Patent Application: US-201214125812-A

Abstract:
the present invention relates to the use of certain polymers as a substrate for stem cell , such as pluripotent stem cell growth and / or culture . the present invention also relates to articles such as tissue culture materials and cell culture devices comprising at least one polymer hydrogel as described herein .

Description:
the present invention will now be further described with reference to the following figures which show : fig1 : ( a ) high throughput screening of polymer hydrogels for long - term support of hesc growth , control of differentiation and enzyme free passaging . ( i ). hydrogel microarrays with 609 different polymers ( in quadruplicate ) were fabricated by in situ polymerisation on a glass microscope slide . the microarrays were treated with human escs for 24 hours before fixing and staining with dapi . ( ii ). mosaic image of the top 120 hydrogels ( with nine copies of each ) re - synthesized and cultured with human escs for 2 , 4 and 7 days before fixing and immunostaining for oct4 / nanog . the expansion shows a single polymer feature with human escs cultured for 7 days ( upper = dapi , middle = oct4 and lower = nanog ). ( iii ). four candidates ( showing good cellular binding and thermal - release properties and the highest number of oct4 / nanog positive cells ) were synthesized on glass coverslips . the cells were cultured for over 20 passages using , at each step , temperature triggered detachment . briefly , once 70 %- 80 % confluency was reached , cells were incubated at 15 ± 3 ° c . for 30 minutes , most of the cells were detached and could be broken down to smaller cellular aggregates by gentle pipetting . then the cells were plated onto fresh polymer hydrogel coverslips . ( b ) structure of the hydrogels hg9 ( hpma / deaea ( n = 1 , m = 3 ) and hg19 , 20 and 21 ( aetma - cl / deaea ( n = 3 , n ′= 1 ; n = 1 , n ′= 1 and n = 1 , n ′= 3 ). ( c ) images of cell colonies showing expression of hesc markers oct4 and nanog after culture on hydrogel hg21 after 20 passages . ( d ) flow cytometry analysis of human escs thermo - detached from hg21 and enzymatically harvested from mg respectively . ( e ). qrt - pcr comparison of expression levels of the pluripotency markers oct4 , nanog , sox2 , tet1 and tet3 at passages 10 and 20 of culture of cells grown on hg21 , normalised to the “ housekeeping gene ” gapdh ( shown as a ratio with respect to rh1 cells cultured on mg ) fig2 : fluorescent images of in vitro differentiated rh1 human escs harvested from hg21 after 20 passages cultured in mtesr1 medium and subjected to a standard embroid body mediated differentiation protocol . images depict immunoreactivity for β - iii tubulin ( ectoderm ), α - fetoprotein ( endoderm ) and smooth muscle actin ( mesoderm ) demonstrating the pluripotent state of rh1 cells . ( a ) thermo - detachment of rh1 from hg21 by incubation of the cells at 15 ° c . for 30 minutes . ( b ) thickness change of bulk hg21 ( in pbs ) with temperature under different compressive strengths . relative thickness measurements were determined in the range of 1 . 6 - 11 . 3 kpa ( only two lines shown ). extrapolation of this data was used to give the properties of the hydrogel under physiological conditions (± sd , n = 3 ). ( c ) x - ray photoelectron spectroscopy ( xps ) analysis of dried hg21 showing that the polymerisation proceeded according to the starting monomer ratios . fig4 : a ) ratio of drops of monomer printed at each position . b ) the pattern of the printed hydrogel microarray . the pitch between polymer spots was 0 . 8 mm . the 6 monomers in first column in table 1 are the main monomers . during printing , each forms two - monomer combinations with all other monomers respectively . briefly , each line along the width direction of a slide , two monomers overprinted at each spots , was printed with 7 ratios in drops , which are 14 / 2 , 12 / 4 , 10 / 6 , 8 / 8 , 6 / 10 , 4 / 12 and 2 / 14 respectively . fig5 : average rcm1 hesc binding on the top 50 hydrogel polymers after 2 , 4 and 7 days culture in medium with bfgf . fig6 : ( a ) comparison of the number of cells stained with nanog ( rhod ), oct4 ( fitc ) and dapi on top 25 hydrogels after 7 days culture in mtesr with bfgf . ( b ) fluorescent and bright field images of human escs bound on polymer spots after 7 days culture stained with hoechst 33342 ( dapi ), oct4 ( fitc ) and nanog ( rhodamine ). compositions are the merging images of three colours . culture medium was mtesr1 with bfgf . small colonies were formed on the spots . fig7 : hesc