AU2014256400A1 - Methods, surface modified plates and compositions for cell attachment, cultivation and detachment - Google Patents

Abstract

The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of 0 and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity.

Description

METHODS, SURFACE MODIFIED PLATES AND COMPOSITIONS FOR CELL ATTACHMENT, CULTIVATION AND DETACHMENT FIELD OF THE INVENTION The present application is a divisional application of Australian Application No. 2009215516, which is incorporated in its entirety herein by reference. [0001] This application claims priority to provisional application serial number 61/030,544, filed February 21"t 2008. [0002] The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on, and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of 0 and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity. BACKGROUND [0003] Cultivation of mammalian cells is one of many processes in the life and health sciences. Vessels for mammalian cell culture and analysis involving anchorage dependent cells are often made of glass or a polymer, such as, for example, polystyrene, that frequently requires additional surface treatment to allow the cells to attach to the surface of the vessel. Such treatments may include applying an adlayer on the surface, for example, by adsorption, grafting or plasma polymerization techniques. Alternatively, the surface treatment may be via chemical modification of the vessel surface itself, which can be achieved by, for example, atmospheric corona, radio frequency vacuum plasma, DC glow discharge, and microwave plasma treatments. These surface treatments change the composition of elements and chemical groups in the surface. The particular chemistry that results depends on the surface treatment method, energy, and time, as well as the composition of the gasses used. 1 [0004] For example, US5449383 discloses a substrate comprising a bulk polymeric material; and a thin polymeric layer which is suitable for supporting cell growth, comprising a la reorientation resistant polymer coipri sing plasma-polymerized amide ronomers presenting aide groups for the attachment of eelIs, wherein said anide monomers are selected from the group of dimethyl famie and anides having the formula R -CON(RP wherein is an aliphatic, alieycli, or aromatic groureach of which axy be optional Isubstituted by halogen atoms or hydroxyl group, and R and R" are each independently hydrogen or an alkyl group, and wherein said thin polymer layer promotes attachrnen. and proliferation of said eels 00015 In another exampleT P0348969A discloses a method for endothelialization of a polymeric suroe comprising contacting a polymerid with a plasna generated from a gaseous material comprising nitrogen whereby said poymeric surface is modified to contain surte amino groups and applying to said modified surface sufficient endothelial c eens to form a confluetilayer of cells on saId amino group containing surface without a requirement foi cell proliferation. 100061 In another example EPO(92C302A2 discloses a method for infliencing the growth of cell culture in a growth media on a substrate, characterized in that the surface chemistry of' he substrate is modied hy subjecting the surface of the substrate to a plasiahich is produced from carbon, hydrogen oxygen nitrogen sulphur phosphorus, a halogen, or a compound of any one ofthese elements. n00071 in another example, US 6,61TI2B2 discloses an alpparatus tor treating a polymeric substrate surace comprising: a) a gas inlet, a microwave energy source atnd a pasnma mixing chanber, the plasma mixing chamber in fluid communication with both the gas inlet and the micmwave energy sotrce;(b) a dual chambered treatment area having an inner treatmIent chamber contained, within an outer treatment chamber, said inner treatment chamber having an opening in fuid comnunication with said outer chamber; (c) said plasma mixing chamber in fluid communication with said outer treatment chamber by means of an aperture; d avauum outlet line attached to said outer chamber; and (e) whereby said opening in said inner treatment chamber is aligned ith said aperture. said opening being spaced from said aperture at predetermined distance. [0008] In on.e example, U2O3/) 80903A 1 discloses a polymeriC substrate having a working surface upon which cells can be cultured wherein the surface oxygen content 2 is at least 25 percent as measured by electron microscopy for chemical analysis at depth about 50 Angstroms. [00091 In one exanple, WO2006114098 discloses a microestructured biocompatible material for surgical implants and cell guiding tissue culture surfaces The microstructure of the biomaterial surface is selected to promote growth of undifferentiated ES cells; promote neuronal differentiion of' Ecells; or promote diferentiation of Eas cells. [00101 ID another eXample. Bigdel a ,iotcchnoi 13146-153, 2008) describes a method of adaptation and/or selection of human ES cels to be cultivated without differentiation under feeder-cel -Ifree conditions and without prior treatment of the solid substrate surface with extracelular matri prote involving (i)changing media from nedium conditoned by hurnan diploid embryoni c lung fibroblasts to medium conditioned by neonatal ehondrocytes; (i then passaging the cells enyrancaly from the mouse embryonic feeder cel layer to Matrigel rteted plates, then to Costarr plates and finally, to rimari plates, and (iP changing back to the first used medium again Very few of the humanFS cells subjected to this method gave rise to established cell lines, suggesting that this method involves selection oflhuman ES CelIs to the culture conditions. 10011 Surface treatment that change the composition of elerentis and chemical groups in the surface itself have successfully been used for preparing polymer solid substrates for the culture of many types of mammnalian cell Howevr there are significant limitations in terms of poor attachment and/or cultivation using certain types of mammalian cells for example, pluripotent stem cells and human embryonic kidney (HIK) 293 cells, [0012] Graham et , M Gen, Virol 36:5902 1977) disclose the generation of the cell line HEK293. [0013] lIE.K293 cell attahmenmay enhanced by making an adlayer on the solid substrate surface using, for example, extracelular matrix proteins, polylysine, polyornithine, or tene before adding the IHEK293 cells to the culture vessel. Preparing the adlayer iS however time-consumng, and typical results in a non-sterile solid substrate with a shorter shelf life than the bare solid substrate. 3 Th erefore there is a significant need for methods and materials for enhancing the attachment of HEK293 cels to solid substrates lacking an adlayer. 10014} Current methods of culturing piuripotent stem cells, in particular, etbyonic stem (ES) cells require complex culture conditions, such as. for example. culturing the embryonic stem cells on a solid substrate surface with a feeder cell layert or on a solid substrate surface with an adlayer of extraceliuar matrix protein Culture systems fnat employ these methods often use feeder cells or extraceliular matrix proteins obtained frm a different species than that of the stem cells being cultivated (xenogeneic material). Media obtained by exposure to feeder cels that is media conditioned by cells other than undifferentiaed ES cells, may be used to culture the ES cells, and media may be supplemented with animal serum. 10015] For example, Reubinoff et' al (Natur BioteclnoL 18399-404 2000) and Thompson etah (Science 282:1145-1147,1998) disclose the culure ofES celllines from human blastocysts using a mouse embryonic tiboblast feder cell layer, 100161 In another example, Xu et at (Nature BiitechPnolgy 19.971-974, 2001) discloses the uise of Matrig-eW and laminin for treating solid substrate surfaces before feeder-cell free cultivation of human ES cells without differentiation [00171 in another example Vallier el al. ( Cell Sci i 1844954509 2005) discloses the use of fetal bovine serum ior treating solid suNstre surthees before feeder-cell free cultivation of human ES cells without differentiation. 100181 In another example WO2005014799 discloses conditioned medinin for the maintenance, prol iferation and differentiation of mammalian cells, W)200501.4799 state: 'The culture medium produced in accordance. with the present invention is conditioned by the cell secretion activity of mrie cells in particular. those differentiated and immortalized transgenic hepatocyzes, named MM]H (Met Murine Hlepatocyte) 100.91 I another example Wanatabe eat (Nature fBiotechnol 35:68-686, 2007) state "a ROCK inhibitor emits survival of dissociated human embryonic stem cells, and demonstrate reduced dissociation-induced apoptosis, increases cloning efficiency (from approximately 1 to approximately 2 )and fciitation of subeloning after 4 gene transfer using mouse embrynic fibroblasts as feeder cells, collagen and Matrigel as etracelludar mnatrix protein.and Y-27632 or Fasudil tor inhibition of ROCK, Furthemore dissociated hunan ES cells treated with -276 were protected iom apoptosis in serun-free. suspension culture [00201 In another example, Peerni er a! (EMSO Journal 26:4744-4755, 2007) state "Complexity in the spa organization of human embryonic stem cell (hEiSC cultures creates beterogeneous incroenvironnents cies) that influence hESC fate This study demonstrates that fhe rate and trajectory of hESC differentiation can be controlled by engineering hESainiche properties. Niche size and composition regulate the balance between diffrentauion-inducin and -inhibiting factors, Mechanisticaly 1, a niche size-dependent spatialgradient of Smadi signaling is generated as a rest of antagonistic interactions boeen IESCs and hES-Cderived extra-embryonic endoderm{n (xE These iteractions are mediated by the localized seeretion of bone nmorphogenetic protein-2 (MP2) by ExE and its antagonist, growth diderentia(tion faetor-3 (GDF3by hESCs. Micropatterning ofhESCs treated with small interfering (si RNA against GDF3, BviP2 and Snmadas well treatments with a Rho-associated kinase (ROCK) inhibitor demonstrate that independent control of Smadl activation can rescue the colony size-dependent differention of ESCs. Our results illustrate, for the first time, a role for Snadl in the interaction of' spatial iforrmaton and in the niche-size dependent control of hESC selfrenewa, and differentiate ion {0211 Jn another example Koyanagi N1 Mt al (J Neurosi Res 2007 Sep 7 [Epub ahead of print]) state "Rho-GTPase has been inplicated in the apoptosis of many cel tye including neurons, but the mechanism by which it acts is not Fully understood. Here, we investigate the roles of Rho and ROCK. in apoptosis duing tranplanation of embryonic stem cell-derived neural precursor cells, We find that dissociation of neural precursorsactivates Rho and induces apoptosis. Treatment with the Rho inhibitor C3 exoenzyme and or the ROCK inhibitor Y-27632 decreases the amount of d'issoci ation-nduced apoptosis (anoikis) by 20-30%. Membrane bI ebbing. which is an early morphological sign of apoptosis; l of aspase.; and release of cytochrome c fom the mitochondria are also reduced by ROCK inhibition. These rests suggest that dissociation of neural. precursor cells elicits an. intrinsic pathway of cell death that is at least partally mediated through the Rho:/ROCK pathway. Morovr,in an animal transpiantadon model, inhibition of Rho and/or ROCK suppresses acute apo tosis of grafted oells, After transplantationtumor necrosis factoripha and pr rowth factor are strongly expressed around the graft. R.OCK inhibition also suppresses apoposis enhanced by these infammatory cytokines, taken together these results indicate that inhibition of Rho/ROCK signaling may urve survival ofgatdcellx icel epacmet herapy,) 0022] In another example Yoneda etat (I Cell Biol. 170: 443-453, August 3, 2005) states "he nomokgous mammalian rho kinases (ROCK I and 11) arc assumed to be functionary redundant, based largely on kinase construt overexpression. As downstream effectors of Rho GTPases. their major substrates are mnyosin light chain and myosin phosphatase. Both kinases are implicated in microfiament bundle assembly and smooth muscle contractility Here analysis of tibrobiastadheson to fibroneetin revealed that although ROCK if was more abundant, its activity was alWays lower than ROCK L Specfic reduction of ROCK I by siRNA resulted in loss of stress fibers and focat adhesions, despite persistent ROCK 11 and guanne triphosphate-boumd Rhok In contrast, the microfilament cytoskeleton was enhanced by ROCK I I down-regulatin. Phagocytic uptake of tbronectm-coated beads was strongly down-regulated in ROCK ilAdepleted cells but not those lacking ROCK 1. These effects originated in par from distinct lipid-indig preferences of ROCK ple.kstrin homology domains ROCK 11 bound phosphatidylinositol 3 and was sensitive to its levels. properties not shared by ROCK I. Therefore endogenous ROCKs arc distinctly regulated and in turn are involved with different myosin compartments. [00:23] In another exampc. Harb et al (P10S ONE 3(8): e300i oi 10.1371/journalpone000001, August 2008) discloses an essential role of the Rho-RockMyosin signalinigaxis tor he regulation of basic cellell communications in both mouse and human ES cells and would contribute to advance (sicj in medically compatible aeno-fae environments for human pluripotent stem cells. [0024] The use of xenogeneic material may be unsuitable for certain applications utilizing pluripotent stem cells. Alternative materials may be used. or example, Stojikovic et A (Stem Cells In23895-902 2005) discloses the use of human serun for treating solid 6 substrate surfaces before feeder-cell fre cultivation of human E$ cells without differenttation. [00251 An alterative culture system employs scrumfree medium supplemented with growth factors capable of Promoting the proliferation of ES ceils 100261 For example, Cheon et atL ioReprod D1030. /bioireprod3 0504687019 Oct 2005) disclose a &eder-cell free, serunfree culture system in which ES cells are maintained in tucondujoned serum replacement medium supplemented with diuerent growth factors capable of triggering ES cell self-renewal 100271 In. another example, Levenstein a d (Stem Cells 24:56854 2006) disclose methods for the long-term culture of human ES cells in the absence of fibroblasts or conditioned mcdiur sing media supplemented with basic fibroblast growth factor (FGF.). 100281 in another example US2005014 8070 discloses a method of culturing human E3S ce Is in de eed media without senma and without tbroblast felder els the method comprising: ulturing the stem cells in a culture medium containing albumin amino acids, vitamins, minerals, at least one transferring or transferrin substituteatilest onc insulin or insulin substitute, the culture medium essentially free otmammaiian fetal serum and containing at least about 100 rig/mI of a FOF capable of activating a FGF signaling receptorwherein the growth factor is supplied rorn a souIce oier thanpjut a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder celIs or corditioned medium. {0029] In another example, US20050233446 discloses a defined media useful in culturing stem cels including undifferentiated primate primordial stem cells In solution, the media is substantially isotonic as compared to the stem cells being cultured, In a given euture, the particular medium comprises a base medim and an amount of each of basic FGh3 i znsuln and ascorbicacidneessary to support substantially undifferentiated growth of the primordial stem cells. [00301 In another example, O6800480 states "n one embodiment a cell culture medium for growing primatederived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure ow endotoin basic medium that is effective to support the growth of primate-deived primordial stern ce Is, The basic medium is combined with a nutrient serum effective to support the growth of primate derived prnordial stem cells and a substrate selected from the group consistig of feeder eelIs and an extracelular matrix component derived from feeder cells The mnediumn further mIetdes nonessential armno acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt" [0031 iIn another example S20050244962 states: -in one aspect the invention provides a method of cudturig primate embryonic sten clIs. One cutes othe stern celin a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum ) and in the presence of fibroblast growth factor that is supplied from a source other than just a PfIroblast feeder layer, In a preferred fbrm, the fibroblast feeder layer, previously requed tosustain a stem cell culture is rendered unnecessary by the addition of sufficient Ibrobiast growth factor 100321 In another example, W02005065354 discloses a defined isotonic cultre medium that is essentially feeder-free and seruim-free. comprising. a, basal medim; b. an amoun of basic fibroblast growth factor sufflient to support growth of substantial undifferentiated mammalian stem cells; c.an amount of insulin sufficient to support growth of substantially undifferentiated mamnmlian stem cells; and d. ar. amount of ascorbic acid suffnient to support growlI of substantially undiferenated mammalian stem cells, [00331 In another example W02005086845 discloses a method for maintenance of an undiffereniated stem cell, said method composing exposing a stem cell to a member of the transforming growth factor-beta (TGF) family of proteins member of the fibroblast growth factor (EG F) family of proteinsor nicotnamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state or a sufficient amount of tine to achieve a desired result. [00341 Puripotent stem cells provide a potential resource fbr research and drug screening. At present iargesscale culturing of human cell lines is problematic and provides substMtial challenges. Apos solution to these challenges is to s and culure the human ES cells as single cells Single cells are more amenable to standard tissue Culture. techniques. such as, for example, cOUnng transfection, and the like, 8 10035] For example Nicolas t al provide a method for producing and expanding hmnan ES cell lines from single cells that have been isolated by fluorescence-actiated cell sorting fol lowing genetic nodificatijon by lentiviras vectors (Stem Celts Dev, 16109 118, 20071 [0036] In another example, US patent application US2005158 2 discloses a method "for iprovimg growth and survial of single human embryonic ster cells The method includes the step of obtaining a single undifferentiated hES cell; mixing the single undifferentiated cdI with an extracclinar matrix to encompass the cdcI, and inoculating the mixture onto feeder ceus with a nutrientmedium in a growth environmentt~ [00371 In another example, Sidha a at (St Celis Dev, 15:61-69, 2006) describe the first report of three human ES cell clones, hES 3 1, 3.2 and 3 den ved from the parent line hES3 by sorting of single-cell preparations by flow cytometry. [00381 However, passage aid culture of human ES cells as single ceisea to genetic abnormaliies and the loss of pilupotenv. Cyiture conditions are important i he maintenance of piuripotency and genetic staibity Generall, passage of humanES cell lines is conducted manually or with enzymatic agents such as collagenase. liberase or dispase. [00391 For example. Draper et al, note the presence of "karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem cel lines on fivendependent occasions" (Nature Biotechnol 22:53-54, 2004). [0040] in another example, Buzzard et at state, "we have only ever detected one karyotvpe change event the culture methods used may have had some bearing on our results, given that our methods are distinctly different frmm those used by most other groups. Typically we passage human ES eelIs after 7 days by first dissecting the colony with the edge of a broken pipette... No ennZymaie or chemical nthods of ce dissociation are incorporated into this method, We speculate that this may explain the relative cyiogenetie resiHnce of hES (human ES) cells in our hands," (Nature Riotechnol 22:381382 2004). 