Newby, A.C. Preparation of human saphenous vein for coronary artery bypass grafting impairs its capacity to produce prostacyclin. Cardiovasc. Res. 21:28-33; 1987. 3. Belsen, T.A.; Schmidt, S.P.; Falkow, L.J.; Sharp, W.V. Endothelial cell seeding of small-diameter vascular grafts. Trans. Am. Sot. Artif. Intern. Organs 28:173; 1982. 4. Bond, M.D.; van Wart, H.E. Characterization of the individual collagenases from Clostridium histolyticum. Biochem. 23:3085-3091; 1984. 5. Cambria, R.P.; Megerman, J.; Abbot, W.M. Endothelial preservation in reversed and in situ autogenous vein grafts. Ann. Surg. 202(1):50-55; 1985. 6. Choi, E.T.; Callow, A.D.; Ryan, U.S. Interventional Techniques to accelerate healing: Gene therapy. In: White, R.A.; Hollier, L.H., eds. Vascular surgery: Basic science and clinical correlations. Philadelphia: JB Lippincott Co.; 1994:81-86. 7. Douville, E.C.; Kempczinski, R.F.; Birinyi, L.K.; Ramalanjaona, G.R. Impact of endothelial cell seeding on long term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft. J. Vast. Surg. 5:544; 1987. 8. Forster, R.I.; Bernath, EAnalysis of urokinase immobilization on the polytetrafluoroethylene vascular prosthesis. Am. J. Surg. 156:130-132; 1988. 9. Graham, L.M.; Vinter, D.W.; Ford, J.W. Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts. Early studies with autologous canine cells. Arch. Surg. 115:1289-1294; 1980. 10. Herring, M.; Compton, R.S.; Gardner, A.L.; LeGrand, D.R. Clinical experiences with endothelial seeding in Indianapolis. In: Zilla, P.; Fasol, R.; Deutsch, M., eds. Endothelialization of Vascular Grafts. Base]: Karger; 1987:218-224. 11. Herring, M.; Gardner, A.; Glover, J. A single staged technique for seeding vascular grafts with autogenous endothelium. Surg. 84:498-504; 1978. 12. Jarrell, B.E.; Williams, S.K. Interventional techniques to accelerate healing: Endothelial seeding. In: White, R.A.; Hollier, L.H., eds. Vascular surgery: Basic science and clinical correlations. Philadelphia: JB Lippincott Co.; 1994:71-80. 13. Jarrell, B.E.; Levine, E.M.; Shapiro, S.S.; Williams, S.K.; Carabasi, R.A.; Mueller, S.N.; Thornton, S.C.
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Human adult endothelial cell growth in culture. J. Vast. Surg. 1:757-764; 1984. Jarrell, B.E.; Williams, S.K.; Stokes, G.; Hubbard, EA.; Carabasi, R.A.; Koolpe, E.; Greener, D.; Pratt, K.; Moritz, M.J.; Radomski, J.; Speicher, L. Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery. Surg. 100(2):392-399; 1986. Kempczinski, R.F.; Rosenman, J.E.; Pearce, W.H.; Rodersheimer, L.R.; Berlatzky, Y.; Ramalanjaona, G.R. Endothelial cell seeding of new PTFE vascular prostheses. J. Vast. Surg. 2:424-429; 1985. Kesler, K.A.; Herring, M.B.; Arnold, M.P.; Glover, J.L.; Park, H.M.; Helmus, M.N.; Bendick, P.J. Enhanced strength of endothelial attachment on polyester elastomer and polytetrafluoroethylene graft surfaces with fibronectin substrate. J. Vast. Surg. 3:58-64; 1986. Lewis, W.H. Endothelium in tissue culture. Am. J. Anat. 30:39-59; 1922. Maciag, T.G.; Hoover, G.A.; Stemerman, M.B.; Weinstein, R. Serial propagation of human endothelial cells in vitro. J. Cell Biol. 91:420-426; 1981. Madri, J.A.; Williams, S.K. Capillary endothelial cell cultures: Phenotypic modulation by matrix components. J. Cell Biol. 97:153-165; 1983. Nojiri, C.; Park, K.D.; Grainger, D.W.; Jacobs, H.A.; Okano, T.; Koyanagi, H.; Kim, S. In vivo nonthrombogenicity of heparin immobilized polymer surfaces. Trans. Am. Sot. Artif. Intern. Organs. 36:Ml68-M172; 1990. Park, P.K.; Jarrell, B.E.; Williams, S.K. Thrombus-free, human endothelial cell surface in the midregion of a Dacron vascular graft in the splanchnic venous circuitobservations after nine months of implantation. J. Vast. Surg. 2:468-475; 1990. Potzsch, B.; Grulich-Henn, J.; Rossing, R.; Wille, D.; Muller-Berghaus, G. Identification of endothelial and mesothelial cells in human omental tissue and omentum derived cultured cells by specific cell markers. J. Lab. Invest. 63:841-852; 1990. Quist, W.C.; Haudenshield, C.C.; Logerfo, F.W. Qualitative microscopy of implanted vein grafts: Effects of graft integrity on morphological fate. J. Thorac. Cardiovast. Surg. 103:671-677; 1992. Richardson, J.V.; Wright, C.B.; Hiratzka, L.F. The role of endothelium in the patency of small venous substitutes. J. Surg. Res. 28:556-562; 1980. Rosenman, J.E.; Kempczinski, R.F.; Pearce, W.H.; Silberstein, E.B. Kinetics of endothelial cell seeding. J. Vast. Surg. 2:778-784; 1985. Schmidt, S.P.; Hunter, T. J.; Sharp, W.V.; Malindzak, G.S.; Evancho, M.M. Endothelial cell seeded four millimeter Dacron vascular grafts. J. Vast. Surg. 1:434-441; 1984. Schmidt, S.P.; Hunter, T.J.; Hirko, M.Small diameter vascular prostheses: Two designs of PTFE and endothelial cell seeded and nonseeded Dacron. J. Vast. Surg. 2: 292-297; 1985.
