Patent Application: US-201414889453-A

Abstract:
the invention pertains to a combinatorial method of identifying the hydrogel formulations controlling phenotype and fate of difficult - to - culture cell types .

Description:
hereinbelow , embodiments of the invention will be presented in greater detail , by means of embodiments . generation of arrays of 3d signaling cell culture microenvironments from a tool - kit of molecular building blocks to realize the three - dimensional screening of cellular microenvironments according to the present invention it has proven useful to engineer a biomaterials system composed of a library of molecular building blocks which can be independently mixed and then cross - linked in the presence of cells to potentially form a huge diversity of three - dimensional cell microenvironments with distinct and independently controllable properties . it was found that the invention can preferably be carried out on the basis of synthetic hydrogels as biomimetic three - dimensional cell microenvironments with very well defined biochemical and mechanical properties ( lutolf and hubbell , 2005 ). preferably , the coagulation enzyme activated transglutaminase factor xiiia ( fxiiia ) is used in the context of the present invention to crosslink multiarm poly ( ethylene glycol ) ( peg )- based pre - polymers into 3d hydrogel networks under physiological conditions ( cf . fig1 a ) ( ehrbar et al ., 2007 ). indeed , it was found that single mouse esc encapsulated within such peg - based hydrogels show a very good viability ( 89 . 1 ± 7 . 3 %) that is not significantly different ( p = 0 . 44 ) from standard culture conditions on gelatin - coated plastic ( 92 . 5 ± 3 . 6 %); results not shown in detail . gel - encapsulated single escs are physically separated from each other from the beginning of an experiment and remain so over time , thereby expanding as clonal entities . to allow for efficient growth in three dimensions , the gels can be rendered susceptible to cell - secreted proteolytic remodeling by designing gels bearing a protease substrate site for degradation ( lutolf et al ., 2003 ; patterson and hubbell , 2010 ) ( cf . fig1 a ). moreover , biologically active molecules such as oligopeptides or proteins can be site - specifically attached to the matrix during cross - linking ( fig1 a ). this molecular “ tool - kit ” for making various hydrogel matrices effectively permits to probe a very large combinatorial space of different 3d cellular microenvironments . as a proof - of - principle , five key signal types of the three - dimensional in vivo microenvironments were prepared where each of these characteristics could be independently varied ( cf . fig1 b ): matrix mechanical properties ( abbreviated with “ mp ”), susceptibility to proteolytic degradation (“ dg ”), extracellular matrix - derived proteins (“ ec ”), cell - cell interaction proteins (“ cc ”) and soluble factors (“ sf ”). by varying the polymer content , the stiffness ( represented by young &# 39 ; s moduli , e ) of the gels were specified between ca . 300 and 5400 pa ( ehrbar et al ., 2011 ) ( fig1 b ) which is in the physiologically relevant range of soft tissues ( engler et . al ., 2006 ). the gels were rendered differently degradable by matrix metalloproteinases ( mmp ) via the incorporation of peptides of different sensitivities ( i . e . k cat / k m ) to cell - secreted mmps ( lutolf et al ., 2003 ). importantly , to perfectly match the mechanical properties of gels that are crosslinked by different mmp substrate peptides , the precursor content was adjusted . thus , key mechanical and biochemical properties of this synthetic gel system were independently controlled . to systematically modulate the cell signaling properties of the matrices , a set of ecm and recombinant growth factor proteins was chosen which had previously been implicated in regulating esc pluripotency , and which were enzymatically tethered to the gels in the context of the present invention ( cf . fig1 b ). esc interaction with laminin , fibronectin and collagen iv had previously been associated with a loss of pluripotency in 2d ( prudhomme et al ., 2004 ). however , ligation of their integrin homodimers by peptide analogs in a three - dimensional culture system indicated that they could instead promote maintenance of self - renewal ( lee et al ., 2010 ). in addition , an increasing body of work has pointed to transmembrane proteins involved in cell - cell signaling as mediators of self - renewal : e - cadherin , epcam and jagged have been chosen here as representative examples ( andrews et al ., 2008 ; gires et al ., 2009 ; soncin et al ., 2009 ). finally , the soluble esc regulatory factors leukemia inhibitory factor ( lif ), bone morphogenetic protein 4 ( bmp4 ) and fibroblast growth factor 4 ( fgf4 ) ( prudhomme et al ., 2004 ; qi et al ., 2004 ; ying et al ., 2003 ) were employed to a serum - free medium formulation such that they could reach the encapsulated cells via the diffusive properties of the gel network . in the present embodiments , four levels of modulation for each of the five categories were specified , leading to a total parameter space of 1024 unique conditions ( cf . fig1 c ). additionally , cell density , increasingly recognized as an important modulator of esc fate by autocrine effects , could be prescribed in this experimental setup as well as retrospectively imaged as demonstrated below . this level of complexity in formulating three - dimensional cell - containing matrices , as well as the required repetitions , is preferably reduced to practice by miniaturizing the sample volumes and adapting automated methods for gel fabrication ( cf . fig1 c ) and cell fate analysis ( cf . fig1 d ). specifically , a commercially available liquid