9 n0041) another example italIpova :t state ibu& passage methds.can perpetuate aneuploid cell populations aher extended passage in culture, but may be used for shorter periods up to at least 3 passages) without compromising the karyotypes it may be possible to maintain a normal karyotype in hES eells under longderm mnuital propagation conditions followed by lirnted buk passagig experiments requiring greater quantities of hES cells than manual passage methods, alone, can provide". (Nature Biotechnol 23 19-20h05), 0042] In another example eng g aL state "the results demonstrated thath second protocol (trypsinization with gentle pipetting) is much less detrimental to Cellular viability than is the first protocol (ollagenase treatment with scratch " This in turn translated to higher freeehaw survival rates tBioteehnology and Applied Biochemistry 47:3 >37. 2007). [00431 In another example, Hasegawa el uwstate we have established hESC sublines tolerant of' compl etc dissociation. These cells exhibit high repeating effi.cieney and also high cloning effiency and they maintain their ability to differentiate into the tree gera ayers;" (Stern Cels 242649-2660. 2006) 10044] Therefore, there is a significant need for methods and compositions for the cultivation oftmammalian Cells, including ctivation of' pluripotent stem cells in the absence of feeder cels and an adlayer,1hile maintaining the pluripotency of the cells SFI MMARY [00451 In one embodiment the present invention provides methods and compositions for the attachment cultivation and detachmnent of es to a solid substrate suriace containing from at least about O.5% N. a stun ofO and N of greater than or equal to 17,2% and a contact angle of at least about 13.9 degrees, lacking a feder cell layer and lacking an. adlayer 10046 In one embodiment, the present invention provides a method to enhance the attachmnt of cells to a surface containing from at least about 0>% NS a sum of ( and N of ureater thaun or equal to 172% and a contactangle of at least about 13.9 degrees. lacking a feeder cell layer and lacking an adlayer; comnprising the steps of: 10 a, Obtaining a suspension of eelIs b, 'Treating the suspension of CelIs dith at least one comnpoutnd selected from the group consisting of: a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho acti vity and c, Adding the suspension of celIs to the surface and allowing the cells to attach 0047] In one embodieent, the cells are maintained in cdiure afte the cells attach to the surface. In an alterate embodiment the at least one compound is removed. [00481 in one embodiment the cells are detached from the surface by removing the at least one compound [00491 In one embodiment the suspension of eeLs is a suspension of. users of cells. In an. ahernate embodiment, the suspension of cells is a. suspension of single eIs, 0050) In one embodiment the cells are pluripotent stem cells, In an alternate embodiment the cells are stem cells, [0051] In one embodiment, the present invention provides a method to enhance the attachment of oells to a surface containing from at least about 0% N a sum of O and N of greater than or equal to 22m3% and a contact angle of at least about 13,9 degrees lacking a feeder cell layer and lacking an adlayer, comprising the steps of: a, Obtaining a suspension of ells, and b, Adding the suspension of cells to the surface and all owing the cells to attach. BRIEF DESCRIPTION OF THE DRAWINGS [0052] Figure 1 shows phase contrast micrographs (4A) of cells of the human ES cef line Hi that were passaged twice aN cluster with UBER;ASE on srtace modified plates .2 or 4I mages of cellS of thc human ES cl Iine H il cultured on plates treated with a 1:30 dilution of Manrgel Nuodon Delta plates are also shown. [00531 Figure 2 shows the ettect of 10 pMi 21 32 on the attachment ofhurnan ES cells to surface mod ified p1 ates. The figure shows phase contrast micrographs (x) of cells of the human ES cell line H1-11 that were passaged twice as clusters on surface modified plNes 3 and 4, Cens were then passaged onto surface modified plates 2, 3 or 4, in MEF conditioned medium containing 10 pM Y-2762. Cells were cultured for fur days prior to taking the photographs. Cells cultured in. the absence of Y 27632 were itseluded as Cotilidls. 100541 Figure 3 shows a schematic of the timencourse of treatment of compounds on human ES cells cultured on the surface modified plates of the present invention Cells of the human ES cell line 1i-i were passaged four times as chsters with I3BERASE treatment on sauce modified plates 3, or 4, and cultured in M EF conditioned medium. Cells were treated for the first two days after passage with either 10 pM of the Rho Kinase inhibitor Y-27632, or with 0.5 ngmi of the Rho inhibitor a cell permeable form of exoenzyme C3 transferase. Cells that were treatedwith the Rho Kinase inhibitor,2o Y07632 and were thereafter treated for thc first two days afer each passage with -27632 on surface modified. plate 3 are referred. to as "7s", Cel Is that were treated with the Rho Kinase inhibitorW27632 and were thereafter treated for the first two days after each passage with Y227632 on surface modified plate 4 are referred to as "3s". Cells that were treated wh.he Rho inhibitor for two days and were then treated with the Rho Kinase inhibitor, Y27632 for two days afr each passage and thereafter treated for the first two days after passage with Y 27632 on surface modified plate 3 are referred to as "58" Cells that were treated with the Rho inhibitor fbr two days and were. then treated with the Rho kinase inhibitorY27632 for two days ater each passage and thereafter treated. for the first two days after passage wAh V-27632 on surface modified plate 4 are referred. to as "Is"' [0055] Figure 4 shows the expression of markers associated with pluripotency and differentiation in human ES cells treated according to the prtool outlined in Figure 6 as determined by qRT4)CR. 0056 Figure 5 shows the expression of pluripotency markers in cells of the human ES cell line lt as determined by flow cyrometry at passage 4 (p4), passage 9 (p9 , and again. at passage. 10.1 or 12 (plO p1 orpl2t {0057] liiire 6 shows innmoiUfluorescent images of cells of the human ES cel line I 11 were passaged serially as clusters with LIBERASE treatment on surface modified 12 plate 4. and cultured in MEF conditioned mediaim Expression of proteins associated with markers of pluripotency was detected in cells cultured for 1 passages on surface modified plate 4. Cels were treated with 10 pM Y-27632 for two days after each passage. [0058] Figure 7 shows the ability for human ES cells to form definiive endoderm after culture on surface modified plates, CeNs of the human ES cell line H1 were passaged iI times as dusters with LIBERASE treatment on surface modified plates 3, or 4 and cultured in M EF conditioned medium At passage 8 (p8) and again at passage 10 or II (p101 1) els were treated with DMNEMi2 media containing (5% FBS 100 ng/ml Activin A, and 20ng/ml Wnt3a for two days and thentreated with DMEM:F 2 media containing 2'. FB and 100 ng/ml Activin A for three more days, The y-axis on the graph shows the percent posidve CXCR4 cells obtained by flow cytonotry; See also Table S. 100591 Figure 8 shows the ability for human ES cells to form pancreatic endoderm after culture on surface modified plates. Cells of the human ES cell line Eli were passagced eighttinesas clusters with IBERAS E treatment on surface modified places 3. or 4 and cultred in MEF conditioned mcdim At passage 8 (pS) cells were subjected to differentiation to definitive. endoderm by treatment with DMEMF12 media containing 0.5% FBS. 100 ng/ni Activin A and 20 ng/nt Wnt3a for two days and then treated with DMEM :F12 media containing 2%t FBS and 100 nganl Aetivin A for three more days. The eelIs were then fArther differentiated to embryonic foreigut with four days of treatment with DMEM:F12 media containing 2% FBS. 100 ng/ml F(F 10, and I ipnM cyclopamine/KAAD. The cells were then differentiated to pancreatic endoderm with four days of treatment with DMEN: 12 media containing I% 3-27 1.0(0 ng/mi FGF-I0, 1 gM cyciopamineKKAAD) and 2 pM retinoic acid. Cells were stained by imnmunofluorescenee for PD- green) and E-cadherin red) and total cell number was ident5id by Hoechst dye (biue4) [00601 Figure 9 shows the ability of human ES cells cured on. surface modified plates to form cmbryoid bodies. [0061] Figure 10 shows the karyotype of human ES cells Cultured on surface modified plate 4, 13 100621 Figure I shows the effet of treatment with Rho kinase nbtors (-27632from EMD biosciences Y-27632 fiom Signa, Fasudil, and Hydroxyfasudil) on the attachment of human ES cells to surface modified plates, Cells were cultured in medium containing the indicated compounds, at the concentrations lstedfor three days Ces were stained with crystal violetund images taken 00631 Figure 12 shows the dose-esponse of-27632 on the attachment ofhuman ES cells to surface modified plates. Various concentrations of the Rho kinase. inhibitorY 27632, was added to the cultures at a specified concentration (0. I 2, 4, or 10 pM Y 27632) for the first day. The cells were then maintained from day 2 onward in media containing 10 gM Y-27632 with daily media changes for five days, Media was removed from the plates on day five and the cels were stained with 05% crystal violet, and images taken [00641 Figure 13 shows the formation of human ES cell colonies fiur days after passage onto surface rnodified plates 2, 3, or 4 with orwithout 10 ofM -27632. I 0065] Figure 14 shows the frimation of human ES cel Icolonies four days after passage onto MIatrigel' treated plates with or without 10 pN Y27632 100661 Figure 15 shows the difftrene. between continual and intermittent treatment of human ES cels with Y-27632, on attachment of cells to surface modified plates. [00671 Figure 16 depicts images of cells fron the human ES cell line H, that were passaged as single cells, seeded on to surface modited plae 3 id1EF conditioned media containing (B) or with out (A) 10 p M -27632, The images were taken 24 hours after 10068] Figure 17 depicts the expression of markers associated with pluripotency in. cells from the human ES cell line F1h9, hat were passaged as single cells for S passages, using TypLTM Express, and plated onto surface rnodified plates 3 and 4, with or with out 10 pM ofY-27632 (). The pluripotency nmrkers are listed on the x-axis and the percentage of positive cells is shown on thevaxis. [0069] Figure 18 depicts the total cel number of cells from he human E$ cell inc H9 that were passaged as single cells, plaed onto surface modified plates 3 and 4. The effect 14 of 10 pM of Y 27632 (Y) on cell number was examined on cells passaged on Matrigel (naive N) and cells passaged 10 times on the surface modified plates (acelinateA The different cell conditions are listed on the a-axis and the number ofclls diided by l0os shown in the axs [00701 Figure 19 depicts the rate of growth of cells from the huiman ES cell line H9 that were passage as single eells on Matrigel treated plates prior to the .de s were seeded at I Oen and cultured ia MEF conditioned media with or with out 10 pM of Y-2632 on surface modifiedplaes 3 and 4 The y-xis shows the number of cells collected 2. 3 or 4 days after seeding (divid by 10, (0071] Figure 20 depicts the rate of growth of cells fom the human ES cel line 9,O that were passage as single cells for 10 passages on surface modified plates prior to che study. Cells were seeded at 10/cm 2 and cultured in MEF conditioned media with or with out 10 pM of Y27632 on surface nodiFied plates 3 and 4, The y-axis shows the number of' cells collected 2.or 4 days after seeding (divided by 10 100721 Figure 21 depicts images of cells from the human ES cell line H9 that were passage as single cells, seeded on to suftce modified plates 2 4 and 13 in a 9 6-well fonnat, The MEF conditioned tnedia contained 10 YM of27632, Iages were taken 48 hours after seeding [00731 Figure 22 shows the ability of cells froT the human ES cell line H9 that were passaged as single cellsseeded on to surface modified plates 3 and 4 to differentiate into definitive endoderm. The tent of formaioni of definitve endodert was determined by measuring CXCR expressio-n by flow cytometry. The effect of 10 pM Y2762 on the formation of definitive endoderm. was investigated. Cells were treated with Y-27632 during expansion. Cells expanded and differentiated on IaYrige were included as a control. The y-axis shows percent positive CXCR4 cells obtained by flow cytom etry; 10074] Figure 23 shows the ability of cells from the human ES cell line H9, that were passaged as single cells, seeded on to surface modied plates 3 and. 4 to differentime into pancreatic endodern Cells were plated onto the surface modified plates and cultured in MEF conditioned medium containing 10 pM Y-27632, and passaged 8 15 times on the surface modified plates prior to diffrentiation. T11e y-ais snows the fold increase of pancreatic differennation marker expression (Ngn3 Pdxl, Insulin) by q-PCR at the posterior foregut stage (PI) and the hormone expressing endocrine cell stage (EN). [0075] Figure 24 shows the attachment of human ES cells to surface modified plates. Passage 50 1-19 human ES cells were Plated at a 1;2 dilution on Surfiaces 3 and 4, CellIIN D and PrimariaY 5 '. Media was removed from the plates 24 hours after plating and he cells were stained with 05% crystal violet, and images taken Arrows indicate colonies. [00761 Figure 2$ shows the attachment of human ES cells to surface modified plates. Passage 50 H9 human ES cells were plated at a,2 dilution on Surfaces 3 and 4, CellBINI and Primaria ' in the presence of various concentrations of Y-27632 (0. 1, 2410 and 20 micromolar> Media was removed from the plates 24 hours after plating and the cells were stained with 0.5% crystal violet, and images taken. Colonies are dark spots on the well. Anows are used to highlight colonies on the untreated wells. [00771 Figure 26 shows the attachment of human ES cells to surface modified plates Passage 53 119 hnman ES cells were plated at a 13 dikition on Surfices 2-4 and 13, CelliND anltd PImaria in the absence or presence of Y27632(O or 20 rrnicromolan) Media was removed from the plates 48S hours afie plating and the cells were stained with 05% crystal violet, and images taken. Colonies are dark spots on the well Arrows are used to highlight colonies on the untreated wells. [00781 Figure 27 shows the first attempt (Octobert) and second attempt (December to attach human H9 ES cells to surface modified plates 14 and 15 and an attempt to attach human. 1 ES eelIs to surface modified plates14 and 1.5 Passage 42 and passage 53 H9 human ES, and passage 57 il human ES cells were plated at a12 or 1:3 dilution to the rmodifed surfaces in the presence of 20 micromolarY-27632 Media was removed from the plates 24-48 hours after plating and the cels were stained with 05% crystal violet, and images taken, Colonies are dark spots on the well., Arrows are used to highlight colonies on the plates, 16 0079) Figure 28 shows the attachment of human ES cells to a surface modified plate 4 in defined media. mTeSRA K Passage 50 H9 human ES cells were plated at a 1:2 dilution to the. mIodified surfaces in the absence or presence of 27632 (( or 20 micromolar) in we ls that were untreated or treated with proteins (di% gelatin, 2% BSA, 04mg/m rat Collagen 1 1:1000 diluted Mairrge or 1,5000 diluted Matri gel h>.i Media was removed from the plates 48 hours after plating and the cells were staned with 05% crystal violet, and images Colomies are dark spots on the well. 0080] Figure 29 shows the water contact angles of surface modified plates measured over II weeks using the static sessile drop method. The first measurement was done one week after surface treatment and sterilization. Each data point represents the mean. contact angle (one measurement on each of? drops). The contact angle.s on Nucion Deltan and CellBiNDM plates were measured under the same experimental conditions as Suefaces 1-4 and 13, but the surface treatment and sterilization was done more than 12 weeks before the first rmeasurement (Nuc ion D~eltaiki* as sterilized one week before the first measurement), [0ul Figure 30 shows the density of negative charges on surface modified plates measured as reactivity of surfaces with positively charged crystal violet Three samples of each surface were tested, and absorbarce measurements on desorbed ctystalviolet from each sample were performed in triplicate. Mean and standard deviation ofnine measurements are gven, [00821 Figure 3 1 shows the effect of solid substrate surfaces and Y-27632 on attachment and growth of HEK293 celIs in chemicaly defined serumfree Pro293-CDM medium (A) or E MEM medium supplemented with 10% fital bovine serum (H) 1 EK293 cells were seeded in 96-well plates with Cell BNDM surface. Nuncon Delta surface or Surface 4 The number of HiEK293 cells attached to tese surfaces is shown as a function of culture conditions and concentration of Y-27632, Cels received either: (i) 96 hours of constant treatment in culture with Y-27632 (Y~27632 96h on); or (ii) 48 hours of. constant treatment in culture with Y -27632 followed by a change of medium and then 48 hours in culture without Y-27632 (Y-27632 48h oni48h offl HIEK293 cells cultured without Y27.632 in the medium (No Y-27632) were handled the same way as cells cultured with Y-27632, that is, for either 96-hours 17 without a change of medium, or wit a change of medium after 48 hours, Y-27632 enhanced attachene-t of HEK293 cells on Surface 4 and the CellBIND surface when applied at concentrations of 2(0 and 0 pN. Removing Y-27632 after 48 hours of incubation resulted in detachment of a significant number of cels from Surface 4 and the Cell BINDM surface Mean and standard deviation ofbree measurements are shown. [00831 Figure 32 shows the effect of solid, substrate surfaces and Rho kinase inhibitors Y 27632 and H-i152 t on growth of I Ek293 cells in EMEM niedhm1 wuppemernted wulh 10 fetal bovine scrumA HEK293 cells were seeded in atdlih 24well plates with either Surface 4 (A) or a non treated (but gamma irradiated 25 kG2 poivstyrene surface (B [00841 Figure 33 shows the effect of FA 1152 ard SurfaeW 4 on HEK293cell attachment and morphology. H4EK293 Cells were seeded in Muitidish 12-well plates in EMEM uedi urn supplemented wi thi .N0% fetal bovine serur and - 1152 and incubated for 67 hours in an automated in-incubator microscope. Growth curves in A and photorndcrographs in FT show the general efect of H- 152 on IK293-cell attachment and growth on surface 4, and the effect of a change of medium on HEK293-cell attachment and morphology on surface 4 in the presence or absence of 0085] Figure 34 shows gowth curves over 3 passages for 1-EK293 els grown n source 4 and Nunelon Delta' surface in the absence or presence of2$ y 27632, HEK293 cells in EMEM median supplemented with. 10% fetal bovine serum were passaged3 times by trypsinization, [0086] Figure 35 shows the effect of Rho kinase inhibition on the attachment of cells of the human. embryoc stem celline I-11 to surface modified plates 4, 1.8 and 19. and Primariat Wells A&B were control wells on all surfaces. Wells C&D contained 10g-M Vt27632, Wels E&F contained 3pM H 1152~gtycy. Wells G&H contained 10pM 1f1152-glycyl [00871 Figure 36 shows the effect of Rho kinase inhibition on the attachment of cells of the human embryonic stem ce line F-I to surface modified plate 30, (d no treatment 18 (RI)= 3 pM H1 152-glycyl, (MG)= adyer of :30 dilution of MATRIG EL. (MG+RI)= adlayer of 1:30 dilution of MATRIGEL + 3 1M H1152-glycyl. [00881 Figure 37 shows the effect of Rho kinase iibition on the attachment of cels of the human embryonic stem cell lne H I to surace modfed plate 31( no treatment (RI) -3 pM H] 52gycyL (MG)= adlayer of -130 dilution of MATRIGO EL. (MG+RI) adlayer of 1:30 diltion of MATRJGEL+$ M H il2-cl. O009) Figure 38 shows the effect of Rho kinase inhibition on the attachment of cels of the human embryonic stem Cell ine HI to surface niodified plate 32. (-) = no treatment. (RI)= 3 pM Hi i152glycyl, (MG) ailayer of 130 dilution of MA TRIiGEL (MGRI) = adlayer of 1:30 dii ution of MNATRIGEL + 3 pM F1 i 152glycyl [0090] Figure 39 shows the effect of Rho kinase inhibition on the anachimen t of celns of the human emb nic stem cell line H I to surface modified plate 33. G-) no treatment, (R) 3 pM HI i2glycyl. (MG) = adlayer of 1:30 dihution of MATRIGEL (M 4RI) = adlayer of 1:30 dilution of MATRIGEL 3 pM HI I 52glyeyl 1091] Figure 40 shows the effect of Rho kinase inhibition on the attachment of cells of the human erbryonic sten cell line H I to surface modified plate 34, C)= no treatment, (RI):= 3 pM H 52-glycyi (MG)= adhlyerof 1:30 dilution of MATR3GEL. (MG+RI):: adlayer of 1:30 diiution of MA I RIGEL + 3 pAM H1 152-glvcyl [0092] Figure 41 shows the water contact angles of surface modified plates measured over 40 weeks using the static sessile drop method, [00931 Figure 42 shows the water contact angles of surface modified plates using the static sessile drop method (094] Figure 43 shows the density of negative charges on suface modified plates measured areciity of sumae wXpositvely charged crystal vioet [O95] Figure 44 shows the density of negate charges on source modiied plates 4, 22-24 and 29 measured as reactivity ofisurfaces with positively charged crystal violet Three samples of each surtbce were tested, andabsorbance measurements on desorbed crystal violet from each sample were performed in. triplicate The negative 19 charge density for surfaces 4, 21,24 and 29 was normalized to the negative charge density of the Nunclon Ddta 1 I' surface, Mean and standard deviation o inne mneasurenmentIs are gi ven, DETAILED DESCRIPTION 100961 For clarity of disclosure, and not by way of limitation, the detailed diescri pton of the invention is divided into the following subsections that describe or illustrate certain matures, enbodiments or applications of the preseninvention Definitions 0097] "Adlayer" as used herein refers to a layer that is formed on a surfac of a solid substrate by attaching molecules to the surface by eiter covalent (also known as grafing) or non-covalent (also known as adsorption) bonds Moleculesused in making an atiayer can, for example, be protei.naceous molecules, hich may include, for example. extracelhdar matrix proteins, amino acids and the like, and non biological molecules, such as, fr example; polyethyleneiiniDe 100981 "heellineage" refers to cells with positive gene expression for the transcription factor PDX-I and at least one of the following transcription factor NGN-3 Nkx2.2, Nkx6. NeuroD, lIi 142F43 beta, MAFA, Pax4, and Pax6, Cells expressing ina rkers characteristic of the 3 cell ineae incude fi clls, 100991 "(lis expressing markers characteristic of the definitive endoderm lineage" as usetid herein refers to cells expressing at east one of the following markers: SOXw-175 GATA -4. HNF-3 beta OSC,Cerl Nodal, FGF-8 rachyry Mix-ike homeoMx protein IGF-4 ('48, comesodernin (EOMES), DKK4, FOEi17 GATA-6. CXCR4, C Kit CD99. or OTX2. Cells expressing markers characteristic of the definitive endoderrn lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cels and definiive endoderm cells. 