Patent Application: US-201213407900-A

Abstract:
scaleable , vascularised tissue constructs that are composed of a multiplicity of cell containing , discrete and separable modules , methods of fabricating same and uses thereof . the tissue construct is a tissue substitute used in tissue transplantation or substitution or for the purpose of in vitro mimic of normal tissue .

Description:
reference is first made to the figures , a detailed description of which is as follows : fig1 illustrates modular construct design and fabrication . ( a ) collagen with or without hepg2 cells is drawn into the lumen of a sterilized polyethylene ( pe ) tube and incubated at 37 ° c . for 30 minutes to allow gelation . the pe tubing containing the gel is fed through an automated tubing cutter and sectioned into 2 mm lengths which are collected in a sterile centrifuge tube . cell culture medium is added and the tube is vortexed to release the collagen - cell modules from the lumen of the sectioned pe pieces . pe sections float , while collagen modules sink . the collagen cylinders with embedded hepg2 cells are subsequently seeded with huvec . once complete coverage of the collagen surface with huvec has been achieved ( typically 2 - 3 days ), the cell - seeded cylinders are assembled into a larger structure ( here a tube ) to form the construct . assembly of the modules creates a network of interconnected channels that permeate the construct . medium or blood is perfused through this network to supply nutrients to the cells within the construct . ( b ) light micrograph of a collagen - hepg2 module before huvec seeding . ( c ) confocal microscopy image of ve - cadherin stained module indicating a confluent layer of huvec over the module surface at 7 days after seeding . ( d ) modular construct in the flow circuit being perfused with phosphate buffered saline . ( e ) confocal microscope image of a collagen - hepg2 - huvec module retrieved from a construct after 7 days of medium perfusion with hepg2 cells labeled with vybrant cfda se . fig2 : ( a ) flow and shear profiles through two collagen modular constructs . flow rate of ( pbs ) through two separate constructs ( construct length , 0 . 5 cm ; construct diameter , 0 . 3 cm ) as a function of applied pressure difference ( hydrostatic head ); open and filled points represent different constructs . each point is the mean of two flow rate measurements made at each pressure difference . the slope of the fitted line was used to calculate bed porosity using the ergun equation from which the shear stress on the surface of the modules was calculated ( insert ) for each construct . ( b ) microct image of microfil cast of a poloxamine modular construct ( without huvec ). light coloured regions correspond to the microfil ( ie the channels ) and dark regions correspond to modules , illustrating the interconnectedness of the flow channels that are normally lined with endothelial cells . porosity based on the number of light pixels was 22 . 6 %. fig3 : characterization of module thrombogenicity using whole blood studies . ( a ) clot formation times . the presence of huvec on the modules significantly increased the time to clot formation ( p = 1 . 4 × 10 − 5 ) of slightly heparinized whole blood ( 0 . 75 u / ml ) in a clotting test . in some cases clot formation never actually occurred and the test was terminated between 4500 and 5400 seconds ; in these instances the recorded time was the test termination time . mean clot time is represented by the thick central line within the box . open circles and stars represent outliers and extreme outliers respectively . ( b ) fresh whole blood ( 0 . 75 u / ml heparin ) perfused through an huvec - covered modular construct ( solid circles ) maintains platelet levels no different to those measured in the absence of modules ( open circles , flow circuit blank ; includes polypropylene mesh required to keep modules in place ). blood perfusion through a control modular construct in which huvec have been removed by dispase - collagenase action ( open squares ), however , results in significant reductions in platelet number indicating platelet activation and the thrombogenic response that occurs in the absence of huvec . error bars indicate the standard error of the mean ( n = 3 , 4 and 7 for background , dispase treated modular constructs and huvec covered modular constructs respectively ). fig5 : scanning electron micrographs of baec seeded modules 1 week after seeding . scanning electron micrographs of baec seeded modules one week after seeding showing the classic cobblestone morphology . good coverage of the modules with ec is achieved after 7 days in culture . fig6 : methacryloyl groups were added to the ends of poloxamine ( poloxamine methacrylate ) and then a solution of the poloxamine and collagen was photo - crosslinked to create an interpenetrating network . greater attachment of ec was obtained with a quaternized ( methylated ) poloxamine , to which methacrylolyl groups were then added . fig7 : live ( calcein am ) huvec seeded on methylated poloxamine - collagen modules ( left ) and film ( right ) 1 day after seeding . stiffness enables shape retention . the methylated poloxamine was combined with collagen ( as a semi - interpenetrating network ) and this resulted in very good ec attachment to modules . fig8 : masson trichrome staining of collagen modules in omental pouch in nude rats , showing channels in presence of huvec ( left ) but not in absence of huvec ( right ) fig9 : uea - 1 staining of huvec lined channels , day 7 , nude rat . left ( 7 wk rat ); right ( 5 wk rat ; high mag ). arrow shows vessel in cross - section fig1 : ( a ) ve - cadherin stained rat aortic ec on collagen modules , showing good coverage at day 7 after seeding ( b ) cfse staining of rat microvascular ec at day 11 . fibronectin was added to collagen gel to enable ec proliferation fig1 : bioluminescent images of luciferase transfected cho cells embedded in huvec covered modules in an omental pouch in nude rats at day 7 . collagen - hepg2 modules were fabricated ( fig1 a ) by gelling a solution of endotoxin - free collagen , containing suspended hepg2 cells , within the lumen of a small bore polyethylene tube . the tubing was then cut into 2 mm lengths using an automated cutter and gently vortexed to remove the cell - containing collagen modules from the tubing lumen . modules with different dimensions can be produced by using different tubing diameters and sectioning lengths . hepg2 cell viability within individual modules ( fig1 b ) was greater than 90 % and even with perfusion for 7 days ( fig1 e ) viable cell numbers were similar , if not greater than those of modules cultured under static conditions . after 7 days of culture , cell densities reached high values ( 0 . 