handling robot was used to accurately synthesize one μl of each of the unique conditions in triplicate , in a completely automated manner , onto glass slides ( not shown ) or into standard 1536 - well plates ( not shown ). the latter format was chosen as it , presented an ideal surface to volume ratio for the selected hydrogel drops and represented a standard format which could be adapted to various experimental setups . once the three - dimensional gel array is generated , the system can function as a multimodal assay platform , where multiple readouts can be obtained in parallel . esc fate is highly dependent on the composition of the 3d cell culture microenvironment in order to obtain quantitative information on esc self - renewal and differentiation in response to the above three - dimensional cell culture microenvironment array , an oct4 - gfp reporter cell line was used , in conjunction with automated imaging and image analysis ( cf . fig1 d ). the transcription factor oct4 is widely considered as a marker of esc pluripotency ( kiwa et al ., 2000 ). cells embedded in three - dimensional matrices were imaged the day following seeding to obtain the actual initial number of cells for each well . in the most permissive conditions , these cells formed spherical colonies within three days , and kept proliferating until fixation after five days ( cf . fig1 d , left panel ). confocal microscopy confirmed that colonies growing in these conditions - were in a true three - dimensional space of approximately 500 μm in thickness ( cf . fig1 d , middle panel ). automated imaging of the complete 3d architecture of the hydrogel was combined with an efficient image analysis pipeline to obtain a number of morphological readouts . colony area was taken as a measure of proliferation , and gfp intensity as a measure of esc pluripotency ( cf . fig1 d , right panel ). for every unique combination of matrix effectors ( i . e . mp , dg , ec , cc and sf ), colony area and gfp intensity were calculated as the average of three replicates . each averaged value was represented as a square arid giver , a color ( with red representing relative high values and blue representing relative low values ) and all values / squares were organized by input condition thereby generating a heat map representation ( cf . fig2 a ). in general , soluble factor modulation was found to be the strongest predictor of heat map intensity , with the lif condition leading to both high proliferation and self - renewal , with the opposite effect for fgf4 and bmp4 , and an intermediate regime appearing for the conditions with no soluble factors . the top ten self - renewing and proliferating conditions were all within the lif condition , and most tended to be in the conditions of low mechanical properties and absence of cell - cell interaction proteins . indeed , when all cell culture microenvironments were plotted as colony area versus gfp intensity ( cf . fig2 b ), three populations emerged that were identified as cell culture microenvironments containing lif , no factors “ none ”) and bmp4 / fgf4 , with very little overlap between them . the lif dependence proved to be a strong biological validation of the platform , as lif is known to be a critical signal to maintain esc pluripotency and self - renewal via phosphorylation of the transcription factor signal transducer and activator of transcription ( stat ) 3 ( niwa et al ., 1998 ). additionally , distinct area vs . gfp relationships emerged out of such a representation , suggesting that the correlation between self - renewal and proliferation is linked to a particular soluble factor regime ; for fgf / bmp , irrespective of growth characteristics , self - renewal was lost , while with lif proliferation was strongly correlated to self - renewal , in cell culture microenvironments devoid of any exogenous soluble factors , self - renewing colonies were observed . this is in marked contrast to lif - free two - dimensional cultures that result in rapid loss of esc pluripotency and points to the potential role of other factors involved in maintaining esc in three dimensions . interestingly , the most self - renewing and proliferating ( i . e . high gfp , high area ) matrix characteristics in all subpopulations were found in conditions of lower matrix stiffness ( cf . fig2 c and 2 d ). systems - level analyses reveal relative and combinatorial effects of esc fate determinants in three dimensions to quantify in a systematic way how self - renewal and proliferation varied as a function of the signaling cell culture microenvironment , generalized linear models ( glm ) were utilized which encompassed all of the five determinants or . the cell culture microenvironment and their interactions ( cf . fig3 ). this approach allowed to explain more than 70 % of the variability in the system . by decoupling subtle effects from the more dominant ones , the relative importance of various factors could be quantified ( cf . fig3 a ), establishing a global hierarchy of components affecting esc fate in three dimensions . soluble factors accounted for more than 60 % of the model variance . physical properties , including matrix degradability and stiffness , accounted for approximately half of the remaining model variance , and tethered protein and initial cell density effects each accounted for at most 15 %. the role of individual factors was also investigated within these categories ( cf . fig3 b ). bmp4 and fgf4 , when presented as single factors in serum - free medium as in the context of the present invention , impaired self - renewal and no other microenvironmental factor could overcome this effect , to any significant degree . degradable matrices favoured self - renewal , but not necessarily proliferation . thus , the physical parameters