1010] "Cels expressing markers characteristic of the pancreatic endoderm lineage" as used herein refers to cells expressing at least one ofthe following markers: PDX- HlNF .beta, PTF~I alpha, HNF-6. or HB9 Cellsepressing markers characteristi of the pancreatic endoderm lineage include pancreatic enidoderm ceus, 20 f01011 "Cells expressing markers characteristic of the pancreatic endocrine lineage" as used herein refers to cells expressing at least one of the following markers: NGNS Neuro IsletPDX~KX61, Pax4 Ngn. or PT-I' alpha, Cells expressing markers Characteristic of the pancreatic endocrine lineage include pancreauc endocrine cells, pancreatic hormone expressing cellsand pancreatic honone sccretin cells. and cells of the ceillineage. [0102] "Definitive endoderm" as used herein refers to cells which bear the characteristics of cells arising frm the epiblast during gastrIation and which form the gastrointestinal tract and its derivatives Definitive endoderm cells express the following markers: CXCRA HN F3 beta, GATA-4, SOX7 Cerberus, OTX2 goosecoid eKitCD99.and 10103] itaclluiar matrix proteins" refers to proteinaceous molecules normaly found between cels in the body or in the placenta. Extracelllar matrix proteins can be derived from tissue, body thds such as, for example, blood, or media conditioned V non recombinant cells or recombinant cells or bacteria, [0 1041 xtraenryonic endoderm" as used herein refers to a population of cells expressing at least one of the following markers SOX 7AP and SPARC, [01051 "H1EK293 cells" refers to a cell line generated by transtomation of a culture of normal human embryonic idnebyCelS Graham et al. (. Ger. VirL. 369:592 1977 and any cells derived from this parent cell line, 0106] fvarker" as used herein are nucleic acid or polypeptide molecules that are differentially expressed in a Cell of interest. n this context, diffirential expression. means an increasedievel for a osit e marker and a decreased Ievel for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identifed and diAsinguished fron other cells using any of a variety of methods known in the art. [01071 "Matrix" as used herein refers to a 3-dimensional support to which cells may attach 10108] Cesendodenn col" as used herein refers to a Ce expressing at least one ofte following markers: CD, comesodermin (EOMES> SOX-1. DKK4, HNF-3 beta. GSC, FGF17, (IATA-6 101091 "Pancreatic endocrine cell" or pancreatic hormone expressing cell as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, ghicagonm somatostatin and pancreatic polypeptide. [01101 "Pancreatic hormone secreing cell" as used herein refers to a cel.l capable of secreting at leat one of the following hormones: insulin, glucagon, somatostain. and pancreatic polypept ide. 101111 "Pro-prinitive streak cellas used herein r"fes to a cell expressing at least one of the foliowingnmarkers:Nodalor F83 101121 "Primitive streak ell" as used herein refers to a. cell pressing at leastone of the following markers: Brachvury, Mix-like homeolbox protein, or FGP-4. 101131 "Surfiace") as used herein refers to the outermost layei of molecules of a solid substrate vessel or matrix intended fir use in cell culture or analysis The elemental composition e the roughness, and the wettability of the surface can be analyzed by X-Ray Photoelectron Specto)scopy (XPS), Atomic Force Microscopy (AFI), and contact angle muesurement, espectively 101141 'Surface modified plate" reters to a vessel containing any one of sur-faces 1-34, described in Examples j 6, 17 and 26 or plates containing surfaces that are sold under the trade names Nunclon a C B"' nND ad Primaria'l The vessel can, for example, be made of a poyner, such as polystyrene (PS), cyclic oleina copolymer ((OIO), pol.ycarbonat (P(), polymnedthyl methacrylate (PMMA.), or styrene acryIonitrile. copolymer (SAN), [0115j Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renewm and differentiate to produce progeny cells including self-renewing progenitors, nonrenewing progenitors and terminally differentiated cells, Stem cells am also characterized by their ability to diffrentiate in wirn into functional cells of various ell lineages firom multiple germ layers (endodern, mesodem and ectodernA as 22 well as to giVe nse to tissues ofnmliple germ layers fohowing transplantation and to contribute substantially to most, if not alL is allowing injection into blastocysts. [01161 Stem cells are classified by their developmental potential as: (i) tiapotent, meaning able to give rise to all embryonic and extraentryonic cell typesji i pluripotent, meaning able to give rise to all embryonic cell types. (iii)nltipoteni meaning able to give rise to a. subset Of cell iineages, b)ut all wit(un a particular tissue.organ or physiological system (for eXample, hematopoietic stem ells (SC)can produce progery that include HSC (self- renewal) blood cell restricted oligopotent progenitors and al ell types and elements (eg, platelets) that are normal components of the blood) (iv) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than nutipotent stem cells; and (v) unipotent, meaning able to give rise to a single cell lineage (egspermatogeniestenm ceilsj 171 Differentiation is the process by which an unspecialized ("uncommitted) or less specialized cell acquires the features of a special.ed cell such as, fotr example, anerve cell or a musce cell A differentiated or diffirentiation-induced cell is one that has taken on a more specialized (committedposition within the lineage of a cel The terIri committed". when applied to the process of differentiation, refers to a ce that has proceeded in the differentiation pathway to a point where under normal circumstances it will continue to differentiate into a specific cell type or subset of cell types, and cannot. under normal circunstances, differentiate into a different cell type or revert to a less diferentiated cell type. Dedifferentiation refrs the process by which a cell reverts to a less speciahzed (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, that is. Which cells it came nwhat cells it can giv rise to The lineage of a cell places the cell within a hereditary scheme of development and differentiation, A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted celto the lineage of interest. (181 Various terms are used to describe cellshi cuhure "Maintenance" refers generally to cells placed in a growth medium under conditions that facilitate cell growth and/or division hat may or may not result in a larger population. of the cells. <Passaging" reirs 23) to the process of removing the cells from one culture vessel and placing them in a second cuhure vessel under conditions that facilitate cell growth and/or division. [01191 A specific population of cells, or a cell lines sometimes referred to or characterized by the number of times it has been passage. For example, a cultured celi population that has been passaged ten times may be referred to as a RI0 culture. The primary culture, that is, the first culture folnowilig the isolation of cels from tissue. is desi grnated PG. Following the f&st subcuhture. the cells are described as a secondary culture (P1 or passage 1. After the second subculure, the cells bec(rme a teary culture (P2 or passage 2) and so on, it will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doubling of a culture is greater than the passage nunboer The expansion of cells at is, the number of population doubling) during the period between passaging depends on many factors, mcluding but not limed to the seeding densitysubstrate medium, growth conditions, and time between massaging, [11201 In one embodnent, the present invention provides a method to enhance the attachment of cells to a surface containing roma t l about 0.9% N, a sur of' O and N of greater than or equal to 22.3% and a contact angle of at least about 1. degrees, lacking a feeder cell layer and lacking an adlayer., comprising the steps of: a, Obtaining a suspension of cels, and b, Adding the suspension of cells to the surface and allowing the cells to attach. [01211 In one enubodiment the present i nvention provides a method to enhance the attachment of cells to a surface containing frorn at least about 0,5% N a sum of 0 and N of greater than or equal to 172% and a contact angle of at least about 1 3.9 degrees. lacking a feeder cell layer and lackINg an adlayer, composing the steps o a Obtaining a suspension of cells, b, Treating the suspension of cells with at least one compound selected from the group consisting of a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activityand c, Adding the suspension of cells to the surface and. allowing the cells to attach. 24 101221 :In one embodient the suspension of eIs is a suspension of clusters of cells. in an alemnate embodiment the suspension of cells is a suspension of single cells. [01231 In one embndimnt the cells are pluripotent stem cells, In an alermate embodiment. the cells are stem cells. 101241 In one embodiment, the surface has an adlayer, In one embodimentthe adlayer is an extracellular matrix component, such as, for example, those derived from basement membrane or that nay flon part of adhesion moiecu crceptor-lgand couplings. h One embodiment, the adlayer is made from MATRIGEL (Becton Dickensont MATRIGlL is a solubl preparation from Engeibreth-Holm Swarm tnnor cells that gels at room temperature to fram a reconstituted basement menbrane. The proteinaeeous adlayer may also be foned from laminin; fibronectin, proteoglycan, entactin, heparan sufate, and the like alone or various combination. 101251 In one embodiment. the cells are maintained in culture after the cells attach to the surface. i an alternate embodiment the ail east one compound is removed aier the cells attach to the surface. In one em xodianentthe cells are detached frn the swrac by removing the at least one compound. 101261 In one embodiment,the suspension of cells is treated with at least one compound capable of inhibiting Rho kinase activity In an atenate embodimentthe suspension ot ceils is heated with at least one compound capable ofinhibiting Rho activity in an altemate embodimentthe. s son of cells is treated with at least one compound capabe of ihibiting Rho kinase activity and at least one compound capable of sibiting Rho activity [0127j The at least one compound.icapable of inhibiting Rho kinase activity is selected from the group consisting of, Y-27632, Fasudi. and Hydroxyfasudil [01281 In one embodiment the at east compound capable of inhbtng Rho kinase activity is W27632. 101291 The at least one compound capable of inhibiting Rho kinase activity may be used at a concentrationfromi about (Ill l- ) about 100pM In one embodiment, the at least one 25 compound capable of inhibiing Rho kinase actvty is used at a concentration of about 10piv. [01301 In one embodmntar , the at least one compound capable of hibiting Rho activity is a Rho GTPase inibitor 101311 In one etnent the atleast one compound capable of inhiting Rho adcvity is exoenzymeCi Transferase, 101321 The at least one compound capable of nhibiting Rho activity miay be used at a concnentraton from about 0.01 p/mIl to about 5jpgjrI l.a one enmodime nctthe at least one compound capable of inhibiting Rho activity is used at a concentration of about I : pg/mi. Surface Modified Plates [ 33}] Surface modified plates suitable for use in the present invention may be vessels whose surfaces have been modified to contain from at least about 0,5% N, a stun O and N of greater than or equal to 17.2% and a contact antgl. of at least about 9 degrees. Aterativel, the suibce may be adim ensional marix, such as, tor example. a porous scaffold to which celIs can attach. 101341 In one embodiment, the surface modified plate comprises a plate whose surface contain s rom at least about 0 % N a sum of ) and N of greater than or equalto 17.2% and a contact anleof at least about 13 degrees, In an alternate embodiment the surface modified plate comprises a plate whose suace contans from. at least about 0.5% N, a sum of 0 and N of greater than or equal to 19.5% and a contact angle of t least about 13 9 degrees. [0135] In one embodiment the surface modified plate comprises a plate whose surface contains from at least about 1 .3% N, a sum of () and N of at least about 24.9% and a contact angle. of at least about 20.3 degrees. ich is revered herein as surface mTIodiied Plate 1 [01361 In one embodiment, the surface modiefid plate comprises a plate whose surface contains from at least about 1 .7% N. a sum of Oand N of at least about 29.6% and a 26 contact a e of at least about 43 trees which is referred herein as surface modified plate 2. [0137] in one embodiment the surfer modified plae comprises a plate whose surface contains frm at least about 2A)% N1 a suTn ofO( and N of at least about 30,7% and a contact angle of at least about 8.4 degrees which is refered herein as surtace modified plate 3 [01381 In one embodiment, the surface modified plate compi5S a plate whose surface contains from at least about 2.1% N, a sum of ( and N of at least about 30,2% and a contact angle of at least about 17.4 degrees, which is refered herein as surface modified plate 4. 10 139] In one embodimentthe surface miodificd plate comprises a plate whose surface contains from at least about 1.8% N num of t and N of at least about 28,2% and a contact angle of at least about 8 8 degrees, which is revered herein as surtace modified plate 13, 101401 In one embodiment, ie surface modified plate comprises a plate whose surface contains from at least about 1.0% N. a sum of 0 and N of at least about 27.8% and a contact angle of at least about 44.3 degrees, which is sold under the trade name CELLB3IND. 101411 in one embodiment, the surface modified plate comprises a plate whose surface contains from at least about 10.2% N a sum of () and N of at least about 23.0% and a contact angle of at least about 395 degrees, which is sold under the trade natne PRIMARIA, Charatekation ofhe Smtde Moidified -Plates [01421 In one embodinent. the elemental composition of the surface ofthe surface modified plates may be analyzed by X a-Ry Photoelectron Spectroscopy (XPSj XPS also known as Eectron Spectroscopy for Chemical Analysis (ESCAY is used as a method to detemine what elements or atoms are present in the surface of a solid substrate tl elements in concentrations less than 0 1 atomic perce can be detected except hydrogen and helium), and to determine the bonding environment of such elements or atonis. As an example; an X PS analysis of a polystyrene(contains only carbon and hydrogen) solid sample would typically give greater than 97% carbon, lesshan 3% oxygen and 0% nirogen (hydrogen is not detected; different levels of oxygen may be detected due to oidation of the polystyrene cins at the surface, for example, as a result of sterilization by irradiation) (Brevig t AB, Biomnais 263039~3053 2005; Shen and Horbett Biomed. iater Res. 57:336 5 2001), 01431 in one enbodi nenrt, the roughness of the surface of the surface modified plates may be analyzed by Atomic Force M icroscopy (AFM}. Surface atoms or molecules with a lateral resolutiori down to IA and a vertical resolution down to 0.1A can be imaged by AM. [01441 In one embodiment, the wettability of the surz'ee of the surbee modifed plates may be analyzed by measuring the contact angie For examplei contact angle measurement by the static sessile drop method provides information on theinteractio between the surface of a solid substrate and a liquid. The contact angle describes the shape of a iquid drop resting on the surface of the solid substrate, and is the angle of contact of the liid on the surface of the solid substrate/ measured within the liquid at the contact line where liquidsolid, and gas meet.A surface with a water contact angie larger than 90is termed hydrophobic. and a surfaec with water contact angle les than 90* is termed hydroplhilie On extremely hydrophilic suifces that is surfaces that have a high affinity for water, a water droplet will completely spread (an effective contact angie of (r), [01451 In one embodiment the negative charge density of the surface of the surface modified plates may be analyzed by measuring the reactivity of the surface with crystal violet, Crystal violet carries a positive charge, which enables it to bind to negatively charged miecules and parts of molecules, fo example negatively charged functional groups present on a polymer surface. A. surface with a high crystal violet reactivty has a higher densty ot negative charges than a surace with a low crystal violet reactivity given that the surfaces have the same roughness and thus area, Pluripotent Stem Cells Characterization of Phaatent Sm Ce2 11461 Pluripotent stem cels may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4. and markers detectable using antibodies designated Tra- I 60 and TraI81 (Thomson et aScience 282 -45 [998 Differentiation of phiripotent stem celIs in vitOresults in the loss of SSEA~4 Tra- 160 and Tra- 141 expression presente) ani Increased expression of SSEA- Undiieretiated pluripotent stem cells typically have alkaline phosphatase aetiviy.which can be detected by fixing the cells wih 4% paratormaldehyde and then developing with Vector Red as a substrate, as described by the manufacturer (VetorLaboratories Burldingame Callf Undifferentiated piuripotent stemn cells also typically express Oct-4 and TERT, as detected by RT-PCR, O1471 Another desirable phenotype of propagated luripotent stem cels is a potential to differentiate into cells of all three gerninalayers: endodem mesoderm and ectoderm tissues. Phipoteney of stem cellsan be conimed, for example, by injecting cells into severe combined immunodeficient (SCW) mice fixing the teratomas that form using 4% paraformaldehyde and then exanining them histologically foI evidence of cell types from the three germ layers Altematively. pluripotency nmay be determined by the nation of embryoid bodies and assessing the ernbryoid bodies for the presence of markers associated with the three germinai layers. 