Patent Application: US-201113703495-A

Abstract:
the present invention relates to the use of 3 - dimensional synthetic or animal - derived bioscaffolds as substrates for the improved growth and differentiation of hps ; these scaffolds being adapted for use in conjunction with existing cell culture lab plastic - ware . more specifically , it relates to the seeding of these scaffolds , either alone or in conjunction with various biologic matrix coatings , with hps cells for the improved differentiation of said hps cells into hepatocyte or hepatocyte - like cell types . the invention also relates to the seeding of partially - differentiated hepatocyte progenitors onto scaffolds for further differentiation into more mature hepatocyte - cell types .

Description:
one aspect of the present invention relates to a method for differentiating human pluripotent stem cells ( hps ) or hepatocyte precursor cells into mature hepatocyte or hepatocyte - like cells , the method comprising the steps : i ) seeding of hps or hepatocyte precursor cells on a 2 - dimensional surface to initiate differentiation ii ) transferring the initially differentiated cells of step i ) to a 3 - dimensional ( 3d ) scaffold for further differentiation and maturation . the cells may be seeded at a higher density on the 3d scaffolds than on the 2d cultures , such as threefold higher or fivefold higher or tenfold higher however . further , rho kinase ( rock ) inhibitors may be added and / or the method can be performed under feeder - free or xeno - free conditions . the cells used in the methods according to present invention may be xeno - free cells , such as a xeno - free hps cell line or cells derived from a xeno - free hps cell line , or the cells may be human embryonic stem ( hes ) cells or induced pluripotent stem ( ips ) cells . the hepatocyte precursor cells according to present invention may be definitive endoderm ( de ) cells or not and may have characteristics of fetal endoderm or hepatic endoderm . the transition from step i ) and ii , i . e . the transfer of the cells obtained in step i ) are transferred to a 3d - scaffold ( step ii ) may be performed after 2 to 25 days , such as 4 to 15 days . the 3 dimensional scaffold may be chosen from one of the following types : i ) a porous alginate sponge , ii ) a biodegradable poly ( urethaneurea ) ( puur ) polymer , iii ) an emulsion - templated polystyrene , iv ) a synthetic nanofibrillar composite v ) a microwell device with at least one non - vertical side - wall , vi ) a porous sponge fabricated from poly ( l - lactic acid ) [ plla ] or the 3 dimensional scaffold may be of another suitable type . the porous alginate sponge may have a pore size of 50 - 200 μm , whereas the pore size of the emulsion templated polystyrene scaffold is in the range of 0 . 1 - 1000 μm , such as 15 - 45 μm . further , present invention relates to a method as described above , wherein the 3d - scaffold is uncoated . in another aspect , the 3d - scaffold is pre - coated e . g . with one or more extracellular matrix components . examples of such extracellular matrix components comprise : gelatine , laminin , fibronectin , collagen , polylysine , vitronectin , hyaluronic acid , hyaluronan hydrogels , silk fibroin , chitosan or a composite of any of the forementioned . the 3d - scaffold of present invention may be contained within a suitable culture vessel , such as a multiwell plate including but not limited to a 6 - well plate , a 12 - well plate , a 24 - well plate , a 48 - well plate , a 96 - well plate , a 384 - well plate , a 1536 - well plate or other suitable cell culture vessel . hence , the invention detailed herein comprises a process used to improve the differentiation of hps cells towards cells having a more hepatic phenotype or cells ultimately having a partially or fully mature hepatic phenotype , this process requiring the cells to be initially cultured on a 2 - dimensional culture surface before being transferred one of six possible 3 - dimensional scaffolds . this improvement in hepatic phenotype is independent