growth on top 10 hydrogels and the viability after thermodetachment treated under 15 ° c . for 30 min . fig8 : markers / morphology of rh1 hesc on 3 hydrogels at p10 . fig9 : immunostaining of rcm1 hesc at p20 harvested from hg9 and hg21 . fig1 : immunostaining of h9 hesc at p5 harvested from hg9 , hg19 , hg20 and hg21 . fig1 : immunostaining of differentiated cells from rcm1 hesc after 20 passages on polymer hydrogels revealed expression of markers for the three embryonic cell layers : ( a ) endoderm , afp and alb , ( b ) ectoderm , nestin and tuj1 and ( c ) mesoderm , von kossa - stained calcium phosphate . fig1 : cumulative population of human escs on hydrogels compared with cells on matrigel . ( a ) rcm1 cells on mg , hg21 and hg9 ; ( b ) rh1 cells on mg and hg21 . fig1 : ( a ) temperature dependence of strain and storage modulus ( g ′) during heating and cooling of hydrogel samples at 2 ° c ./ min with 2 minute equilibration time . arrows show the start of the temperature sweeping . the results showed that the strain of the hydrogel underwent shrinkage for about 11 % at 1n when the temperature increased from 10 ° c . to 22 ° c . corresponding to a larger storage modulus ( g ′) at higher temperature . ( b ) the mechanical properties of hg21 showing the results of storage modulus ( g ′) and loss modulus ( g ″) versus strain (%). ( a ) kidney capsule teratomas , induced by injection of rh1 cells cultured for 20 passages on hg21 into nod / scid mice , contain tissue derived from all three primary germ layers . from left to right : islands of undifferentiated ec / es cells , neural rosettes ( ectoderm ), gut epithelium ( endoderm ), and cartilage ( mesoderm ). sections were stained with masson &# 39 ; s trichrome . ( b ) schematic summary of copy number variations detected by comparative genome hybridisation analysis of rh1 cells using a nimblegen ™ 135k probe array following culture on hg21 for 10 and 21 passages ( p10 , p21 ) and contemporaneously age matched matrigel ™ ( mg ) for 21 passages . depicted are micro deletions and duplications and their size in kilobase pairs (& gt ;#& lt ;, & lt ;#& gt ;, respectively ) in relation to their location on chromosomes for each genomic dna sample . no gross chromosomal aneuploidies were detected between hg21 and mg grown cells . copy number variations occurring in the former also occurred in the latter on chromosomes 8 , 9 , 13 and 20 . fig1 . characterisation of h9 cells cultured long term on hg21 . a , h9 cells cultured on hg21 - coated cover slips for 6 passages retain expression of transcription factors oct3 / 4 and nanog as assessed by immunocytochemistry . b , h9 cells cultured on hg21 - coated coverslips for 6 passages retain rna expression of transcription factors oct3 / 4 , nanog and sox2 . rna levels are expressed as fold change compared with the expression of respective genes in h9 cells grown on matrigel ( mg ) for 6 passages c , hematoxylin & amp ; eosin staining of sections of teratomas formed in testes of fox chase scid beige mice following injection of h9 cells cultured on hg21 for 9 passages shows that the teratomas contain derivatives of all three germ layers : mesoderm ( cartilage and muscle ), ectoderm ( neural rosette ) and endoderm ( glandular structures ). all chemicals used for hydrogel microarray fabrication were purchased from sigma - aldrich except tridecafluoro - 1 , 1 , 2 , 2 - tetrahydrooctyl - dimethylchlorosilane ( fds ) ( abcr gmbh co , kg ). n - acryloyl - n ′- propylpiperazine ( nanppa ), 2 , 2 ′-( ethylenedioxy ) bis ( ethylamine ) mono - acylamide ( eoa ) was synthesized in house while n - isopropylacrylamide ( nipaa ) was purified by recrystallization as described previously . 17 , 20 other chemicals were used as received . tertiary polymer blend hydrogel microarrays ( 28 × 87 spots ) were fabricated using inkjet printing . initially masked glass slides were generated by printing sucrose solution ( 20 % wt ) on cleaned glass slides and followed by treatment with fds for over 4 h followed by rinsing with acetone and water to remove excess fds and sucrose . slides were treated with 3 - trimethoxysilane propylmethacrylate overnight before rinsing with acetone . for the array fabrication , the redox initiators ammonium persulfate ( aps ) and temed were used as previously reported . 17 in the printing / fabrication procedure , an aqueous solution of the crosslinker 2 , 2 ′- methlenebisacrylamide ( mba , 3 % wt / wt ) was added to the solutions of the 18 main monomers ( see table 1 ) before printing , with each combination of two monomers printed in quadruplicate . the monomer ratios varied in steps of 14 % ( fig4 ). initially , 6 drops of the initiator ( aps , 1 % wt / wt in distilled water ) were printed . these were overprinted with the two monomers ( 16 drops in total ) and finally 6 drops of temed ( 28 . 