0148] Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technigue and compared to published karyotypes of the corresponding primate spedes It is desirable to obtain cells that have a " normal karyotype Which means that the cells are euploid, wherein all huna. hmmosomes are present and not noticeably altered, Sources qf P i potert MSen Cells [0149] The types of pWuripotent stem cells that may be used include established lines of pinripotent Cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example a blastocyst embryonic tissue, or fetal tissue taken any time durig gestation, typically but not necessarily before approxiatly 1.0~2 weeks gestation- Non-imniting examples are established lines of human ES eels or human enbryonic germ cells, such as, for example the human ES cell lines H I H7, and. H9 29 (Wi(ell Also contemplated is use of the compOsitions of this disclosure during the initial establishment or .stabillzation of such cells, in which Case the source cells would be primary pluripotent cells takendirectly froi the source tissues. Also suitable are cells taken from a plurpotem stem cell population already cuhdured in. the absene of feeder cell as well as a pluripotent stem cell population already cultred in the presence of feeder cells. Also suitable are mutant human ES cell lines such as, for example, [3001 v (BresaGe Athens GA) Also suitable are cells derived from adult human somatic cells, such as for examplescells disclosed in Takahashi et a Cell 131: 12 (200) [01501 In one embodiment, human ES cells are prepmed as described by Tlmnson at (U.S. Pat. No. 5'84378); Science 282:114 1998; Curr. Top, Dev. Biol. 38:133 f, 1998; Proc. Na. Acad Sci U.S.A. 92:7844, 1995, Culture iPluripoten Stem Cells 011511 in one embodiment, pluripotent sten cells are cultured on a layer of feeder cels or extracel I ar matrix protein that support th piuripotent stem cells in various ways prior to culturing according to the methods of the present invntion. For example, pluripotent stem eelIs are cultured on a feeder cell layer that supports proliferation of puripotent stem cells without udergoi ng substantial differcntation. The growth of pluripotent sten cells on a feeder cell layer without differentiation is supported using (i Obtaining a culture vesse I containing a teeder cel layer; and (ii) a medium conditioned by culturing previously with another cell type, or a noniconditioned medium for example, free of serum or even chemically defined. [01521 In another example. pluripotent stem cells are cultured in a culture system that is essenti ally free of feeder cells, but nonetheless supports proliferation of pluri potent stern cells without undergone substantial differentianon. Thcgrowth of plurpote nt sten cels in tedcr.cell free culture without differentiation is supported usig i) an adlayer on a solid subustrate surface with one or more extracel.lular matrix proteins; and (i) a medium conditioned by ctdituring previously with another cell type, or a nton-cnditioned medim, fbr example, free of serum or even chemically defined. 30 101531 In an alterate embodiment pluripotent stem eis are cultured on a surface modified plate containng front at least about 0.5% N, a sum of 0 and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees in a mcdiurn conditioned by culturing previously wth another cell type, or a non-conditioned median for example tree of serur or even cemically defined. 101541 &dturc enc n: An example of cel culture meditun suitable for use in the present invention may be found in tJS2002007 2 1 Another example of cell culture medium suitable for use in the present invention may be found iT US664204, Another example of cell culture medium suitable for use in the present invention may be found in W02005014799 Another example of cell culture medium suitable for use in the present invention may be found in Xu et (Stem Cels 22:972<980 2004), Another example of ell culture medium suitable for use in the present invention may be found in US20070001 1Anotheexample of cell culture medium suitable for us in the present invention may be fouad in Choon et al. (BioReprod DO: 101 095/bioireprodl 0504%M6870; 19 Oct 2005 Another example of cell culture medium suitable for use in the present invention ma be found in Levenstein et al. (Stem Cels 24: 568574, 20061. Another example of cell culture medium suitable for use in the present invention mxa be fod in US20050 148070, Another example ofi celledtare medium suitable for use in the present invention may be found in US20050233446, Another example of cell cudure rnedum suitable for use in the present invention ma> be found in US6800480 .Another example of cell culture medium suitable for use in the present invention may he found in US20050244962. Another example of cell culture medium suitable for use in the present invention may be found in W02005065354. Another example of. el culture medium suitable for use in the present invention may be found in W02005086845. 101551 Suitable culture media may also be made from the following components, such as. for example. Dulbeetis modified Eagle s rnedium (DM EM> Gibco # 11965-092; Knockout Dulbecco's modified Eagids medium KC DMEMv, Gibco 4 10829-018; Hamslw F12/5 DMAEM basal medium; 200 mM iglutamine, Gibco # 15039~027 non-ssential amino acid solution Gibco 111401)50 mecapoethanol Sigma 4 M47522; human recombinant basic fibroblast growth factor (bFG (ibco i 13256 029, DIINre'ndation offizt;4iowent Si(te d/s (0156] Inone cinbodineont o r t inventio pluripotent stein ces arc propagated in culture, while maintaining their piuripotency. Changes in phuripotency of the cells wth tinne can be determined by detecting ehangesithe levels of expression of markers associated with phAripoteney Ahematively changes in pluripotency can be monitored by detecting changes in the levels of expression of markers associated with differentiation or markers associated with another cell type [0157 In an alternate embodiment, pluripotent stem cells are propagated in culture and then treated in a manner that promotes their diffrentiadon into another cell type. The other cell type may be a cell expressing markers characteristic of the definitive endodern Wiel type may be a cel expressing markers characterisic of the pancreatic oiddernt lineage Alernatively; the cel type may be a cell expressing markers characteristic of the pancreatic endocrine lineage, Aiternatively, the cell type may be a cell expresig markers characteristic of the {3 cell lineage, 0 158] Pluripotent stem cells treated in accordance with the methods of the present invention nmay be differentiated into a variety of other cell types by any suitable method int the art [01591 For example pluripotent stem cells treated in accordance wi the methods of the present invention may be differentiated into neural cells cardiac cells, hepatoeytes, and the like. [0-160] For example, pluripotent stem cells treated in accordance with. the methods of the present invention may be differentiated into neural proenitors and cardiomvocytes according to the methods disposed in. WO200703 0870. [01611 In another example, pluripatent stem cclls treated in accordance with the methods of the present invention may be differentiated into hepatoeytes according to themethods disclosed in US patent 6138,589. 32 0162 For example, plitripotent stem cels may be diferentated into cells expressing tarlkers charateristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et a. Nature Biotechnol 234-1541,2005. 01631 For example, pluripotent stern cells may e differentiated into Cels expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki eat ed Development 131:16511662. 2004. [01641 For example. pluripotent stem \els may be differentiated into eieI expressing markers characteristic of the definitive endodern lineage according to the methods disclosed in McLean et a Stem Cells 25:2918, 2007. [01651 For example, phiripotent stem cells may be differentated into cells expressing markers characteristic of the definitive endodermlneage according to the methods disclosed in D'Amour w at, Naure Biotecnol. 24,1392-1401, 2006. [0166] varkers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX1T GATA4, Itif-3eta, C Ceti Nodal, FGF-S Bachyu Mixdlike homeobox proteinFFA CD48, comesodermin(EOMES DKK4. FGF 11,GATA6 CXC R4,C-Kit, CD99. and OTX2. Stable foruse in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endodernm lineage., In one aspect of the present invention, a cell expressing markers characteristic of the deini tive endoderrnlineage is a primite streak precursor cell lnean alternate aspect, a cell expressing nmarkers characteristic of the detuitive endoderrm lineage is a rnesendodermn cell. In an alternate aspect. a cel expressing markers characteristic of the dennitive endoderm lineage is a definitive endoderr cell. [01671 For exampi. plurpotent stem cells may be diferentiatd into cela expressing markers characteristic of the pancreatic endoderm jiinecage according to the methods discIosed in D'Amuretir at, Nature Bioechnol. 241392-140 1, 2006. [01681 Markers characteristic of the. pancreatic endoderm lineage are selected frm the group consisting of Padx L, HN F- beta. TF la FIN F-6C 18B9 and PROX I Suitable for use in the present invention is a cel that expresses at least onef o the markers characteristic othe pancreatic endoderm. lineage. In on of the present irendon, a MI 33 expressing markers characteristic ofhe pancreatic endodern lineage is a pancreatic endodernt cell. [01691 Pluripotent stem cels may be ddferentiazed into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art 01 701 For exane, pluripotent stMt cells tay be diftereindated into cels expressing markers characteristic of the pancreatic endocrin iicage according to the methods disclosed in D'Armour atNature Biotechnol. 24 39241401 . 2006 101711 For example. Mripotent stern cells may be diffenrentd into cells expressing markers characteristic of the pancreatic endocrine lincage, by the methods disclosed in 1) Amour et al. Nature Biotechnol. 24,1392 i401 2006. 10172] Markers characteristc of the pancreatic endocrine lineage are selected fom the group consisting of NGNJ NeuroDsleti, Pdx.-i NKX6,l Pax-4, Ngn and PTF1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: i lucagon soratostannand pancreatic polypeptide. Suable for use in the present invention is a cell that expresses at least one of the markets characteristic of the pancreatic endocrine lineage I one aspect of present invention, cell expressing m arkers characteristicof the pnrai endocrine lineage is a pancreatic endocrine eIL The pancreatic endocrine cell nay be a pancreatic hornoneexpresung cell. Alternativelythe pancreatic endocie cell may be a pancreatic hormone-secretng cell. [0173] In one aspect of the present ivention, the pancreatic endocrine cell is a ccli expressing markers characterisaic of the 3 cell lineage. A cell expressing markers characteristic of the p cell lineage expresses Pdxi and at least one of the following transcription fators: NGN2Nkx24 Nkx6. , NeuroD Lb 1, HNF c3 beta MAFA, Pax4, and Pax In one aspect of the present invention, a cell expressing markers Characteristic of the 13 cell lineage is a 1 cell [01741 The present invention is further illustrated, but not limited by. the following examples.

EXAMPLES

Example I Passage and Maintenance of Human Embryonic Stem Cells as Cel Clusters 11751 The human ES cel lines H I and HN were initially maintained on litoimyiCin C inactivated primary mouse embryonic fibroblasts (IEF), The human ES eells were switched from ME F eeders to aige (Becton-Didinson, Bedford MA) over repeated passages, 10176] Treatment of surfaces with MaWriel Growth Factor Reduced atrigel'was awed at 4C and then diluted 130 in cold DM EM/12 (nwitrogen Carsbad. CA), Volumes sufficient to cover the surface were added to each 6cn dish (2 an) or each well of a 6-wel plate (I mlt and incubated I hr at rom temp Treated surfaces were used within a few hours or stored at 4C up to two weeks, [01771 Human ES cell culture: Undifferentiated human ES cell colonies (from either the 19 or H I ines were harvested from feeder layers by incubation in Imwnl collagenase V (Sima-Adrich St s Lous, M) in DMETE12 for 10 minutes. fbIowed by scraping with a pipette; Cell clumps were pelleted by centrifugation at 600 x g for for minutes and the pellet dispersed gently with a 2-mi pipette to break colonies into small busters of cells. These ell clusters were seeded onto Matrigel '<reated dishes inl media conditioned with mouse embryonic fibroblasts (MEF-CM. further supplemented with bFGF (8 ng/d; R&D Systems. Minneapolis, MN). at 50150 colonies per 6-ci dish in 5 m gwbth mediumdiumium was changed daily. Colonies on Matrig T in MEFKM became large and were passed when they occupied 70-80% of the surface areaapproximately every 3-4 days. The human ES cells in the colonies had a high nucleus to cytoplasm ratio and had prnmnent nucleoli similar to human ES cells maintained on feeders (Figure ) Differentiated cells represented less than 5% of total cells in culture. [01781 For routine passage of ells in MEF~CM on Matrigel - els were incu'atId in I mg/mi collagenase IV in DM EM 1/Fl2 for up to 60 minutes and removed from the dishes by forceful streams of DMEM/FI 2 with scraping, Cells were pelted, dispersed and seeded at a 13 or 1:4 ratio Example 2 35 Passage of Human Embryoic Stem Cells as Single Cells (01791 HamanES cells of the cell linie 119 were grown as single cells acorditig to the methods disclosed in US Patent Application LFS5163U8PSP assigned to LifeScan Inc. Cls were passaged by treatment with TypLn Express fo five nuates at 37(C, and seeded at 10,000 cels/emi substrate surface, Example 3 Attachment, Cultivation and Maintenance of Pluripotency of Human Embryonic Stem Cells Using Surface Modified Plates Lacking Extracellular Matrix Protein/Components and Feeder Cells 101 nluman ES cells of the line HI i at passage 49 were maintained in M EF conditioned niedia on, Nunclon Deltar" pltes treated with a 1:30 dilution of growth factor reduced Matriget prior to study. Cells were dissociated from the surface for passage by 1mg/nIl collagenase dissociation or by nanua scraps [01811 These cells were then seeded onto two untreated welIs of the surface modified plates (6-well formnat) Additionally one well of each paite was treated with 0.1% xeno-dree human geiatmn as a control. CeIs were also plated directly onto treated and gelatin~ treated wells of Costar""" (cat. no. 3516; Corning, Corning Y Falcon (oat. no: 354146 Beton Dickinson, Franklin Lakes. NJ and Nunclon Deta (cat, no. 1 40675; Thermo Fisher Scientific, Roskilde, Denmark) 16well plates fornegative contls and plated onto wells treated with 130 edition of growth factor reduced to provide as positive controls In all treatments Cells were maintained in N iF conditioned media. (01821 We observed that after two passages surface modified plates 2. 3, and 4 had attached ES Cell colonies, which reattached to the plates and grew following enzynatic dissociation. There was no apparent diffrene in rate of attachmentor growth in gelatin or untreated wels from surface modified pates 2, 3 or 4, 101831 Cells mechanically dissociated fom plates treated with T:30 dilution of growth factor reduced Matrigd vwere poorly attached to surface modified plates 2,3 and 4, whle 36 Cells enzymnatically dissociated with mg/ml collagenase were well attached in. gelatin or unzoreated wells fron sorfce modified plates 23or 4. [f184] H1p49 ES cells added to surface modified plates I and 512 and to untreated or gelatin treated Nunlcion Delta plates Falcon plates, and CostarM plates did not attach, The same cells did attach to plates treated withI :30 dilution of growth factor reduced lviatriget indicating that the cells were competent to attach to a substrate surface. 101851 Normal passage time for ES cells of the 1iU line plated on 1:30 dilution of growth factor reduced Matrigel ' was 34 days, however cells plated on surface moditied plates 3 and 4 took 7 days of culturing before they were ready for passage, This was probably due to the reduced rate of attachment on the treated surfaces, since more sta-rtint colores were apparent on Natrigel Nttreated surfaces immediately after plating than on Surfaces 2 3 and 4. [0186j The passag (p) 50 Cells were splt at a I to 2 ratio and halfof the sample was coleced for RNA purifeation ad tested f&W expression of Iuipotency makes (Table I) The other hal f of each sample was repeated to source modified plates. Colonies that formed at this passage (p 1) also required 7 days of culturina before they wereready to be passed and the small colonies that deveped after only 4 days of caltuing are shown in Figure I. These colonies maintained dassical ES cell colonyw mnorphology 101871 Cultures were stopped at passage 4 on surface modifed plates 2. 3 and 4 and samples were assayed for pluripotency markers by qRT-PCR (Table 2) and. differentiated to a defnitive endoderm fate (DE). Cells at Passage 4 maintained expression of the classical pluripotency markers: Oet4, Nanog; Sox2, and TERT Furthermore the cells were able to differentiate to a definitve endoderm fate upon exposure to a media containing DME M/F12 [0ng/m Activin A. 20 ngfl Wnnt3a, and C,5-2,0% F13S (Table 3) indicating that pluripotency was aaintaned in the cells through passage 4. Example 4 Attachment, Cultivation and MI aintenance of Pluripotency of &mian Embronic Stem Cells on Surface Modified Plates Lacking Extracellular Matrix .)7 Protein/Components and Feeder Cells: Effects of Rho Inhibition and Rho Minase Inhibiion [0188] Human ES cells of the tine H, at passage 49 were maintained in MEF conditioned media on Nunelon DeItta plates treated with a -30 dilidon of growth fiactor redeed MAatrigel t prior to study, Cells were dissociated front the surface for passage by I mgmI collagenase dissociatioa. [01891 These cells were then seeded onto untreated wells of surface modified plates (6~well format) Cells were also plated directly onto untreated and gelain-treated wells of Costa" Eacontand Nunclon Delta 1 6-ell plates for negative controls and plated onto wells treated with 1:30 dilation of growth factorreduced Matrigel to provide as positive controIs In all treatments cells were maintained in MEF conditioned media. 10190] Iluman ES cells of the line HI, at passage 49 added to sude modified plates I and 512 and to untreated or gelatin treated Nundon Deltatm plates and Costar - plates did not attach, however, they did attach to surface modified plates 2, 3. and 4. The same cels did attach to plates treated with 1:30 dilution of growth factor reduced Mlatrigelt indicating that the cells were competent to attach to a substrate surface. [01911 Normal passage ine for H' ES cells plated on 1:30 dilution of growth factor reduced Matrigel was 3-4 days, however eel s plated on sIflace modifed plates 2, 3 and 4 took 7 days of culturing before they were ready for passage. This was probably due to the reduced rate of attachment on the surface modified plates, since more starting coIonies were apparent on NIMatrige created surfaces immediately after plating than on surface modified plates 2, 3 and 4. 10192] The passage(p) 50 Cels were split at a 1. to 2 ratio and half of the sanm-plc was collected for RNA purification and tested for expression of pluripotency markers (Table 1, The other haf of each sample was related to surface modified pates Colonies that forced at this passage (p5 1) also required 7 days of culturing before they were ready to be passage and the small colonies thatdeveloped afer only 4 days of culturing are shown in Figure 1. These colonies mainta-ned classical ES cell coony morphology, 101931 Due to the delay in passage the cels were split at aI to 2 ratio and half of the passage 4 samples were plated in MEF conditioned media or MEF conditioned media supplemented with the Rho kinase (ROCK) inhibitor, W27632, at a 10 pM concentration in. aT attempt to improve cell growth kinetics. Cells were kept in the plating media for 48 hours after passage at which tine the media was charged to fresh unsupplemnted IfF conditioned media, [01941 The addition ofY.T2632 at a -0pM concentration significantly increased plating etciency of the ces (p2) and the improve n in colony growth was apparent after 4 days osa igre 2 Ate actively prior to colagenase dissociation hunan ES cells of the line H I were also treated with 0. 