0963-6897/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Cell Transplantation, Vol. 1, pp. 293-298, 1992 Printed in the USA. All rights reserved.
Original Contribution A PORCINE MODEL F O R ADIPOSE TISSUE-DERIVED ENDOTHELIAL C E L L TRANSPLANTATION C A R L T O N Y O U N G , * B R U C E E . J A R R E L L , * ! JAMES B . H O Y I N G , * ! AND S T U A R T K . WILLIAMS
*tt
tDepartment of Surgery, University of Arizona, Health Sciences Center, Tucson, AZ 85724, USA, and 'Department of Surgery, Jefferson Medical College, Philadelphia, PA 19107, USA
However, during preliminary work to establish a por cine model for fat-derived microvascular endothelial cell transplantation we encountered difficulty in iden tifying a suitable source of fat for subsequent micro vascular endothelial cell isolation. Development of a porcine model of endothelial cell transplantation is de sirable because the pig exhibits similarities to humans with respect to cardiovascular disease. While pigs exhibit copious quantities of fat, the ma jor deposits of fat in this animal are not suitable for microvascular endothelial cell isolation. During sub sequent studies of several anatomically distinct fat sources, we identified porcine preperitoneal fat as a potential source. We report that this fat exhibits a morphology similar to human subcutaneous fat and contains predominantly adipocytes and microvascular endothelial cells. Procedures for the isolation and characterization of microvessel endothelial cells from porcine preperitoneal fat are described.
• Abstract — The transplantation of endothelial cells repre sents a technology which has been suggested for applications ranging from improvement in function of implanted vascu lar devices to genetic therapy. The use of microvascular en dothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from por cine fat. A variety of anatomic sites within the pig were eval uated to determine the appropriateness of different sources of fat for endothelial cell isolation. The preperitoneal fat was determined to be optimal due to the predominance of endo thelium in this tissue and the ease of isolation of microvas cular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissuederived microvessel endothelial cell isolation. • Keywords — Endothelium; Vascular graft; Adipose tissue; Ovine. INTRODUCTION
Vascular endothelial cells have been isolated and cul tured from a variety of animal species (including man), a variety of tissue types, and from both large vessel and microvascular sources (13). Our laboratory has fo cused on developing methodologies for the isolation and subsequent transplantation of endothelial cells to create new vascular linings on implantable prosthetic devices (5,6,14). Microvascularized fat-derived endo thelial cells have been of particular interest because these cells are abundant in fat and the procedures for their isolation are relatively simple and compatible with the operating room. Animal models of fat-derived microvascular endothelial cell transplantation have es tablished the efficacy of these procedures for the cre ation of new cell linings on implants (8,10,12,15).
MATERIALS AND METHODS Microvascularized
Fat
Procurement
All animal studies were performed following ap proval of protocols by our animal review committee. Yorkshire pigs were premedicated with acepromazine (1 mg/kg i.m.), atropine (0.5 mg/kg i.m.) and ketamine (20 mg/kg i.m.). Prior to intubation, pentobar bital (15 mg/kg i.v.) was given. Pentobarbital was infused i.v. as necessary to maintain anesthesia. After intubation, a midline abdominal incision was made. Approximately 10 g of fat was removed from subcu taneous, perirenal, and omental fat deposits. In addi tion, a 10 g quantity of fat was obtained from a fat
ACCEPTED 5 / 1 / 9 2 . { C o r r e s p o n d e n c e s h o u l d be addressed t o D r . Stuart K .
Williams, Department o f Surgery, University o f A r i z o n a , H e a l t h Sciences C e n t e r , T u c s o n , A Z 85724. 293
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deposit we identify as properitoneal fat. This fat was abundant in the region between the transversalis fas cia and peritoneum. Fat samples were immediately placed in separate beakers containing harvest media (media 199E; 10% fetal bovine serum; 50 jtg/mL Lglutamine; 2.5 ug/mL fungizone; 80 ug/mL gentami cin). They were then processed for histology or endothelial cell isolation. Histology
Upon removal, properitoneal fat was immersed in 4% paraformaldehyde in 0.5 M PIPES at 4°C for 1 h. Fixed samples were processed for paraffin embedding and 4-6 um sections were taken using a Spencer 820 microtome. Sections were stained with modified Gomori trichrome. They were examined and photo graphed with a Nikon Optiphot microscope. Microvessel
Endothelial
Cell
Isolation
Samples of fat were minced with scissors to produce fat segments approximately 2 mm in dimension. Minced fat was washed twice with divalent cation-free Dulbecco's phosphate-buffered saline (DCF; 2.7 mM KC1, 1.47 mM K H P 0 , 139 mM NaCl, 8.1 mM Na HP0 , pH 7.4) supplemented with 0.1% bovine serum albumin (BSA). The tissue was then incubated in 0.4% (w/v) crude clostridial collagenase (Boehringer Mannheim) and 0.4% (w/v) human serum albu min (HSA; Red Cross Co.) for 30 min at 37°C in a shaking water bath. The cellular slurry was centrifuged at 300 x g for 7 min and the pellet was resuspended in DCF-PBS and centrifuged at 300 x g for 4 min. The final pellet was resuspended in buffer appropriate for culturing or cell transplantation as described below. Cell numbers were determined by using a Coulter Counter (Coulter Electronics, Hialeah, FL). 3
2
2
4
4
Microvessel
Endothelial
Cell
Culture
Isolated endothelial cells were resuspended in com plete culture media containing media 199E, 5 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine, 80 ug/mL endothelial cell growth factor (ECGF) and 100 ug/mL heparin. The cells were plated at 1 x 10 cells/cm on gelatin coated T-25 polystyrene tissue culture flasks and maintained at 37°C and 5% C 0 in a water-jacketed incubator. At confluence, cells were split using trypsin at 1:4 split ratios. 5