of the matrix may not only dictate colony growth but also coordinate stemness . these processes do not always act in parallel and may be mediated by interacting factors . indeed , ecm proteins tended to increase the size of colonies but decreased oct4 expression , which matched previous findings in two - dimensional analysis , where laminin and fibronectin was shown to enhance differentiation of esc ( prudhomme et al ., 2004 ). surprisingly , cell - cell interaction proteins such as epcam tended to decrease both proliferation and self - renewal in the present system , although all three chosen proteins had been previously implicated in maintenance of esc self - renewal in two - dimensional studies ( andrews et al ., 2008 ; gires et al ., 2009 ; soncin et al ., 2009 ). this suggests that certain pathways could be differently activated or overridden by other factors in a three - dimensional environment . synergistic or antagonistic effects between factors which were statistically significant were represented by a network interaction map ( cf . fig3 c ) and as clustered heat maps showing significant pair - wise interactions ( fig3 d - g ). while some interactions such as those involving soluble factors were involved both in self - renewal and proliferation , others were self - renewal - specific ( cf . fig3 c ). for example , the presence of epcam with all ecm proteins reduced colony growth ( fig3 e ), while the presence of collagen in gels with low cell density enhanced colony growth ( fig3 f ). thus , ecm proteins , with their role in activating cell adhesion complexes via integrin engagement ( lee et al ., 2010 ), are involved in modulating the purely physical components of the matrix . overall , the interaction scheme centered around the soluble factors , underscoring their key role in regulating esc fate even in a three - dimensional environment . to study key aspects of esc regulation in 3d identified by this large - scale screen in more detail , the platform was rendered compatible with complementary downstream cell assays including flow cytometry and quantitative rt - pcr ( cf . fig4 ). the novel read - outs were implemented in more targeted experiments ( cf . fig4 a ) and in a time - lapse mode ( cf . fig4 b ), in order to also shed light on the dynamics of 3d esc behavior . for instance , the potency of lif was simultaneously investigated by performing a dose - dependence study , and the effect of mechanical properties was evaluated by widening the stiffness range and retrospectively measuring the stiffness of every gel ( not shown ), and a wider range of pre - set cell densities was explored ( cf . fig4 a ). proliferation was maintained in all conditions for the first , two days , followed by a strong dependence on all three parameters ( cf . fig4 c ). notably , lif maintained its proliferative role in 3d even at a concentration three orders of magnitude less than the 2d standard . it was confirmed that proliferation was directly related to lower matrix stiffness , while an intermediate stiffness was found to be optimal for self - renewal and colony - forming efficiency ( cf . fig4 d ). this finding is in line with data suggesting that 3d solid stress can control cellular growth at both the fluoroscopic and cellular levels in multicellular aggregates such as tumor spheroids ( helmlinger et al ., 1997 ). more recent work has shown that the elasticity of the in vitro substrate can direct stem cell fate ( engler et al ., 2006 ; gilbert et al ., 2010 ). the present results obtained in the context of the invention suggest that mechanical properties could play a similar and fundamental role in regulating esc maintenance in 3d , where optimal properties in the range of those measured in the early blastocyst might be most appropriate ( khalilian et al ., 2010 ; murayama et al ., 2006 ). in order to corroborate these findings at the colony - level with single - cell flow cytometry and gene expression data , hydrogel matrices within each well were digested with protease solution while maintaining cellular integrity . total cell counts by flow cytometry were moat closely correlated to a measure of total colony area by imaging ( not shown ). to better define the role of cell density , the total colony area was normalized by initial cell density : while higher initial cell densities led to larger colonies , this was not reflected in actual cell numbers counted by flow cytometry ( cf . fig4 e ), suggesting that cells within larger colonies may have died . as with image - based data , each condition could be visualized individually ; a heatmap representation ( cf . fig4 f ) indicates a clear trend towards a graded bidirectional influence of lif and mp , an observation reinforced by a global analysis showing the near - equal role of these two factors in influencing cell proliferation and the reduced role of cell density . an additional advantage of dissociating cells from the gels is the possibility to readily perform immunocytochemisty . for example , staining for ssea1 , a surface marker commonly used as a complementary marker of pluripotency to transcription factors such as oct4 , indicated a wide range of ssea1 expression even in cases of high oct4 ( cf . fig4 g ). ssea1 expression generally reached higher levels in softer matrices , and a ssea1 high / oct4 low subpopulation emerged in cases of high lif and high mp , suggesting that , as a result of changes in mechanical properties and lif concentration , some cells have undergone early commitment steps which is reflected by subtle changes in intracellular and extracellular markers of pluripotency . to demonstrate that cells collected from the gels could be used for essentially any complementary downstream assay , quantitative rt - pcr on selected samples was performed . such analyses showed how the expression of some genes associated with pluripotency was significantly downregulated rex1 ) while others ( nanog ) remained largely unchanged as a result of changes in the physicochemical environment , whereas changes in 3d matrix stiffness resulted in significant upregulation of map2 , a gene associated with early neuroectodermal differentiation ( cf . fig4 h ). taken together , the possibility to perform these multimodal readouts , including time - lapse imaging , flow cytometry and pcr , opens up broad avenues for looking at cellular systems in microarrayed 3d cell culture microenvironments according to the invention . systematic cell culture microenvironmental factor analysis reveals a set of factors marking a signature of neuroepithelial differentiation to demonstrate another embodiment of the invention , a similar methodology as described above was used to investigate factors modulating early neural development , particularly esc - derived neuroepithelial differentiation . importantly , beyond quantifying read - outs of proliferation and differentiation , the latter assessed by gfp intensity reporting sox1 expression , conditions were sought which could recreate the morphological features of neuroepithelial cysts , which have been defined as tissues comprised of bent epithelial cell layers enclosing a lumen ( gin , 2010 ), including their spherical and neo - polarized features . in order to maintain a wide - ranging parameter space ( 5 categories × 4 factors per category ) but optimize the number of required experimental conditions , the full factorial experimental design was modified to focus on two rounds of 4 - category modulation . in the first array , mp , dg , ec and sf were assessed in a full factorial design ( with no cc proteins ), and in the second , mp , dg , ec , cc were assessed in a similar way , with the soluble factor fgf4 in all conditions . quantification of the results was performed using the previously described glm - based modeling strategy . soluble factors , dominated by the effect of lif , contributed more than 74 % of the variability observed for colony area , with only small contributions from mechanical properties and mmp - sensitivity ( 5 %) and nearly negligible effects from other factors ( data not shown ). in contrast , the variability in gfp intensity was attributed to a much wider set of factors : while soluble factors still contributed the largest proportion ( 32 %), cell - cell interaction proteins accounted for 16 % of the variance and physical properties ( mechanical properties and mmp - sensitivity ) accounted for 9 % each ( cf . fig5 a ). the contribution of individual factors to sox1 - gfp intensity was further analyzed to determine which factors would have roles as positive or negative regulator of differentiation . fgf4 was the soluble factor which had the strongest positive effect ; on gfp , with lif and bmp4 both strong negative regulators of neuroepithelial fate ( cf . fig5 b ). the lack of any soluble factor led to only a small negative effect on sox1 - gfp expression , indicating that the baseline condition produced a generally neutral effect on cell fate within the tested conditions . for mechanical properties an intermediate - soft range in elasticity promoted highest sox1 - gfp expression . indeed , a slight negative effect on differentiation appears in soft matrices and a more pronounced negative one in the stiffer conditions ( cf . fig5 b ). non - degradable matrix was significantly more permissive to the formation of gfp + colonies than mmp - sensitive matrices ( cf . fig5 b ). it can be noted that no change in bulk mechanical properties of the matrix over time in any of the synthetic matrices is observed over the 5 days , suggesting that the effect seen is independent of bulk changes in mechanical properties and is therefore experienced et the local cell and colony scale ( data not shown ). the effects of ecm proteins on sox1 - gfp expression in this synthetic system were not significant when presented individually , in contrast to expected changes in differentiation profile expected based on ecm - rich natural matrices such as matrigel . finally , cell - cell interaction proteins were found to be strong negative regulators of differentiation , in particular jagged , a ligand activating the notch pathway , was seen as the strongest negative regulator of gfp expression ( cf . fig5 b ) suggesting that the addition of a factor present at the desired cell fate does not necessarily function as the right cue to lead an undifferentiated cell towards that lineage , and in fact may promote the opposite behavior . interactions between factors were seen to contribute of the overall variance of the gfp model , and , based on the mathematical model of all possible category interactions , 5 pair - wise interactions were determined to be statistically significant ( cf . fig5 c ). soluble factors ( sf ) interacted with three other categories , both biophysical ( mp and dg ) as well as biochemical ( ec ) ( cf . fig5 f , g , h ). these sf interaction effects seem to be systematic across the three categories : fgf4 , for example , has an additive effect on all other factors , which could be interpreted simply as a non - linear effect , which is not captured as a main effect in the linear model but whose non - linearity can be captured by such interaction analysis . similarly , the non - degradable condition also exhibits such a non - linear effect , but the ecad condition had a positive influence in the presence of the highly degradable condition ( cf . fig5 d ). similarly , ecad has a more pronounced positive effect in one of the stiffer conditions ( cf . fig5 e ). screened conditions provide a basis for mechanistic insights into neuroepithelial