5 ng/ml of a cell permeable form of the Rho inhibitor; ('3 eotnsferase. hich also increased the plating efficiency of the cells; [0195] While cells plated in 10 pM Y27632 could be assuaged 4 days aer plating cells plated without tie ROCK inhibitor were not ready to be split 4 days after plating. Cells treated with-, Rho inhibitor,03 exotransferase were also not ready for passage 4 days after plang and cells exhibited increased differentiation to a fibroblastlike mnorphoiogy( onsequently, elis treated with Rho inhibitor at passage 4 were treated with Y22 a it all subsequent passagesO(Figure 3) [0196j Cells were further passage tat least 10 passages on surface modifed plates 3 and 4 and wee tested for ~te presence of markers associated with piupotency genes by qRT PCR cell surface marker expression by flow cytometry; and ~nnmunofluoreseence of cciiLurface and nuclear poteins (Figures 4-6), Cellular phuripotency was also confirmed by testing their capacity to differentiate to defnitive endodem. pancreatidoddoder and fibrin eobyroid bodies composed of the trece germ layers (Figures 79. Cells were also tested for karyotypic stabiliyand we observed that cels could mantain a normal karyotype (Figure 10), Example 5 Attachment and Detachment of HuNman Embryonic Stem Cells Through Rho Kinase Inhibition 39 0 -197 1Inan ES cels of the line -9. at passage 40 were maintained in MEF conditioned niedia on Nunclon Delta" plates treated with a 1:30 dilution of growth- factor reduced MatrigeK" Pan to study Cells were dissociated rom the surface for passage by I mgmi collagenase dissociation or by manual scraping. [01981 These ce lswere then seeded onto surface modified plates 2, 3,4 and 13 (1 2well forna in the presence of increasing amounts one of the foilowig Rho Kinase inhibitors: Y-27632 (from Sigma. St. Louis, MO or EMI), San Diego CA). Fasudil (Sigma), or Hydroxyasudii and maintained fhr days, each day replacing the media and compound At the end of day there, media. Was removed and the plates were staned with Crystal Violet (0.5% in water) to visualize colonies. [01991 We observed tha by day three. surface modified plates 2, 3 4 and. 13 had attached ES eecolonies in the presence of increasing amounts of Rho kinase inhibitor. Best results were obtained through the use of Y-2732 (10 pM) although some colonies coukd be observed to attach and grow with the Rho kinase inhibitors, Fasudil and Hydroxyfasudil(Figure 111 [0200] Wt. attempted to determine the optimal dose of Y-27632 to promote cell bindingiby treating cells with a range of phting concentrations of Y27632 for the first day of culture. After the first day in culture cells were treated on subsequent days with a 10 pM concentration of Y27632. We observed that the maximal concentration to stnulate attachment and growth of ES cells was 10 pM (Figure 12) and that this occurred on surface modified plates 2 3 4, 13 and CellBINDTM (Corning, Coming, NY). 102011 The effect of treating the cWlls continuously wh a single dose of Y27632 on attachment and growth was also tested, The cells were dosed with 0, 1, 4 or 10 pM Y27632 thQ 4 days. Some binding was observed on surface modited plates without treatment ( 01'M, however the optimal concentration to stimulate attachment and growth of ES cells was 10 M Y~27632 (ab 4) on surface modified plates 2, 3, 4, 13, 10202] Since the addition of ROCK inhibitor significantly enhances the plating and growth kinetics on surface modified plates 2 3 and 4 versus untreated cells Figeure 13), we 40 wished to determine if his was due to maintenance of proper cell attachment or due to increased cel proliferation. We observed that Rho Kinase inhibinon does not increase cell prolifertion. because cells treated withY2762 grow at a similar density as untreated cells (Figure 141I treatment mnta is the attachment of cels to the surface and allows them to grow with normal proliferaion kinetics (Figure 1 5 Removal of a Rho Kinase inhibitor from the growth media of ES cells plated in the presencof Rho kinase inhibitor results in detachment of the cells fn the surface. The formation ot ernbryoid bodies with differentiation to the 3 germ hineages is accomplished by culturing ES cells in a suspension. Consequently although later reapplication of a. Rho kinase inhibitor restored attachment of cells (Figure i as expected, substantial dierentiation of the ES cell culture was observed in sanmles where Rho kinase inhibitor was withdrawn for 24 hours of culture and cells were allowed to detach, grow in suspension for 24 hours and. Rho kinase inhibitor was then reapplied, Example 6 H9 human ES cells passaged with TrypLEN' Express on Surface Modified Plates Show Improved Adhesion with Y-27632 t0203) initial pssaging of 19 hunan ES cels onto surface .nodifed plates, Adhesio is improved with continuous II pM of 6&32. This is true for the four surface odied latest tested 2,3, 4 and 13. Images of H9 cells 24 hours after seeding on Surface 3 are shown in Figure 16, Example 7 H9 single Human Embryonik Stem Cells Passaged with TypL E "Express on Surface Modified Plates Remain Pluripotent [02041 Human ES cells are piuripoterin and have the ability to diffrentiate into all cell. lineage The pluripotent state of the cells must be mltaned by the surface on which they grow. To determine if the surface modiied places can maintain human ES ell pluripotency, the human ES cells were passage 3 times with coliagenase and 38 tines with TrypLE Express followed by 5 passage or' surface modified plate 3 (Surfacesurface modified plate 4 (Surface 4) or Marig at 1 30 dilution. 10 41 iA of *Y-27632 was added to the media of indicated samples. The expression. of pluriote.ncy markers Tra-l 60 Tra41-81 SSEA-3 and SSEA4. was evaluated by flow tometry. Resuhs are shown in figure 17 The percentage of positive eells is indicated on the y-si e human ES cels grown on surface modified paes 3 ,and 4 cai maintain thei p1 potency Example 8 Rho Kinase inhibition Promotes Adhesion and growth of Cells from the Human Embryonic Stern Cel line H9, Grown. as Single Cells on Surface Modified Plates upon Transfer from Matrigel< [02051 The role of Y-27632 in human ES cell adhesion and cell growth was studied in relation to the surface modified plates. U9 human ES cells were passage 38 times with coliagenase and 50 times with TrypLEm Express followed by seeding onto Suaces modified pMates 3 or 4 (naive cells> Aternatively H9 human ES cels passage 38 times with collagenase and 38 times with. Triple Express followed by 9 passages on surace modified plate 3 (Surface 3, aclimated cells), orsurface modified plate 4 (Surface 254 acclimated cells). Cells were seeded at a density of I0 4 cm in MEF conditioned media and grown for two days with or without the presence of 10 l.M of Y2732 Results are shown in figure 8 Y-27632 improves attachment of naive cels to surface modified plates 3 and. . 27632 did not improve anachment of acclited cells to surface modified pltes 3 or 4, surface modified plate 3 improved attachnetnt and/br growth of naive cells, Surface modified late 4 improved atachment and/or growth of acci-mated human ES cels The cells were followed fbr a total of 4 days (Fiures 19 and 20). The naive single cells exhibited an increase growth rate when cltured with 1.0 ,ptM Y27632 with surface modified plate 3 showing a slight advantage (Figure 19). The acclimated single els exhibited improved growth rates with out the 10 M of Y-27632 (Figure 20). Example 9 Surface Modified Plates can be used to Screen Compounds 102061 Surface modified plates in 96-well conguration and in the Society for Biomolecular Screening SBS) standard format can be used for growing single i uman ES cels in 42 the presence of 10 pM -272. images of 119 single cells plated in 96wel I plate wells are shown in Figure 21. This would allow ifor the screening of cormpounds direct in 96well plates with no interfering eel s or adayers such as mouse embryonic fibroblasts or M atriget, respectivelv Example 10 Single Embryonic Stem Cells Cultured on Surface Modified Plates are able to Differentiate into Definitive Endoderm 102071 One goal is to differentiate human ES cells do difterentell lineages. To deterne if surface modified plates can support differentiationriuman ES cells were passaged 38 times with collagenase and 38 times with TrypLE Express followed by 9 passages on surface modified plate 3 (Surface 3'j or surface modified plate 4 (Surice 4) As a positive control, hunan ES cel s were grown on Matrigel at 130 dilution, 1.0 pM of -27632 was added tohe rnedia during expansion of indicated cell samples Mfer cell expansionthe ability of the cultured cel to form definitive endoderm was evaluated. Briefly. 70% confluent cultures were treated with 100 ng/il Activin A. 10 nagnl Wntaa and 05% FBS in DMEM-FI2 media for two days. The treatment was followed by 3 days i t4 100 n'g/nil Aetivin A and 2% FBS in DMEM/ , ClIs differentited into definitiv endoderi are identified by CXCR4 protein exrceo via flow cytonetty (gure 22). The percentage of positive cells is indicated on they-axis. Human ES cells cutred as sindlecelIs can differentiate into definiti ve endodern in the presence. or absnc of Y-27632 on surface modified lates 3 and 4. Example H. Single Embryonie Stem Cells Cuhured on Surface Modified Plates are able to Differentiate into Pancreatic Endoderm 02081 After completion of the definitive endoderm protocol, the ells were incubated fr 3 days with FGF- 0 ngml; R&D Systems, the sonic hedgehog inhibitor.KAA.D cyclopamine (25 pM M; Si ldrh} and 2% FBS in DMEM-F12 medium At this point, cels not treated with Y7.62 during expansion detached frn the surface modified plates 3 and 4. The ceIl treaed witI-7632 during expansion were 43 incubated an additional four days with FGF-7 (50 ngrnil, KAAD cyclopamine (2.5 pM). Retinoic Acid ( giM; Sigma-Aldrich) and 1% B2 -(trogen) in DMEM-12 (posterior Ireat stage P ) A fter this time, cels were ineubated an additional foiur days in Exendin 4 50 ng m SigmaAldrichl) DAPT pM; Calbiocheru4 and 1% 1127 in DMEMP12 DiZfrianon was contain ed to the paneatic endodern stage (EN). This entailed a -hreday treatment with CM medium (iavitmgen) containing Song/nvI HF ItGF (R&D Systems) and Exeadin 4 (S0ng/m.) and 1% $27. RN A samples were taken at stages PE and EN from one well of the surface modined plates 3 and 4. These samples were then analyzed by realime PCR at this step for pancreatic markers Pl l Nkx6 1 Nkx22, Pax4, NeuroD, HINFb Ptfla Insulin and AFR. Evaluation of the same pancreatic endoderm markers was repeated at this stage RNA samples from untreated human ES cels of the same liNe were subjected to reaLtime PCR in parallel to treated samples. Treated samples were normalized to untreated controls set to a fold change of . Pdx I and insulin expression was monitored and compared between surface ndified plates [02091 Induction of pancreatic endoderm markers was observed from eelIs treated on surface modified plates 3 and 4 although expression was higher witcll treated on surface mthfied plate 3 (gure 23) Both surface modified plates in the presence of V 27632 during expansion can support the differentiation of single human ES cells to posterior foregut and pancreatic endoderrn whereas single cells not treated with Y 27632 during expansion detached prior to posterior toregut differentiation. Example 12 HI and H9 Hunman ES Cells Adhere to Surface Modified Plates and Adherence is Enhanced by Treating Cels with Y-27632 {021.0] Passage 49 [19 hurnan ES cells preiously plated to 1:30 Matrigel treated lastieware and grown in MEF conditioned media supplemented with 8ng/mI of bFGF were LIBERASE treated and plated to surface modified plates in MIEF conditioned media supplemented with 8ngmi of bFF and not otherwise treated or supplemented with increasing concentrations of Y~732s We observed that 24 and 48 hours alter plating H9 hUnan ES cells to surface modified plates small colonies could be observed on Surfaces 2-4 and i3,iand CellBIN Dr U and Prima TN(cat, no. 44 353846, lecton Dickinson Franklin Lakes, NQ with crystal violet stain (Figures 24 261 Furthermore, dhe adherence of H9 human ES celcolonies was improved by de addition of W27632 and the effet was dose responsive(Figure 25). Low concentrations of Y-27632 0 to 2 nicromary showed a minimal improvement in human ES cell attachment verus untreated human ES cells (Figure 25) while higher concentrations oft27632(4 to 20 mieromolar) promoted adherence of human ES cells to surface modified plates as measured by crystal violet stai (Fugure 25 and 26). 10211] In addition to the dynamic regulation of' human ES cell attachment by addition of Y 27632 to the cell culture media, We observed different rates of adhesion of human ES cells to variousurface modified plastics in die presence of 27632. For example cells were less adherent to Cell-,IND plates and were more likely, over time to detach front CellBIND plates even in the presence of sustained Y-2762 treatment while MA C were more adherentmd lesikely to detach from surface modified plates 3 4. or 13 or Primaia w hen treated with the Rho kinase inhibitor, 1-2.632 (Figure 25 and 26) Example 13 Cels from the Human Embryonic Stem Cell Lines H and 19 Attach and Form Colonies at Different Rates on Surface Modfied Plates in the Presence of W 27632 [02121 HI and H9 human ES cells previously plated to 1;30 Matrigelnereated plastieware and grown in MEF conditioned media supplemented with 8 kgl of' bFF were LIBERASE treated and plated to surface modified plates in M EF conditioned media suppiemen ted with ng/ml of FGand not otherwise treated or supplemen ted with 20 micromolar Y -27632 Fortyeight hours after plating H9 human ES cells to surface modified plates 14 and 15, small colonies were observed when the media. was supplemented with 20 micromolar Y-27632 (attachment and colony frmation was variable from experiment to experiment) (Figire .27. H human ES els also attached to and formed colonies on both Surface 14 and 15 in media supplemented with .20 micronolar Y27632. and this was more prevalent than te binding observed with 11-Whmnan ES cells. These data indicate that there is human IFS cell lined-oline variability in attachment to and colony formation on solid substrate surfaces, 45 Example 14 Human ES CeH Attachment to Surface Modified Plates Using Defined Media 02131 Passage 49 19 hwuan ES cells were passaged twice in the define media, mrTeSR M on Matrgetreated plasticware. The cells were then LIBERASE treated and plated onto the surface modified plate Nunc4 in mTeSP edia Cells were either plted in media with or without 20 micromolar YCn2 ells were also treated with various proteins for 30 minutes prior to seeding e is (no treatmet 0, 1% g latin 2% ESA, .34mg/mi rat Collgen L :1000 Matrigel or 1:50 atriel)to determine if those proteins could promote human ES cell adhesion in defined media with or without Y27632 (Figue 28. We observed that inte absence ofY2632 human ES cells plated onto a surface modified plate in defined media did not attacb even in thepresence of extraceilular matrix proteins suchfas Colagen I or 1: 000 Matrielt However, when 20 nicromolar Y27632 was added to define m;TeSR

T

~ media, h urnan ES cells adhered to surface Nunc4. Furthenore, this adherence w as equivalent in untreated wells and wells treated with O 1% gelatin, 2% BSA. and 034 mg/mI rat Clagen There was a modest increase in human ES ci attachment in wells with ow concentrations of Matrieelr(1:1000 and 1:5000 dilutions), however these concentrations of Matrige were insufficient to promote adhesion in the absence of Y2763i These results demonstrate that in the presence of the ROCK inhibitor, Y -27632 huian ES cells can be cultured on modified plastic substrates in def'ined media and that low concentrations of Miatrige of about 1000 or 1:5000 can improve this adhesion. Example 15 Surface Modified Plates in a Flask Format can Pro)mote Human ES Cell Attachment and Differentiation to Definitive Endoderm and Pancreatic Eadoderm 0214] H I and 19 human ES cells previously plated to 1:30 Narig plastieware and grown in NIEF conditioned media supplemented with 8ng/il of bFGF were LIRERASE treated andplated to T25, T75 Ti50 and''175 flasks at a 12 or 1 :3 seeding density onto various size flasks with modified surfaces. The cells were 46 seeded in MEF conditioned media supplemented with ngmil of b F and 20 mieromolar Y-27632. Hutan ES cell colonies wethen allowed to growvith daily media changes of N EF conditioned media supplemented with Snu/l of bFGF and 20 mIcroNmoar I-27632. unil the plates were approxinmiely 50% confluent. At this time the miediav as changed to DMEM/NIF12 media contain 2% BSA. 100ngrd Activin A20ngniil Wnt3a, and 20 micromolar Y27632 and the eelIs were naintained in this aedia for 2 days with dai media changes On day 3 and 4 the media. was changed to DMEM/F 12 media containing 2% BSA, 100ng/m Aetivin A, and 20 ricromolar Y-27632. Cells were then released frnom the surface with TrypLE and assays by flow etometrvy Or expression of the definitive endoderm (DE) surface marker. CXCR4. We observed that under these conditions, human ES cells differentiated to a highly CXCR4 positive population, that was as high as almost 90% CXCR4+, indicating that the cels were mostly differentiated to definitive endodernm (Table 5), Furthermore, the attachment of the cells to the culture surface during growth or during differentiation was dependent on maintaining ROC K inhibition, since withdrawal of Y-27632 from the culture media resumed in cell detachment from the plastic. 021 ] We wished to determine if pancreatic endoderm could be formedrom the defintve endodernn derived on surface modified plates in flask formal. To do so, we incubated the cells foR an additicnl four days with Y-27632 (20 mi(molar) FGE750 ng/nI), KAAD eyclopamine (2 meMmolar) and 1% B2 (lnvitrogewn in DMEM F 12 and then an addition four days in this mediasupplemented with Retioic Acid (1 mieromolar; Sigm&Aldrich) to differentiate the cells to a panereatic endodem stage; RNA samples were then takenand analyzed by realtime PR for the pancreatic marker Pdx Treated samples were normalized to unheated controls set to a.id change of I. We observed that samples had increased levels of PDX1 versus undifferentiated hunan ES cells, with mRNA levels at least 256 fold higher in the differentiated cells than that observed in undifferentiated human ES cells Example 16 Surface Treatment and Sir face Modified Plates 47 102161 Surface modified plates were prepared by treating inleeon molded items using a corona plasma treatment or a microwave plasma treatment (Table 6). The polymer materials used im injeeion molding were poysone. poyearbonate at bend of polycarbonate and polystyrene, and cyclic olefin copolymer The surface modified plates were mdividualy packed in plastic bags then sterilized by gamnira irradiation (25 kuy! and finally stored at room temperature until used in cell culture or surface charactrization experiments. Surthecamodified plates 18. 30 and 3.