3 - 1 × 10 8 cells / cm 3 ), depending both on cell growth and on module contraction , within an order of magnitude of cell densities within tissues ( 10 8 - 10 9 cells / cm 3 ). one day after fabrication , modules were seeded and incubated with huvec under static conditions . full surface coverage and shrinkage was achieved within 3 days ( fig1 c ). in some cases , huvec bridging of modules in close proximity was observed . huvec densities reached 4 . 5 ± 1 . 5 × 10 5 cells / cm 2 (& gt ; 90 % viable ) within 7 days , consistent with confluent huvec densities observed on tissue culture polystyrene . the quiescent , non - thrombogenic ec lining within the channels of the tissue construct is critical to enable whole blood to percolate around the modules with a significantly lower level of thrombosis than that associated with biomaterial surfaces . huvec are useful in this context since they express low basal levels of tissue factor , a potent coagulation initiator . collagen was selected for the module base material in the prototype as huvec naturally reside on a type iv collagen membrane , albeit not the same type as that ( type i ) which is readily available . it is possible to incorporate other extracellular matrix components , such as type iv collagen , elastin peptides and glycosaminoglycans , during module fabrication to further enhance the function of the modules . modules were randomly assembled into a tissue construct by pipetting a suspension of modules into a larger tube , acting as the enclosure ( fig1 d ). the modules produced from four meters of collagen filled pe tubing were sufficient to assemble a 0 . 5 - 1 . 0 cm long × 0 . 3 cm diameter construct ( construct volume of 0 . 038 - 0 . 075 cm 3 ). the interstitial spaces , formed between the assembled modules , constituted huvec - lined interconnected channels , on the order of a few hundred microns in size , that permeated the modular construct enabling fluid and particularly blood perfusion . the seeded endothelial cells were expected to control the dynamic balance of pro - and anti - thrombogenic factors to maintain continuous blood flow without thrombosis . we envision the modular construct , in an appropriate organ - like shape , will be connected to the vascular supply of the host using appropriate host vessels or artificial vascular grafts . tissue constructs were perfused at physiological pressure differences ( i . e & lt ; 100 mm hg ) with cell culture medium to simulate the flow of blood through a fully functional construct . pressure difference versus flow rate flow profiles , obtained for two separate modular constructs ( fig2 a ), were used to estimate construct porosity and shear levels within the channels of the constructs . analysis of these profiles using the ergun equation indicated construct porosity was 22 % and 24 % ( lower than expected , see below ) in the two constructs shown in fig2 a . using these porosity values , the average shear stress on the huvec ( fig2 insert ) was calculated to be in the range of 3 to 30 dyne / cm 2 depending on the flow rate . the channels within a similar modular construct ( prepared with a stiffer material ) are shown in a microct image in fig2 b . this illustrates the interconnectedness of the channels and the laminar , well - defined percolating flow profile in a modular construct . exposure to flow ( 24 hours at a flow rate 0 . 08 - 0 . 11 ml / sec / cm 2 ; shear approximately 2 - 3 dynes / cm 2 ) in the construct increased f - actin levels , and elongated and flattened the huvec on the module surface . the seeded endothelial cells maintained their non - thrombogenic phenotype as demonstrated by various assays , including ones involving whole blood perfusion . the tissue factor activity ( factor xa generation chromogenic assay ) of huvec seeded modules cultured under static conditions was low . huvec covered modules produced significantly longer times to clotting ( 0 . 75 u / ml heparin ; rocking platform arrangement ) than collagen only modules ( fig3 a , p = 1 . 4 × 10 − 5 ). in 9 out of 14 trials using huvec modules clotting had not occurred at test termination compared to 1 out of 15 trials for collagen only modules . the presence of the huvec significantly reduced the thrombogenicity of the module surface . lastly and most significantly , slightly heparinized ( 0 . 75 u / ml ) whole blood was perfused through the constructs at a rate of 0 . 334 ml / min ( equivalent to ˜ 7 dynes / cm ) and the effluent analyzed for platelet concentration ( fig3 b ). when constructs assembled from huvec covered modules were perfused , there was no reduction in platelet concentration relative to the background changes associated with the flow circuit itself ( i . e . measured in the absence of modules ). blood perfusion through collagenase - dispase treated huvec modules ( to remove the huvec layer after module shrinkage ) significantly reduced platelet concentration in the collected perfusate . the reduction in effluent platelet concentration is an indicator of thrombogenicity in the absence of huvec ; the absence of this reduction ( relative to the background ) is an indicator of the functional efficacy of the huvec seeded modules in inhibiting platelet activation . obvious thrombus formation ( at 30 minutes ) was seen in the majority of flow circuits without endothelial cells , but not when endothelial cells were present . the presence of the huvec significantly reduced the thrombogenicity of the construct . the potential for scaleability arises because , unique to the modular approach , the underlying design principles can be delineated . the three main constraints that influence the design of the modular construct are : nutrient supply ; incorporating clinically significant numbers of cells within a construct of implantable volume ; and the shear force on the huvec layer . nutrient supply , determined by mass transfer within the construct , was estimated not to be a significant design constraint . channel dimensions are expected to be of the same size as the modules ( i . e ., on the order of a few hundred microns ) allowing good oxygen mass transfer , the likely limiting nutrient , within the construct channels . moreover , hepg2 cells remained viable within an assembled construct over 7 days , suggesting mass transfer to the encapsulated cells was sufficient , at least for the cell seeding density and module size used . since , it has been