morphology and polarity high - throughput screens such as the one presented here can serve as hypothesis - generating tools for more targeted studies . for example , the putative non - linear effect of fgf4 , identified by factor interaction analysis , was shown to be cell - density mediated : at relatively high cell density ( 3 million cells / ml ), the addition of fgf4 to the medium had little effect , whereas at lower cell density ( 1 million cell / ml ), the lack of fgf4 led to loss of proliferation and gfp intensity ( cf . fig6 a ), underscoring the role of this growth factor as an autocrine regulator of neural differentiation . analysis of the image set from such a screen also outputs additional metrics beyond average colony area as a measure of proliferation and sox1 - gfp expression as a measure of differentiation , with morphological metrics being particularly relevant to relate observed phenotypes to input cell culture microenvironments . by employing a clustering approach to combine multidimensional outputs into clusters of linked morphology , proliferation and differentiation ( not shown ), a phenotypical signature can be constructed which provides a more complete picture of cellular behavior . as such , it was possible to determine that characteristic smooth , round colonies were generated almost exclusively in non - degradable conditions , whereas colonies maintain high gfp expression but with more eccentric and stellate shapes were present in the degradable matrices ( cf . fig6 b ). in further experiments ( not shown ) using broadband mmp inhibitors , it was ascertained that this phenomenon was indeed protease - mediated . thus , by broadening the metrics of interest to include morphological features , it was possible to identify particular microenvironmental conditions inducing different morphologies which may be linked to specific morphogenetic pathways . one of the hallmarks of neural cyst formation is the development of a lumen . confocal 3d reconstructions clearly showed that colonies were indeed well distributed throughout the thickness of the gel , and demonstrated no particular planar bias ( cf . fig6 b ). colonies growing in close proximity in 3d configuration in the non - degradable gels did not fuse but maintained a thin hydrogel boundary between each other ( cf . fig6 b ), suggesting that growth in such matrices was accomplished by outward force against the matrix and not by any remodelling process . most significantly , a measure of apico - basal polarity ( cf . fig6 c ) and the beginnings of possible involution was observed in selected conditions characterized by non - degradable matrix and fgf4 . further experiments revealed that , while in conditions where single ecm factors were present only infrequent cyst polarity was evidenced , when all three ecm components ( laminin , collagen iv and fibronectin ) were all incorporated into the matrix , robust and frequent polarity was established ( cf . fig6 d ). in this high - throughput approach , the response of esc in arrays of 3d cell culture microenvironments yielded new insights into the regulation of neuroepithelial differentiation . as in the esc self - renewal study , soluble factors played a predominant role , particularly in determining proliferation , and had a clear effect in either promoting or impeding differentiation . matrix effects , notably matrix mmp sensitivity , played an equally significant ; role in specifying cell fate . indeed , spherical neuroepithelial colonies were only observed in non - degradable matrices , while in degradable matrices , the extent of differentiation and colony morphology were significantly altered . furthermore , proteins involved in cell - cell interactions impaired neuroepithelial differentiation , while ecm proteins played a significant role in establishing apico - basal polarity only presented in combinatorial manner . a high - throughput platform such as the one deployed here is therefore seen not only as a tool for understanding cell culture microenvironmental influences on developmental pathways , but also as a tremendous hypothesis - generating process which can lead to elucidation of more complex mechanisms . additional embodiments of the invention include the possibility of conducting migration and morphological assays . furthermore , such assays can be performed starting from single cells ( as seen previously ) or from cell aggregates formed in vitro ( e . g . embryoid bodies ) or isolated directly from live tissue . as an example , mesenchymal stem ceils were aggregated to a size of 300 cells , incorporated in combinatorial cell culture microenvironments , and their migration was assessed after 16 hours ( in the presence of pdgf as a soluble factor in the medium ). both matrix - tethered ecm - mimicking peptides ( fibronectin derived rgd motif ), as well as matrix mmp sensitivity modulated the migration response of cells outward from the cluster ( cf . fig 7 a ). a variety of epithelial cell clusters , including for example pancreatic , endometrial , intestinal or mammary , could be incorporated into the screening platform , and assessed for stem cell markers , polarity or other morphological features ( cf . fig7 b ). peg vinylsulfone ( peg - vs ) was produced and characterized as described elsewhere ( lutolf and hubbell , 2003 ). in a second step , peg - vs was functionalized with factor xiiia - peptide substrates via a michael - type addition . a glutamine - containing peptide ( nqeqvspl - ercg - nh 2 ) and different types of lysine - containing peptides with various mmp sensitive sequence were used : acfkgggpqgiwgq - ercg - nh 2 ( peptide w ), acfkgg - gdqgiagf - ercg - nh 2 ( peptide a ), acfkgg - pqgiagq - ercg - nh 2 ( peptide g ), acfkgg - vpmsmrgg - ercg - nh 2 ( peptide v ). consequently , one glutamine - peg precursor ( q - peg ) and four different lysine - peg precursors were obtained : w - peg , a - peg , v - peg , g - peg . functionalization and characterization of these precursors was performed as described elsewhere ( ehrbar et al ., 2007 ). in brief , peptides were added to peg - vs in a 1 . 