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28. Sharefkin, J.B.; van Wart, H.E.; Williams, S.K. Enzymatic harvesting of adult human endothelial cells for use in autogenous endothelial vascular prosthetic seeding. In: Herring, M.B., ed. Endothelial seeding in vascular surgery. Grune and Stratton; 1987:79-101. 29. Shepard, A.D.; Eldrup-Jorgensen, J.; Keough, E.M. Endothelial cell seeding of small caliber synthetic grafts in the baboon. Surg. 99:318; 1986. 30. Shindo, S.; Takagi, A.; Whittemore, A.D. Improved patency of collagen impregnated grafts after in vitro autogenous endothelial cell seeding. J. Vast. Surg. 6:325; 1987. 31. Stanley, J.C.; Burke], W.E.; Ford, J.W. Enanced patency of small diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells. Surg. 92: 994; 1982. 32. Stanley, J.C.; Burkel, W.E.; Linbald, B. Endothelial cell seeding of synthetic vascular prostheses. Acta Chir. Stand. Suppl. 529:17-27; 1985. 33. Tannenbaum, G.; Ahlborn, T.; Benvenisty, A.; Reemstma, K.; Nowygrod, R. High density seeding of cultured endothelial cells leads to rapid coverage of PTFE grafts. Curr. Surg. 222:623; 1983. 34. Thornton, S.C.; Mueller, S.N.; Levine, E.M. Human endothelial cells: Cloning and long term serial cultivation employing heparin. Science 222:623-624; 1983. 35. Visser, M.J.P.; van Bockel, J.J.; van Muijen, G.N.P.; van Hinsbergh, V.W.M. Cells derived from omental fat tissue and used for seeding vascular prostheses are not endothelial in origin: A study on the origin of epitheloid cells derived from human omentum. J. Vast. Surg. 13: 373-381; 1991.
36. Wagner, R.C.; Matthews, M.A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10:286-297; 1975. 37. Walker, M.G.; Thomson, G.J.L.; Shaw, J.W. Endothelial cell seeded vs. nonseeded ePTFE grafts in patients with severe peripheral vascular disease. In: Zilla, P.; Fasol, R.; Deutsch, M., eds. Endothelialization of vascular grafts. Basel: Karger; 1987:245-248. 38. Williams, S.K.; Jarrell, B.E.; Rose, D.G.; Pontell, J.; Kapelan, B.A.; Park, P.K.; Carter, T.L. Human microvessel endothelial cell isolation and vascular graft sodding in the operating room. Ann. Vast. Surg. 3(2):146152; 1989. 39. Williams, S.K.; Rose, D.G.; Jarrell, B.E. Microvascular endothelial cell sodding of ePTFE vascular grafts: Improved patency and stability of the cellular lining. J. Biomed. Mater. Res. 28:203-212; 1994. 40. Williams, S.K.; Wang, T.F.; Castrillo, R.; Jarrell, B.E. Liposuction derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type. J. Vast. Surg. 19:916-923; 1994. 41. Williams, S.K. Isolation and culture of microvessel and large-vessel endothelial cells: Their use in transport and clinical studies. In: McDonagh, P.F., ed. Microvascular perfusion and transport in health and disease. Basel: Karger; 1987: 204-245. 42. Zilla, P.; Fasol, R.; Deutsch, M. Endothelial cell seeding of PTFE vascular grafts in humans: A preliminary report. J. Vast. Surg. 6:535; 1987.
Pergamon 0963-6897(95)00023-2
Original Contribution ENDOTHELIAL
CELL TRANSPLANTATION STUART
Section
of Surgical
Research,
K.