predicted that a patient could survive on 10 % of normal liver function , an engineered liver with the cell densities achieved in our construct ( 3 - 10 % of tissue densities ), could conceivably have sufficient cell mass to support patient survival . we have demonstrated the use of microscale modular components in a biomimetic fashion to assemble uniform , potentially scaleable ( micro ) vascularized tissue - engineered constructs containing multiple cell types which were perfused with whole blood . the current prototype enabled maintenance of cell viability , at high cell densities and whole blood perfusion with minimal blood activation . modular tissue assembly is a biomimetic alternative to traditional scaffold based strategies , which offers many advantages for engineering whole organ and large tissue grafts and potentially transforms the conventional cell seeding / porous scaffold paradigm of tissue engineering . in an alternative embodiment , gelatin modules (˜ 120 μm diameter × 1 mm long ) containing hepg2 spheroids were prepared inside a glass micropipette ( 0 . 282 mm id , drummond microcap ) prewashed with pluronic l101 . hepg2 spheroids were prepared by culture in αmem with serum on bacteriological polystyrene culture dishes for 4 days ; at this time spheroids were approximately 100 μm in diameter and contained roughly 300 cells each . spheroids were suspended in 55 μl of 300 bloom , type a gelatin ( 25 wt %) liquid (˜ 40 ° c .) and a droplet of the gel - spheroid suspension was placed onto a sterilised glass slide , from which it was drawn into the glass micropipette . after 20 - 30 minutes refrigeration , ( enough time to ensure gelation ) the gel - spheroid modules were expelled from the glass capillary into a sterile solution of very dilute glutaraldehyde ( 0 . 05 %) in pbs . after 20 minutes the modules were washed twice in pbs followed by a 1 - 2 hour wash in cell culture medium . a 20 minute exposure time was sufficient to cross - link the gelatin so that the rods did not fall apart when incubated at 37 ° c ., yet avoided prolonged exposure of the cells to glutaraldehyde . modules were cut by hand under the microscope into 1 mm lengths , although the automated cutting device used for collagen gel ( above ) could be used here as well . despite the various manipulations and especially the brief exposure to very dilute glutaraldehyde , the cells appeared to remain largely viable based on mtt conversion ( the spheroids became purple ) and confocal microscopy with the live / dead cell assay , at least for 9 days after fabrication . not surprisingly the central core of the spheroids remained viable while the outer rim had dead cells , presumably reflecting the effect of the glutaraldehyde . modules were randomly packed without difficulty in 2 mm id pe tubes , capped with a mesh . packing gelatin rods into the pe tubing ( the enclosure ) was done by pouring a slurry of the gel rods suspended in pbs ( phosphate buffered saline ) into the larger diameter tube . a nylon mesh filter ( millipore , pore size 100 μm ) was used at the bottom of the tubing to retain the rods ; a similar one was mounted on the top to create the tubing construct . endothelial cells were seeded onto the gelatin rods after loading the rods into the polyethylene tube much as vascular grafts are seeded . the assembled construct was filled with an ec suspension ( 2 - 4 × 10 5 cells / cm 2 of gelatin or 1 . 2 - 2 . 5 × 10 7 cells / cm 3 for a 2 mm diameter × 5 cm tube with 70 % porosity ) and the construct “ soaked ” in cell suspension for 2 hours to enable the ec to settle and adhere to the gelatin . after gentle rinsing , subsequent static culture for 1 to 4 days was sufficient to reach confluence . the soaking conditions ( time , ec concentration ) is optimised based on the number of cells retained by the gelatin rods and the uniformity of coverage . alternatively , ec were seeded on the gelatin rods prior to assembly into the pe tube ( the enclosure ) under static conditions in 24 well non - tissue culture plates ( to minimise adhesion to the plate itself ). the cells are allowed to adhere to the gelatin ( 1 - 4 hour incubation ) and then cultured to reach confluence over 4 days . some agitation of the rods within the 24 well plates is needed to obtain reasonably uniform coverage . the cells readily adhered as expected . the ec covered modules were then loaded into the pe tube as above ; some loss of ec may occur during this loading step , necessitating a brief incubation (& lt ; 1 day ) to restore a monolayer . another means of preparing modules was to push a film of gel containing embedded cells or spheroids , through a 250 μm sieve ( fig4 ). gelatin modules containing cells or spheroids were produced in this way . cross - linking ( 25 minutes , 0 . 025 % gta ) resulted in approximately 200 × 200 μm × 500 μm long rectangular modules and was sufficient to maintain module integrity upon incubation at 37 ° c .. and prevent agglomeration within the culture dish . gelatin ( 300 bloom , type a , sigma - aldrich canada , oakville on ) was cast from a 20 wt % solution at 40 ° c . onto a teflon disk inserted in a 30 mm petri dish . hepg2 cells or spheroids were added to the gelatin prior to casting ( 1 . 4 - 1 . 8 × 10 7 cells / ml ). the cast gel was chilled for 5 minutes at 4 ° c . in a refrigerator and then removed from the petri dish mold and placed on a rubber sheet ( fig4 ). the cast gel and rubber sheet were then inverted over a 60 mesh ( 250 μm gap size ) stainless steel sieve ( ws tyler , st . catherines on ) and a glass rod was rolled over the top of the rubber sheet to push the gelatin through the mesh . a glass cover slip was used to collect the extruded gel modules from the lower side of the mesh and transfer them into a 0 . 025 % glutaraldehyde ( gta , sigma - aldrich canada , oakville on ) aqueous solution . modules were cross - linked for 25 minutes and then washed 3 times in phosphate buffered saline ( pbs , university of toronto tissue culture media preparations ), incubated in medium for 1 hour and then transferred to fresh medium after which module cultures were fed every two days . embedded cells or spheroids remained in the gelatin and no obvious migration out of the modules was observed . in some cases , modules did not separate completely from adjacent ones during the sieve fabrication step . the resulting large agglomerates did not cross - link fully during the gta treatment and dissolved on incubation at 37 ° c . pipetting the modules several times through a disposable 1 ml pipette tip before cross - linking improved module separation and reduced the number of agglomerates . the hoechst dna assay indicated that 1 ml of modules contained ˜ 8 . 