red; 2 mM CaCI , 2 mM MgCl , 5.5 mM glucose, 2 mM EGTA, 10 mM MES; pH 7.2). Human large ves sel smooth muscle cell (HSMC) and Madin Darby ca nine kidney (MDCK) epithelial cell lines were used as staining controls for a SMC actin and cytokeratin, respectively. 2
2
von Willebrand factor. Due to the dispersed nature
of vWf staining in MVEC as compared to large vessel EC, we utilize peroxidase staining to increase the sen sitivity of the assay. Cells were fixed with 20% meth anol at room temperature for 20 min. Cells were washed with IB containing 1% dry milk (Alba) three times for 5 min each wash. Cells were incubated with a primary polyclonal antibody against human antigen (1:100 dilution in IB with 1% milk; DAKO Co.) to vWf for 1 h at room temperature. Cells were washed three times with IB in 1 % milk using 5-min incuba tions at room temperature between washes. The sec ondary antibody (1:200 dilution) conjugated with biotin was incubated for 25 min at room temperature. Following a rinse, cells were incubated sequentially in avidin peroxidase, 25 min, washed three times with PBS, diamino benzidine with hydrogen peroxide, dis tilled water, and copper sulfate. The stained cells were then visualized with a Nikon Optiphot-2 microscope. Cytoskeleton. Cell cultures were fixed with 4% paraformaldehyde for 10 min, washed three times with IB using 5-min incubations, and treated with 0.5% Triton-X 100 for 2 min. The cell cultures were then washed three times with IB containing 1% milk using 5-min incubations. Cultures were incubated with pri mary antibody (monoclonal, anticytokeratin or antialpha smooth muscle cell actin, (Sigma, St. Louis, MO) diluted 1:100 with IB and 1% milk for 1 h. The cell cultures were washed three times with IB and 1 % milk using 5-min incubations, and incubated for 1 h with secondary antibody (either FITC and TRITC-labelled rabbit antimouse IgG) diluted 1:32 in IB and 1% milk. The cells were then washed three times in IB and three times with DCF-PBS (pH 7.4) and viewed under epifluorescence illumination using water immer sion objectives.
2
2
Immunochemistry
Cultures were characterized using antibodies specific for von Willebrand factor (vWf), alpha smooth mus cle cell actin and cytokeratin, peptide 18. Cultures were washed with immunochemistry buffer (IB; Hanks Balanced salt solution, HBSS without phenol
Graft
Sodding
Freshly isolated microvascular endothelial cells were used immediately to treat the lumenal surface of ePTFE prosthetic grafts. Following isolation, MVEC were resuspended in sodding media which contained: media 199E and 15% autologous porcine serum. The concentration of cells was determined by electronic counting using a Coulter Counter (Coulter Electron ics, Hialeah, FL). A 3 mm i.d. ePTFE graft was pre pared for sodding by securing Leur locks to each end
Pigfat microvessel isolation • C . YOUNG ET AL.
of the graft with silk ties. One end was sealed with a cap while the other end was connected to a 60 cc sy ringe which contained sodding media. The graft was then wet with sodding media by pressurizing the graft to 5 PSI. Pressure was monitored by means of a pres sure gauge placed in line between the graft and sy ringe. Approximately 10 volumes of sodding media were forced through the graft interstices, and the graft was incubated in sodding media for an additional 30 min at room temperature. After 30 min, sodding media was aspirated from the lumen of the graft and replaced with MVEC in sodding media at 37°C at a concentration to provide 2 x 10 cells per cm . The syringe containing sodding media was again attached to the graft with an interpositioned pressure gauge and the graft pressured to 5 PSI. This pressure was main tained for 5 min or until four graft volumes of sodding media had passed into the graft. After sodding, the graft lumenal surfaces were analyzed for the presence of cells. 5
295
2
Sodded graft analysis. Immediately, sodded grafts
prepared for either scanning electron microscopy (SEM) or staining with the fluorescent nuclear dye bisbenzimide (BBI; Hoechst Co.). Surfaces to be ex amined via SEM were fixed overnight in 2% glutaraldehyde in 0.5 M PIPES at 4°C, serially dehydrated in acetone, and critical point dried. Prepared samples were visualized using a JEOL 820 scanning electron microscope. Samples to be stained with BBI were fixed in 4% paraformaldehyde in 0.5 M PIPES overnight at 4°C. Samples were washed twice with PBS pH 7.4 and stained with 5 ug/mL BBI in PBS for 10 min at room temperature. Stained samples were viewed using epifluorescence illumination.
were
RESULTS
Fat samples were surgically removed from anesthe tized pigs from a variety of anatomic sites for sub sequent analysis. Based on cell yield as well as cell morphology, fat obtained from the preperitoneal area was considered preferable. This fat was globular in texture and was removed as an intact piece of tissue. Histological evaluation of preperitoneal fat revealed this tissue to be comprised of predominantly adipose and vascular endothelial cells (Fig. 1). While collagen and reticular fibers were present, we did not observe large dense deposits. The complete removal of the se rosal, mesothelial cell-rich, lining was also confirmed morphologically. Following digestion of extracellular matrix using a collagenase digestion and the separation of vascular elements from adipocytes by centrifuga tion, primary isolates of pig microvascular endothelial cells (PMVEC) were obtained. The primary cultures
F i g . 1. L i g h t m i c r o g r a p h o f a p a r a f f i n section o f p r e p e r i t o n e a l p o r c i n e fat stained w i t h H & E s t a i n . T h e p r e d o m i nate cell types present a r e a d i p o c y t e s a n d v a s c u l a r cells. B a r = 50 urn.