5 % wt / wt aqueous solution ph 7 ) at 20 ± 2 ° c . with the humidity in the printing chamber controlled at 70 ± 5 %. in total 87 lines were printed giving 609 different polymers ( in quadruplicate = 2436 polymer spots ). the printed slides were kept at 37 ° c . for 8 h before gently rinsing with water and ethanol . the slides were uv sterilised for 20 minutes and washed with pbs ( x2 ) prior to cell culture . the same procedure was used for the preparation of the “ hit ” hydrogel microarrays . a ) monomers were dissolved in water with the ph adjusted to ph 7 using naoh ( 2n ) or hcl ( 2n ). the ph of the ceao solution was adjusted using a na 2 co 3 ( 20 % wt ) solution . hydrogel coated cover slips and slides were prepared as described previously . errort bookmark not defined . briefly , solutions of the monomers , crosslinker and photoinitiator in n - methyl - 2 - pyrrolidone were coated on treated coverslips and exposed to 365 nm uv light for 30 minutes and placed in a 50 ° c . oven overnight . the polymer coated coverslips were then washed with ethanol , acetone and dried in air . rcm - 1 , rh1 and h9 hes cell lines were used for the high - content screens . the cells were cultured on plastic tissue culture well plates ( corning inc . ny ) coated with matrigel ( bd ) in a feeder - independent environment with chemically defined mtesr1 medium ( stemcell technologies ) at 37 ° c ., 5 % co 2 . cells were passaged using a collagenaseiv ( invitrogen )/ scraping regime ( or thermally — see below ). human esc were seeded at a density of 60 , 000 cells / cm 2 on microarray slides in 5 ml mtesr1 containing 1 % penicillin / streptomycin ( invitrogen ), 1 % fungizone ( invitrogen ) and 10 μm rock inhibitor ( calbiochem ). cells were incubated for 2 days before fixing and staining with dapi . to optimise the evaluation of the cell number analysis on hydrogel spots , we introduced a 4 level cell counting system , which has the benefit in the initial screening of identifying possible polymers . level 0 was used if less than 10 % of the area of a spot was covered with cells ; level 1 , 10 % to 40 %; level 2 , 40 % to 70 % and level , 3 70 % to 100 %. combining the results from two independent screens , 120 ‘ hit ’ polymers were chosen because of their high levels of binding ( table 2 ). some monomer combinations showed very good cellular affinity in all ratios such as nanppa / nipa . in this case , three ratios ( 4 / 12 , 8 / 8 and 12 / 4 ) were arbitrarily chosen as hit polymers . the ‘ hit ’ microarrays were printed with 9 replicates per polymers and incubated with rcm1 for 2 , 4 and 7 days respectively . after fixation the arrays were stained for oct4 and nanog and treated with dapi before scanning . to count the bound cells per unit area on hydrogel spots , the scanned images were analysed automatically using pathfinder software and then corrected manually . the top 50 hydrogels are presented in fig5 , showing that the cells proliferated on the polymer spots . some hydrogels showed most cells on the 4 th day which could mean detachment of cells ( maybe because colonies were washed away during medium refreshment as they become too over - crowded ). most rcm1 cells on polymer spots expressed oct4 / nanog after 7 - day culture ( fig6 a ). some small stem cell colonies could be observed on hydrogel spots as shown in fig6 b . the top polymers were scaled up for analysis of longer - term culture . hydrogel - coated coverslips were sterilised for 20 min under uv light and were then washed with dulbeccos phosphate buffered saline ( pbs ) ( sigma ), before cells were seeded at a density of 90 000 cells / cm 2 . once 80 % confluency was reached , cells were split at a suitable ratio ( 1 : 1 . 5 or 1 : 2 ) by thermodetachment at 15 ± 3 ° c . for 30 minutes ; they were then collected , pelleted , resuspended in mtesr1 and replated accordingly . cells were maintained by daily replacing ⅔ of the media with fresh mtesr1 . in this study the top 35 hydrogels were coated on glass coverslips ( 13 mm ) as described earlier ( table 3 ). after 7 day culture in mtesr1 , hydrogels were judged by the number of adherent cells and their morphology . it was observed that cells on some hydrogels became & gt ; 75 % confluent with good morphology colonies . after low temperature treatment , most of the cells were detached and could be broken down to smaller cellular aggregates by gentle pipetting . then the cells were plated onto fresh polymer hydrogel coverslips . during the second passage , 10 hydrogels which showed the best colony morphology , easiest thermo - detachment and high cellular viability were chosen for scale - up ( fig7 ). cells were split at 1 : 1 . 