9 ± 0 . 4 × 10 5 cells when a cell suspension was embedded or 5 ± 0 . 4 × 10 4 cells when spheroids were used . manual counting lead to higher numbers , (˜ 14 . 0 ± 1 . 4 × 10 5 cells or 9 . 1 ± 1 . 8 × 10 5 cells respectively ), although of the same order of magnitude . glutaraldehyde cross - linking was necessary to generate dimensionally stable modules but it was necessary to minimize gta exposure in order to minimize the loss of cell viability . we tested the viability of cells encapsulated within gelatin during cross - linking ( direct exposure to gta ) and the viability of cells cultured on the surface of gelatin films that had previously been cross - linked with gta and subsequently washed to remove any gta solution ( indirect exposure due to residual leakage ). for cells embedded in cross - linked gelatin films viability was 11 ± 8 %, using cck - 8 , after cross - linking with 0 . 025 % gta for 10 - 20 minutes . in modules ( 0 . 025 % gta , 25 min cross - link ) viability was 40 ± 5 % relative to non cross - linked modules , measured using the alamar blue assay . a more significant loss of viability was seen for spheroids embedded within the gelatin during cross - linking . using cck - 8 , viability was 5 - 10 % for spheroids after cross - linking ( 0 . 025 % gta , 10 - 20 minutes ) and was even lower at higher gta concentrations . using the alamar blue assay , spheroids in modules ( 0 . 025 % gta , 25 min cross - linking ) had a viability of 30 %± 1 % relative to non - encapsulated spheroids . endothelial cells cultured on top of 0 . 025 % gta cross - linked gelatin films had greater than 80 % viability ( cck - 8 or alamar blue ) for films cross - linked for 30 min , independent of passage number ( p5 - p9 ). thus leaching of residual gta did not appear to be a significant problem under the cross - linking conditions ( 0 . 025 % gta , 30 min ) necessary to stabilize module integrity . the characteristic baec cobblestone morphology was observed using sem ( fig5 ). baec adhered well to gelatin films . centrifugation resulted in detachment of & lt ; 30 % at 400 or 1000 rpm for one clone . results were slightly different for passage number and were slightly greater at higher cell densities ( 50 , 000 cells per well in a 96 well plate ). there was no significant difference relative to cells cultured on tcps . furthermore the action of pipetting the modules through a pipette tip , which was expected to generate significant shear on the module surface , did not detach the ec or beak up agglomerates of modules bridged by ec , indicating significant adhesion to the gelatin substrate . in another embodiment modules were prepared using a synthetic collagen - mimetic material that was stiffer than collagen ( and therefore resistant to compaction ) but that like collagen allows both cell encapsulation and cell growth on the surface . this collagen - mimetic material was a poloxamine - collagen semi - interpenetrating network error ! bookmark not defined . ; poloxamine is a four - arm peo - ppo block copolymer derivative , tetronic ™ 1107 . methacryloyl groups were added to the ends of the poloxamine ( fig6 ) and a solution of the poloxamine with collagen also in the same solution was photo - crosslinked . cells ( hepg2 ) were embedded easily and at high viability ( sosnik et al ., tissue eng ., 2005 ). the poloxamine - collagen material was much stiffer ( 2 , 000 to 7 , 000 pa for polymer concentrations between 6 to 8 %) than collagen alone (˜ 50 pa ) as was evident also in the cylindrical shape of these modules which was preserved through many weeks of culture . a positively charged poloxamine hydrogel was also prepared by grafting quaternary ammonium groups in the poloxamine network through a photo - initiated free radical copolymerization of mixtures of poloxamine - methacrylate and ([ 2 -( methacryloyloxy ) ethyl ]- trimethylammonium chloride ( maetac ) ( sosnik et al ., j . biomed . mater . res . part a , 2005 ). the modification resulted in good huvec attachment and confluent monolayers were achieved on films and modules . this material was not suitable for cell encapsulation due to acute cell death associated with exposure to maetac during embedding . following the same quaternization strategy , but with a focus on reducing the cytotoxicity for cell encapsulation by reducing the concentration of reactive methacryloyl derivatives , the tertiary amine groups of poloxamine were methylated with iodomethane — eliminating the need for maetac . this derivative was subsequently reacted with methacryloyl isocyanate , producing positively - charged materials ( fig6 ) that were further crosslinkable by a photointiated free radical polymerization error ! bookmark not defined . . a gradual increase of both the storage modulus ( g ′) and the loss modulus ( g ″) resulted from increasing the polymer concentration : for example , g ′ values were as high as 23 , 000 pa for 18 % methylated poloxamine - methacrylate hydrogels ( at 1 hz , 100 pa of oscillatory stress ), compared again to ˜ 50 pa for collagen gels . hepg2 cells embedded in different compositions and exposed to u . v . light displayed good viability levels after the crosslinking , unlike the maetac approach . a well - spread endothelial cell morphology was apparent on methylated poloxamine films after pre - incubation in serum containing medium . the methylated poloxamine was also combined with collagen ( as a semi - interpenetrating network ) and this resulted in very good attachment to modules ( fig7 ). the methylated poloxamine displays the attributes that make it a useful material for modular tissue engineering . degradable versions of these modified poloxamines can be prepared by introducing lactic acid groups into the poloxamine prior to the addition of methacroyl groups . this results in stiff poloxamine based modules , that shrink and change in shape ( over a few days ) as the poloxamine derivative degrades . human umbilical vein smooth muscle cells ( uvsmc , a cell - line , atcc , manassas , va .) were embedded in collagen gel modules as described in embodiment 1 . the cells were cultured in 10 % fetal bovine serum ( fbs , sigma , st . louis , mo .) supplemented medium consisting of f - 12k kaighn &# 39 ; s modified medium ( gibco , burlington , on ) further supplemented with 0 . 1 mg / ml heparin , 0 . 