2 - fold molar excess over vs groups in 0 . 3 m triethanolamine ( ph 8 . 0 ) at 37 ° c . for 2 h , followed by dialysis ( snake skin , mwco 10k , pierce ) against ultrapure water for 4 days at 4 ° c . after dialysis , the salt - free products ( q - peg , w - peg , a - peg , v - peg , g - peg ) were lyophilized to obtain a white powder . factor xiii ( fibrogammin p , csl behring ) was reconstituted in water from lyophilized powder to a concentration of 2000 / ml . 1 ml of factor xiiia was activated with 100 μl of thrombin ( 20 u / ml , sigma - aldrich , switzerland ) for 30 min at 37 ° c . aliquots of activated factor xiiia were stored at − 80 ° c . for further use . precursor solutions to give hydrogels with a final dry mass content ranging from 1 . 5 to 4 % were prepared by stoichiometrically balanced ([ lys ]/[ gln ]= 1 ) solutions of q - peg and each of the four lysine - pegs in tris - buffer ( tbs , 50 mm , ph 7 . 6 ) containing 50 mm calcium chloride . the cross - linking reaction was initiated by 10 u / ml thrombin - activated factor xiiia and vigorous mixing . to obtain disc - shaped matrices , the liquid reaction mixtures ( 50 μl ) were sandwiched between sterile hydrophobic glass microscopy slides ( obtained by treatment with sigmacote , sigma ) separated by spacers ( ca . 1 mm thickness ) and clamped with binder clips . the matrices were then incubated for 30 min . at 37 ° c . three 50 μl gel disks at 3 . 5 % w / v peg were made for each of the 4 peptide sequences and allowed to swell for 12 hours in 50 mm tris , 100 mm nacl , 10 mm cacl 2 buffer at ph 7 . 5 . they were then weighted and placed in a 40 mm mmp - 1 solution , dissolved in the same buffer . the gel mass was recorded at 2 - hour intervals for the first 12 hours , then at t = 18 , 24 , 48 and 72 hrs . the time to complete degradation was determined either directly or by linear regression ( for g peptide ) and inverted to give a measure of degradability . gel disks ( n = 3 for each mmp sensitivity ) were allowed to swell in buffer for 12 hours and small strain oscillatory shear rheometry was then performed . swollen hydrogel discs of 1 to 1 . 4 mm thickness were sandwiched between the two plates of a bohlin cv 120 rheometer ( bohlin instruments ), with compression up to a range between 85 % to 75 % of their original thickness to avoid slipping . measurements were then conducted in constant strain ( 5 %) mode . shear stress was recorded over the frequency range of 0 . 1 to 1 hz and average storage moduli g ′ over the frequency range were obtained . storage modulus ( g ′) was plotted as function of hydrogel % peg w / v for each of the 4 mmp - sensitivities . linear interpolation was performed on the g ′ vs . % peg data points . the % peg corresponding to 0 , 600 , 1200 and 1800 pa was determined for each degradability . to bind cell - cell interaction proteins to the hydrogel network , a fc - tag / protein a conjugation strategy was used . to render protein a susceptible for factor xiiia - catalyzed crosslinking , a q - containing peptide was linked to the protein a using nhs - peg - maleimide , a heterobifunctional peg linker . the modification of protein a was achieved in a two - step reaction : functionalization with maleimide group by reaction of nhs - peg - maleimide in 10 - fold molar excess , followed by q - peptide attachment via its cysteine side chain . consequently , the q - protein a - functionalization was qualitatively assessed by sds - page . a fluorescent counter - reactive substrate for factor xiiia , lysine - containing probe , lys - tamra , was chosen for detecting if a factor xiiia - mediated crosslinking reaction can occur . upon mixing of protein a and lys - tamra in the presence of factor xiiia , a fluorescent signal corresponding to protein a was detected on sds - page and in - gel fluorescence scanning , demonstrating successful bioconjugation . in order to achieve covalent tethering of cell - cell interaction proteins into factor xiiia - based hydrogels , fc - tagged e - cadherin , epcam and jagged ( r & amp ; d systems ) were premixed with q - protein a in a 1 . 66 molar excess ratios for 30 min at room temperature . considering the fact that protein a has five fc - binding sites , we have afforded 3 times molecular excess of each fc - binding sites with respect to a fc - protein , which should ensure optimal immobilization of morphogens . the obtained solution of fully functional proteinaceous constructs were aliquoted and stored at − 20 ° c . until further use . based on the hypothesis that large - sized ecm proteins could be natural substrates for factor xiiia and would tether to the hydrogel network without further conjugation , the following ecm proteins of interest in solution were used : laminin , collagen i ( bd biosciences ), fibronectin ( r & amp ; d systems ). a fluorescent binding assay was performed , in which proteins were mixed with each of fluorescent factor xiiia - substrates ( q - peptide - alexa647 or lys - tamra ) in the presence of factor xiiia . the reactions were qualitatively analyzed by sds - page and in - gel fluorescence scanning , demonstrating that indeed the proteins are susceptible for factor xiiia - based crosslinking . all ecm proteins were aliquoted at 4 ° c . and stored at − 20 ° c . in all esc self - renewal experiments oct4 - gfp mouse embryonic stem cells ( mesc ) ( r1 line provided by zandstra lab ) were routinely cultured on gelatin - coated dishes in medium