were routinely maintained for 10 passages without loss of morphology and proliferative capacity. These cells exhibited typical microvessel endothelial cell morphol ogy, displaying a compact cell shape and dense cyto plasm (Fig. 2). PMVEC cultures stained positive for the presence of vWf (Fig. 3) and negative for the epi thelial specific cytokeratin peptide 18 (Fig. 4). Von Willebrand staining consisted of a diffuse, punctate staining distributed across the cell. There was a gen eral lack of a smooth muscle cell actin detected in cells of confluent cultures; however, an occasional cell ex hibited a light staining as compared to the smooth muscle cell controls (Fig. 5). BBI nuclear staining of ePTFE surfaces, sodded with freshly isolated PMVEC, demonstrated the pres ence of nucleated cells on the surface of these grafts (Fig. 6). SEM analysis revealed these cells to be asso ciated with the surface of the ePTFE material via cy toplasmic extensions (Fig. 7). However, following this brief incubation of cells with the ePTFE, cells had not begun to spread or develop contact with each other by typical junctional processes. DISCUSSION
Transplantation of cells onto or within prosthetic devices has been suggested as one method to improve the healing and function of these devices following their implantation (3,4,9). Microvascular endothelial cell transplantation has been previously evaluated as a method to improve the biocompatibility of pros thetic implants (2,8,10,14). Furthermore, the proxim ity of endothelial cells to the blood stream has made this cell an attractive choice for genetic engineering
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F i g . 3. M o n o l a y e r s o f (a) P M V E C a n d ( b ) H S M C stained f o r the presence o f v o n W i l l e b r a n d f a c t o r ( v W f ) . v W f w a s detected b y i m m u n o p e r o x i d a s e staining o f c o n f l u e n t cultures u s i n g a p o l y c l o n a l a n t i b o d y p r e p a r e d against h u m a n v W f . P M V E C exhibited a diffuse, homogeneous staining, while H S M C displayed b a c k g r o u n d staining. Bar = 1 3 ^ m .
F i g . 2. Phase contrast light m i c r o g r a p h o f confluent cultures o f ( a ) P M V E C , ( b ) M D C K , a n d (c) H S M C . P M V E C ex h i b i t e d little cell o v e r l a p p i n g as w e l l as a m o r e c o m p a c t c y t o p l a s m relative t o t h e H S M C . F u r t h e r m o r e , P M V E C m o n o l a y e r s d o n o t display the t y p i c a l epithelial m o r p h o l o g y o f M D C K m o n o l a y e r s . N o t e the r o u n d e d , tightly packed cells o f the M D C K c u l t u r e s w h i c h w a s n o t o b s e r v e d i n the P M V E C c u l t u r e s . B a r = 70 j a n .
studies (7). Our laboratory has been evaluating the clinical feasibility of microvessel endothelial cell trans plantation with particular emphasis on developing methodologies compatible with the operating room using devices and solutions currently approved for hu man use. The availability of animal models is vital to preclinically evaluate the safety and efficacy of endo thelial cell transplantation techniques. While no animal model individually duplicates the physiologic conditions and prosthetic implant response observed in humans, individual animal models do du plicate certain human responses and physiologies ( 1 ) . Our interest in developing a pig model for endothelial cell transplantation stems from the similarities between
the pig and human with respect to the development of atherosclerosis. The pig model affords the opportunity to evaluate not only the mechanisms underlying the development of atherosclerosis, but also to evaluate the function of prosthetic devices used to replace vas cular structures damaged due to atherosclerosis. Fur thermore, the development of intimal hyperplasia in the pig is also similar to humans providing a model of postimplant device function with respect to the hyper plastic response. Our evaluation of pig fat indicates that, as previ ously observed with human and canine fat ( 1 4 ) , a striking difference exists between adipose tissues de rived from different anatomic sties. Pig fat is unique with respect to the highly fibrous nature of most of the common sources of fat we have evaluated in other spe cies. The subcutaneous fat in pigs is course and fibrous and contains few adipocytes. While microvasculature is present, subcutaneous tissue in the pig contains a large number of other cell types. For this reason this fat was not appropriate for our studies. Other ana tomic sources of fat were examined including perire nal and omental. While omental fat has been used previously by other investigators, including our labo ratory, for endothelial cell transplantation ( 8 , 1 1 , 1 3 ) ,
Pigfat microvessel isolation • C. YOUNG ET AL.
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F i g . 6. F l u o r e s c e n c e m i c r o g r a p h o f freshly isolated P M V E C sodded o n t o e P T F E . Sections o f sodded e P T F E were stained f o r n u c l e a t e d cells w i t h the D N A intercalating d y e , B B I . B a r = 50 um.
F i g . 4. I m m u n o s t a i n i n g f o r the presence o f c y t o k e r a t i n pep tide 18 i n cultures o f (a) P M V E C a n d ( b ) M D C K . A m o n o clonal a n t i b o d y developed against b o v i n e antigen c o n j u g a t e d t o F I T C was used i n the staining p r o c e d u r e . T h e M D C K cells e x h i b i t e d the t y p i c a l p e r i p h e r a l b a n d l o c a l i z a t i o n associated w i t h the c y t o k e r a t i n c y t o s k e l e t o n , w h i l e the P M V E C d i d not e x h i b i t c y t o k e r a t i n specific s t a i n i n g . B a r = 5 t u n .
the pig exhibits very limited supplies of omental fat. The highly microvascularized omentum is extensive in the pig. However, this tissue was not evaluated due to the high density of mesothelial cells in this tissue (11). Preperitoneal fat exhibited a physical consistency
similar to human liposcution-derived subcutaneous fat, the source of fat used in our human studies. His tological evaluation of this fat revealed the presence of both microvascular endothelial cells and adipocytes. Careful dissection of this fat from the peritoneal sero sal covering effectively eliminated the presence of me sothelial cell contaminants. Collagenase digestion using a crude collagenase chosen for human fat disso ciation resulted in the rapid and effective dissociation of the pig fat. Following centrifugation to remove buoyant adipocytes, we observed a microvascular-rich vascular pellet. The cells in this pellet were viable and primary cultures could be established. Using culture conditions defined for human microvascular endothe-
F i g . 5. F l u o r e s c e n c e light m i c r o g r a p h s o f ( a ) P M V E C a n d ( b ) H S M C s t a i n e d f o r t h e presence a S M C a c t i n . P M V E C e x h i b i t e d b a c k g r o u n d staining f o r a S M C a c t i n u t i l i z i n g a f l u o r e s c e n t a n t i b o d y c o m p l e x . H S M C c o n t a i n e d dense b u n d l e s o f actin d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m . B a r = 10 um i n (a) a n d 4 um i n ( b ) .