03 mg / ml endothelial cell growth supplement ( ecgs , bd bioscience , franklin lakes , n . j . ), 1 % penicillin and streptomycin solution ( gibco ). to induce uvsmc quiescence , the medium for a confluent uvsmc layer was replaced with quiescence medium ( qm ), identical to that used above for uvsmc but without serum . modules containing both uvsmc and huvec were cultured in the egm - 2 medium supplemented with the bullet kit and 0 . 03 mg / ml endothelial cell growth supplement . embedded smooth muscle cells ( smc ) showed normal morphology ( f - actin staining ) and protein expression ( calponin , sm myosin heavy chain by western blot ). smc contractile state was sensitive to serum withdrawal ( leung , 2005 ): embedded smc phenotype ( as determined by presence or absence of serum ) affected huvec junction morphology ( ve - cadherin expression ) consistent with the predictions from other smc - huvec co - culture systems ( armulik et al ., 2005 ). smc phenotype also appeared to affect subsequent huvec proliferation rate as assessed by brdu uptake assay . hepg2 cells were useful as model cells in early studies and the smc - huvec system enabled further exploration of the modules as a co - culture system . huvec covered modules were implanted into an omental pouch , an enclosure to be filled with modules , in nude rats . the omental pouch is prepared by folding the omentum up towards the stomach and suturing ( 7 ‘ o ’ silk sutures ) along the left and right edges of the omentum and along the top of the pouch but leaving an opening for the placement of the modules . modules , suspended in pbs are placed into the omental pouch using a sterile 1000 μl micropipette tip , while preaggregated modules ( e . g ., prepared by incubation at high density in a small well ) are placed into the pouch with tweezers . the opening is sutured closed to completely enclose the modules . fig8 shows that collagen gel modules ( in green ) coated with huvec have channels ( see arrow , order of 100 μm in “ width ”) that persist up to 21 days after implantation in the omental pouch . without huvec the collagen modules remodel and do not appear to form channels . some of these channels ( fig9 right ; uea - 1 lectin staining , ulex europaeus agglutinin i , vector laboratories ) appear to have erythrocytes within the lumen . to avoid the apparent immune response to xenogeneic ec microvascular rat ec are seeded onto collagen gel modules . a simple modification ( inclusion of fibronectin into the collagen gel ) has resulted in good rat ec attachment to and junction formation on collagen modules ( fig1 ), although these must be incubated for 11 days instead of 3 - 7 before confluent modules are obtained . in order to track the viability of the cells without sacrificing the animal we prepared ( by retrovirus ) luciferase stably - transfected cho cells , embedded them in collagen modules , seeded them with huvec and implanted them in an omental pouch in the nude rat . injecting luciferin ip , the xenogen cooled ccd camera was used to detect , through the skin , the weak emitted light ( fig1 ). modules can fill a liver enclosure through intraportal infusion similar to clinical islet transplantation methods ( bottino et al ., 1998 ). in a rat , a midline incision exposes the peritoneal cavity and the underlying portal vein . modules are loaded into a catheter attached to a 1 ml syringe and injected into the vein via a 25 - gauge needle . manual compression is used to minimize bleeding at injection site . it will be understood that , although various features of the invention have been described with respect to one or another of the embodiments of the invention , the various features and embodiments of the invention may be combined or used in conjunction with other features and embodiments of the invention as described and illustrated herein . although this disclosure has described and illustrated certain preferred embodiments of the invention , it is to be understood that the invention is not restricted to these particular embodiments . rather , the invention includes all embodiments that are functional , electrical or mechanical equivalents of the specific embodiments and features that have been described and illustrated herein . the human hepatoma cell line , hepg2 ( american type culture collection , rockville , md .) was cultured in 25 cm 2 tissue culture flasks in rpmi 1640 culture medium with l - glutamine ( invitrogen canada , burlington , on ) supplemented with 15 % bovine calf serum ( hyclone , logan , utah ) and 2 % penicillin and streptomycin ( invitrogen canada , burlington , on ) at 37 ° c . in a 5 % co 2 / 95 % air humidified atmosphere . human umbilical vein endothelial cells ( huvec , cambrex bio science walkersville , inc ), were cultured in 75 cm 2 tissue culture flasks in egm - 2 medium suggested by the suppliers supplemented with egm - 2 bullet kit ( cambrex bio science , walkersville , inc ) at 37 ° c . in a 5 % co 2 / 95 % air humidified atmosphere . in modules where both cell types were present , both cell types were cultured in huvec culture medium . vitrogen collagen solution ( type i , bovine dermal , 3 . 1 mg collagen per ml ; cohesion technologies , palo alto , calif .) was mixed with 10 × minimum essential medium ( invitrogen canada , burlington , on , 125 μl 10 × medium per ml collagen ) and neutralised using 0 . 8 m nahco 3 ( sigma - aldrich canada , oakville , on ). pelleted hepg2 cells were mixed with the neutralised collagen ( 2 × 10 6 cells / ml ) and the solution drawn into the lumen of an ethylene oxide gas sterilized polyethylene tube ( 0 . 76 mm id × 1 . 22 mm od ) connected to a syringe at one end . after 30 minutes incubation , to allow collagen gelation , the gel - filled tubing was cut into 2 mm lengths using a custom - built automated cutter ( fig1 a , fcs technology , london on ). sections were vortexed gently in cell culture medium to remove the gel - cell module cores from the tubing lumen . the collagen - cell modules were allowed to settle , separated from the polyethylene tubing and cultured in petri dishes under static conditions . collagen only modules were fabricated identically ( same collagen concentration ) without the addition of the hepg2 cell pellet . huvec ( p1 - 6 , 1 . 5 - 2 . 0 × 10 6 cells per ml of settled modules ) were added to modules with or without encapsulated hepg2 cells in a 15 ml centrifuge tube and incubated for 60 minutes with gentle shaking every 10 minutes . modules were then transferred into a non - tissue culture polystyrene petri dish . medium was replaced every 1 - 3 days . after incubation overnight a sample ( n = 96 ) of modules containing hepg2 cells was selected and light microscopy images were taken of each module in a 96 well plate ( one module / well ) using an olympus microscope . modules were then seeded with approximately 1 . 