of any particular cell culture medium composition ( see fig9 for details of cell culture protocols employed ). based on gene expression and metabolic studies , the inventors of the present invention have found that 3d scaffolds provide a better environment for the maturation and further differentiation of hps - derived hepatocyte cells or hepatocyte progenitors compared to more traditional 2d substrates ( fig1 - 6 ). further , the inventors have found a further effect when culturing the cells on a 2d surface before seeding the cells in the 3d environment . this can be seen by comparing the expression of several hepatic marker genes such as cyp2c9 , albumin , cyp3a4 , and ugt2b7 which are all expressed more highly in cells cultured on 3d scaffolds than on 2d surfaces , regardless of cell line , matrix coating , days in culture or scaffold chosen . specifically , a large number of hepatic transporters ( bsep , fabp1 , mrp2 , ntcp , oatp2 , oct1 ) are expressed in much higher levels when cultured in 3d indicating more mature and functional hepatocytes . this can be seen for a range of different scaffold types including topographical scaffolds such as ultraweb ( fig3 ) and aggrewell ( fig5 ), porous sponge types such as algimatrix ( fig2 ) and poly - l - lysine ( fig6 ) and on synthetic polymer - based scaffolds such as artelon ( fig1 ) and alvatex ( fig4 ). the 3d scaffold may be pre - coated with a matrix component to better resemble in vivo microenvironment normally encountered by hepatocytes . specifically , the matrix components gelatine and matrigel have been shown to improve the expression levels of some hepatic markers when used in conjunction with the scaffolds . expression of several markers ( tat , cyp2c9 , oatp2 ,) is higher on matrigel - coated artelon ( artimplant ) compared to uncoated ( fig1 c & amp ; e ; fig1 a & amp ; d ). a number of markers ( tat and ugt2b7 ) are significantly more highly expressed on matrigel - coated alvatex compared to uncoated ( fig4 f & amp ; g , analysed at day 18 ) and this is also true for several other markers ( such as tat , ugt2b7 ) which are expressed to a higher degree on gelatine - coated alvatex compared to uncoated ( fig4 e & amp ; g ). this particular embodiment of the invention can be optionally applied depending upon which markers it is desired to see expressed to a high degree . since expression of a particular set of markers will imply a certain phenotype or stage of hepatic lineage , application of this aspect of the invention may allow a finer degree of control over the functionality of the hepatocytes generated . an extension of this aspect could be the addition of one or more different cell types to the hps cells or hps - derived hepatic progenitors which are seeded onto the 3d scaffolds , allowing co - culturing of cell lines on the 3d scaffolds . notably , the cells may be seeded at different relative densities on the 3d scaffolds . the relative density may be a standard × 1 density ( i . e . the same density as cells seeded on the 2d control surface ) or a three - fold higher initial density ( designated as x3 ) or a ten - fold higher density or any density there in between . fig1 h & amp ; i show that expression of markers oatp2 , oct1 and tat is higher in cells seeded at the lower x1 initial density when using uncoated artelon ( artimplant ). expression of certain markers ( e . g . cyp7a1 ) are also lower on cells seeded at × 3 on uncoated alvatex compared to cells seeded at × 1 , ( fig4 n & amp ; o ) but other markers ( such as oct1 ) had higher expression in cells seeded at × 3 on uncoated alvatex ( fig4 i & amp ; k ). therefore application of this particular embodiment is again dependent upon the desired phenotype of the hepatocyte cells which is required by the user . to permit control of the differentiation , the stage of differentiation at which the cells are seeded on the 3d scaffolds is relevant , i . e . cultured for longer being of a more mature phenotype before seeding in 3d scaffold . the hps cells can be seeded in the scaffolds at several different