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i c a l l y d e r i v e d a n d c u l t u r e d c a n i n e e n d o t h e l i a l cells. S u r g e r y 91:550-559; 1982. 4. H e r r i n g , M . ; G a r d n e r , A . L . ; G l o v e r , J . A single staged t e c h n i q u e f o r seeding v a s c u l a r g r a f t s w i t h a u t o g e n o u s e n d o t h e l i u m . S u r g e r y 84:498-504; 1987. 5. J a r r e l l , B . E . ; L e v i n e , E . ; S h a p i r o , S . ; W i l l i a m s , S . K . ; C a r a b a s i , R . A . ; M u e l l e r , S.; T h o r n t o n , S. H u m a n adult endothelial cell g r o w t h i n c u l t u r e . J . V a s e . S u r g . 1:757764; 1984. 6. J a r r e l l , B . E . ; W i l l i a m s , S . K . ; S t o k e s , G . ; H u b b a r d , F . A . ; Carabasi, R . A . ; Koolpe, E.; Greener, D.; Pratt, K . ; M o r i t z , M . ; R a d o m s k i , J . U s e o f f r e s h l y isolated c a p i l l a r y endothelial cells f o r the i m m e d i a t e establish ment o f a m o n o l a y e r o n a vascular g r a f t at s u r g e r y . S u r g e r y 100:392-399; 1986. F i g . 7. S c a n n i n g e l e c t r o n m i c r o g r a p h o f a s o d d e d e P T F E s u r f a c e . N o t e the cell extensions associated w i t h the p o l y m e r s u r f a c e . B a r = 10 am.
lial cells, the PMVEC cultures were routinely passaged. Finally, morphological evaluation and immunocytochemistry established that cultures derived from pig properitoneal fat maintained a phenotype typical of endothelial cells in culture. Freshly isolated PMVEC were amenable to vascu lar graft sodding techniques we have previously used with both canine and human EC. These cells remained adherent to the vascular grafts when exposed to a physiological shear (data not shown). The availability of PMVEC will permit the evaluation of the effective ness of endothelial cell transplantation towards the im provement in function of vascular prosthetic devices in a pig model. Future studies will evaluate PMVEC sodding of vascular prosthetic devices and the forma tion of new cellular linings on these devices following sodding techniques. REFERENCES 1. D i d i s h e i m , P.; D e w a n j e e , M . K . ; F i s k , C . S . ; K a y e , M . P . ; F a s s , D . N . A n i m a l models u s e f u l f o r p r e d i c t i n g clinical p e r f o r m a n c e o f b i o m a t e r i a l s f o r c a r d i o v a s c u l a r use. I n N o r e t o s , J . W . ; E d e n , M . , eds.: Introduction o f bioma terials t o clinical c a r e . P a r k R i d g e : N o y e s P u b l i c a t i o n s ; 1984. 2. D o u v i l l e , E . C . ; K e m p z i n s k i , R . F . ; B i r i n y i , L . K . ; R a m a l a n j o a n a , G . R . I m p a c t o f endothelial cell seeding o n long-term patency and subendothelial proliferation i n a small-caliber h i g h l y p o r o u s p o l y t e t r a f l u o r o e t h y l e n e g r a f t . J . V a s e . S u r g . 5:544-550; 1987. 3. G r a h a m , L . M . ; B u r k e l , W . E . ; F o r d , J . W . ; V i n t e r , D . W . ; K a h n , R . H . ; Stanley, J . C . E x p a n d e d polytetra f l u o r o e t h y l e n e vascular prostheses seeded w i t h e n z y m a t -
7. N a b e l , E . G . ; P l a u t z , G . ; B o y c e , F . M . ; S t a n l e y , J . C ; N a b e l , G . J . R e c o m b i n a n t gene expression i n v i v o w i t h i n e n d o t h e l i a l cells o f the arterial w a l l . Science 244:13421344; 1989. 8. P e a r c e , W . H . ; R u t h e r f o r d , R . B . ; W h i t e h i l l , T . A . ; R o sales, C ; B e l l , K . P . ; P a t t , A . ; R a m a l a n j o a n a , G . S u c cessful endothelial cell seeding w i t h o m e n t a l l y d e r i v e d m i c r o v a s c u l a r e n d o t h e l i a l cells. J . V a s e . S u r g . 5:203210; 1987. 9. S c h m i d t , S . P . ; H u n t e r , T . J . ; S h a r p , W . V . ; M a l i n d z a k , G . S . ; E v a n c h o , M . M . E n d o t h e l i a l cell-seeding f o u r - m i l limeter D a c r o n vascular grafts. J . Vase. S u r g . 1:434-441; 1984. 10. Sterpetti, A . V . ; H u n t e r , W . J . ; S c h u l z , R . D . ; S u g i m o t o , J . T . ; B l a i r , E . A . ; H a c k e r , K . ; C h a s a n , P.; V a l e n t i n e , J . Seeding w i t h e n d o t h e l i a l cells d e r i v e d f r o m the m i c r o vessels o f the o m e n t u m a n d the j u g u l a r v e i n : A c o m p a r ative s t u d y . J . V a s e . S u r g . 7:677-684; 1988. 11. T a k a h a J i i , K . ; G o t o , T . ; M u k a i , Hata, J . Cobblestone monolayer o m e n t a l adipose tissue are possibly dothelial. I n V i t r o Cell D e v . Biol.