5 × 10 6 huvec per ml of settled modules , and incubated for 4 days , after which they were re - imaged . measurements of module diameter and length , before and after endothelial cell seeding , were made using imagepro software ( media cybernetics , san diego calif .). cell metabolism of encapsulated cells was measured using the alamar blue ( ab ) assay at days 1 , 3 and 7 . briefly a micropipette was used to add 10 modules ( 3 replicates ), containing hepg2 cells , in a 200 μl volume , into a 24 well plate . 10 % ab ( biosource international , inc . camarillo , calif .) was added and the sample incubated for 7 hours . supernatant samples were transferred into a 96 well plate and read using a sunrise elisa plate reader ( tecan , maennedorf , switzerland ) at 570 nm and 600 nm . module samples were then digested using collagenase ( sigma - aldrich canada , oakville , on , final concentration 0 . 236 mg / ml in culture medium ), incubated overnight and stained with trypan blue . the numbers of live and dead cells were counted manually using a hemocytometer . to assess cell viability , within the assembled construct , modules containing hepg2 cells or collagen only modules seeded with huvec were cultured under static conditions for 6 days and then within a flow circuit ( see below ) for 24 hours . modules were retrieved from the circuit and tested immediately for viability by digestion and staining with trypan blue as above . the viability of hepg2 cells cultured within an assembled construct for 1 week was assessed using vybrant cfda se prelabelled cells ( 10 μm , carbofluorescein diacetate succinimidyl ester , molecular probes , burlington , on ). one day after fabrication the hepg2 modules were seeded with huvec and then after 2 days incubation , to allow module shrinkage , were assembled into a construct within a flow circuit . after 1 week of medium perfusion modules were retrieved from the flow circuit , fixed in 3 . 7 % paraformaldehyde - pbs ( electron microscopy science , hatfield , pa .) for 30 minutes , washed in pbs and observed using fluorescence microscopy ( zeiss , axiovert 135 ). fifty ml centrifuge tubes with two holes punctured in the cap through which to thread masterflex l / s - 13 and l / s - 16 tubing ( labcor , anjou , qc ) and approximately 0 . 015 g of glass wool (˜ 0 . 075 cm 3 ) ( to hold the modules in place ) were assembled into a continuous loop flow circuit with a number of other connectors and stopcocks ( various suppliers ). a masterflex peristaltic pump was used to circulate medium through the flow loop from a 19 ml reservoir . modules ( 0 . 5 - 1 . 0 ml ) were loaded into the circuit ( total circuit volume 20 ml ), within a laminar flow hood , using a 10 ml pipette via a luer lock connector . modules were maintained in the flow circuit at 37 ° c . in a 5 % co 2 / 95 % air humidified atmosphere for 24 hours or 1 week . medium was added to the reservoir every 1 - 2 days . pressure difference across the construct was recorded using low pressure gauges ( h . o . trerice , oak park , mich . ), inserted on either side of the construct . duplicate measurements of flow rate through the construct were measured for a range of pressure differences , by the timed collection of 0 . 5 ml medium from the circuit via a t - connector output . the gradient of flow rate versus pressure difference ( darcy &# 39 ; s permeability ) was calculated and the gradient of similar curves measured in the absence of a construct ( i . e ., with glass wool only present ) was subtracted to isolate the pressure difference contribution from the construct . the ergun equation was then solved for porosity by iteration using the solver program in microsoft excel . the values used for constants present in this equation were , length of construct 0 . 5 cm , fluid viscosity 0 . 01 g / cm 3 , module diameter 0 . 0411 cm and shape factor 0 . 874 . a construct of the same diameter and length was prepared using modules of a stiffer material ( 20 % poloxamine - collagen modules error ! bookmark not defined . ), and so enable perfusion with the viscous microfil solution (“ low viscosity ”, flow tech , inc . carver , mass . ; component : diluent ratio of 4 : 15 , 10 % curing agent ) used for microct ( mice imaging centre ( mice ), hospital for sick children , toronto ). using microview software the number of pixels above the threshold corresponding to the microfil was used to calculate the volume fraction of microfil and hence the construct porosity . fresh whole blood ( 10 ml ) was collected from consenting donors ( with ethics approval by the university of toronto ), who had not taken medication within 72 hours of phlebotomy , into a syringe containing heparin ( final concentration 0 . 75 u / ml ), after discarding the first ml . a 350 μl sample of slightly heparinized blood was mixed with 200 μl of collagen modules or huvec coated modules in a microcentrifuge tube . a 400 μl sample of this was then pipetted into a 25 cm length of polypropylene tubing ( 1 . 57 mm id ) connected at either end via silastic ™ tubing ( 1 . 57 mm id ) to 200 μl pipette tips connected to a rocking platform ( gemmell et al ., 1995 ). rocking was initiated and the time until blood motion ceased or significant clot deposition occurred within the tubing was recorded as the clotting time . constructs were assembled from huvec covered modules or huvec covered modules treated for 15 minutes in 100 mg / ml collagenase dispase solution ( roche , mississauga , ontario ) to remove all huvec from the surface ( for control modules ), yet retain the size and stiffness of the contracted collagen . the short treatment time ensured module dissolution did not occur and microscope observation confirmed removal of the huvec layer . constructs were assembled within a 0 . 2 ml length of a 1 ml graduated pipette and held in place at both ends with 1 cm 2 sections of polypropylene mesh ( ppm - 3 , biomedical materials , slatersville , r . i .). silastic ™ tubing ( 10 cm , 3 . 18 mm id ) was used to connect the pipette section to the syringe pump ( 824e infusion pump model a - 99 , razel scientific instruments inc ., fairfax vt .). the construct was pre - filled with pbs to prevent air bubble formation . blood was collected in a 10 ml syringe from consenting volunteers that had taken no medication into 0 . 75 units / ml heparin . it was necessary to use a small amount of heparin ( 0 . 