containing 15 % serum ( hyclone ) and 106 u / ml lif ( millipore ). twelve hours prior to the experiment , the medium was changed to serum - free knock - out medium ( ko ). in all neuroepithelial differentiation experiments sox1 - gfp mouse embryonic stem cells ( 46c cell line provided by tanaka lab ) were routinely cultured in medium containing 15 % serum ( hyclone ) and 10 6 u / ml lif ( millipore ). before the experiment , cells were trypsinized and resuspended in neural differentiation medium devoid of any induction cues . the n2 / b27 formulation was as reported elsewhere ( ying , 2003 ). in all mesenchymal stem cell migration experiments , primary multipotent mesenchymal stem cells from human placenta ( cells used at passage 8 , provided by ehrbar lab ), were routinely cultured at in medium containing 15 % serum ( invitrogen ). aggrewell plates ( stemcell technologies sarl ) were used to generate cell aggregates of 250 cells each , which were then harvested and used in gel encapsulation experiments . to stimulate migration in the assay , 50 ng / ml pdgf ( peprotech ) was added to the basal medium . experiments with mammary epithelial cells were carried out using mcf10a cell line , routinely cultured in growth medium and differentiated as described elsewhere ( debnath , 2003 ). primary epithelial cell aggregates were isolated and differentiated using standard techniques and reagents , as outlined elsewhere ( jin , 2013 ; schatz , 2000 ; li , 2012 ; pancreatic , endometrial , intestinal , respectively ). a viability assay was carried out to compare cell behaviour in 2d and 3d conditions . wild - type mescs were trypsinized and seeded on gelatin - coated tissue culture dish ( 2d ) or encapsulated in a 600 pa non - degradable ( a ) hydrogel disk ( 3d ) at 1m cells / ml . incubation at 37 ° c ., 5 % co 2 was carried out for 4 hours in + lif serum - free conditions , followed by staining with live / dead cell viability assay , following the manufacturer &# 39 ; s instructions . conventional ( 2d ) or confocal ( 3d ) fluorescence imaging was carried out , followed by manual counting of the proportion of live ( green ) vs . dead ( red ) cells was carried out on three independent samples . in order to achieve the combinatorial complexity of this approach a hamilton microlab starplus automatic liquid handling robot with nanopipettor head was used . all automated steps were programmed with microlab vector software version 4 . 1 . 1 ( hamilton eonaduz ag , switzerland ). stock solutions of premixed stoichiometrically balanced peg solutions corresponding to the four peptides were prepared by mixing glutamine - peg precursor ( q - peg ) with the four different lysine - peg precursors : w - peg , a - peg , v - peg , g - peg . each of these four stock solutions was diluted to the rheology - determined four corresponding concentrations required to achieve matched target stiffnesses . the dilution and all subsequent steps of the process were performed robotically , with fluid handling parameters optimized for every material class and checked by mass measurement ort a balance ( data not shown ). importantly , 30 % of the total final volume was left empty ( spare volume ) to account for subsequent addition of proteins , cells and factor xiiia ( 10 % of total volume for each component ). these 16 combinations were aliquoted into 256 wells of a 384 - well plate . ec and cc proteins were thawed on ice , diluted to a concentration of 500 nm , and placed into the wells of a cooled 384 well plate . the four ec proteins ( including blank control ) were dispensed into the 256 gel precursor - filled wells , followed by the four cc proteins , in an orthogonal manner such as to obtain at the end of this step 256 unique combinations of mp , dg , ec and cc ( 4 × 4 × 4 × 4 ). a 96 well plate was prepared with the mediums containing three soluble factors and a blank control : fgf4 , bmp4 at 10 ng / ml ( r & amp ; d systems ) and lif at 106 u / ml ( millipore ). cells were trypsinized and resuspended in a serum - free medium at a concentration of 1 × 106 cells / ml and kept on ice . simultaneously , frozen aliquots of factor xiiia were thawed and also kept on ice . then in a sequential fashion , cells were dispensed into eight wells of the mixed gel precursors , quickly followed by dispensing and robotic mixing of factor xiiia . immediately following the addition of factor xiiia , and , before onset of gelation ( circa 2 - 3 min ), the 8 - channel nanopipettor was used to aspirate 12 . 5 μl from each well and dispense 1 μl in 12 wells of a 1536 well plate . this process was repeated a total of eight times per 1536 plate ( 12 × 8 channels × 8 times × 12 drops /( channel . time )= 768 drops ) to fill half of a 1536 well plate . throughout the process the 1536 well plate was cooled to 4 ° c . to prevent evaporation . at the end of the gel dispensing round , the 4 different mediums in the 96 well plate were dispensed into the 1536 experiment plate . the medium dispense steps were also carried cut sequentially and were synchronized such that all gels were allowed to cross - link for approximately 30 minutes . overall the process from trypsinization to completion of the medium dispense took two hours , and was carried out four times for the entire experiment ( 4 × 4 half - plates of 1536 wells = 3072 wells ). for the neuroepithelial differentiation experiments , in a first array , soluble factor modulation was performed with all other combination except , cell - cell interaction proteins . in a second array , all cell - cell interaction proteins were tested against all other conditions other than soluble factors , which was limited to the fgf4 condition . this soluble factor regime was chosen as it has been reported to be most favorable for neuroepithelial differentiation 2 , particularly in situations of low cell density where autocrine feedback mechanisms are reduced . in this study , in order to identify the role of extrinsic factors independently of autocrine mechanisms , we imposed a relatively sparse initial cell density of 200 cells / μl , with daily medium exchanges . in experiments focusing on mechanical properties , a technique was developed to measure stiffness by indentation in each of the 384 wells ( not shown ). a compression tester ( ta . xtplus texture analyze , stable micro systems ltd ) was fitted with a custom - made 1 . 