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12. W a n g , Z . ; D u , W . ; L i , G . ; S h a r e f k i n , J . B . R a p i d c e l l u lar l u m i n a l c o v e r a g e o f D a c r o n i n f e r i o r v e n a c a v a p r o s thesis i n d o g s b y i m m e d i a t e seeding o f a u t o g e n o u s endothelial cells d e r i v e d f r o m o m e n t a l tissue: Results o f a p r e l i m i n a r y t r i a l . J . V a s e . S u r g . 12:168-179; 1990. 13. W i l l i a m s , S . K . I s o l a t i o n a n d c u l t u r e o f m i c r o v e s s e l a n d large vessel endothelial cells: T h e i r use i n t r a n s p o r t a n d clinical studies. I n : M c D o n a g h , P . , e d . M i c r o v a s c u l a r p e r f u s i o n a n d t r a n s p o r t i n health a n d disease. B a s e l , S w i t z e r l a n d : K a r g e r P u b l i s h i n g C o . ; 1987:204-245. 14. W i l l i a m s , S . K . ; J a r r e l l , B . E . ; R o s e , D . G . ; P o n t e l l , J . ; Kapelan, B . A . ; Park, P.K.; Carter, T . L . H u m a n microvessel e n d o t h e l i a l cell i s o l a t i o n a n d v a s c u l a r g r a f t s o d d i n g in the operating r o o m . A n n . V a s e . S u r g . 3(2): 146152; 1989. 15. W i l l i a m s , S . K . ; C a r t e r , T . ; P a r k , P . K . ; R o s e , D . G . ; Schneider, T . ; J a r r e l l , B . E . F o r m a t i o n o f m u l t i l a y e r cel lular lining o n a p o l y u r e t h a n e v a s c u l a r g r a f t f o l l o w i n g endothelial cell sodding. J . B i o m e d . M a t e r . R e s . 26:103117; 1992.
Cell Transplantation, Vol. 1, pp. 293-298, 1992 Printed in the USA. All rights reserved.
Original Contribution A PORCINE MODEL F O R ADIPOSE TISSUE-DERIVED ENDOTHELIAL C E L L TRANSPLANTATION C A R L T O N Y O U N G , * B R U C E E . J A R R E L L , * ! JAMES B . H O Y I N G , * ! AND S T U A R T K . WILLIAMS
*tt
tDepartment of Surgery, University of Arizona, Health Sciences Center, Tucson, AZ 85724, USA, and 'Department of Surgery, Jefferson Medical College, Philadelphia, PA 19107, USA
However, during preliminary work to establish a por cine model for fat-derived microvascular endothelial cell transplantation we encountered difficulty in iden tifying a suitable source of fat for subsequent micro vascular endothelial cell isolation. Development of a porcine model of endothelial cell transplantation is de sirable because the pig exhibits similarities to humans with respect to cardiovascular disease. While pigs exhibit copious quantities of fat, the ma jor deposits of fat in this animal are not suitable for microvascular endothelial cell isolation. During sub sequent studies of several anatomically distinct fat sources, we identified porcine preperitoneal fat as a potential source. We report that this fat exhibits a morphology similar to human subcutaneous fat and contains predominantly adipocytes and microvascular endothelial cells. Procedures for the isolation and characterization of microvessel endothelial cells from porcine preperitoneal fat are described.
• Abstract — The transplantation of endothelial cells repre sents a technology which has been suggested for applications ranging from improvement in function of implanted vascu lar devices to genetic therapy. The use of microvascular en dothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from por cine fat. A variety of anatomic sites within the pig were eval uated to determine the appropriateness of different sources of fat for endothelial cell isolation. The preperitoneal fat was determined to be optimal due to the predominance of endo thelium in this tissue and the ease of isolation of microvas cular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissuederived microvessel endothelial cell isolation. • Keywords — Endothelium; Vascular graft; Adipose tissue; Ovine. INTRODUCTION
Vascular endothelial cells have been isolated and cul tured from a variety of animal species (including man), a variety of tissue types, and from both large vessel and microvascular sources (13). Our laboratory has fo cused on developing methodologies for the isolation and subsequent transplantation of endothelial cells to create new vascular linings on implantable prosthetic devices (5,6,14). Microvascularized fat-derived endo thelial cells have been of particular interest because these cells are abundant in fat and the procedures for their isolation are relatively simple and compatible with the operating room. Animal models of fat-derived microvascular endothelial cell transplantation have es tablished the efficacy of these procedures for the cre ation of new cell linings on implants (8,10,12,15).
MATERIALS AND METHODS Microvascularized
Fat
Procurement
All animal studies were performed following ap proval of protocols by our animal review committee. Yorkshire pigs were premedicated with acepromazine (1 mg/kg i.m.), atropine (0.5 mg/kg i.m.) and ketamine (20 mg/kg i.m.). Prior to intubation, pentobar bital (15 mg/kg i.v.) was given. Pentobarbital was infused i.v. as necessary to maintain anesthesia. After intubation, a midline abdominal incision was made. Approximately 10 g of fat was removed from subcu taneous, perirenal, and omental fat deposits. In addi tion, a 10 g quantity of fat was obtained from a fat
ACCEPTED 5 / 1 / 9 2 . { C o r r e s p o n d e n c e s h o u l d be addressed t o D r . Stuart K .