75 u / ml is much less than the 5 u / ml needed to stop all coagulation ) to prevent premature clotting during the blood draw or while the blood was sitting in the syringe pump . the silastic ™ tubing was filled with blood from the syringe before being connected to the pbs pre - filled pipette / construct section . the syringe was then placed on the syringe pump located on a rocking platform ( to minimize blood settling ) within a 37 ° c . oven and blood was perfused through the construct at a rate of 0 . 334 ml / min . at regular intervals during perfusion , 400 μl samples of the perfusate were collected in 0 . 6 ml graduated microcentrifuge tubes containing 8 μl of 200 mm edta . an initial sample was collected from the syringe before connecting it to the construct . the experiment was terminated when all the blood from the syringe had been used or if circuit blockage occurred . constructs were removed and dissected for evidence of thrombus formation within the construct or the polypropylene mesh . the students t - test was used to determine significant difference when only 2 treatment groups were being compared . analysis of variance ( anova ) was used to test for significant differences among multiple test groups . q - q plots were used to assess the normality of the data . the levene &# 39 ; s test for homogeneity was used to test for equal variance among samples . when equal variance could be assumed the tukey hsd post - hoc test was used to identify significant differences among multiple test groups . when equal variance could not be assumed the games - howell post - hoc test was used to identify significant differences among multiple test groups . in all tests were two - tailed and a p - value of 0 . 05 was considered significant . the human hepatoma cell line , hepg2 ( american type culture collection , rockville , md .) was cultured in 25 cm 2 tissue culture flasks using a - mem culture medium ( university of toronto tissue culture media preparations ) supplemented with 10 % fetal bovine serum , 100 u / ml penicillin and 100 ng / ml streptomycin ( invitrogen canada , burlington on ), at 37 ° c . in a 5 % co 2 / 95 % air humidified atmosphere . spheroid cell aggregates were formed by the addition of 0 . 5 ml of cell suspension ( 3 × 10 6 cells / ml ) to 4 . 5 ml cell medium in 60 mm non - tissue culture polystyrene dishes , ( fisherbrand ). bovine aortic endothelial cells ( baec ) were harvested using the method of jaffe el al ., 1973 , in the laboratory of . dr . p . marsden , and cultured on 60 mm tissue culture polystyrene dishes coated with 0 . 2 % gelatin ( sigma - aldrich canada , oakville , on ) using rpmi 1640 culture medium with l - glutamine ( invitrogen canada , burlington on ) supplemented with 15 % bovine calf serum ( hyclone , south logan , utah ) and 10 % penicillin and streptomycin at 37 ° c . in a 5 % co 2 / 95 % air humidified atmosphere . within a week of cell harvest from the animal , single clones were selected using a cloning tube and then expanded . cells were fed every two days and sub - cultured once per week with a 5 : 1 splitting ratio . two or three clones , each from different aortas , were used for each set of experiments to highlight differences among clones . the cell density within the modules was determined by measuring the amount of dna in proteinase k digested gelatin samples using hoechst 33258 ( molecular probes , eugene , oreg .) with a gemini xs fluorescent plate reader . gelatin modules were frozen in cryovials using liquid nitrogen , freeze dried and digested in proteinase k solution ( 0 . 5 mg / ml proteinase k and 0 . 1 mg / ml sds in a buffer solution of 50 mm tris - hcl , 0 . 1 m edta , 0 . 2 m nacl , ph 7 . 4 ) for 15 h at 55 ° c . with gentle shaking . samples were aliquoted with hoechst dye solution ( 10 mm tris , 1 mm edta . na 2 2h 2 o , 0 . 2m nacl , 0 . 1 μg / ml hoechst 33258 dye ) in equal volumes into a black fluorescence plate and fluorescence was read at an excitation wavelength of 360 nm and an emission wavelength of 465 nm . samples containing known numbers of cells were used for calibration . dna content was also calibrated with calf thymus dna ( sigma - aldrich canada , oakville on ). for comparison cell density was also determined manually by digesting gelatin modules with collagenase ( 1 . 25u / ml , molecular probes , eugene , oreg .) at 37 ° c .. for 6 - 12h . the released cells or spheroids were pelleted , washed in pbs , incubated with trypsin and counted using a hemocytometer . cell viability was assessed using either the tetrazolium based cck - 8 assay ( dojindo molecular technologies , inc ., gaithersburg , md .) or alamar blue assay ( ab , biosource international , inc . camarillo , calif .). briefly , the test solution was added to the cells , ( 10 % test solution for both cck8 and ab ) and incubated for 3h ( cck - 8 ) or 6 . 5h ( ab ). aliquots of the medium were transferred into a fresh 96 well plate and solution absorbance read in a versa max plate reader at 450 nm ( cck - 8 ) or 570 nm and 600 nm ( ab ). the viability of cells cultured on cross - linked gel films was assessed to evaluate the indirect effect of any long term gta leaching from the gels . gelatin films , ( cross - linked with different gta concentrations and reaction times ), were washed 3 times in pbs , incubated for 1 h in medium and then incubated in fresh medium overnight . baec or hepg2 cells were seeded on the cross - linked films at densities between 5 × 10 3 and 50 × 10 3 cells per well and incubated overnight . viability was measured using cck - 8 or alamar blue and reported relative to tissue culture polystyrene ( tcps ). the direct effect of gta was assessed by measuring the cck - 8 viability of hepg2 cells ( as a suspension or as spheroids ) within gelatin films which were cross - linked with different gta concentrations for different times while the cells were present . the effect of gta cross - linking on a gelatin module ( not a film ) was tested using alamar blue . viability was calculated relative to monolayers of cells on tcps or to non cross - linked modules . baec ( 1 - 2 × 10 6 cells ) were added to 5 ml of settled gelatin modules ( no hepg2 cells ) suspended in culture medium in a 60 mm non - tissue culture polystyrene petri dish . cultures were incubated and observed daily using optical microscopy . after 7 days or when a confluent layer of ec had formed , modules were prepared for sem analysis using a method slightly modified from one described previously by wissemann el al ., 1985 . modules were washed in pbs , fixed in 4 % gta on ice for 1 h and then transferred to a gelatin coated glass cover slip . after a 1 h treatment on ice with 10 % gta , samples were serially dehydrated in ethanol , frozen in liquid nitrogen and freeze dried . after gold coating , samples were examined using a hitachi s - 570 scanning electron microscope at an accelerating voltage of 20 kv . baec growth on gelatin films was assessed both manually and using the ab assay . baec were seeded ( 10 - 40 × 10 3 cells per well ) on tcps and gelatin films ( cross - linked for 30 min or 120 min with 0 . 