stages of differentiation , e . g . as undifferentiated cells ( day 0 ) or after treatment with activin a for definitive endoderm - induction ( day 4 - 7 ) or as hepatic precursors ( day 9 , 11 , 13 , 14 and 15 ) for maturation in the 3d - scaffolds . characterisation of such precursor cells can be carried out to determine that they are on embryonic endodermal lineage prior to seeding on scaffolds ( fig1 ) or the more advanced hepatocyte precursor ( fig1 ), based on expression of defined marker genes characteristic for both stages . differences are readily observed on cells cultured on artelon ( artimplant ), where for example , expression levels of oct1 are almost double in cells seeded at 14 days compared with those seeded at day 11 ( fig1 f & amp ; h ). in contrast to this , levels of oatp2 are decreased in cells seeded at 14 days on artelon ( artimplant ) compared to those seeded day 11 ( fig1 j & amp ; m ). this trend can also be seen in cells cultured on alvatex , where again the 14 day old seeded cells possess lower expression levels of oatp2 than the 11 day cells , possibly indicating that oatp2 is a marker for more immature hepatocyte phenotypes ( fig4 i & amp ; j ). in a similar manner it can be seen that levels of oct1 are also higher in cells seeded when younger ( day 11 ) than in more mature ( day 14 ), ( fig4 n & amp ; p ). thus length of cell culture and differentiation time is an important factor of this invention in terms of controlling final phenotype of the cells produced and can be altered accordingly . as outlined in fig9 , and in the table below , the duration of culturing and the media compositions may be adjusted to optimize the maturity and developmental stage of the cells obtained by the methods disclosed herein . a related aspect concerns the differentiation regime to which the cells are subjected prior to seeding onto the scaffolds , as detailed in example 2 , in particular whether the cells are exposed to medium containing an inhibitor of the rho - kinase rock , such an inhibitor being used to improve survival and re - attachment of cells after passaging and seeding both in the 2d and 3d stages . an improvement in function and metabolic activity has been observed in the hepatocytes seeded onto 3d scaffolds compared to those grown on conventional 2d surfaces . hesc - derived hepatocytes , matured in 3d scaffolds according to example 6 , showed greater induction of cyp activity than 2d control cultures ( fig7 & amp ; 8 , 10 ) when exposed to the drugs phenacetin ( apap ), midazolam or diclofenac , with the alvatex scaffold showing the greater induction than the ultraweb . a further examination of cells cultured on alvatex ( reinnervate ) showed that at both 23 and 29 days after initiating differentiation , those cells had a much greater metabolic activity than cells cultured on 2d surfaces as measured by cyp2c9 activity . cyp2c9 activity was already high in cells cultured on 3d surfaces but was also observed to be elevated when cells were treated with diclophenac ( fig8 ). this again shows that hepatocytes which are differentiated on 3d scaffolds have a superior metabolic hepatocytoc function when compared to 2d cultured cells . these results are borne out by the findings that levels of the crucial hepatic marker cyp3a are also higher in cells cultured on matrigel - coated alvatex ( reinnervate ) and slightly higher in cells cultured on ultraweb compared to the 2d control when cells were assayed for metabolic breakdown products of the drug midazolam ( fig8 ). thus the invention has shown several examples of improved hepatic metabolic activity in cells cultured on 3d scaffolds . hence as discussed above an aspect of the present invention relates to a method wherein the hepatocyte cells or hepatocyte progenitors display elevated metabolic activity as shown by increased expression of one or more cyp markers , such as cyp2c9 or cyp3a and / or elevated levels of hepatocyte - associated genetic markers selected from the list comprising : cyp1a1 , cyp1a2 , cyp3a4 , cyp2c9 , cyp3a7 , cyp7a1 , mrp2 , albumin , ugt2b7 , aat , tat . further , an aspect of present invention relates to a method wherein the hepatocyte cells or hepatocyte progenitors display increased expression of one or more hepatic - associated transporter proteins such as bsep , fabp1 , mrp2 , ntcp , oatp2 , oct1 . the present method may as discussed above , stimulate that the proportion of hepatocytes or hepatocyte progenitors compared to non - hepatocyte cells is greater than 5 %, such as 10 %, such as 20 %, such as 30 %, such as 40 %, such as 50 %, such as 60 %, such as 70 %, such as 80 %, such as 90 %, such as 100 %. a method according to any of the preceding claims wherein the hepatocytes or hepatocyte progenitors are co - cultured on the 3d scaffolds with at least one other cell type chosen from , but not limited to : stellate hepatic cells , hepatic immune ( kuppfer ) cells , hepatic endothelial cells , biliary epithelial cells or fibroblasts accordingly and as discussed above , an aspect of the present invention relates to an hps derived hepatocyte cell or hepatocyte progenitor cell or a composition comprising an hps derived hepatocyte cell or hepatocyte progenitor cell obtainable by a method described herein . further aspects of the present invention relates to the use an hps derived hepatocyte cell or hepatocyte progenitor cell or a composition comprising an hps derived hepatocyte cell or hepatocyte progenitor cell obtained by the methods herein in therapy , regenerative medicine , for drug screening , toxicity testing or drug delivery . the composition may further comprise a 3d scaffold , such as but not limited to a porous alginate sponge , a biodegradable poly ( urethaneurea ) ( puur ) polymer , an emulsion - templated polystyrene or a synthetic nanofibrillar composite . all hps cells ( as defined above ) can be used as staring material for this invention . for the examples below hepatocyte - like cells were derived in vitro from undifferentiated human embryonic stem cells ( hesc ) cultured on mef cells ( heins , n . et al . 2004 , stem cells ). the cell lines used for this experiment could be , but is not limited to the hes cell line sa002 , sa121 , sa181 , sa461 ( cellartis ab , göteborg , sweden , http :// www . cellartis . com ) and they can be propagated as described heins , n et al . 2004 . these cell lines are listed in the nih stem cell registry , the uk stem cell bank and the european hesc registry and are available on request . along with hps obtained from hesc , hps obtained from hips ( induced pluripotent stem cells ) have been used for the derivation of hepatocytes for the examples of this invention . in this case , “ hips - hep ” is intended to mean a cell type derived from induced pluripotent stem cells which is expressing mature hepatic markers such as albumin , cyp3a4 , ugt2b7 , oatp - 2 , adh1a , ugt1a6 , cyp2c9 , cyp2c19 and cyp2d6 ( see also “ definitions ”). use of xeno - free ips cells or a xeno - free derived hes cell line such as sa611 ( cellartis ab , goteborg , sweden , http :// www . cellartis . com ) could optionally allow the entire invention to be used under xeno - free conditions to produce a xeno - free cell product . derivation of hepatocytes from human pluripotent stem cells using 3d scaffolds . hepatocytes were derived from both hes cells and human hips cells according to the protocols a - e , as detailed below . before start of differentiation , the cultures were washed twice with pbs . the different media ( table 1 ) were prepared freshly and added from day 0 and afterwards according to the protocol overview . medium was changed every day or every second day during the initial differentiation ( id ) step , and every second or every third day afterwards . only one cell line was used for each experiment , but optionally a second ( or more ) cell line could be added when the initial cell line is seeded onto the 3d scaffolds allowing co - culturing of the cells . 2d cultures were seeded on matrigel coated culture vessels , except in experiment 114 and 116 when 2d cultures were seeded on gelatin coated culture vessels . 