Williams, Department o f Surgery, University o f A r i z o n a , H e a l t h Sciences C e n t e r , T u c s o n , A Z 85724. 293
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deposit we identify as properitoneal fat. This fat was abundant in the region between the transversalis fas cia and peritoneum. Fat samples were immediately placed in separate beakers containing harvest media (media 199E; 10% fetal bovine serum; 50 jtg/mL Lglutamine; 2.5 ug/mL fungizone; 80 ug/mL gentami cin). They were then processed for histology or endothelial cell isolation. Histology
Upon removal, properitoneal fat was immersed in 4% paraformaldehyde in 0.5 M PIPES at 4°C for 1 h. Fixed samples were processed for paraffin embedding and 4-6 um sections were taken using a Spencer 820 microtome. Sections were stained with modified Gomori trichrome. They were examined and photo graphed with a Nikon Optiphot microscope. Microvessel
Endothelial
Cell
Isolation
Samples of fat were minced with scissors to produce fat segments approximately 2 mm in dimension. Minced fat was washed twice with divalent cation-free Dulbecco's phosphate-buffered saline (DCF; 2.7 mM KC1, 1.47 mM K H P 0 , 139 mM NaCl, 8.1 mM Na HP0 , pH 7.4) supplemented with 0.1% bovine serum albumin (BSA). The tissue was then incubated in 0.4% (w/v) crude clostridial collagenase (Boehringer Mannheim) and 0.4% (w/v) human serum albu min (HSA; Red Cross Co.) for 30 min at 37°C in a shaking water bath. The cellular slurry was centrifuged at 300 x g for 7 min and the pellet was resuspended in DCF-PBS and centrifuged at 300 x g for 4 min. The final pellet was resuspended in buffer appropriate for culturing or cell transplantation as described below. Cell numbers were determined by using a Coulter Counter (Coulter Electronics, Hialeah, FL). 3
2
2
4
4
Microvessel
Endothelial
Cell
Culture
Isolated endothelial cells were resuspended in com plete culture media containing media 199E, 5 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine, 80 ug/mL endothelial cell growth factor (ECGF) and 100 ug/mL heparin. The cells were plated at 1 x 10 cells/cm on gelatin coated T-25 polystyrene tissue culture flasks and maintained at 37°C and 5% C 0 in a water-jacketed incubator. At confluence, cells were split using trypsin at 1:4 split ratios. 5
red; 2 mM CaCI , 2 mM MgCl , 5.5 mM glucose, 2 mM EGTA, 10 mM MES; pH 7.2). Human large ves sel smooth muscle cell (HSMC) and Madin Darby ca nine kidney (MDCK) epithelial cell lines were used as staining controls for a SMC actin and cytokeratin, respectively. 2
2
von Willebrand factor. Due to the dispersed nature
of vWf staining in MVEC as compared to large vessel EC, we utilize peroxidase staining to increase the sen sitivity of the assay. Cells were fixed with 20% meth anol at room temperature for 20 min. Cells were washed with IB containing 1% dry milk (Alba) three times for 5 min each wash. Cells were incubated with a primary polyclonal antibody against human antigen (1:100 dilution in IB with 1% milk; DAKO Co.) to vWf for 1 h at room temperature. Cells were washed three times with IB in 1 % milk using 5-min incuba tions at room temperature between washes. The sec ondary antibody (1:200 dilution) conjugated with biotin was incubated for 25 min at room temperature. Following a rinse, cells were incubated sequentially in avidin peroxidase, 25 min, washed three times with PBS, diamino benzidine with hydrogen peroxide, dis tilled water, and copper sulfate. The stained cells were then visualized with a Nikon Optiphot-2 microscope. Cytoskeleton. Cell cultures were fixed with 4% paraformaldehyde for 10 min, washed three times with IB using 5-min incubations, and treated with 0.5% Triton-X 100 for 2 min. The cell cultures were then washed three times with IB containing 1% milk using 5-min incubations. Cultures were incubated with pri mary antibody (monoclonal, anticytokeratin or antialpha smooth muscle cell actin, (Sigma, St. Louis, MO) diluted 1:100 with IB and 1% milk for 1 h. The cell cultures were washed three times with IB and 1 % milk using 5-min incubations, and incubated for 1 h with secondary antibody (either FITC and TRITC-labelled rabbit antimouse IgG) diluted 1:32 in IB and 1% milk. The cells were then washed three times in IB and three times with DCF-PBS (pH 7.4) and viewed under epifluorescence illumination using water immer sion objectives.
2
2
Immunochemistry
Cultures were characterized using antibodies specific for von Willebrand factor (vWf), alpha smooth mus cle cell actin and cytokeratin, peptide 18. Cultures were washed with immunochemistry buffer (IB; Hanks Balanced salt solution, HBSS without phenol
Graft
Sodding
Freshly isolated microvascular endothelial cells were used immediately to treat the lumenal surface of ePTFE prosthetic grafts. Following isolation, MVEC were resuspended in sodding media which contained: media 199E and 15% autologous porcine serum. The concentration of cells was determined by electronic counting using a Coulter Counter (Coulter Electron ics, Hialeah, FL). A 3 mm i.d. ePTFE graft was pre pared for sodding by securing Leur locks to each end
Pigfat microvessel isolation • C . YOUNG ET AL.
of the graft with silk ties. One end was sealed with a cap while the other end was connected to a 60 cc sy ringe which contained sodding media. The graft was then wet with sodding media by pressurizing the graft to 5 PSI. Pressure was monitored by means of a pres sure gauge placed in line between the graft and sy ringe. Approximately 10 volumes of sodding media were forced through the graft interstices, and the graft was incubated in sodding media for an additional 30 min at room temperature. After 30 min, sodding media was aspirated from the lumen of the graft and replaced with MVEC in sodding media at 37°C at a concentration to provide 2 x 10 cells per cm . The syringe containing sodding media was again attached to the graft with an interpositioned pressure gauge and the graft pressured to 5 PSI. This pressure was main tained for 5 min or until four graft volumes of sodding media had passed into the graft. After sodding, the graft lumenal surfaces were analyzed for the presence of cells. 5
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2
Sodded graft analysis. Immediately, sodded grafts
prepared for either scanning electron microscopy (SEM) or staining with the fluorescent nuclear dye bisbenzimide (BBI; Hoechst Co.). Surfaces to be ex amined via SEM were fixed overnight in 2% glutaraldehyde in 0.5 M PIPES at 4°C, serially dehydrated in acetone, and critical point dried. Prepared samples were visualized using a JEOL 820 scanning electron microscope. Samples to be stained with BBI were fixed in 4% paraformaldehyde in 0.5 M PIPES overnight at 4°C. Samples were washed twice with PBS pH 7.4 and stained with 5 ug/mL BBI in PBS for 10 min at room temperature. Stained samples were viewed using epifluorescence illumination.