Department of Surgery, University of Arizona 1501 N. Campbell Ave., Tucson, AZ 85724
Biomaterials;
INTRODUCTION
Historical Perspectives While the function of blood vessels in maintaining body function are described in the earliest investigations of modern physiology and medicine, the function of the endothelial cell which lines these blood vessels has only become evident during the last 25 yr. The need to replace lost organ function has resulted in clinically applicable methods for whole organ transplantation as well as cell transplantation. Yet the need for viable endothelium on vascular transplants (e.g., coronary artery bypass grafts, saphenous vein grafts) remained overlooked during the development of other types of cellular transplantation. The development and first clinical use of synthetic vascular grafts provided initial evidence of the importance of the lumenal endotheha1 cell lining on native blood vessels. These synthetic ACCEPTED
Health
Sciences
Center,
grafts functioned satisfactorily in large diameter positions such as abdominal aorta interposition grafts. On the other hand, smaller diameter synthetic grafts, especially those used to bypass arteries to distal positions below the knee, exhibit reduced long term patency. In general, synthetic vascular grafts with internal diameters less then 6 mm exhibit patencies that are usually deemed clinically unacceptable. The lack of satisfactory long term patency of small diameter grafts has been attributed to the inherent thrombogenicity of their lumenal surface. While all synthetic grafts exhibit thrombogenicity, large diameter grafts (i.e, > 6 mm in diameter) do not exhibit significant thrombosis related failure. Smaller diameter grafts have less tolerance for lumenal thrombosis especially when there is limited flow to the peripheral tissue being revascularized. Several polymer based strategies have been proposed and evaluated including the bonding of heparin, streptokinase or other antithrombogenic molecular species on the lumenal surface (8,20). These methods do exhibit improved short term patency, however these effects are measured in terms of days and not years necessary for improved polymer function. Small diameter vessel bypass surgery has therefore been limited to autologous native blood vessels and most predominantly saphenous vein. These vessels do permit revascularization of ischemic tissue with bypass to the level of the peroneal/tibial artery in the leg and coronary artery bypass. The satisfactory function of these autologous vessels has been attributed to the presence of viable endothelial cells on the lumenal surface. The importance of maintaining the integrity of endothelium during vein harvest has been extensively stressed (2,5,23,24). Clearly the lack of viable endothe-
0 Abstract - Endotbelial cells line the lumenal surface of all elements of the vascular system. These cells exhibit numerous metabolic functions necessary for the maintenance of homeostasis. The critical role of endothelium in maintaining normal blood vessel function is exemplified by the poor clinical performance of small diameter polymeric vascular grafts which fail due, in part, to the lack of a functional endothelium on the lumenal surface. Extensive research has explored the potentiality of transplanting endothelial cells onto polymeric vascular grafts to improve graft function. Several critical issues have been explored including the source of endothelial cells for transplantation, the interaction of endothelium with polymers and the healing process of endothelial cell transplanted grafts. The future of endothelial cell transplantation will also include the use of these cells as vehicles for genetic engineering. 0 Keywords - Endothelium; Collagenase; Thrombogenicity; Gene therapy.
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lial cells on the lumenal surface of polymeric grafts is a significant cause of continued synthetic graft thrombogenicity. Development of Seeding Technologies In 1978, Dr. Malcolm Herring first reported the successful isolation of endothelial cells from segments of vein and their subsequent transplantation onto synthetic vascular grafts using a method he termed endothelial cell seeding (11). The seeding method involved the mixing of freshly isolated cells with autologous blood and the use of this cell-blood mixture to preclot the graft. This procedure resulted in a coagulum of cells intermixed in clotted blood on the surface of the graft. The expectation was that endothelium would subsequently migrate to and proliferate on the lumenal surface resulting in the establishment of a contiguous endothelial cell lining. Subsequent work from the laboratories of Graham (9), Stanley (32), and Schmidt (26) confirmed the ability to isolate large vessel endothelium from segments of vein and established in implant studies in dogs that endothelial cell linings formed on seeded grafts. With these pioneering studies the evaluation of methods for endothelial cell transplantation onto vascular grafts focussed on several critical issues including source and methods for cell isolation, methods for cell deposition and the preparation of polymer surfaces to enhance cell attachment. Numerous other questions needed to be addressed before expanded clinical use of endothelial cell transplantation could be realized. Source of Endothelium for Transplantation The first source of cells for graft seeding were venous derived and used without further culturing. The yield of cells from a viable piece of vein was considered relatively low equaling approximately 1 X lo4 cells for the entire vein segment harvested (16). This number of cells would provide only a sparse coating on the lumenal surface of a polymeric vascular graft used for distal bypass in the leg and a significant cell proliferation would be necessary to establish monolayers. Studies by Kempzinski et al. (25), determined that even if a significant number of seeded cells could be obtained, a significant number of cells would be lost from the graft during the restoration of blood flow necessitating even more cell proliferation. For this reason several investigative groups began to evaluate alternate sources of cells especially sources which could increase the number of cells present in the initial inoculum. The use of culturing techniques to expand the number of endothelial cells available for transplantation has been possible for non human endothelial cells since the 1920s (17) however, human endothelial cell prolif-