5 mm diameter indenting tip . force was recorded between 0 and 70 % strain . young &# 39 ; s modulus was calculated from the slope of the curve between 20 and 30 % strain ( elow ), using the equation : e = 2ad / f ( 1 - v 2 ), where a is the radius of the indentor tip cylinder ( 0 . 75 mm ), d is the indentation depth in mm , f is the recorded force in n , and v is poisson &# 39 ; s ratio , taken to be 0 . 5 . gels in the 384 well plate were washed with pbs for 30 minutes , then incubated at ; 37 ° c . with tryple express ( invitrogen ) cell dissociation solution for three cycles of 30 minutes . after each cycle , cells were collected , transferred to a round bottom 96 well plate and kept at 4 ° c . at the end of the process wells were washed with serum - containing medium . in cases where antibody staining was carried out , ssea - 1 - alexafluor647 ( ebiosciences ) was used according to the manufacturer &# 39 ; s instructions . cells were analyzed using an accuri c6 flow cytometer ( bd biosciences ). for pcr , cell dissociation was carried out with tryplee express as above , rna was isolated using tripure isolation reagent ( roche ) according to the manufacturer &# 39 ; s instructions , cdna was synthesized using iscript select cdna synthesis kit ( biorad ) and rt - pcr was carried out with iq sybr green supermix ( biorad ) on a applied biosystems 7500 machine . all imaging was carried out on a bd pathway 435 automated imaging system ( bd biosciences ). imaging was performed on plates with live cells at d1 in the gfp channel . plates were fixed with paraformaldehyde and stained with dapi at d5 , followed by imaging in the gfp and dapi channels . a 4 × objective ( olympus up - lan fln n . a . 0 . 13 ) was used such that an entire well could be captured in a single field of view . even at this low resolution , single cells at d1 could be distinguished . at every xy position , i . e . for every well , six images were captured across a z - stack height of 800 μm . for each well , these six images in each channel were collapsed into a single additive image . the 3d information content of an entire experiment was obtained within less than four hours . all images from the esc self - renewal study were processed using algorithms developed in cellprofiler v . 9777 ( broad institute ). for d1 analysis : collapsed image stacks for each well in the gfp channel were input . images were thresholded and segmented . the number of cells per well was the only readout of interest here . all segmentations were validated by visual inspection . for d5 analysis : collapsed imago stacks for each well in the gfp and dapi channel were input . dapi images were thresholded and segmented . identified colony areas in dapi were used as masks for the gfp images . for each colony , area ( in pixels ), average ( across colony pixels ) dapi intensity and average gfp intensity was recorded . all images from the neuroepithelial differentiation study were processed using algorithms developed in cellprofiler v . 11710 ( broad institute ). in brief , collapsed image stacks for each well in the gfp and dapi channel were input . dapi images were thresholded and segmented using the otsu adaptive segmentation algorithm . colonies were identified as those covering an area above 8 pixels ( i . e . smaller colonies were discarded from further analysis ). identified colony areas in dapi were used as masks for the gfp images . for each colony , shape metrics were obtained from this segmentation process and the dapi and gfp colony intensity metrics for these colonies were obtained from the original , unprocessed images . ail segmentations were validated by visual inspection . matlab r2010b ( mathworks ) and matlab r2010b was used to process and visually explore the data . number of colonies at d1 and d5 , as well as the number of colonies , average colony area ( in pixels ) and average gfp and dapi intensity ( in arbitrary fluorescence units ), all at d5 , were calculated by averaging single colony data for each well ( 3072 data points ) and for each unique condition ( 1024 data points ). gfp intensity was normalized to dapi intensity , and the area was converted from pixels to mm 2 . the data was further centered around the mean and rearranged by input conditions to obtain the heat maps represented in fig2 . data from the individual conditions was input into r v2 . 11 . for colony area and normalized gfp intensity , glm ( generalized linear models ) models , which took into account all possible interaction terms were specified . the step aic procedure was run to obtain optimal models based on the akaike criterion . the glm procedure of sas v9 . 0 software ( sas institute ) was used to test the significance of colony area variation . differences of ls means ± standard errors with the control were tested for significance . the same procedure was used to explain the variability of gfp intensity . the used models considered the effects of mp , dg , cc , ec and sf , as well as interactions determined to be significant . for all parametric tests , normality of the residues and homogeneity of the variance were examined in qq and tukey - 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