2d cultures were seeded in 150 . 000 - 200 . 000 cells / cm 2 . the table below shows an overview of the different experiments , which cell lines were used as starting material , the variants of differentiation protocol as outlined in fig9 , time of the differentiation protocol when the cells were seeded in 3d scaffolds , time of differentiation when cells were analyzed , and whether 10 mm rockinhibitor was added or not to the medium at time of seeding in scaffolds ( see also fig9 for schematic details of protocols employed ): artelon ( artimplant ); & lt ; 150 μm poresize , formatted for 48 well plates , kindly provided by artelon ( artimplant ) ( västra frölunda , sweden ) [ see wo0035507 ; also blumenthal , b . et al . ( 2010 )] alvatex ; size for 48 wells , kindly provided by stefan przyborski reinnervate , ( durham , uk ) ( see wo07125288 , wo10038013 ) algimatrix ; invritogen / gibco , cat no 12684 , lot nr 731950 , 96 wells ( exp088 + 089 ) [ see wo08112904 ; also shapiro , l & amp ; cohen , s . ( 1997 )] ultraweb ; corning , cat no 3873xx , lot no 09207043 , 96 wells ( exp088 + 089 ) [ see wo09032117 ; wo10060066 , also piryaei , a . et al . ( 2010 )] aggrewell ; stem cell technologies , cat 27865 , approx . 300 microwells , [ see wo2008106771 ] highly porous polymer matrices as a three - dimensional culture system for hepatocytes . the artelon ( artimplant ) and alvatex scaffolds were soaked in 70 % etoh , the etoh was removed and the scaffolds were washed twice with dpbs . matrigel : growth factor reduced matrigel from bd , lot no 0934 was diluted to 0 . 016 mg / ml in dpbs , and added to scaffolds and 2d culture dishes , incubated at rt for at least 1 h rt , then matrigel was removed immediately before use . gelatine : gelatin from production was added to the scaffolds and / or 2d culture dishes , incubated at rt for at least 30 min , then the gelatin was removed immediately before use . media was removed from the cell cultures , and the cultures were washed 2 × in dpbs −/− ( 37 ° c ., 0 . 5 ml / cm 2 ). tryple select was added ( rt , 0 . 1 ml / cm 2 ) and incubated at 37 ° c . ( 4 - 5 min for undifferentiated cells or cells after activin a treatment , 10 - 45 minute for cells at progenitor stage ). the cells were then detached by flushing the cells with a p1000 . vitrohes was added ( 37 ° c ., 0 . 1 ml / cm 2 ) and the cell suspension was transferred to centrifuge tubes and centrifuged at 330 × g 5 min at rt . the cell pellet was resuspended in culture medium , cells were counted and diluted to appropriate seeding suspension . in some experiments , 10 mm rock inhibitor was added to the culture medium at day of seeding ( exp113 , 114 , 116 , 118 , 119 , 120 ) hesc - hep were derived from cell - line sa002 , sa121 , sa181 , sa46 cultured from the def cultures system or sa002 cultures on mef - layer as indicated in figure legends . total rna was collected and isolated from the hes - hep cultures at day 18 , 22 , 24 , 25 and 30 by using rna isolation kit from qiagene . quantitative reverse transcriptase pcr , qrtpcr , by using taqman probes , was performed for the following hepatic marker genes : phase i drug metabolizing enzymes ; cyp ( cytochrome p450 ) 1a2 , 2c9 , 3a4 , 7a1 , phase ii drug metabolizing enzymes gsta1 ( glutation - s - transferas a1 ), ugt2b7 ( udp glucuronosyltransferase 2b7 ), phase iii , transporters ; mrp2 ( multi - drug residence protein 2 , also called abcc2 ), bsep ( bile salt export pump ), ntcp ( solute carrier , sodium / bile acid cotransporter ) oct1 ( solute carrier , organic cation transporter ), oatp2 ( solute carrier , organic anion transporter ), fabp1 ( fatty acid binding protein involved in fatty acid uptake , transport , and metabolism ), and general hepatic markers ; aat ( alpha - 1 antitrypsin ), adh1a ( alcohol dehydrogenase 1a ) alb ( albumin ) and tat ( tyrosine - amino transferas ). all data was normalised to the house - keeping gene crebbp , except when other is indicated . data is presented as fold change of 2d controls , except when other is indicated . data is