0 Volume 4, Number 4, 1995
eration in culture has been comparatively slow. Several breakthroughs in the 1980s permitted the increased proliferative capacity of human endothelial cells in culture. Maciag (18) identified an endothelial cell growth factor later classified as heparin binding growth factor which stimulated, albeit in a very limited way, growth of human umbilical vein endothelial cells. In 1983, Dr. Susan Thornton working with Dr. Elliot Levine identified heparin as a necessary cofactor with heparin binding growth factor for the stimulation of human umbilical vein endothelial cell growth (34). Jarrell and coworkers quickly reported that heparin and heparin binding growth factor would stimulate human adult endothelial cell growth in culture (13). These endothelial cells included vein derived endothelium analogous to the cells to used for graft seeding. For the first time autologous endothelial cells could be procured and expanded in culture permitting high density seeding of vascular grafts. The use of large vessel derived endothelium for transplantation posed several questions which affected clinical acceptance. First, derivation of these cells required a separate surgical procedure to derive a segment of suitable vein for cell isolation. Since patients requiring the vascular procedure were suffering from systemic vascular disease, the inclusion of an additional surgical procedure involving the removal of a segment of native vein was not universally acceptable. In addition, the yield of endothelial cells from segments of vein was limited to a maximum of 1 x lo5 cells per cm2 surface area of vein harvested. This value represents the density of endothelial cells on a vein segment and procurement of 100% of the endothelium from the lumenal surface of veins is rarely achieved. Hence, the actual seeding density of endothelial cells using vein derived cells is low requiring the seeded cells to undergo significant proliferation to create a monolayer on seeded grafts. While culturing of cells before seeding can dramatically increase cell number, culturing raises several other concerns. Among these concerns are the fact that the endothelial cells are being treated with growth factors to stimulate their growth. Since endothelial cells have one of the lowest inherent proliferation rates in normal blood vessels, stimulating their growth by addition of growth factors forces them into a condition they rarely undergo under normal conditions. For these reasons investigators have actively pursued other sources of endothelial cells for transplantation. In 1984 Jarrell and Williams (13) reported methods for the isolation of autologous microvessel endothelial cells from adipose tissue for use in cell transplantation. The first anatomic site reported as a source of adipose tissue was fat deposits associated with omentum. This
Endothelial cell transplantation
fat is highly vascularized with an endothelial cell density capable of theoretically providing > 1 x lo6 endothelial cells/gm fat (14). The methods described by Jarrell and Williams (14) represented modifications of the methods originally described by Wagner in the earlier 1970s reported for the isolation of endothelial cells from rat epididymal fat (36). These rat derived microvascular endothelial cells have undergone extensive characterization and evaluation of cellular function (19,41). The isolation methods for endothelial cells from human omental fat were similar to the earlier methods reported by Wagner in that the fat is treated with crude Clostridial collagenase to digest extracellular matrix and release cells. The separation of endotheha1 cells from adipocytes is relatively easy based upon the density differences in these two cell types. During centrifugation at relatively low speeds (100 x g force) adipocytes rise to the top of an aqueous buffer and dense endothelial cells are pelleted. The pelleted endothelial cells are subsequently washed by centrifugation to remove collagenase. Methods for the isolation of endothelial cells from autologous human fat have undergone significant refinement since these earliest reports as well as extensive characterization of the cells derived. The major methodological improvement in these methods has been the use of liposuction derived SC fat as a source of tissue (38). A patient undergoing a vascular procedure therefore does not need to undergo a laparotomy to remove omental or perirenal fat, but rather removal of a small amount (50 cc) of fat can be achieved using a hand held syringe/cannulae device. Removal of fat is achieved in less then 5 min requiring a small (~1 cm)
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skin incision. Liposuction derived fat is also advantageous since the tissue is essentially minced into small pieces during the liposuction process accelerating the digestion process. Figure 1 illustrates the morphology of liposuction derived fat. At this lower magnification scanning electron micrograph the individual adipocytes with average diameters of approximately 100 microns are easily identified. Much higher magnification (Fig. 2) illustrates the presence of microvascular elements in association with the surface of adipocytes. Identity of Cells in Human Fat A significant controversy was established in the field of endothelial cell derivation from fat when Visser, et al. reported that omental tissue derived from humans contained predominantly mesothelial cells and not endothelium (35). This report cast significant doubt on the types of cells derived from adipose tissue and whether mesothelial cells and not endothelium were actually being used for transplantation. Since the original report by Visser, et al. additional reports have clarified this controversy (22,40). First, investigators have erroneously used the terms omentum and omental fat as interchangeable terms to describe tissue harvested. Omentum and omental fat are histologically and anatomically distinct tissues. Omentum represents a vascularized tissue with a thick, multilayered serosal surface composed predominantly of mesothelial cells (35). Visser, et al. were correct in stating that this tissue exhibits a predominance of mesothelial cells. Adipose tissue derived from sites in association with the omentum exhibits a very different histology and is composed of adipocytes and microvascular endothe-
SIZE BARFig. 1. Scanning electron micrograph of human liposuction-derived low magnification (x78; bar = 100 pm).).