-32 were molded using the same polymer matters asrace modified plates 19, 33 and 34. respeCtively, but were not plasma treated. Surfaces 14 and 31 were not gamma irradiated, 102171 Corona plasma treatment was carried out in a metal vacuum chamber with only one electrode inside the camber and electrically isolated fom the inside of chamber (C Lab Plasma; 'eta phone A/S TDenmark The metal walls served as counter electrode ground) , A self-tuning corona generator generated the electrical field giving sutliciemt energy to genemie plasma in the entire chamber. A iem to be treated was placed at the bottom of the chamber. The chamber was closed and evacuated to a pressure of 02 mbar At this pressure the valve to the vacuum pump was closed and the corona generator engaged. The generator was set to generate an o Ouputof2000 XV'. The plasma was energized for 5 to 60 seconds. The gas inlet valve (air) was then opened, and the pressure in the chamber retuned to atmosphere level, 10218] The microwave plasna treatment was carried out in a quarts vacuum chambers (Model 300-E for surface modified plates 5-12 and Model 440 for surface modified plates 14 and 15; both from. Technics Plasma Gm Germany), T he energy to generate the plasma was supplied by a 2A3 OHz microwave generator outside the chamber. An item to be treated was placed on a glassplate inside the chamber. TIe chamber was closed and evacuated to a pressure between 023 and 0.5 mbar. The valve to the vacum ptup was kept open, and the pressure was maintained h desired vaMe by adjusting gas (air or oxygen) How with thie gas inlet valve, The microwave generator was then engaged. The generator was set to generate an output of 500 or 600 W The pump valve was then closed, and the air inlet valve was opened, in order to bring the pressure in the chamber to atmospheric level, 48 10219) Table 6 shows power, time pressure. and gasses used in preparing surface modified plates by corona plasma or rmicrowave plasma Example 17 Surface Characterzation of the Surface Modified Plates of the Present IAvention Waiter Con tact Anagles 102201 Surfae modified plates I 4 and 13 were individual packed in plastic bags, sten ized, and stored at roon temperature throughout the test peiod. Contact angles were. first measured one week after surface treatment and sterilization and then again at the time points given in Figur 2 A ta nts were done using the static sessile drop method and a (i-X measuring lead from BR Systems AB Sweden [gonioneter consisting of video camera and computer sof (Q. 3) The tangent leaning method was used for calculation of the contact angles Drops of 4,0 .pL MiiliQ water was applied using automatic drop application in static mode, according to the manufactures instruedons, The contact angle of each drop was measured once (7 drops were appied to each sample per time point). For each tie point, a new sample was used in order to avoid any influence from earlier measurements. Measurements on Nunclon IeM and Ccl NDI surfaces was performed under the same experimental conditions as measurements on Surface 1-A and 13, but the surface treatment and sterilizdin was done rer than 12 weeks before the frst measurement (Nunelon Deha * was sterilized one week before the first measurement Figure 29 shows that surface modied plates ' A and 13 were of similar hydrophiiicity and more hydrophilic (lower water contact angles) than Nunciaon Delta and CelUBINDrv surfaces. The hydrophilicity of surfee modified plates 1-4 ad I 'was stabe for at least 12 weeks aer surface treatment and sterilization 10221] CelltBN<D ias previously been described as having a contact angle of 1.3 degrees (standard deviation of4 degrees) [Corning Technical Report2005) Comi ng CeIlfBiND® Surface An improved Surface foir Enhanced Cell. Attachment (CLS-AN 057 REVI) on 49 tp#adgiotn o/Cececs/ei dtell B NI) ImprovedSurtace CLS AN 057.pdf], Native Charge Dens ity 10222] The density of negatve charges on surface mod Ned plates 1-4 and 13, Nunclon Delta)' surface. CelBIND s c r surfacPce. [aleon? 5 ' surface, and a non-treated (but sterilized) polystyrene surface (all in 3-cm dish format) was determined. Three inl of aqueous crystal vAiolet solution (0015% wn) was dispensed in each dish, and dishes were incubated for 60 minutes at room temperature under gentle shaking (50 rpm). In order to remove crystal violet not bound to the surfaces, the dishes were washed three times with3lMilliQ water, and then dried over night at 60. The crystal violet bound zothe surface was desorbed by addition of Lm5 ml of 0,1 M HC1 in EtOH solution (99%) and incubating the dishes for 2 minutes at room temperature under gentleshaking (SOrpm Absorbance of the HCI:EtOH solution with desorhed crystal violet was measured at 590 nrn using an EnViion 2100 nicrolate reader (Perkin Elmer; Waham A A, USA), Absorbance values were corrected for background absorbance of HCEEtO solution. The negative charge density was measred on three dishes per surface and absorbance measurement was performed in triplicate for each dish. [02231 The negative charge density for surface modified plates is shown in. Figure 30, The negative charge densities of Surfaces 1-4 and 13 were sui ar, but longer surface treatment time in the interval of 5-60 seconds tended to result in a lower surface negative charge density, Surfaces 1-4 and 13 had slgniicantly tower negative charge densities than CellBIND surface and a NunIon D-e3ltaaM surface treated in 2007. Surfaces 1-4 and 13 had negative ehare densities at the same level as a Nunedon ha surface treated in 2005, and significantlv higher negative charge densities than PrinariiaM surfac P lotn surfaceN and nantreaed (but sterilzed) polystyrene surface. The lower negative charge density of Nunclon Delta>surface treated in 2005 than of Nuncon Deta surface treated in 2007,suggest that surface treated polystyrene becomes slightly less negativdy charged over tone. The high level of negative charge density of CellBIN is not because of higher surface roughness and thus surface area (Sec A FM analysis in this Example) 50 102241 Surface modified plates 1-4 and 1 L 15, and plates with Nunclon Delta Costart Falcon 1 CeU 131 N, and. Primariak surfaces were analyzed using XPS. Sample was presented to the xray source by cutting sections from the plates and mounting them with spring clips onto a stainless steel sample holder. Samples were irnadiated. with A Ik radiaion (1486 e1) The analysis was performed th an angle of 4Y between the sample and nayz spectra were curve fit using the software package provided by the instruments vendor, Physical Electronics. The software utiized commercal Matlab"" routines for data processing. The instrament used for the analysis was a Physical Electronics Mode400 X-Ray Photoelectron Spectrometer The outermost two to five nanometers in depth in a. region of about one millimeter in diameter frn the surface treated part of e plates was analyzed in each of two plates per surface. 10225] Suace elemental compositioi in. units of atomic percent is shown in TabLet 7, All surface modified plates contained carbon, oxygen and nitrogen (hydrogen is not detected in XPS) in the surface. Surfaces 1 -4 Surface 13 and CellIND surface contained more oxygen than the other surfaces analyzed Sufaces 1-4 and Surfaces 13-15 contained less nitrogen than Primaria but more nitrogen than the surfaces of N tnclon Dehlta. Custard Falin an-d C-llBIN D plates. Oxygen and itrogen levels correlated positively with longer surface treatment time (Surfaces 1-4 and 13), and the highest levels of both of these &ci'nents wre obtained using 30 or 60 seconds of corona plasma. treatment (Surface 3 and Surface 45 respectively) Sufaces and 4 were similar in e lemental composition. Surfaces 2 and 13 were similar in eiemen.ntal. composition and more like Surfaces 3 and 4 than Surface 1 in eemental composMon, [02261 C s spectra peaks were curve fit (best chi-squared tfi in order to identify and quantify the barding enviroments or carbon in the surfaces by using peak widths and energy locations for species as found in the literatureFable 8). The concentrations are reported in units of atomic percent, which were obtained by nmutiplying the area percent by the atomic concentration Sufaces 24 and 13 were similar in terms of the carbon bonding environments. The proportion of carbon in C* )0C-C bonding environment was lower in Surfaces 2-4 and 1.3 than in the other surfaces analyzed. The proportion of carbon in 0-[C-0]-0 bonding 51 e-nvironment was higher in Surefacs 24 and 13 than in the other surfaces analyzed. SimiIarites between Surfaces 2-4 and 13 and Surface I. CenllBIND u and/or Primnaria " surfacee were also identified. The proportion of carbon in (0C or CI NH3E.bonding environment (same energy location in spectra) was higher in Surfaces 14 and 3 thanr in the other surfaces analyzed, The proportion of carbon in 0-0 C*G0 bonding envimoment was higher in Surfaces 2-4. SurIace 13 and Primaria 1 surface than il the other surfaces analyzed The proportion of carbon in CO bonding environment was higher in Surfaes 2-4 Surface 13, and CellBlND IM surface than in the other surfaces analyzed The proportion of carbon in. C bonding environment was higher in SurfTaes 1-4, Surface 13, and (elBINID surface than in the other surfaces analyzed. The proportion of carbon in -[PC bonding environment was higher in Surfaces 1-4 SutAWfae 1C3el BIND" surface, and Primaria'"surfiace than in the other surfaces analyzed. The energy loss peak resulted frm an aromatic fi-4 ransitin, and is an indicator of surface aromati~city. [02271 The Ol s spectra peaks were almost Gaussian and could not be curve fi N specta peaks were curve fit (best chi-squared fit in order to identify and quantify the bonding environments for nitrogen in the surfaces by using peak widths and energy locaion fo speiesas oun in the literature (Table 9). The concentradons are reported in units of atomic percent, which were obtained by rmutplying the area percent by the atomic concentratin The N Is signals rn Nuni on Dela CelBliNDU Costar and Falcon "surfaces were weak.and it was, therefore, not possible to do identification of the bonding environments for nitrogen in these surfaces. N is spectra were indistinguishable for surface modified olates l-4 and 13 and data resulting from curve fiting of two representative e Ns spectra is shown. The propononl of nitrogen n H-N, bonding environment was higher in Surfaces 1-4 and 13 than in Surfaces 14 and 15 and Primarif surface, Nitrogen in --NI-b bonding environment was detected only in Strfaces 14 and 15 and Priai surfae Nitrogen in -N0 bonding environieni was detected only in Surfaces 1-4 and 1 3, and in a single sample of Surface 15 Nitrogen in N0g bonding environment was detected only in Surface 15 and Primnargii surfce. [0228S CeIIBIND has previously been described as having an elemental composition of 70.4% carbon. 29.0% oygen 0.6%nitrgen, and <.01% other elements, and a 52 relatively high concentration of C40-{] C05 and COO .R roups as analyzed by ESCA [Corning Technical Report (2005), Corning@ CelBIND@ Surface: An Improved Surface fo Enhanced Cell Attaebment (CLS-AN-057 REV 1) on htp: /catalog2 comnin gcom/Itesciencesmediapd ft CClQiMND Iimproved SurfcC CES AN 057 pdfl. 10229] Primaria has previously been described as having an elemental opposition of' 74,6% carbon. 14 4% oxygen, 11.1% nitrogen, and 02% other elementswith mainly nitrideQ(DN) and urea [FIN(C=0: )NI-] carbon-to-nitrogen bonding e'nvi onmnents, as analyzed byi iSCA. A IUni Forcr' MNroscopy (ARM)t 0230 Surface modified plates 14 and 13, and plates with Nunclon DeltaM and Ce]BIND surfamcs were analyzed using AFlM SampLes were analyzed using a Digital Instruments Multimode Atomie Force Microscope in tappneg mode. The tip used was a tapping mod tip type IESP Samples were attached to the sample disks with double sticky tape. Regions of 10 ymn x 10 pum and 500 nm x 500 nm of the surfaceareated part of the plates were analyzed. Surface mean roughness (Ra) and maxinun height (Rrnax in units of nanometers are shown in Table 10 Like the plates with NunekIon Dt ha and (llBI1ND surfers surface modified plates 1-4 and 13 were relatively smooth, and Ra and Rmax did not correlate with surface treatment time in either of the two scans Analysis of nontreatd polystyrene ard oxidized polystyrene surfaces intended for cell culure, and Primaria surface has been described by Shen and Horbett (J. Biomcd. Mate Rc 57:336-345, 2001); surface roughness approximately 4 nm for all three surfaces. Example 18 Surface Elemental Composition and Contact Angle in Relation to Human ES Cell Attachment and Colony Formation [02311 A summary of the results of the .XPS analysis of surface elemental composition, the surface contact angle measurements, and human ES cell attachment and colony formation experiments is given in Table I 53 1O2321 Hunan ES cel attachment to and colony formation (at least 1 colonies per 10 cm surfaceion a sofid substrate surface in the absence of a compound capable of inhibiting Rho or Rho kinase was observed on only surface modified plates 2-4 and 3, CellBIND plates, and Primaria> plates (cells were presented to the surfaces as a suspension of clusters of cells), Surface modified plates 2-4 and 13 supported celi attachment colony formation and passaging. After about three passages, the growth rate of human ES cells on surtce modified plates 2-4 and 13 decned pontneously (only in the absence of Rho inhibition and Rho kinase inhibitions although cell morphology indicated that the cells were not d ifferentia ting Furthermore, phlripotency marker expression was maintained in cells passaged four ties on SurfAce Cel1BIND' plates supported human ES cell attachment and colony formation, but diffehrentiation ofthe cel s was observed prior to the first passage. Based upon cell morphology observations, Prin i plates supposed human ES cell attachment and colony formation without signs of dibrcntiation (passagig was not tested) Both oxygen (for example Surface 2 versus Surface 14) and nitrogen (for example, Prinnria versus Costar and Surfaces 2 and 13 versus CellBElNDEIM content of surfaces had an effect on the ability of the surfaces to support human ES cell attachment and colony formation in the absence of Rho inhibition and Rho kinase inhibition. Surfaces with a nitgen comeni of a least about 09,9(1/Na sum of nitrogen and oxygen content of at least about 223%, and a water contact angle o at least about 13.9 degrees supposed human ES ceiiattachment and colony lot 0tuon in the absence of Rho inhibition or Rho kinase inhibition. [0233] Unian ES cell attachment and colony fbmniation (at jeasti colonie per 10 cm surface on a solid substrate surace in tie presence of a compound capable of inhibiting Rho or Rho kinase was observed on surface modified plates I 15 surface modified plate 19, surface modified plate 33, surface modified plate 34, CeliND and Primariai ellss were presented to the surfaces as a suspension of custers of cels} We noted that surfaces 2-4 and 13 and Primania "were better than surfaces 1 19. 33 and 34 and ClBINDM. which again were better than surfaces 5-12, 14 and 15, at promoting human ES cell attachment and colony formation. On surface modified plates 3 and 4 and in the presence of a Rho kinase inhibito human ES cells attached and torned colonies that expanded and could be passaged at least 10 times, gilgln rise to phuipotent cells with normal karyotvpe (karyotype tested only in cells 54 rown on Surface 4), Both oxygen (for example, CeBIND versus Nunclon and roge(fo aple, PrimariaM versus Costar"; and Surfatees 2 and 13 versus CelBlNDt)} content of surfaces had an effect on the ability of the surfaces to support human ES cell attachment and colony formation in the presence of Rho kin.ase inhibition Su es wih a inhogen content of at least about 0.%.a sum of nitrogen and oxygen content of at least about 17.%, and a water contact angle of at least about 13:9 degrees supported hman ES el attachment and colony formation in the presence of Rho kinase inhibition, Surfaces with a nitrogen content of at least aboui 0.5%. a sum of nitrogen and oxygen content of at least about 17.3% but less than 19.9%. and a water contact angle of at least about 9A degrees supported human ES cel attachment and colony formation in the presence of Rho kinase inhibition in some cases (surface 14), but not in others (surfaces 2>24 10234] We noted that removal of Rho kinase inhibitor from huian ES cell cuhures captured on surface modified plate resulted in detachment of the human ES cells fmm the surface ofthe solid substrate The cels codd then be reattached to the surface by re treatment with a Rho kinase inhibitor. Given that enzymatic passage of human ES cells is a potential stressor and may cause kargotypic instability using temporary removal of Rho kinase inhbitor to passage human ES el could eliminae the stresses of enzymatic passage. 10235] Human ES cell attachment and colony formation was also demonstrated using aninal componenvtree medium, Rho kinase inhibition and surfcc unodified plate 4,rer treatment of surface modified plate 4 with eracelilar matrix proteins resulted in more colonies, but only in the presence of Rho kinase inlhibinon. [0236] In addition to passaging human ES cells with enzymatic niethods that maintain colony style culture conditions by passaging ells as chsters, human ES cells could also be passaged as single cells using enzymes like Tryp.E T 5 ' or Accutaseoht in the presence or in the absence of Rho kinase inhibitor, human ES cell colonies dissociated into a suspension of single. cells using TrypLETmi attached to surface modified plates 3 and 4, and formed colonies that could be passaged at least five times and give rise to cells with pluripotency markers, 5 102371 Removal of Rho kinase inhibitor from the human ES cell cultures prepared by passaging the cells as a suspension of sige cclls did not result in detachment of the human ES cells from the surface of the sohd substrate, but resulted irr colonies that grew fester than if the Rho kinase inhiitor was not removed Example 19 Treatment with Y27632 Enhance HE K293 Cell Attachment to Surface Modified Plates 102381 Human emTbryonic kidney celis 293 (l EK293,CACC no 85120602) were maintained in Eagle's Mininium Essential Medium (EMEM, Lona Verviers Belgim) containing 1W% fetal bovine serum fBS; Lonza}. The cells were adapted to,. Pro293a4-DMd rueditin (Lonza), a chemically defined. serurnvfreeciu optimized for cultivation of adherent HEK293 by gradually and over several passages usingte sequentiaraios of 3:, 11, 1:3. 1, and finally 0:l of seruni-suplenmented EMEM and Pro293aCDM iadin. For maintenance and adaptation. HEK293 cells were seeded at 240 ax 104 eelIsm in 75-c) flasks with Nunclon Deita surface (Thermo Fsher Scientic Roskilde, Denmark) and passaged at70-0% confluence using TrypsinDTA for dissociation. [0239] Pro293a-CDM medium(100 pl) supplemented with Y-27632 a Chemical Co, St.Louis, MO) in concentrations of 1 0 40 or 10 p.M was dispensed in fiat-bottomed, 96well plates with Surface4 Nunclon Deha surface or CelIBINi surface: Another 100 I of Po293a aCD medium with [EKX293 cels was added to the wells (4.0 n04 cells/cn Ihe culures were then incubated at 37'C in a humidified atmosphere of 5% i (i) 96 hours; or (i;48 followed by cultures onc with 200 pl Dilbecco4 Phosphate Buffered Saline (DBTS Lonzat then adding 200 p. of Pro293a-(DM medium without Y-27632, and finally incubating cultures for another 48 hours. 