were
RESULTS
Fat samples were surgically removed from anesthe tized pigs from a variety of anatomic sites for sub sequent analysis. Based on cell yield as well as cell morphology, fat obtained from the preperitoneal area was considered preferable. This fat was globular in texture and was removed as an intact piece of tissue. Histological evaluation of preperitoneal fat revealed this tissue to be comprised of predominantly adipose and vascular endothelial cells (Fig. 1). While collagen and reticular fibers were present, we did not observe large dense deposits. The complete removal of the se rosal, mesothelial cell-rich, lining was also confirmed morphologically. Following digestion of extracellular matrix using a collagenase digestion and the separation of vascular elements from adipocytes by centrifuga tion, primary isolates of pig microvascular endothelial cells (PMVEC) were obtained. The primary cultures
F i g . 1. L i g h t m i c r o g r a p h o f a p a r a f f i n section o f p r e p e r i t o n e a l p o r c i n e fat stained w i t h H & E s t a i n . T h e p r e d o m i nate cell types present a r e a d i p o c y t e s a n d v a s c u l a r cells. B a r = 50 urn.
were routinely maintained for 10 passages without loss of morphology and proliferative capacity. These cells exhibited typical microvessel endothelial cell morphol ogy, displaying a compact cell shape and dense cyto plasm (Fig. 2). PMVEC cultures stained positive for the presence of vWf (Fig. 3) and negative for the epi thelial specific cytokeratin peptide 18 (Fig. 4). Von Willebrand staining consisted of a diffuse, punctate staining distributed across the cell. There was a gen eral lack of a smooth muscle cell actin detected in cells of confluent cultures; however, an occasional cell ex hibited a light staining as compared to the smooth muscle cell controls (Fig. 5). BBI nuclear staining of ePTFE surfaces, sodded with freshly isolated PMVEC, demonstrated the pres ence of nucleated cells on the surface of these grafts (Fig. 6). SEM analysis revealed these cells to be asso ciated with the surface of the ePTFE material via cy toplasmic extensions (Fig. 7). However, following this brief incubation of cells with the ePTFE, cells had not begun to spread or develop contact with each other by typical junctional processes. DISCUSSION
Transplantation of cells onto or within prosthetic devices has been suggested as one method to improve the healing and function of these devices following their implantation (3,4,9). Microvascular endothelial cell transplantation has been previously evaluated as a method to improve the biocompatibility of pros thetic implants (2,8,10,14). Furthermore, the proxim ity of endothelial cells to the blood stream has made this cell an attractive choice for genetic engineering
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F i g . 3. M o n o l a y e r s o f (a) P M V E C a n d ( b ) H S M C stained f o r the presence o f v o n W i l l e b r a n d f a c t o r ( v W f ) . v W f w a s detected b y i m m u n o p e r o x i d a s e staining o f c o n f l u e n t cultures u s i n g a p o l y c l o n a l a n t i b o d y p r e p a r e d against h u m a n v W f . P M V E C exhibited a diffuse, homogeneous staining, while H S M C displayed b a c k g r o u n d staining. Bar = 1 3 ^ m .
F i g . 2. Phase contrast light m i c r o g r a p h o f confluent cultures o f ( a ) P M V E C , ( b ) M D C K , a n d (c) H S M C . P M V E C ex h i b i t e d little cell o v e r l a p p i n g as w e l l as a m o r e c o m p a c t c y t o p l a s m relative t o t h e H S M C . F u r t h e r m o r e , P M V E C m o n o l a y e r s d o n o t display the t y p i c a l epithelial m o r p h o l o g y o f M D C K m o n o l a y e r s . N o t e the r o u n d e d , tightly packed cells o f the M D C K c u l t u r e s w h i c h w a s n o t o b s e r v e d i n the P M V E C c u l t u r e s . B a r = 70 j a n .
studies (7). Our laboratory has been evaluating the clinical feasibility of microvessel endothelial cell trans plantation with particular emphasis on developing methodologies compatible with the operating room using devices and solutions currently approved for hu man use. The availability of animal models is vital to preclinically evaluate the safety and efficacy of endo thelial cell transplantation techniques. While no animal model individually duplicates the physiologic conditions and prosthetic implant response observed in humans, individual animal models do du plicate certain human responses and physiologies ( 1 ) . Our interest in developing a pig model for endothelial cell transplantation stems from the similarities between
the pig and human with respect to the development of atherosclerosis. The pig model affords the opportunity to evaluate not only the mechanisms underlying the development of atherosclerosis, but also to evaluate the function of prosthetic devices used to replace vas cular structures damaged due to atherosclerosis. Fur thermore, the development of intimal hyperplasia in the pig is also similar to humans providing a model of postimplant device function with respect to the hyper plastic response. Our evaluation of pig fat indicates that, as previ ously observed with human and canine fat ( 1 4 ) , a striking difference exists between adipose tissues de rived from different anatomic sties. Pig fat is unique with respect to the highly fibrous nature of most of the common sources of fat we have evaluated in other spe cies. The subcutaneous fat in pigs is course and fibrous and contains few adipocytes. While microvasculature is present, subcutaneous tissue in the pig contains a large number of other cell types. For this reason this fat was not appropriate for our studies. Other ana tomic sources of fat were examined including perire nal and omental. While omental fat has been used previously by other investigators, including our labo ratory, for endothelial cell transplantation ( 8 , 1 1 , 1 3 ) ,
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F i g . 6. F l u o r e s c e n c e m i c r o g r a p h o f freshly isolated P M V E C sodded o n t o e P T F E . Sections o f sodded e P T F E were stained f o r n u c l e a t e d cells w i t h the D N A intercalating d y e , B B I . B a r = 50 um.