SC fat illustrating
the predominance
of adipocytes
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SIZE BARFig. 2. Higher magnification dothelial tube on the surface bar = 10 pm).
scanning electron micrograph of human liposuction-derived fat illustrating a microvascular enof an adipocyte. This microvessel is surrounded by a dense layer of extracellular matrix (x600;
lial cells. This adipose tissue is covered by a serosal layer rich in mesothelial cells, however, careful dissection of the thick adipose deposit will significantly reduce the contribution of mesothelial cells to the final isolate. The use of adipose tissue derived from SC fat using liposuction has provided an easily obtainable source of tissue devoid of mesothelial cells. Recently a complete characterization of cells found in SC fat was performed and reported (40). The conclusion of this work was that SC fat contains predominantly adipocytes and endothelial cells. In fact, prior to any tissue digestion human SC fat contains in excess of 85% endothelium based on the total cells present per unit volume of fat. Following tissue digestion and separation of adipocytes by centrifugation, the final cell isolate is highly enriched in pure endothelium.
cell isolation has resulted in a general acceptance of crude collagenase for cell isolation, including methods resulting in human cell transplantation. However, concerns with the numerous components present in crude collagenase have prompted investigators to evaluate methods to purify those components present in crude collagenase necessary for cell isolation. Purification of collagenase for use with endothelial cell isolation has been achieved (4,28). Investigators have reported that two major components of crude collagenase, specifically trypsin and pure collagenase, are necessary for successful endothelial cell isolation from both large blood vessels and microvascularized fat tissue. In conclusion, numerous sources of endothelial cells for subsequent cell transplantation have been described including large vessel and microvascularized fat. Cell culturing techniques have been described to increase the number of endothelial cells available.
Collagenase- Critical Enzyme in Endothelial Cell Isolation A method common to all processes described for endothelial cell isolation has been the use of collagenase to digest tissue prior to cell isolation. Use of the term collagenase suggests a degree of enzyme purity, yet this is not the case. The collagenase used in most endothelial cell transplantation studies reported to date exists as the crude lyophilized powder that results from the culture of Clostridium histolyticum (4,28). This powder contains numerous other components in addition to specific collagenases. Among the other components are a variety of proteases, proteins, metals, ions and toxins as well as residual components of C. histolyticum cell walls. The wide spread use of collagenase for
Preclinical Animal Trials of Endothelial Cell Transplantation With the availability of methods for the isolation of endothelial cells, their deposition onto grafts and an understanding of the conditions necessary to establish shear resistant endothelial association with graft surfaces, numerous investigators have performed animal trials to assess the efficacy of endothelial cell transplantation toward improved function of small diameter (<6 mm) prosthetic grafts (1,3,7,15,27,29-31,33,39). A majority of these studies have been performed in canine models due to the general acceptance of this model as being most similar to humans with respect
Endothelial
cell transplantation
to graft healing characteristics. The precise method of graft placement (carotid, aortofemoral or femoral), use of antiplatelet drugs, type and use of control grafts (paired carotid or femoral grafts as compared to unpaired aortic grafts) varies significantly among investigators. The rationale for different methods of graft preparation and placement are equally justified. For example, authors argue that while the use of paired grafts (e.g., carotid or femoral artery) permits comparison of cell transplanted grafts with control grafts on the contralateral side, the failure of one of the paired grafts could result in altered function of the graft on the contralateral side. This effect cannot occur in unpaired grafts. On the other hand a significant disadvantage of unpaired models is the known variability of thrombotic state of animals especially related to platelet reactivity, coagulation and fibrinolytic capacity. No universally acceptable model of small diameter graft evaluation has been established. Table 1 illustrates a compilation of the results of published trials using 4 mm inner diameter polymer grafts in animal models of endothelial cell seeding. The analysis presented in this table has been performed using nonpaired statistical analysis within each study. The significance of endothelial cell transplantation can be evaluated first by assessing fractional increase in patency between seeded and nonseeded grafts. In this column a positive fractional increase indicates a benefit of seeding, a zero increase indicates no benefit and a negative fractional increase indicates seeding has a negative influence on graft patency. The 95 % confidence limits have been provided for each study to indicate variability. The major conclusions from this table are the consistently observed positive benefit of endothelial cell seeding with respect to graft patency. While the benefit of seeding is not always statistically significant, seeding is the most common predictor of improved patency. While all of these reports claim a positive patency effect of endothelial cell transplantation, only two reports provide evidence of a statistically significant improvement in patency. Another common feature of most previously reported animal trials of endothelial cell transplantation is the duration of the implant phase for no more then 3 mo. Recent reports have established the durability of endothelial cell linings on cell transplanted grafts for periods extending to one year (39). The long term behavior of endothelialized grafts with respect to improved patency has not been definitively established. A definitive conclusion of nearly all endothelial cell transplantation studies in animals has been the observed ability to accelerate the formation of an endothelial cell lining on the lumenal surface of polymeric grafts (Fig. 3). This consistent finding has prompted
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SIZE BAR (4
SIZE BAR @I Fig. crk
3. Scanning electron micrographs (Meadox;
x60;
bar = 100 $j.