presented in fig1 - 4 and shows that hepatic genes are expressed at higher levels in hesc - hep when cultured in 3d compared to 2d . the improved expression levels of the other hepatic markers in 3d cultures are supporting the finding that 3d cultures are important in hepatic differentiation of hesc . at day 16 , 18 , 20 , 21 and 25 hesc derived hepatocyte cultures were analyzed for cytochrome p450 1a , 2c and 3a activity by incubating the substrates phenacetin , diclophenac and midazolam to a final concentration of 26 μm , 9 μm and 3 μm respectively in phenol red - free williams medium e , supplemented with 0 . 1 % penicilline - streptomycin 2 mm l - glutamine and 25 mm hepes . a volume of 100 μl diluted substrates were added per cm 2 of the well surface ( e . g . 75 ul pr 48 wells ). hesc derived hepatocyte cultures with substrates were incubated over night . after 16 h , medium was collected and subsequently , centrifuged at 10 000 g , 4 ° c . for 20 min . samples were analysed by liquid chromatography - mass spectrometry ( lc - ms ) lcms for presence of the metabolite paracetamol , 4 - oh - diclophenac and 1 - 0h midazolam , biotransformed by the cytochrome p450 enzymes cyp1a2 , 1a1 ( phenacetin ) cyp 2c9 , 2c8 ( diclophenac ) and cyp 3a4 , 3a5 ( midazolam ). improved cyp2c9 activity and improved induction of cyp2c9 activity in cells cultured in reinnervate scaffolds cell line sa461 ( cellartis ab ) was cultured and differentiated according to conditions in examples 1 and 2 , experiment 083 and then according to the scheme below : differentiation was carried out up to day 15 , and on day 15 scaffolds were cut in small circles to fit into 24 well plates . the alvatex ( relnnervate ) scaffolds were washed with 70 % etoh , the etoh was removed and the wells were washed twice with pbs +/+. matrigel : gfr matrigel lot no 6741 diluted to 0 . 016 mg / ml in dpbs , and added to scaffolds , 500 ul pr well and pr scaffold . the scaffolds were incubated for ˜ 1 hour rt with the matrigel . the matrigel was removed immediately before use . the aim was to seed in 2d wells with a split ratio of 1 . 5 : 1 . in alvatex ( reinnervate ), 5 times as many cells were seeded , which is a split ratio of 7 . 5 : 1 . starting out with 27 wells of 9 . 6 cm 2 , that is 260 cm 2 . for seeding 100 ul pr cm 2 in the alvatex ( reinnervate ) scaffold the cells were resuspended in ( 260 / 7 . 5 × 0 . 1 = 34 . 6 ) 3 . 46 ml . 27 wells of cells were washed 1 × in dbps +/+ and 2 ml tryple select was added pr well , incubation 23 min at 37 c . the spin down medium was added and cells were flushed to detach them . cells collected and spun 5 min 400 × g . resuspended in 3 . 46 ml seeding media . 200 ul of this suspension was seeded pr alvatex ( reinnervate ) scaffold . 600 ul of the suspension was diluted 5 ×, and of this , 200 ul was seeded pr 2d well . then medium was added to a total of 800 ul pr well in all wells . counting cells : 27 . 9 × 10 6 cells / ml ( 87 % viability ), 3 . 46 ml total , 96 × 10 6 cells total . cells were induced with 1 mm phenobarbital , 10 μm dexamethasone and 5 μm β - naphthoflavone . to obtain these solutions 100 μl 64 mm dexamethasone solution was diluted with 540 μl dmso to obtain a 10 mm dexamethasone solution 11 . 4 mg β - naphthoflavone was dissolved in 8 . 446 ml dmso to obtain a 5 mm solution 1m phenobarbital was diluted 1 : 1 with pbs to 35 ml m6 ( w / o dex ) 35 μl 10 mm dex , 35 μl 5 mm β - naphthoflavone and 70 μl 0 . 5m phenobarbital . control medium ( non - induced medium ) did not contain any dmso to be able to compare the enzyme activity with the baseline enzyme activity . to each well , either induction or control medium was added ( 400 ul / well 2d cultures , 2000 ul / well in reinnevate scaffolds ) and the plate was incubated 24 h . after 24 hours of induction , all wells were washed 2 × in dpbs +/+ and activity assay medium was added , 200 ul / well in 2d controls and 1000 ul / well in reinnervate scaffolds . activity assay medium : a protein - free medium without phenol red consisting of we with 0 . 