F i g . 4. I m m u n o s t a i n i n g f o r the presence o f c y t o k e r a t i n pep tide 18 i n cultures o f (a) P M V E C a n d ( b ) M D C K . A m o n o clonal a n t i b o d y developed against b o v i n e antigen c o n j u g a t e d t o F I T C was used i n the staining p r o c e d u r e . T h e M D C K cells e x h i b i t e d the t y p i c a l p e r i p h e r a l b a n d l o c a l i z a t i o n associated w i t h the c y t o k e r a t i n c y t o s k e l e t o n , w h i l e the P M V E C d i d not e x h i b i t c y t o k e r a t i n specific s t a i n i n g . B a r = 5 t u n .
the pig exhibits very limited supplies of omental fat. The highly microvascularized omentum is extensive in the pig. However, this tissue was not evaluated due to the high density of mesothelial cells in this tissue (11). Preperitoneal fat exhibited a physical consistency
similar to human liposcution-derived subcutaneous fat, the source of fat used in our human studies. His tological evaluation of this fat revealed the presence of both microvascular endothelial cells and adipocytes. Careful dissection of this fat from the peritoneal sero sal covering effectively eliminated the presence of me sothelial cell contaminants. Collagenase digestion using a crude collagenase chosen for human fat disso ciation resulted in the rapid and effective dissociation of the pig fat. Following centrifugation to remove buoyant adipocytes, we observed a microvascular-rich vascular pellet. The cells in this pellet were viable and primary cultures could be established. Using culture conditions defined for human microvascular endothe-
F i g . 5. F l u o r e s c e n c e light m i c r o g r a p h s o f ( a ) P M V E C a n d ( b ) H S M C s t a i n e d f o r t h e presence a S M C a c t i n . P M V E C e x h i b i t e d b a c k g r o u n d staining f o r a S M C a c t i n u t i l i z i n g a f l u o r e s c e n t a n t i b o d y c o m p l e x . H S M C c o n t a i n e d dense b u n d l e s o f actin d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m . B a r = 10 um i n (a) a n d 4 um i n ( b ) .
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i c a l l y d e r i v e d a n d c u l t u r e d c a n i n e e n d o t h e l i a l cells. S u r g e r y 91:550-559; 1982. 4. H e r r i n g , M . ; G a r d n e r , A . L . ; G l o v e r , J . A single staged t e c h n i q u e f o r seeding v a s c u l a r g r a f t s w i t h a u t o g e n o u s e n d o t h e l i u m . S u r g e r y 84:498-504; 1987. 5. J a r r e l l , B . E . ; L e v i n e , E . ; S h a p i r o , S . ; W i l l i a m s , S . K . ; C a r a b a s i , R . A . ; M u e l l e r , S.; T h o r n t o n , S. H u m a n adult endothelial cell g r o w t h i n c u l t u r e . J . V a s e . S u r g . 1:757764; 1984. 6. J a r r e l l , B . E . ; W i l l i a m s , S . K . ; S t o k e s , G . ; H u b b a r d , F . A . ; Carabasi, R . A . ; Koolpe, E.; Greener, D.; Pratt, K . ; M o r i t z , M . ; R a d o m s k i , J . U s e o f f r e s h l y isolated c a p i l l a r y endothelial cells f o r the i m m e d i a t e establish ment o f a m o n o l a y e r o n a vascular g r a f t at s u r g e r y . S u r g e r y 100:392-399; 1986. F i g . 7. S c a n n i n g e l e c t r o n m i c r o g r a p h o f a s o d d e d e P T F E s u r f a c e . N o t e the cell extensions associated w i t h the p o l y m e r s u r f a c e . B a r = 10 am.
lial cells, the PMVEC cultures were routinely passaged. Finally, morphological evaluation and immunocytochemistry established that cultures derived from pig properitoneal fat maintained a phenotype typical of endothelial cells in culture. Freshly isolated PMVEC were amenable to vascu lar graft sodding techniques we have previously used with both canine and human EC. These cells remained adherent to the vascular grafts when exposed to a physiological shear (data not shown). The availability of PMVEC will permit the evaluation of the effective ness of endothelial cell transplantation towards the im provement in function of vascular prosthetic devices in a pig model. Future studies will evaluate PMVEC sodding of vascular prosthetic devices and the forma tion of new cellular linings on these devices following sodding techniques. REFERENCES 1. D i d i s h e i m , P.; D e w a n j e e , M . K . ; F i s k , C . S . ; K a y e , M . P . ; F a s s , D . N . A n i m a l models u s e f u l f o r p r e d i c t i n g clinical p e r f o r m a n c e o f b i o m a t e r i a l s f o r c a r d i o v a s c u l a r use. I n N o r e t o s , J . W . ; E d e n , M . , eds.: Introduction o f bioma terials t o clinical c a r e . P a r k R i d g e : N o y e s P u b l i c a t i o n s ; 1984. 2. D o u v i l l e , E . C . ; K e m p z i n s k i , R . F . ; B i r i n y i , L . K . ; R a m a l a n j o a n a , G . R . I m p a c t o f endothelial cell seeding o n long-term patency and subendothelial proliferation i n a small-caliber h i g h l y p o r o u s p o l y t e t r a f l u o r o e t h y l e n e g r a f t . J . V a s e . S u r g . 5:544-550; 1987. 3. G r a h a m , L . M . ; B u r k e l , W . E . ; F o r d , J . W . ; V i n t e r , D . W . ; K a h n , R . H . ; Stanley, J . C . E x p a n d e d polytetra f l u o r o e t h y l e n e vascular prostheses seeded w i t h e n z y m a t -
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