of (a) ePTFE
.
(Impra;
30 micron internodal
distance;
x240; bar = 10 pm) and (b) Da-
_
a number of investigators to evaluate endothelial cell transplantation in human trials. Human Studies of Endothelial Ceil Transplantation As previously described, Herring was the first to use enzymatically harvested endothelial cells from veins to treat vascular grafts in humans. These studies were continued from 1978 until 1982 and used Dacron grafts in the lower extremity (10). The most definitive finding in these studies was the deleterious effect of smoking on patency. The results did suggest an improved patency in seeded grafts as compared to nonseeded controls. Herring subsequently began a randomized
trial of endothelial cell transplantation using 6 mm i.d. ePTFE grafts in bypasses between the proximal and distal popliteal artery segments. Most impressive was the statistically significant improvement in patency of seeded vs. control grafts after both one and two year follow-up. Again patency was better in nonsmoking patients. Since these pioneering studies several other groups have initiated, albeit small in number, clinical trials of endothelial cell seeding (12,37,42). Significant variation exists in the performance of these trials making comparison of results extremely difficult. Variables such as the size (4 mm to 10 mm internal diameter), graft type (ePTFE vs. Dacron), placement (above knee vs. below knee arterial vs. aorto bifemoral bypasses),
Endothelial cell transplantation
use of cell culture (immediate transplantation vs. culture), type of endothelial cells (venous derived vs. SC fat vs. omental fat vs. omenturn), method of deposition (seeding in plasma or blood vs. gravity deposition vs. pressure deposition-sodding) and use of anticoagulants are controlled within each study but permit very little comparison of data and the inability to conclude definitively on the benefit of endothelial cell transplantation toward improved prosthetic graft patency. Figure 4 illustrates the structure of ePTFE (Fig. 4a) and Dacron (Fig. 4b) grafts and demonstrates the immense difference in their structure. These differences make comparisons between the healing characteristics of these materials difficult. Standardized Method of Endothelial Cell Transplantation for Prosthetic Grafts As just stated there is no standard method universally accepted between investigators for endothelial cell isolation and transplantation for use with polymeric bypass grafts. Our cell transplantation team has spent the past decade optimizing conditions for the use of fat derived microvascular endothelial cells for endothelial cell transplantation onto vascular grafts. The key elements of this process include the following critical steps. First, we have selected liposuction using a handheld liposuction syringe-cannulae system as the most reproducible method to procure SC fat from the abdominal wall of patients (38). Fat digestion is subsequently performed using a partially purified Clostridium histolyticum derived collagenase which has been selected for optimal cell isolation and cell function characteristics (28). The most critical function of the
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cells assessed has been dissociation of fat into a single cell suspension and the ability of cells to rapidly (~20 min) adhere and spread on polymeric surface. During the process of endothelial cell isolation we have found it critical to treat grafts (ePTFE) with autologous serum in an aqueous medium (media 199E). This graft treatment is critical to wet the polymeric surface and thus remove air from the surface, and to permit serum borne proteins, especially fibronectin and vitronectin, to adsorb to the surface of the graft. Endotheha1 cells are resuspended in media 199E containing autologous serum in a volume sufficient to fill the inner volume of the graft. The graft is then connected to a syringe containing excess serum containing medium and the graft pressurized to force fluid through and cells into the interstices of the graft. The pressure gradient is maintained until 4 graft volumes pass through the interstices or until 5 min has passed. Our studies have established a homogeneous deposition of cells results on the lumenal surface of grafts treated this way. Grafts are subsequently implanted while maintaining the inner surface of the graft moist with serum containing medium. Our clinical experience using this method of microvessel endothelial cell isolation and deposition using the process we have termed sodding is ongoing. Morphological evaluations of grafts retrieved from patients has been extremely limited but of great interest since they establish our ability to accelerate the formation of endothelial cell linings on human grafts (21) (Fig. 5). The evaluation of the efficacy of endothelial cell transplantation toward improved graft patency are ongoing in controlled clinical trials.