10240] The number of viable ceUs in the wells was then determined using a lactate dehydrogenase (L Dl) activity kit from Roche, Switzerland. Briefly, wells were washed with Pro293a-CDM medium. and adherent cells were lysed in 100 ld DPBS with 2% (vv) Triton X100 (Signta Chemical Co.) during a 30-min incubation at 37C, Lysate and 100-p catalyst and dye reagent mixture were mixed and incubated in the dark at 25*C for 30 mi. The reaction was stopped by adding 50 p, of 1.0 M SCI. and the absofbance at 490 mu was measured in a microplate reader (Genios Pro; Tecan. Austria The number of cells was calculated using the A490 values from these samples and fom standards containing I DH- rom a known numbeef cells. [02411 The effect of the solid substrate surfaces and Y-27632 on attacIent and growth of FIEK293 cells in Pm293a -CM medium is shown. in Figure 31a, where the 96-hour continuous exposure to0Y-7632 is labeled "Y-2 632 96b on" and the 48-hour continuous exposure to Y-2763 followed by a change of medium and 48 hours of incubation in the absence of Y27632 is labeled "Y-27632 48h on48h off" In the absence of -276321 FEK293 cells attached to allthree surfaces. A change of medium after 48 hours of incubation resulted in significantly fewer clls in the cultures measured after 96 hours of incubation. -27632 enhanced attachment of HEK293 cells on Surface -4 and CeIliFND' surface when applied at concentrations of 20 and 5.0 pM, RemovngY-27632 after 48 hours oflicubation resulted in significant detachment of cells from all three surfaces, [0242] A similar experiment, but using 2J, >, 104 non-adapted HEK293 cells per en and EMEM supplemented with 10% FES throughout, was performed. The effect o the solid substrate surfaces and Y-27632 on attachment and growth of EK293 cells in EMEM supplemented with 10% FS is shown in Figure 3 1b here the 96-hour con ituous exposure to -27632 is labeled 727632 961 on" and the 4b-hour continuous exposure to Y-27632 followed by a chang medium and 48 hours of intabadon in the absence of Y-27632 is labeled "-27632 48h. on/48h off" In the absence of Y-27632, HEK293 cells attached to all three surfaces. A change of medium after 48 hours of incubation resulted in significantly fewer cell in he culturesmeasured after 96 hours of incubation. Y-27632 enhanced attachment of H EK293 cells on Surface4 and CellIND surface when applied at concentration of 2.0 and 5,0 pM. Removing 7-27632 after 48 hours of incubation resulted in significant deiachinentof cells from Surface 4 and CeIBSbND Example 20 Treatment with Y-27632 and H-152 Enhance HEK293 Cell Growth on Surface Modified Plates [0243] HEK293 cells were maintained in EMEM (Lonza) containing 10% FBS F (1 onza) Cens were passaged at 7080% confluence using TrypsinlEDTA for dissociation, and seeded at c 20 x 10Icls/en in 75-em aks with Nunclon Deltin surface (Thermno isher Scienfi Roskilde, Denmark [02441 EMEM (500 i supplemented with 10 % FBS contaisng 1.0 5.0 10 15ior 20 pMi Y127632 (Signa Chemical Co), or 0,4, 1.2, 1A 2.4 or 2.8 M HI152 Calbiochen EMD Chnmicals Inc, Darnistadt, Goermany) was dispensed in Multidish 24-well plates i either Surface 4 or a nonAreated (but gamma irradiated; 25 kGy) polystyrene surface. Another 500 p! of EMIEM supplemented with 10% F BS and containing HEK293 cds were added to the wells (2AO ) 0 cells/cm The cultures were placed in an IncuCyte Plus (Essen instruments, Michigan; USA) and incubated at atniosphere of 5% CO in air The hIcu te Plus is an automated imaging platform, configured to fit inside a CO 2 incubator, anm designed to provide kinetic, non-invasive live cell imaging by acquiring phase contrast images of the cells at user-defined times and locations within the dclures. The primary metric of the instrument is culture confluence, that is, the fraction of the surface that is covered by ells. The M IEK293 cells were ircubated for 72 hours without manipulations, and images were collected every two hours at 9 positions in tiplicate cultures Culure confluence was determined using thenhcuCyt"I Pius software (v 4 34,1L25966), (0245 Increasing concentration of Y-27632 and -1152 enhances attachment and growth of lEK-293 cells on Surface 4 (Figure 32a). The effect of a non-treated ceil culture surface and Y-27632 or H- 1152 on attachment and growth of HEK293 is shown in (Figure 32b), Growth and atiachent of HEK293 cells was slightly enhanced in the presence of 10 ptM Y-27632 and 0 i- 1.2 M H 1152, Hweverthe enhancement of growth and atachment of 1EK293 edis on a non-treated cell culture surface is insignificant in comparison to Surface 4. Example 21 Treatment with hP-152 Enhances H EK293 Cell Growth and Attachment to Surface Modified PhIate [0246] H{EK293 cells were maintained in EMIEM (Lonza)containing 10%BS -onza Cens were passaged at 70580% confluence using1rypsinEDTA for dissociation, and seeded at c 2,0 x 10eells/en in 5-ed flasks with Nunclon Delta' surface (Thermo Fisher Scientn. iRoskilde, Denmrv) [02471 EME N 0 .ml supplementedwith 10% FBS containing OA,0 , 11, 2 A 2,2,4 or 2,8 pM -I 152 was dispensed in Multidish 12well plates with Surface 4, Another 1.0 ml of E MEM supplemented wih 10% FBS and containing HEK293 ed Is were added to the wells (4.0 a 0- cells/emn The cultures were placed in an incuCyte Pius, and incubated at 37 C in a humidified atmosphere of 5% COin air for 42 hours (images were collected every 6 hour One ml of culture medium was then removed by pipetting, and 1 0 ml EMEM supplemented with 10% PBS containing 0.2, 0A 0,6. 0. 1.0 12 and 1.4 AM Hl-I 152 was added. The cuhures were placed inthe IncuCte Plus agah and images were collected every hour over the flowing 25 hours, na ges were colleted at 9 positions in triplicaecuhures, and culture confluence was determined using the lncuCyte 1 Plus software, inages from the lncuCyte M Plus collected at specific positions in HEK293 cell cultures grown in the absence or presence of IN 152 (0 .p.M) was retrieved and presented as phase contrast micrographs for the comparison of H1EK293 culture morphology at the following time poitts- start of incubation (0 hours) just beore medium change (42 hours), 1 hour after the medium change (43 hours), and, finally, after 52 hours ot i ncibation, [02481 In the absence of H-152 and in the presence of 0.2 pM or 0.4 pTM IA-1 152. the change of 50% of lthe medium after 42 hours of incubation resulted in a significant reduction in cuire confluence (Figure 33a) In the presence of 0.6aM0 pM or 14 AM H 1152 the effect of changing the medium was minimal. HEK293 cells gryowu on Surface 4 in the presence of 11-il52 covered the solid substrate surfaces more evenly than E1EK293 cel.is grown on Surface 4 in the absence of 1152 (igure 33b) In the absence of H- 152, HEK293 el formed large clusters, whereas 1E K293 c is in the presence of IA- 2 formed smaller clusters with lower cel density 59 Example 22 Treatment with Y-27632 Enhances 1EK293 Cell Growth Over Three Passages on Surface Modified Plates 102491 EMEM (500 1d) supplemented with 10 % FBS containing50 pM V-26327 was dipened in we s of Mutidish 24-well plateswh Surtee 4 or Nunclon Delta surface Another 500 g1 of EMEM supplemented with 10% Snd containing 14EK293 cels was added to he wels (2,0 x 10 cels/m) and thecultres were incubated at 37 C in a humidified atmosphere of 5% CO in air for 3 days, Cells were passage by treatment with IrypstiEDTA (Lonza Verviers Belgium) for two minutes at 3 C and the total celI number was determined using a NucleoCount Ce Counter (Chemometec S, Allerod, Denmark) For successive passages, HEK293 cells were seeded at20 x 104 cellscn 2 The growth of HEK293 ceis on Surface 4 and Nunelon Delta surface was enhanced by the present of l2. pM 27632 (Figure 34). Example 23 Attachment, Cultivation and Maintenance of Human Embryonic Stem Cells Using Surface Modified Plates 4, 18, and 19 that lack Extracellular Matrix Protein/Components and Feeder Cells 10250] Passage 42 H I hES eelIs maintained on 1:30 MAfRIGEL coated plasticware in MEF conditioned media supplemented with 8ng/a ofbWF were ifted by LIBERASEN enzymnai treatment and plated to surface modi ?d 96 wel format a I to 2 dilution in. MEF conditoned media supplemented with Sg/mI of bF The cells were plated to modified surfaces 4, 1. or 19, or Primaria In order to determine the effect of Rho Kiase inhibition on binding.to the modied sUrface we treated the ce ls with either 10pM of the Rho Kinase inhibitor Y27632 or 3 or 10pM of the Rho Kinase inhibitor i-152glycyL Untreated cells served as controls. After 24 hours in culture the wells were aspirated, the cells were dried, and the wells were stained with Crystal violet, 102511 We observed that after 24 hours in culture. ES cell colonies had attached and spread when eated with Rho Kinase inhibitors on surface modied plates 4 and 19 and the 60 PrimariMplate, however the same effect was not observed on surface modified plate 18 (Fiere3%t Example 24 Attachment Cdutivation and Maintena nce of liuman ml ronie Stem Cells Using Surface Modified Plates 30 31, 32. 33, and 34 that lack Extracellular Matrix Protein/Components and Feeder Cells 10252] Passage 47 H I hES eelIs maintained on 1:30 MATRIGEL coated plasticware in MEF conditioned media supplemented with 8ng/ml of bFGF were lifted by T D enzymatic treatment and plated to surface modified 96 well format plates at a I to 3 dilutition in MF c ioned media supplemented with 8ng/ml of bFGF The cells were plated to modified surfaces 30, 31, 32. 33, or 34, In order to determine the effect of Rho Kinase inhibition on binding to the modified surface we treated the cells with 3pM of the Rho Kinase inhibitor H-L152glyy i Untreated cels served as controls Additionally, cells were seeded to wells in the \udace nmodefied plate that were pre treated with MatrigelP 24 hours after plating the media was changed with fresh ME[ conditioned me diaipplemented with Sngml of WGEV and fOr cells seeded i the presence of the Rho Kinase inhibitor the media was supplemented with 3pM 1 i li2g1ycyl. After 48 hours in culture the wells wee aspirated, the cells were dried, and the wells stained with Crysta violet 10253] We observed that after 48 hours in culture, ES cel colonies had attaclted and spread when treated with Rho bKase inhibitors on surface modified plates 33 and 34 (Figure 39 and 40 respectively),however the same effect was not observed on surfae modifed plates 30, 3 i or 32 (Figures 36 40 respectively Example 25 Attachment, Cultivation and maintenance of Human Embryonic Stem Cells Using Swrface Modified Plates 22,'J, 2 or 29 that lack Extracellular Matrix Protein/Components and Feeder Cells [02541 Passage 46 H t i ES cells maintained on 13.0 MATRlGEL coated plasdware in MEF conditioned media supplemented with 8ngnl. of bFGF were lifted by Liberasemhi 1 enzymatic treatment and plated to surface modified 60mm dishes at a I to3 diluton in M EF conditioned media supplemented with 8ng/mt of bFGF The cells were plated to surface modified plates 3. 4 2~ 23, 24 and 29. n order to determine the effect of Rho Kinase inhibition on binding to the modified surfae we treated the cels with 3uM of te Rho Kinase inhibitor fLI 152glyfil to plate the eels, The meda was changed withesh MlEF conditioned media supplemented with 8ng/mi ofbFOF and IpM of the Rho Kinase inhibitor H131 12glycyl 24 hours after plating the cells. Cells seeded to modified surface 3 4 or matrigel coated plastic served as controls The plates were observed by phase microscopy 24 and 48 hours after plating. We observed that after 48 hours in culture, ES cell colonies had not attached to surface modified plates 22) 23, 24 or 29 played with or without Rho Kimase inhibitor, while cells plated to surface modified plate 3 or 4 in the presence of Rho Kinase inhibitor did attach and spread. Example 26 Further Surface Characterization of the Surface Modified Plates of the Present Invention Warte Coact Angles [02551 Surfc modified plates 14 and 13 were individually packed in plastic bags, sterilized, and stored at room temperature throughout a 40-week test period. Contact angles were first measured one week after surface treatment and sterile izatiorn.and then again at the time points given in Figure 4L ARl contact angle measurements were done as described in Example l7 Measurements on Nuncin Deltaand CeflI i' surfaes was performed under the same experimental conditions as measurements on Surface 1-4 and 13, but the surface treatment and sterilizaton was done more than 12 weeks before the first measurement (N ucon Deta 1 * was sterilized one week before the first measurement Fgre 41 shows that surface modified plates 1-4 and 13 were of similar hydropnity and more hy'drophilic (tower water contact angles) than Nuncion Delaland CeIlD.ND surfers, The hydrophilicity of surface noditied plates 14 andi wasstable for at least 41weeks after surface treatment and sterilization, 62 102561 Contact angles weres measur d on surface modified plates 5-12, 2-24 29 and 33, which were packed in plastic bags sterilzed as described in Example 16, and stored at room temperature for 9 weeks (except for surface modified plate 29 which was stored for 28 weeks Surfacei modified plates 18, 19, 32 and 34 were in single mierwell format and could. therete, not be used rib measurements of contact angles. Surface modified plates 30 and 33 were in a microwell plate format, and contact angle *easuremets were performed n the backside of. he plate and not inside wells Contact angles were measured as descrbed in Example 17 (for the highly hydrophilic surface modified plate 29, a smaller drop of25 pla MilliQ water was applied}. but triplicate samples were analysed, with 7 drops beingapplied per sample, Measurements on plates with (Csta Falcon) Primaria''" and Nuncion Dea surfaces was performed under the same experimental conditions, but the surface treatment and sterilizaton was done more than 12 weeks before the first measurement Figure 42 shows that surface rnodiied plates 5-12 were more hydrophilic (lower water contact an ethan Nunclon Delt> Costas and Falcon surface. The hydrophilicity of surfaces 5- 1.2 was comparable to the hydropihilicity- of the Prinaria 1 :surface. andi than dbe hydropilicity of surfaces 1-4 and 13 (shown in Figure 41) The hvdrophiicity of surface modified plates 2224 and 33 was comparable to the hydrophilicity of surfaces L4 and 13 (shown inFgre 41), whereas the hydrophiyicity of surface 30 was comparable to the ydroophilicity otNumcion Deltar Costar and Falcon surfaces, Surface. modified plate 29 vas signify cantly more hydrophilic than the other surfaces analysed. Vegativc Uhage Densi [0257] The density of negative charges on surface modified plates 5-12 (all 5-cm dish borrnal)1, 18. 9, 30, 32, 33 and 34 (all nicrowel1 format), surace modified plates 22 24 and 29 (all. 6-cm dish formatl. and CellBIND surface (3-cm dishformath Primari surface (maidish~6 fbmnat) and Nundon DehaM surfrce (3-cm dish format) was determined, Aqueous crystal violet solution (0015% w/v) in excess was added to each format (034 rl/ixcn for dish format and 013 mItn for microwell fbrmaa) and was incubated for 60 nnites at mom temperature under gentle shaking (50 rpm). In order to remove crystal violet not hound to the surfaces the dishes were washed three times wah 3 ml lilliQ water for the dish. formats and three times with 63 350 z1 MiflliQ water for microwe formats, and then dryed over night at 60V The crystal volet hound to the surface was desorbed by addition of 0. 7 r/enf of 0.1 MI HC1 in EtOHi soltion (99%) and incubating the dishes fo 2 minutes at room temperarure under gentle shaking (50 rpn Absorbance ofthe fHCEtOH solution with desorbed crysta violet was reasweda 590 nm usingan EnVision 2100 iecroplate reader (Perkin Elmer; Waltham, A, SA> Absorbance values were corrected for background absorbance o~f lHI tG -lf solution. he negative charge density was measured on three dishes with surface modified plates 5-12, 22-24,29, CellB [ND tPriaria 1 and Nuncion Deltat" and absorbance measurements were performed in triplicate for each dish. For surface modified plates 18, 19, 30,32,33 and 34. one sample was tested with triplicate measurements 102581 The negative charge denshies of surface modified plates 5-12 were similar, and these surtaces had significantly lower negative charge densities than Ce1BIND surface and Nanclon Deltarn surface, but significantly higher negative charge densities than he Prinaria surface (Fiure 43y. The negative charge densities of surface rnodiNed plates 19; 33 and 34 were significantly higher than the negative charge densities of surface modified plates 18, 30 and 32. the latter being the respective non-reated surfaces injhe same polymer material. The negative charge densities of surface modified plates 22-24 and 29 were normalized to the negative charge density of the Nuncion DeltaA surface, and Figure 44 shows that surtace rnodinfled plates 22-24 had higher negative charge densities than the Nunclon Deta surface whereas the negative charge density for surface modified plate 29 was significantly lower than the negative charge density of the Nunclon Delta surface (and surface 4x \'Rqv Photoeiecron Sp'ctrocopv tAP [0259] Surface modified plates 12, 18 19, 22-24. 29 30, 31-34 were analyzed using XPS as described in Example7 Sur17elemental composition in units of atomic percent is shown in Table 12 All surfaces contained carbon oxygen and istrogen (hydrogen is not detected in XPS)exceptsurface modified plates 31 and 32(not plasma treated), which did not contain nitrogen. Surface modified plates 5-12 contained less oxygen than surface modified plates 1-4 and 1 Abut significantly more oxygen than Costa( Faleon and Nuncion Delta)" surfaces (shown. in Table 7), Surface modified plates 5-12, were prepared by microwave plasma treatmentyhile surfce modified plates 64 4 and 13 were produced by corona plasma treatment, race modified plates 19, 33 and 34. which were prepared by Corona plasma treatment but injection molded frotm other polyrmers than alstvrene (which was used in the preparation of suae modifed plates 1-4 and 13) contained oxygen levels comparable to those f surface modified plaes 1-4 and 13 Surface modified plates 22-24 contained less oxygen than surface modified plates 1-4 and 13. Surface modified plate 29 contained oxygen at a level comparable to surfceemodfied plates -4 and 13 Surface modified plates 12, 19, 33 and 34 contained less nitrogen than surface modified plates 11-4 and 13. but more nitrogen than Costar ,Falco n and Nucdon Delta surtaces(shown in. Table 7) Surface modified plate 29 contained significantly more nitrogen than the otter surfaces analysed, including the Prinaria Ut surface. 10260] Cs spectra peaks were curve fit (bestehi-squared fit.in order to identify and quantify the borndig environments for carbon in the surface modified pates by using peak widths and energy locations for species as found in the literature (Table 13), The concentrations ae reported in units of atomic percent hich were obtained by nudtipying the area percent by the atomic concentration All plasma-treated surfaces, except surfaces 10, 2-24 and 29, were similar in sterns of the carbon bonding envionmnts. The proportion of carbon in C- [0 -Cas significantly higher in Surfaces 19, 33 and 34 than in surfaces 5-12, 18 30 and 32 and surfaces 1-4 and 13 (hown in Table 8t The proportion of carbon in 0 [:::O}- bonding enirontnent was lower in surfaces 5- 12, 19; 33 and 34 thi in surfaces 1-4 and 13. The proportion of carbon in C*-C-0G*-.vas significantly higher in surfaces 5-9, 11, 12, 192 3 and34 than in surfaces 1-4and Y3, 'ut comparable to Nuncion Delta Ut and CellBIND surfaces. The proportion of carbon in C:-0--C or C- Nil; bonding environment (same energy location in spectra) was lower in surfaces 5-12 19, 33 and 34 than in surfaces 1-4 and 1 but was higher than in Costar Falcon CeIBINDIT and Pryiarif surfaces, The proportion of carbon in C-04*=0 bonding was gh-r in rees 19, 33 and 34 than in surfaces, 5-12, and comparable to the level in surfaces 14 and 13. The proportion otcarbon in C= bonding environment was higher in surfaces Si2 than in surfaces 19,33 and 34k but lower than in surfaces 1-4 and 13, The proportion of carbon in C) bonding environment was higher in surfaces 5-12 than in surfaces 19, 33 and 34, and comparable to the level in surfaces 1-4 and 13 Sufaces 224 were similar in terms 645 of carbon bonding enviroments. The proportion of carbon in (C[] {, =-Of: C-0-C or C-N:H C-tI .- *=:0 and C=--0 for surfaces 2224 was significant lower than Or surfaces ~4 and 13 The proportion of carbon in CO and (>C. .C.QI bonding environments was higher for surfaces 22-24 than for surfaces 14 and 13. The carbon bonding evTninment of surface 29 was different from the carbon bonding environment of all other plasma treated surfaces. The proportion of carbon in (740> C was comparable to surfaces -4 and 13, The proportions of carhon in. 0=] Co3 and C -C bonding environments were lower for surface 29 than for surfaces14 and 13. The proportions of carbon in -0-C or C0-N -H C-0(*=0 and (0 bonding environments were higher for surface 29 than for surfaces 14 and 13 The energy loss peak resulted from an aromatic l -Al transition, and is an indicator of surface aromiati city. [0261] The 01s spectra peaks were almost. Gaussian and could not be curve fit. NIs spectra peaks were curve fit (best chi-squared fit), in order to identify and quantify the bonding environments for nitrogen in the surfaces, by using peak widths and energy locations for species as ftoud in the literature (Table 14) The concentrations are reported in units of atomnie percent, which were obtained by nmltiplying the area percent by the atomic concentration The proportion of nitrogen in N~t bonding enironment in all surfaces, except surface 9, lower than a surfaces 1-4 and 13. Nitrogen in -- NHi bonding environment in surfaces 5412. 19, 33 and 34 varied but was higher than in surfaces IAand 13. Nitrogen in -NOj bonding environment in surfaces $12, 19 33 and 34 varied, but was lower than in surface 4 and 13. Nitrogen in-O- bonding environment in surfaces 12 19, 3 and 34 varied, but was higher than in surfaces I -4-and 13, The nitrogen bonding environment of surfaces 22.4 and 29 were different from the other plasmna-treated surfaces. The proportion of nitrogen in -NU, bonding environment in surfaces 22-24 and 29 varied, but was significantly higher than in surfaces 54 and 13. The proportion of nitrogen in --0. bonding environment was power in surfaces 22-24 and 29 than in surfaces I.