1 % pest , 2 mm l - glutamine , 25 mm hepes and 26 um phenacetin , 3 um midazolam and 9 um diclophenac . after 16 hours incubation at 37 c , the medium was transferred to eppendorf tubes and spun down for 20 min at 4 ° c . and 10000 g . supernatants were analyzed by hplc for the presence of oh - diclophenac , which is the metabolite of diclophenac . verification that cells have differentiated into embryonic endoderm or hepatic precursors at the time of seeding in the scaffolds the protocols used for differentiation towards hepatocytes were analyzed for the formation of embryonic endoderm cells after the “ endoderm induction ” step , cells cultured according to protocol a ) ( fig1 ). at day 7 in the differentiation protocol , cells were harvested for rna and analyzed by real - time pcr and compared to the undifferentiated cells from start of the experiment ( day 0 ). it was shown that the differentiation protocols generated cells expressing embryonic endoderm markers ( foxa2 , sox17 , hhex , cxcr4 , cer1 ). furthermore , it was verified that markers ( nanog and oct4 ) for undifferentiated cells was down - regulated compared to the undifferentiated starting material . additionally , it was checked that cells were expressing extra - embryonic endoderm markers ( sox7 and cdx2 ), as these genes were not increased during the endoderm induction . a similar method was employed to test that cells which were being seeded onto scaffolds as “ hepatic precursors ” did indeed possess this phenotype ( fig1 ). relative expression levels of hepatic - markers on hesc - derived hepatocytes cultured either on 2d culture or 3d artelon ( artimplant ) scaffold . artelon ( artimplant ) is either uncoated or coated with matrix ( matrigel or gelatine ) and cells seeded either at x1 density or × 3 density . figure shows three independent cell lines ( sa461 , sa002 , sa181 ) with cells cultured on scaffolds for either 0 , 5 , 11 , 12 , 14 or 15 days according to experiment numbers shown , protocols a ) and c ) relative expression of hepatic markers on hesc - derived hepatocytes cultured either on 2d or 3d algimatrix scaffold . figures show two independent cell lines ( sa002 and sa121 ) with cells cultured on scaffolds for either 14 or 13 days respectively , protocol d ) relative expression of hepatic markers on ips - derived hepatocytes cultured either in 2d or on 3d algimatrix scaffold . cells were differentiated for 7 days prior to seeding on scaffold and analysed after 32 days culture ; protocol h ( exp169a ) or f ( exp169b ) relative expression of hepatic markers on hesc - derived hepatocytes cultured either on 2d or 3d ultraweb scaffold . ultraweb is either uncoated or coated with matrix ( matrigel or gelatine ). figures show four independent cell lines ( sa002 , sa461 , sal21 and sa181 ) with cells cultured on scaffolds for either 4 , 5 , 7 or 14 days , protocols a ), b ), c ), d ) relative expression levels of hepatic - markers on hesc - derived hepatocytes cultured either on 2d culture or 3d alvatex ( reinnervate ) scaffold . alvatex is either uncoated or coated with matrix ( matrigel or gelatine ) and cells seeded either at × 1 density or × 3 density . figure shows four independent cell lines ( sa461 , sa002 , sa181 and sa121 ) with cells cultured on scaffolds for either 0 , 4 , 9 , 11 , 13 or 14 days according to experiment numbers shown , protocols a ), c ), d ) relative expression levels of hepatic - markers on ips - derived hepatocytes cultured either on 2d culture or 3d alvatex ( reinnervate ) scaffold . alvatex is either uncoated or coated with matrix ( matrigel or fibronectin ) and cells seeded after 7 days 2d culture at x6 density that of 2d cultures from which cells were harvested . figure shows one independent ips line ( imr90 ) with cells cultured on scaffolds for up to 29 days before analysis ; culture protocol f . relative expression levels of hepatic - markers on hesc - derived hepatocytes cultured either on 2d culture or 3d aggrewell scaffold . aggrewell is coated with matrix ( fibronectin ) prior to seeding with cells cultured in 