5 ratio onto hydrogel coated coverslips by thermodetachment . once 25 - 30 % confluency was reached the cells were cultured with 1 ml of rpmi 1640 ( invitrogen ) supplemented with 2 % b27 ( invitrogen )+ 100 ng / ml activin a ( peprotech ) and 50 ng / ml wnt3a ( peprotech ) for 3 d . media was changed every 24 hours . cells were subsequently treated with dmso media consisting of ko - dmem ( invitrogen ), 20 % knock out serum replacement ( invitrogen ), 1 % non essential amino acids ( invitrogen ) , 0 . 5 % l - glutamine ( invitrogen ), 0 . 2 % β - mercaptoethanol ( invitrogen ) and 1 % dimethylsulphoxide ( dmso ) ( sigma ) every other day for 5 d . cells were then fed l - 15 media consisting of leibovitz l15 media modified ( sigma ), 10 % tryptose phosphate broth ( sigma ), 10 % foetal calf serum ( gibco ); 1 μm insulin ( bovine pancreas ) ( sigma ), 10 μm hydrocortisone hemisuccinate ( sigma ), 1 . 2 % l - glutamine ( gibco ) and 50 μg / ml ascorbic acid ( sigma ). this was supplemented with 10 ng / ml hepatocyte growth factor ( peprotech ) and 20 ng / ml oncostatin m ( r & amp ; d systems ) every other day for 9 days . after 17 d of differentiation the cells were collected for analysis . rcm - 1 cells grown on hydrogels for more than 20 passages were neuralised using a dual smad inhibition approach as previously described . at 70 % confluency they were provided with cdm medium ; 50 % imdm ( invitrogen ), 50 % f12 ( invitrogen ), 5 mg / ml bsa , 1 % lipid 100 × ( invitrogen ), 0 . 09 % monothioglycerol ( sigma ), 10 mg / ml insulin ( roche ) and 30 mg / ml transferin ( roche ) supplemented with 0 . 2 μm dorsomorphin ( calbiochem ), 1 μm activin inhibitor ( calbiochem ) and 0 . 1 mm n - acetyl cystein ( sigma ), which was subsequently changed every 2 days . after 7 days the cells were thermodetached and colonies harvested and resuspended in fresh cdm supplemented initially with 5 ng / ml bfgf . after neuroectoderm had been firmly established the concentration of bfgf was increased to 20 ng / ml and 20 ng / ml egf was added to the medium . the emerging spheres were maintained for 2 months by passaging weekly : a uniform suspension of neural stem cell clusters ( neurospheres ) was obtained by roughly dissociating spheres with a razor blade and split 1 : 2 . neurospheres were then plated on matrigel ( reduced growth factor , b & amp ; d ) in neurobasal ( invitrogen ), supplemented with 0 . 04 % b27 ( invitrogen ), 1 % l - glutamine ( invitrogen ), 0 . 01 % neaa ( invitrogen ), 1 % penicillin / streptomycin ( invitrogen ) and 1 % fungizone ( invitrogen ). for the first 48 hours cells were cultured in the presence or absence of gamma secretase inhibitor ( calbiochem ) and grown for a further 12 days before processing . rcm1 cells grown on polymer for more than 20 passages were thermally detached and plated onto polymer coated coverslips . hes - mps were used as positive controls . to each of the treatment wells 0 . 5 ml of stempro osteogenesis differentiation medium ( invitrogen a10072 - 01 ) was prepared and added in accordance with the manufacturers instructions . untreated controls were maintained in standard media ( mtesr and hes - mp medium ). cells were fixed in 4 % pfa after 21 days of treatment and osteogenic differentiation was determined with von kossa staining . hes colonies were detached and dissociated by pipetting until a uniform suspension of cell aggregates was obtained . cells were then plated in low cluster plates ( corning ) and grown in eb medium ; dmem ( invitrogen ), 20 % foetal calf serum ( sigma ), 1 % l - glutamine ( invitrogen ), 1 % neaa ( invitrogen ), 0 . 