4 and 13 The proportion of nirogen in ::C-N :O bonding envronmnent was comparable in surfaces 2224 and 29 and surfaces 14 and 13 [0262] Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspectsofthe invention have been illustrated 66 above by reference to examples and preferred embodiments, it wil be appreciated tat the scope of the invention is defined not by fie foregoing description but by the folkAng claims properly construed under principles of patent law 67 TABLES Table I: Expresion of Pluripotencv Markers in Cells of te Hiun;Emnhryonic Stem Cel. Line HA ai Passage 50, Cuured on the SArWae Modfied Plates of the Presen Invention Marker Couture 'Cl U (IATA2 (JA7 NANOG (C14 SOX2 SOX ITRTI TUBB3 Condition 1:30 MAERIGEL Surface 03 0 0,8 0,9 7 0 Q 9 17 32 Modified Plate 2 Gelatin Swace L. 0.4 0 9 0 9 0 .1 9. 0 23 Modflied Plate 2 No Coating SurfEce 03 0 6 0. 0,8 0 8 4 0 30 Modified Plate 3 Surface 05 0 0 0.8 0,5 0 6- 4 0 3 3. Modified Plate 3 68 No Coaiut Surface 03 0,7 0 1.3 -3. 4 Plate 3 No Coating Surface 04 0.4 0 10 0 0 9 2 0 32 Modified Qlate 4 Sutace 0,5 0.6 12 0) 9 4 2 4 1 \Mdified No Coflaing Surface 1.0 03 XN 1.0 08 13 02 Modified Plate 4 No Coai~n6 Table 2: Expression of Plripotency Markers in Cells of the aman Enbryonic Stern Cell Line -U at Passage 53 Chuled on the Surface Modihed Plates of the Present Invention MARKER Coure CFC1 GATA2 X NANOG CT SOX2 SOX7 TE0 Codition Costar LA 10 0 10 LA 1.0 10 10 130 MATRIGKL - -- --- ---- 4 --------------- 4----- Sudace 6.22 3325 1A2 3.98 0J 01 6 0,42 2L 0.50 Modiied Plate Surace 15.80 168 11 2014 TO L-90 1.25 1.89 1 83 Mod ifid Plate 3 Surfae 1141 1754 86 1406 4A6 2 2 639 91 4A2 70 Table 3: Expression of Markers Carateristie of the Defiitive Endoderm Lineage in Ces of the Ihuman Enbryonic Stem Cell Vie 1-f at Passage 5 (p2) and Passage 53 (p4) Cudured on the Surfiace Modified Plates of the Present a enton Treated with Aefvin A % Expression of the surface marker (XCR4 to Iowing dif ferentation of i i hinan ES cells to definitive endoderm ]2, Surface #4 45.5 748 '71 Table 4- Percient confluence (acquisition area occupied by ojcPand total human H19 embryonic stem CeA colonies greate thn 5K -sq. miconus in the acquisiton area after one passage on the Surface Modifid Plates of the Present In-ven-tion A$UA3kMI'IU iI C, A F -------------- --- --- - ----------- -- - - - - - - - - --- ----- -- - -I- -------------- - -. ts - C I - --- 4 -M -OC1W hO -t - t- - [OW --- . ... ...... ......... xpos ........ - ------- .. ...... r..e: ....----- -ut r -------- ------------- .................. --- Table 5 Expression of Markers (aracteristic of the Definitive Endoderm Lineage in Cels of the H ian Embryonic Stem Cell Liue III at Passage 51 Cuured onD the Surface Modified Plates of the Present hnventn Treated with Activin A % Expression of the surface marker (X(R4 following diffterenaion of' R human ES cells to definitive endodernm US DIN! I Surtace l>a N 55 StlflG5 -2 5)1 NA 55 6 73 Thbe 6: Preparation of the Surface Mdiied Plates of the Present iventon Preparation of Surface Modi"ied Plates Power Tim P Surface Polymer Gas -- (-- -r) Corona Plasma I AA0 ____Pok yrene 2000 5 E~0 Xi 13 Polsyrene 2000 10 1E 0 Air 4 PQlst 2000 15 I1 (fl Air 19_ Cyeoefn p2000 60 IE 02 Air 6 Roh 000 Po yrene o6y00 0 1 E02 33 ~ ~ ~ ~ ~ ) lovChoac Iovtrn 200E1-02 X 39 Po- y.t.rne 6000 6V c ---02 xge Microwae PLase SPoYstoene 600 6 0 OA 0 eu 6 POkgxrne f600 12 0.304 Xir ______ 1 1I01'll 0,4 Ai-0 10POlytn 24 i 03-M4 A- 1 9 6ovtrn 00 I6 0 0 (h 1A~~~1 ltxce00 0 (14 0, Jxu I PO 60 600 .e. .ammaato -- 4- - 'abic 7: Surface Elental (>mpoition of. Surilce Modified Pltsas Determined by XPS Surf'aee Eleincntal Qinpomiiin of Surfitee Modifi ed Pl1ates as Deilermidd by X.PS Measurements on tw-o samples and man ± standard deviation1 (SfD) in units of atomic rerent Carbon ~ xget, %/ Nitrgen Surface--------------- -1 2 Meant I 2 Mean 1--1 2 Meant S D SD oII surfaceb 1601) 73.5 715±I 121± 19 iI I 16 17 0M 0 Surface 3 '2, I 78.6 707'' 0o 200t 141~'1 0.13 De "' 3 0 6 9135 1 2' 0.0 Cc 1.NY 4 2.3 72.2 V 25,8 15 0K 1A 1 110 n aA ml 0,1 N',,musnQ ' 8,4. 84763 0471 '14, '12.9 143.1 119- 0 10 JM_ _ _ _ _ _ _ _ 0 __ _ _ 01 _ _ _ _ _ _ _ 'D' (tbe c o-t eedtce racnetain&0.% Tabic 8i: (Iiarbo. dn Enviromnmentm Cy('i Spectra Curve-k Fhimxi Carbon Bowding Environment by Cis Spectra Curve.lifting Atonic percent of each functional. group is given as muean A sandard dev tn imiut a 2) Functional groups and C(Is bindig energies in eV urae 04O-0C- C. jOj C (287 09 C 4 l = (289.8 j( 01Ol loss ev) C (42 (87. 0 eX) (288.9 eXI 0 Peaik (2I~ NHt,; eV) ev) 0211 (292 eV) C)(286.1 ) eV) SurfacelI 34J .- : 1 14A 4, 4,5 ~7 1.7' , 0> 5 5vw 10,6 0 2.1 01 o.1 1. 0,1 1 o___ IV UA I1 0 0 _ 0 ___ 4- 4,-2 3,,4 4, 014 003 0, 1 - 00 .0 0 U 1 5 Surfc\n 13 33,6 6,+132 4, 4,9~ '-i. 25 2 ,0 0, 15 01 DOt< 6 0.5 O.4 01 00o 00 1 _____ 11~~0 15-' O1 00 0 0 -, 10 AU, 00 0 0 ( (f)ta I% o1 40 9" I 0, L00O00 ____ 0 046 + A 0,0 00 OA ______ 77nai"M1 016 C) 0 1 ~ ~ 0 siO 1X 00-, 0 0" 06 0, 1 0 0 , * ~ ~ 3 3Th fucoa giou LO (d, 1diifc in, cm c.n ------------ -- ---- I---------------------------------------------------- --- Table 9 Nitrogen Bonding Environent by N is Spectra. (Iurve Fiting Nitrogen Bonding Environment by Ns Spectra CArve Fitting Atomic percent of each functional group is given as miean & standard deviation (n 2) Functional groups and NIs binding energies in eV Surface (398 ) 0 (4018 eV) (406 eV) (4010 V) U69 2 4 3 4 43 0 ± 4 1002 ± . 0,0 and I * SurIce 5 40± 14 765 1 0 3 0* 2 0 0 \nrtac4 - 6.±1 -t- 14.0 ± 7. _0 100. Pri maAW o0 ) j 8000 & 0 4 000 00 40' 00 * Nh spectra were distinguhable r urfa&e modii te 1-4 and 13 and data reting from curve fit of two representative N is spectra is given. **bTe functional group was identified in only one sample. 77 Table 10 Surface Roughness of the Surface Modified Plates of the Present Invention as D4etrmined by AFM Surface Roughness of the Surfae Modified Plates o the Present ai Determined by AFI Surface 10 ax 10 kmn scan 500 nmn x 500 mn scan Rnm R (mnn am R (m Surfac 240 20,97 0 13 235 nifiied p1 te Surface 2.27 1738 0.42 4,40 modified plate 2 Sumface 2,49 22,44 0.17 200 niodified plate 3 Surface 1.7 '3 3 0.32 520 Surface 2.14 7.99 0.8 2.30 modified p ate Nunclon 1,75 15.22 017 1.67 Clbin .63 -3.04 0-17 0 78 Table 11: Summary of the Results of the XPS Analysis of Surface Elemental Composition and Human Embryonic Stem Cell Attachment and. (Colony Formation Eperiments on the Surface Modified Plates of the Present nvention h32C Adadns 'u ('.ti(' s A dm t -' i Mi' ------------- ---- --- --- --- -- a s --- -------------- ----- --------- 3* t a --- ------------- 3 r 'e:3 33 -6 4 .... ............ .. ... . .. . 3 . .. . . .. . 3 ...... ...... 444... .. 44.. ...... ...... ...... ... 3. wa NUB 2 aa ss3 soa P s N, P 1 f- 0u 1 05Tl is N ND ND It9 O 21A3 I_______ >> M>' ii NY\ NY~ 114 3;>A w4 > "I>i 4Z, '1 T3) -74 ------ \ 3 ' N 8IN IL ND N11&A N A IV' NA; 41 s tw WWI 3> N34 U-34 33) 5733) 3 43 72 3.6 '4e '6 , t. P3 N) ND ND 3 o. O. M4 31A : 013n 111D N-1b 1A.' 6'.6 20) ')3 cawslW _ is3 __ b __ >) ND ,1) '41 13 1' 3*0 > 460 '3 N=Qa MM. fp ND ND 0- a 11 3 s) . SM N 39 1 ' I Man IA1 a u M h MW MMW wM"Mma n Ss 3 '4. '4.3 44 7 . 3 a4 R ins M e 3 o>me(n3 3x4me~e t- i33> 31 '.3 '3 '4e~4 33 sa' 3,rssamIavd33>i>tal 3$r >8na -. y' i Table 12: Surface Eemental Composition as Determined by XPS Surface Elemental Composition as Determined by' XPS eNAsurenments on two saupes (exept Sudace 22) an mean ±estadard deviation (SD) 6 Carbon % Oxygen % Nitrogen SurfaCe 2 T 1 2 2 .ean2 SD e SD Surface 79 71' 791 2 0 195 9 196 0 2 I4 11 3 an02 Surface 9A 792 79.i0 195 188JU 917065 11 13±0 2 Su 7c ? 6 2 800 8 +2 2+ 2 1,9 3 2n 13 d 0 sde8 778 &7 78 i6 28 19 3 20.0 :LU 14 IS 1.5i±0. LA N ~ ~ ~ --------------- -,- - --- Suface 7 72 780 9 a 8 19 8. 19 3 0 1 . J Iufg 10 80A,79 798 91+0 8 190 9 9I 0.2 0, 0 9 08 0 2 Sudai7ce7 77 8Y I& 2M4 20 20,3 1, M0 0 01 9 8 9 Surface 12 77 8S3 iO1 L9 21t8 203 212 U It 08 09 0 Surface 8 97k 971 A a 1 2A 2 2] 23 1 0 (0 0 0 0 0,0 Surface 9 773 + 4K6 i4 20 1 1 11 Studace 12 82 5~ "'8{0 1 9 6 ~~0" 0ON 0

-~

0 - -- - -- Surface' 3* 8 2 291 80.7+22 V 6~ 2 .9~ 7" 10 -------f 84 0 796 81-8 3 1 816 27 6 A L0 16 19 q0 A Surtacc30* 0 4(*tfw 01 S20 12 104 O 10 0' 01 08 9404 Surace 3* *3 N 8 82 164 ''0 IA 4 0,0 0,0 0000,0 Sudace 33 74 2 73 3 N9 { 03 24 9 2 2 20 0 009 0,0 00,±. Surface 3 747 753 7111 06 .24 2 2,1 11 9 0 1 0 L1 0 * Not pl~~asated Oier ecrvn detected ait a .onlcentration of 0,2-2.0%,g 80 Table 13 Carbon Bonding Environment by C1S Spectra Curve Fitting Carbon Bonding Environment by Cis Spectra Curve Fitting Atomic percent of each functional group is given based on one measurement on Surfaces 5 12 and 22 and as mean ± standard deviation for the other surfaces (n = 2) Functional groups and Cls binding energies (eV) C CCC CAOC C-4Op C=0 C-0- Coll3 0- Energ -846 C C C (287.9 C*=4) (289.8 [(:01- loss Surface eV) C* N'H (287.0 eV) (288.9 eV) 0 peak (285 2 (286.1 eV)eV) (291S0 (292 eV1 eV1 eV) eV, Surface 38.0 2 1 2 4.6 0,6 L4 28 08 00 S.ufe6 43 260 106 5 1 13 1U 01 00 Su3aee? 31 2 9 5 08 1A 2:7 05 05 SurfaceS 8 1 9 6 O '6 08 05 SurfacelO 5f5 96 3 0 00 08 16 0 6 SuIc 1 34 20 8 47 234. 04 1 1 0 0.6 Surne12 36 2 0 28 4,8 32 U 07 05 Ntrace i8 86A 00 98 00 00 N k 00 0.0 00 19 14 0.7 Surface9 3LS 187 10 00 li 00 0.0 00 1 03 ' 01 05 S 22ace22 61 5 04 49 0009 2 IS 00 Sace-3 1 62 4 00 9 0.9 00 4.. 0.7 03 0,6 01 0.0 0.5 Surface24 662 8. 24 00 12 1 13 1. 00 6h 04 0 0.6 0.6 0.6 0.1 0.0 03.00. Suahce30* 8 3+ 00 52 9&* 7,4 0'0 0 9 S 1u 4') OQ ..<J ) 0),6 Surfacc3 * 553*~ 93A 9.A 35 0.0 0.0 0.0 0.0 24i 06 01 00 01 Sudface32 t Le , + 07 3 0.0 0.0 0.0 0.0 2 ___85__ 28__ 1 05 0 0 06 -& ~ c ------ -I ----- 1 -)7 ---- ------- t 0.00.-0.--- Surac3 17 .3 9 11 3,5 0.0 0.0 00 17 03 0 0 004 Surface 34 21 21 09 0 00 3 0 00 00 2 0.1 0.6 0.50. * Not plasna treated, 81 Fable 14: Nitrogen Bonding Environment by Nis Spectra Curve Fitting Nitrogen Bonding Environment by NIs Spectra Curve Fitting Atone prcet o eah~mtioa goupis giVen based on one measrement on Surfaces 5. 12 Ato)mice percent of each kuein ip isanr()UJ3,n-at n 2 and 22. and as mea str deviaion for the other surfaces (a 2) Funedonal groups and NIs bing energies in eV ~,Nal OC -N-CO -N1" -NO - ~NO Surface (39K cY) (400.80 e 1 (401l8eY)- i$06-5 Ql (4070 eV) surtace& 3o 500 30 2.090 Surfae 6 46 0 26 0 1.0 5.0 1 0 S-race 25.0 4 0 2 0 2,0 4 0 Surface 8 13.0 56 0 26,0 LO 4 0 Surface 9 2.0 44 0 45.0 4, S Surface 10 80 1 0 170 Tj) Surfae I 13 0 30 0 4.0 11'0 Sutae 8*' ND* ~ND N ND Ni Sudece19 190 &4 51+6 240 .8 +507 40 0.0 Sud±ace 0 1 0 44 0 0.0 0 0 suria__e_23 |260~ 14 A 5 0 4 130 'U0.0 0 0 Sudace 4 230 0 47.0 5 30 0 1 3 0,0 0 0 9c 1 52 0, 354 + 1 6 28 35& 21 30 1.4 S ee30*' ND N ND NiD ND Suirfce31 ND ND ND ND ND Surface 32 ND ND ND) ND ND S '3ace 7.5 ± 0.7 3 0.7 5.0 It 0.0 4 52 Surface 34 11 5 2 555 6 4 2 07 4I0+ 1A L 7 0 * Not plasma treated \** NI);a Iuid but not detected.

Claims (3)

1. 2. The method of claim wherein the sum of () and N is greater than or equal to
2. 19.5%. 3. The method of claim , whereinthe surface has an adlayer 4. The method of claim i , wherein the cells are maintained i culture after the cells attach to the surface. 5. The method of clain A wherein at least one compound is removed after the (.etlls attach to the surface. 6, Ihe method of claim I wherein the eolis are detached front the surface by removing at east one compound 7. The method ofelam . wherein the suspnesion of cells is a suspension of clusters of cell. S. The method of claim wherein the suspension of cells is a suspension of single cells 9. The method of claim 1, wherein the surface is part of a vessel or matrix. 83 10. A method to enhance the attachment of cells to a surface containing fronm at least about 0.9% N, a sum of' 0 and N of greater than or equal to 22.3% aind a contact angle of at least about 139 degrees, lacking a feeder cell layer and lacking an adlayer, comprising the steps of' a. Obtaining a suspension of the cells and b, Adding the suspension of cells to the surface and allowing the cells to attach. 11 . The method of claim 10, wherein the surface has an adlayer: 12. The method of claim 10, wherein the cells are maintained in culture after the cels attach to the surface. 13. The mthod of claim 10, wherein the uspension of cells is a suspension of clusters of cells, 14. The method of claim10,vherein the suspension of cels is a suspension of singl cells, 15. The method of claim 10 ,vherein the surface is part of a vessel or natrix. 16, A surface that is part of a vessel or matrix intended for use in cell culture or analysis, containing fRnm at least about 09% N, a sum of 0 and N of greater than or equal to 22.3% and a contact angle of at least about 13,9 degrees, lacking a feeder cell ir and Uacking an adlayer wherein the surface allow the attachment and u it vaion of cells. 17. The method ofclaim 16 Iherein the surface has an adlayer; 1 SThe surface of Iaim 16, wherein the cells are maintained in culture after the cells attach to the surface. 19- A opposition comprising: a. A surface that is part of a vessel or natlrix intended for use in cell culture or analysis, contaming from at least about 0.5% N, a sum of 0 84 and N of greater than or equal to 17-2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacing an adlayer, and x At east one compound s from the grolp consistig of: a compound capable of inhibiting Rho kinaisc activiy, and a comipound capable of' t biting Rho activity. 20 The composition of cain 19, wherein the sum of 0 and N is greater than or equal to 19.5%,
3. 21. The method of claim 19,werein the surface has an adlaver. 85