2d for 11 days ; culturing according to protocol f ) and cells seeded at either 100 or 200 aggregates per well . figure shows one independent hesc line ( sa181 ) with cells cultured on scaffolds for up to 25 days before analysis . relative expression levels of hepatic markers on ips - derived hepatocytes cultured either on 2d culture or 3d aggrewell scaffold . aggrewell is coated with matrix ( fibronectin ) prior to seeding with cells cultured in 2d for 7 days . figure shows one independent ips line ( imr90 ) with cells cultured on scaffolds for up to 29 days before analysis ; culture protocol i ) relative expression of hepatic markers of hesc - derived hepatocytes cultured on poly - l - lysine 3d scaffold ( hac ). cells were partially differentiated on 2d for 7 days prior to seeding and cultured until day 24 before analysis of marker expression ; culture protocol g ). cyp activities of hesc - derived hepatocytes in 3d scaffolds . 3d scaffold left = alvatex , 3d scaffold right = ultra web . experiment 24 , sa001 ecd . figure shows relative cyp activity of hesc - derived hepatocytes cultured either on alvatex , ultraweb or 2d control , with cells treated with either apap , midazolam or diclofenac to induce cyp response and mimic hepatic metabolic activity . cyp induction in hesc derived hepatocytes cultured in either conventional 2d culture plates or alvatex scaffolds . figures show cyp response in hesc - derived hepatocytes cultured either on alvatex ( reinnervate ) or 2d control , cells induced with diclofenac to mimic hepatic liver activity . activity assay performed at day 23 and day 29 as indicated . protocols a - e and respective experiment numbers together with duration of each experiment 2 days id1 , 5 days id2 , 2 days p1 , 2 days vh1 , 5 days vh2 , 8 days m1 4 days id3 , 3 days p1 , 2 days vh1 , 3 days vh2 , 2 days vh2b , 4 days or 8 days m2 ( for cell cultures analyzed day 18 and day22 respectively ) 2 days id1 , 2 days id2 , 3 days p1 , 7 days vh1 , 10 days of m4 1 day id4 , 5 days id5 , 6 days p1 , 8 days m5 , 15 days m6 . 1 day id1 , 1 day id6 , 5 days id2 , 7 days vh1 , then m1 till end of experiment 1 day id7 , 1 day id8 , 5 days id9 , 7 days vh1 , then m1 till end of experiment 1 day id7 , 1 day id1 , 5 days id2 , 7 days vh1 , then m1 till end of experiment 1 day id10 , 1 day id11 , 2 days id3 , 7 days vh1 , then m1 till end of experiment detailed growth media composition used in protocols a to e induction of cyp3a and cyp2c9 activity by measuring the metabolic product of midazolam , 4 - oh midazolam after 24 hours induction with or without 1 mm phenobarbital and 10 um dexamethasone ( induced / non - induced ). during the 24 hours induction , dexamethasone , dmso and bio was left out from the medium composition m1 . after induction , the activity assay was performed as described elsewhere ( see example 6 ). verification of embryonic endoderm fate of hesc cells cultured on 2d prior to seeding on 3d scaffolds . induction of endoderm markers ( foxa2 , hhex , cxcr4 , cer1 ) and downregulation of pluripotency markers ( sox7 , nanog , oct4 ) in hesc cells cultured in 2d format for 7 days prior to seeding on 3d scaffolds ; two independent cell lines ( sa121 and sa181 ) cultured according to protocol a ) on matrigel - coated 2d surface . brightfield morphology of cells is also shown . verification of embryonic endoderm fate of ips cells cultured on 2d prior to seeding on 3d scaffolds . induction of endoderm markers ( foxa2 , hhex , cxcr4 , cer1 ) in ips cells cultured in 2d format for 7 days prior to seeding on 3d scaffolds ; cell line imr90 cultured according to protocol h ( exp169a ) and f ( exp169b ) verification of hepatic progenitor fate of hesc cells cultured on 2d prior to seeding on 3d scaffolds . induction of hepatic progenitor markers ( afp , epcam , foxa2 , hnf4 - a , krt19 , krt8 ) in hesc cells cultured in 2d format for 11 days prior to seeding on 3d scaffolds ; cell lines sa121 and sa181 , cultured according to protocol g ). baharvand , h . et al . 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