SIZE BAR Fig. 4. Endothelial cell monolayer established on an ePTFE graft using autologous microvascular endothelial canine carotid artery model. This graft was explanted following 5 weeks of implantation (x480; bar = 20 pm).
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SIZE BAR Fig. 5. Scanning electron micrograph of an endothelial cell monolayer on a human polymeric with autologous liposuction-fat derived microvascular endothelial cells (x 132; bar = 20 pm).
Other Applications of Endothelial Cell Transplantation The origins of the needs and methods for endothelial cell transplantation are attributable to the search for improved small diameter prosthetic vascular grafts. This search has resulted in the development of numerous methods for endothelial cell isolation and culture as well as an understanding of endothelial cell function following transplantation. The use of endothelial cell transplantation to improve the function of small diameter prosthetic grafts has reached the phase of clinical evaluation yet the uses of endothelial cell transplantation are just beginning to be realized. With the advent of methods to transfer genetic material into cells and observe functional protein products from this genetic material, investigators began to explore cells available to be used as a cellular vehicle for gene therapy. While numerous mammalian cells are susceptible to genetic manipulation, the endothelial cell has several characteristics making its choice as a vehicle attractive. Endothelium provides the natural lining of all blood vessels in the body and is thus in constant contact with blood. For this reason a gene product would be directly accessible to the blood stream if endothelial cells were used as a vehicle. Moreover, and most likely to the surprise of many molecular biologists, the methods for endothelial cell isolation and subsequent transplantation back into the body have already been undergoing refinement for years. Methods of gene therapy using endothelium suggested by molecular biologists could thus rely on already established endothelial cell transplantation methods. Molecular
graft.
This graft was sodded
biologists have taken advantage of this extensive foundation in endothelial cell transplantation methodology previously established by others. Numerous reports of successful genetic manipulation of endothelial cells have now been reported with great promise toward replacement of defective genes in patients. The use of endothelial cell transplantation has also begun to expand beyond use in arterial bypass grafts. Again based upon established methods for cell isolation and deposition, investigators have started to explore the use of endothelial cells to improve the function of vein grafts, prosthetic arterio-venous grafts, stents, shunts, patches, ventricular assist devices and the total artificial heart. Interestingly, molecular biologists have suggested that endothelium must be genetically manipulated to improve their anticoagulant activity before their use as cellular coatings on these vascular implants (6). This tenant is not universally accepted since many investigators with long standing interest in endothelial cell transplantation believe transplanted endothelium are not genetically deficient in anticoagulant activity. Moreover, the balance between procoagulant and anticoagulant activity maintained by endothelial cells is delicate and should not be manipulated by genetic modification. The field of endothelial ceil transplantation derived from initial studies of endothelial cell-polymer interaction toward the establishment of antithrombogenic linings on small diameter vascular grafts. With the development of operating room compatible methods to isolate autologous endothelium a wide variety of polymer and native surface implants are being considered.
Endothelial cell transplantation The development
of genetic
new use of endothelial livery
of novel
gene
manipulation
cells as a cellular
provides vehicle
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REFERENCES 1. Allen, B.T.; Long, J.A.; Clark, R.E.; Sicard, G.A.; Hopkins, K.Y.; Welch, M.J. Influence of endothelial cell seeding on platelet deposition and patency in smalldiameter Dacron arterial grafts. J. Vast. Surg. 1:224232; 1984. 2. Angelini, G.D.; Breckenridge, I.A.; Psaila, J.V.; Williams, H.M.; Henderson, A.H.; Newby, A.C. Preparation of human saphenous vein for coronary artery bypass grafting impairs its capacity to produce prostacyclin. Cardiovasc. Res. 21:28-33; 1987. 3. Belsen, T.A.; Schmidt, S.P.; Falkow, L.J.; Sharp, W.V. Endothelial cell seeding of small-diameter vascular grafts. Trans. Am. Sot. Artif. Intern. Organs 28:173; 1982. 4. Bond, M.D.; van Wart, H.E. Characterization of the individual collagenases from Clostridium histolyticum. Biochem. 23:3085-3091; 1984. 5. Cambria, R.P.; Megerman, J.; Abbot, W.M. Endothelial preservation in reversed and in situ autogenous vein grafts. Ann. Surg. 202(1):50-55; 1985. 6. Choi, E.T.; Callow, A.D.; Ryan, U.S. Interventional Techniques to accelerate healing: Gene therapy. In: White, R.A.; Hollier, L.H., eds. Vascular surgery: Basic science and clinical correlations. Philadelphia: JB Lippincott Co.; 1994:81-86. 7. Douville, E.C.; Kempczinski, R.F.; Birinyi, L.K.; Ramalanjaona, G.R. Impact of endothelial cell seeding on long term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft. J. Vast. Surg. 5:544; 1987. 8. Forster, R.I.; Bernath, EAnalysis of urokinase immobilization on the polytetrafluoroethylene vascular prosthesis. Am. J. Surg. 156:130-132; 1988. 9. Graham, L.M.; Vinter, D.W.; Ford, J.W. Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts. Early studies with autologous canine cells. Arch. Surg. 115:1289-1294; 1980. 10. Herring, M.; Compton, R.S.; Gardner, A.L.; LeGrand, D.R. Clinical experiences with endothelial seeding in Indianapolis. In: Zilla, P.; Fasol, R.; Deutsch, M., eds. Endothelialization of Vascular Grafts. Base]: Karger; 1987:218-224. 11. Herring, M.; Gardner, A.; Glover, J. A single staged technique for seeding vascular grafts with autogenous endothelium. Surg. 84:498-504; 1978. 12. Jarrell, B.E.; Williams, S.K. Interventional techniques to accelerate healing: Endothelial seeding. In: White, R.A.; Hollier, L.H., eds. Vascular surgery: Basic science and clinical correlations. Philadelphia: JB Lippincott Co.; 1994:71-80. 13. Jarrell, B.E.; Levine, E.M.; Shapiro, S.S.; Williams, S.K.; Carabasi, R.A.; Mueller, S.N.; Thornton, S.C.