025 % gta ) in 96 well plates and incubated either overnight or for 5 days prior to manual counting ( after trypsinization ) or ab assay . the adhesion strength of baec to gelatin films was compared to that on tcps using a centrifugation assay . baec ( p5 to p8 ) were seeded at 10 to 25 × 10 3 cells / well ( 96 well plate ) and incubated overnight to effect adhesion . wells were then washed and filled with pbs . pressure sensitive film was used to seal the pbs within the wells and the plates were inverted . cck - 8 was used to quantify the number of cells per well after centrifugation at 400 or 1000 rpm for 6 min . adherence was calculated by comparison with a static control . anova analysis was used to test for significant differences ( p & lt ; 0 . 05 ) in experimental parameters for multiple test groups . post hoc analysis was performed using the tukey honest significant difference test ( α = 0 . 05 ). the student t - test ( p & lt ; 0 . 05 , 2 tailed ) was used to test for the significance between groups when only 2 test groups were being compared . modules were fabricated as in embodiment 1 . briefly , acidified bovine type i collagen solution ( 3 mg / ml , vitrogen ™, cohesiontech , palo alto , calif .) was mixed with 10 × mem medium ( gibco ) and neutralized with sodium bicarbonate solution ( sigma ), uvsmc were trypsinized and resuspended in the neutralized collagen solution at the desired cell density and the mixture was drawn into gas sterilized polyethylene tubing ( becton dickson , intramedic ™ brand , pe60 , i . d ./ o . d .= 0 . 76 mm / 1 . 22 mm ) and incubated at 37 ° c . for 1 hour . upon gelation , the tubing was removed from the incubator and cut into 2 mm sections ; using an automated custom built cutter . these sections were collected in a 50 ml polypropylene centrifuge tube containing 30 ml of uvsmc culture medium . the collagen modules were then separated from the tubing by gentle vortexing and were collected at the bottom of the centrifuge tube . collagen - only modules were fabricated similarly without the addition of cells . for endothelial cell seeding , 2 million huvecs were used for every 4 meters of tubing prepared , equivalent to approximately 10 ml of settled modules . trypsinized huvecs were mixed in 10 ml of huvec medium in a 15 ml centrifuge tube with modules . the centrifuge tubes were left at room temperature on a uniaxial rocker ( bellco biotechnology , vineland , n . j ., cat # 7740 - 10010 ) for 30 minutes , after which the modules were transferred into a 100 mm non - tissue culture treated petri dish and left in the incubator for 24 hours . modules were transferred to a new non - tissue culture treated petri dish 24 hours post seeding and cultured with fresh huvec medium , with or without ecgs depending on the presence or absence of uvsmc , respectively , for up to 14 days . to prepare for immunofluorescence imaging , modules were removed from the culture medium , rinsed with pbs ( ph = 7 . 4 ) and fixed for 30 minutes in 4 % paraformaldehyde solution ( sigma ), followed by incubation in 0 . 2 % triton x - 100 solution for 4 minutes ( to permeabilize the cell membrane ) and pbs rinses ( 3 × 10 minutes ). the samples were incubated with primary antibodies at 1 : 50 dilution in pbs , in the dark at room temperature for 30 minutes . to assess huvec junction morphology , the modules were stained with anti - human ve - cadherin polyclonal rabbit igg ( sigma ). smooth muscle cell phenotype was characterized by staining with anti - human smooth muscle alpha - actin monoclonal mouse igg ( sigma ). fitc conjugated anti - brdu monoclonal mouse igg ( santa cruz biotechnology , santa cruz , calif .) was used to assess cell proliferation ( see below ). after primary antibody incubation , the samples were washed with pbs ( 3 × 10 minutes ) followed by complementary secondary antibody staining at 1 : 200 dilution in pbs for 30 minutes . secondary antibodies with alexafluor ™ dyes were purchased from molecular probes , burlington , on ( alexafluor ™ 488 goat anti - rabbit igg and alexafluor ™ 568 goat anti - mouse igg ). samples were visualized using a biorad laser scanning confocal microscope ( max - bell research centre , uhn , toronto , on , model mrc - 1024es ) equipped with a motorized z - plane stage . control of the laser scan head and data collection were managed by the software package provided by biorad ( lasersharp , version 3 . 2 ). typically 20 z - section slices were collected and projected for each composite image . all images were exported as uncompressed . tiff format files for data analysis . to determine the viability of both embedded uvsmc and surface seeded huvec in collagen modules , a two color live / dead ™ assay was used ( molecular probes ). all procedures were performed following manufacturer recommendations . the staining solution consisted of 4 μm of calcein - am and 4 μm of ethd - 1 in 1 × pbs . modules were incubated ( 5 % co 2 , 95 % air , 100 % humidity ) at 37 ° c . for 20 minutes in staining solution followed by pbs rinses ( 3 × 10 minutes ). stained modules were visualized using confocal microscopy as described above . proliferation of embedded and surface seeded cells was determined using 5 - bromo - 2 ′- deoxyuridine ( brdu ) incorporation assay . stock solution of brdu was prepared by dissolving brdu powder ( molecular probes ) in sterile dimethyl sulfoxide ( dmso , sigma ) at a concentration of 10 mm . modules were incubated in a 1 : 1000 dilution of brdu stock solution in ec medium for 2 hrs ( final concentration of brdu is 10 μm ), followed by immunofluorescent analysis as above . cells were counterstained with propidium iodide at 10 μg / ml in 1 × pbs ( sigma ), so that proliferating cells were double stained for convenient identification . 1 . a . armulik , a . abramsson , and c . betsholtz , endothelial / pericyte interactions . circ . res . 97 , 512 - 523 ( 2005 ) 2 . bell e , ivarsson b , merrill c . production of a tissue - like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro . proc natl acad sci u s a . 1979 march ; 76 ( 3 ): 1274 - 8 . 3 . bottino r , fernandez l . a , ricordi c , lehmann r , tsan m - 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