2 % 2 - mercaptoethanol ( invitrogen ) for 7 days as a suspension culture . subsequently , ebs were harvested and incubated with pbs - based enzyme - free cell dissociation solution ( sigma ) for 10 minutes at 37 ° c . dissociated ebs were then plated on matrigel at 30 % confluency in the presence of 10 μm y - 27632 and grown for another 7 days in eb differentiation medium ; advanced rpmi - 1640 ( invitrogen ), b27 ( invitrogen ), penicillin ( invitrogen ) and streptomycin ( invitrogen ) in the presence of differentiation factors : mesoderm was obtained by adding 100 ng / ml activin a ( r & amp ; d systems ) for 1 day followed by 6 days of treatment with 10 ng / ml bmp - 4 at ( r & amp ; d systems ); for endoderm the cells were treated with eb differentiation medium supplemented with 100 ng / mlactivin - a for 6 days ; ectoderm was obtained by incubating the cells 3 μg / ml retinoic acid for 7 days . the present inventors have also shown that the hes cells are capable of forming teratomas following injection under the kidney capsule of immunodeficient mice , which supports the fact that pluripotency of the cells is maintained . see fig1 and 15 . fig1 also provides data showing karyotype maintenance . cells were dissociated by incubation with 0 . 025 % trypsin / edta for 5 - 10 minutes at 37 % degrees , resuspended in facs pbs ( pbs + 0 . 1 % bsa + 0 . 1 % sodium azide ) and incubated with preconjugated antibodies ( ssea - 4 , fitc ; ssea - 1 , phycoerythrin ( pe ); biotinylated tra1 - 60 ( all biolegend ) in facs pbs ) for 20 - 40 min . tra1 - 60 biotin detection was performed with streptavidin - conjugated allophycocyanine ( apc ). data were obtained using flow cytometry ( calibur ) and analysed using flow jo . cells were stained using a standard immunocytochemistry protocol . briefly , hes cells on hydrogels were washed once with pbs and fixed with 4 % pfa . after permeabilisation in 0 . 2 % igepal ( sigma ) and blocking in 10 % normal rabbit serum ( millipore ), cells were incubated with primary antibodies oct - 4 ( santa cruz biotechnology ), nanog ( r & amp ; d systems ), gfap ( daco ), β3 - tubulin ( sigma ), nestin ( millipore ), afp ( sigma ), albumin ( sigma ) for 2 - 4 hours at rt or overnight at 4 ° c . visualisation with secondary antibodies was performed using alexafluor antibodies 488 , 555 , 568 , and 647 ( invitrogen ), and nuclei were labelled using 4 ′, 6 - diamidino - 2 - phenylindole , dilactate ( dapi ). imaging analysis was carried out using a leica spe microscope . dna was extracted using a standard phenol / chloroform precipitation method . briefly , pellets of hes cells were incubated o / n in proteinase k and lysis buffer . dna was precipitated with phenol and chloroform , washed with 5m ammonium acetate , isopropanol and 80 % ethanol and the dna was rehydrated in h 2 o and stored at − 20 ° c . comparative genome hybridisation was performed and analysed at the cytogenetics unit at the western general hospital , edinburgh , uk by a standardised comparative genome hybridisation method using the nimblegen nimblegen 135 k array ( v . 3 ) ( table 5 ). b the array analysis displayed the 20q11 . 21 duplication as series of contiguous duplications ( which represent a single 550 kb duplication overall ), the duplication is not listed automatically on the summary by the nimblegn software . to obtain population doubling rates of hes cells on hydrogels , cells were counted at each passage using a haemocytometer . triplicate counts were gathered at each passage and viability was assesed by staining with trypan blue ( sigma ). rheology analysis was carried out using a ta instrument ( ar - 2000 , 40 mm 4 ° steel cone ) with an oscillation frequency of 1 hz and oscillation stress of 1 pa . hydrogel samples were washed in pbs and refreshed every 24 hours for one week before cutting to discs with a ˜ 3 mm thick and ˜ 2 cm in diameter . xps analysis was carried out on a thermo vg scientific sigma probe , with an al k α x - ray source , a pass energy of 80 ev and 0 . 5 ev steps ( scanning − 10 . 00 to 600 . 00 ev ). although the suitability of thermoresponsive polymers for mouse embryonic stem cell culture 17 , 21 , has been reported previously , loh et al observed reduced growth rates when culturing mouse embryonic stem cells on thermoresponsive co - polymers compared to gelatin controls 21 . although they demonstrated that these gelatin - containing polymers support mouse embryonic stem cell growth and cell release at very low temperature ( 4 ° c . ), cell cultures lasted only 3 - 4 days , without cell passaging . to our knowledge , the work presented herein is the first to report long term culture and gentle serial passaging of human embryonic stem cells using a thermomodulatable synthetic polymer , verified with independent cell lines . the fact that cells maintained an undifferentiated state , did not acquire harmful genetic aberrations , and retained pluripotency strongly suggest that the polymers of the present invention are promising candidate substrates for use in large scale , gmp - compliant pluripotent cell growth , such as hes production for clinical purposes . 1 vazin , t . & amp ; 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