15.
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24.
25.
26.
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Human adult endothelial cell growth in culture. J. Vast. Surg. 1:757-764; 1984. Jarrell, B.E.; Williams, S.K.; Stokes, G.; Hubbard, EA.; Carabasi, R.A.; Koolpe, E.; Greener, D.; Pratt, K.; Moritz, M.J.; Radomski, J.; Speicher, L. Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery. Surg. 100(2):392-399; 1986. Kempczinski, R.F.; Rosenman, J.E.; Pearce, W.H.; Rodersheimer, L.R.; Berlatzky, Y.; Ramalanjaona, G.R. Endothelial cell seeding of new PTFE vascular prostheses. J. Vast. Surg. 2:424-429; 1985. Kesler, K.A.; Herring, M.B.; Arnold, M.P.; Glover, J.L.; Park, H.M.; Helmus, M.N.; Bendick, P.J. Enhanced strength of endothelial attachment on polyester elastomer and polytetrafluoroethylene graft surfaces with fibronectin substrate. J. Vast. Surg. 3:58-64; 1986. Lewis, W.H. Endothelium in tissue culture. Am. J. Anat. 30:39-59; 1922. Maciag, T.G.; Hoover, G.A.; Stemerman, M.B.; Weinstein, R. Serial propagation of human endothelial cells in vitro. J. Cell Biol. 91:420-426; 1981. Madri, J.A.; Williams, S.K. Capillary endothelial cell cultures: Phenotypic modulation by matrix components. J. Cell Biol. 97:153-165; 1983. Nojiri, C.; Park, K.D.; Grainger, D.W.; Jacobs, H.A.; Okano, T.; Koyanagi, H.; Kim, S. In vivo nonthrombogenicity of heparin immobilized polymer surfaces. Trans. Am. Sot. Artif. Intern. Organs. 36:Ml68-M172; 1990. Park, P.K.; Jarrell, B.E.; Williams, S.K. Thrombus-free, human endothelial cell surface in the midregion of a Dacron vascular graft in the splanchnic venous circuitobservations after nine months of implantation. J. Vast. Surg. 2:468-475; 1990. Potzsch, B.; Grulich-Henn, J.; Rossing, R.; Wille, D.; Muller-Berghaus, G. Identification of endothelial and mesothelial cells in human omental tissue and omentum derived cultured cells by specific cell markers. J. Lab. Invest. 63:841-852; 1990. Quist, W.C.; Haudenshield, C.C.; Logerfo, F.W. Qualitative microscopy of implanted vein grafts: Effects of graft integrity on morphological fate. J. Thorac. Cardiovast. Surg. 103:671-677; 1992. Richardson, J.V.; Wright, C.B.; Hiratzka, L.F. The role of endothelium in the patency of small venous substitutes. J. Surg. Res. 28:556-562; 1980. Rosenman, J.E.; Kempczinski, R.F.; Pearce, W.H.; Silberstein, E.B. Kinetics of endothelial cell seeding. J. Vast. Surg. 2:778-784; 1985. Schmidt, S.P.; Hunter, T. J.; Sharp, W.V.; Malindzak, G.S.; Evancho, M.M. Endothelial cell seeded four millimeter Dacron vascular grafts. J. Vast. Surg. 1:434-441; 1984. Schmidt, S.P.; Hunter, T.J.; Hirko, M.Small diameter vascular prostheses: Two designs of PTFE and endothelial cell seeded and nonseeded Dacron. J. Vast. Surg. 2: 292-297; 1985.
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