Name: stabilizing hepatocellular phenotype using optimized synthetic surfaces
Uploaded: 2014-09-26T14:00:00
Description: Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

D.C.H., M.B. and F.K. were supported by an EPSRC Follow on Fund. B.L-V and D.S. were each supported by MRC PhD studentships. K.C. was supported by funding from the UK Regenerative Medicine Platform.

1. Synthesis of PHNGAD (Poly[1,6-hexanodiol/neopentyl glycol/di(ethylene glycol)-alt-adipic acid]diol)
<img alt="Scheme 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" />
Scheme 1: Synthesis of PHNAGD. Schematic representation of the synthesis of PHNAGD. PHNAGD was prepared by the reaction of 1,6-Hexanodiol, diethylene glycol, neoppentyl glycol and adipic acid. PHNAGD, Poly[1,6-hexanodiol/neopentyl glycol/di(ethylene glycol)-alt-adipic acid]diol.
<ol>
...

<ol>
	<li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMOylation+of+HNF4&#945;+regulates+protein+stability+and+hepatocyte+function.">SUMOylation of HNF4&#945; regulates protein stability and hepatocyte function.</a> J Cell Sci. 125 (15), 3630-3635 (2012).</li><li>Banerjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+hydrogel+modulus+on+the+proliferation+and+differentiation+of+encapsulated+neural+stem+cells.">The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells.</a> Biomaterials. 30 (27), 4695-4699 (2009).</li><li>Shanbhag, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Progenitor+Cells+Grown+on+Hydrogel+Surfaces+Respond+to+the+Product+of+the+Transgene+of+Encapsulated+Genetically+Engineered+Fibroblasts.">Neural Progenitor Cells Grown on Hydrogel Surfaces Respond to the Product of the Transgene of Encapsulated Genetically Engineered Fibroblasts.</a> Biomacromolecules. 11 (11), 2936-2943 (2010).</li><li>Battista, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+matrix+composition+of+3D+constructs+on+embryonic+stem+cell+differentiation.">The effect of matrix composition of 3D constructs on embryonic stem cell differentiation.</a> Biomaterials. 26 (31), 6194-6207 (2005).</li><li>Tian, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+hydrogel+as+Nogo-66+receptor+antibody+delivery+system+for+the+repairing+of+injured+rat+brain:+in+vitro.">Hyaluronic acid hydrogel as Nogo-66 receptor antibody delivery system for the repairing of injured rat brain: in vitro.</a> Journal of Controlled Release. 102 (1), 13-22 (2005).</li><li>Keshaw, H., Forbes, A., Day, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+angiogenic+growth+factors+from+cells+encapsulated+in+alginate+beads+with+bioactive+glass.">Release of angiogenic growth factors from cells encapsulated in alginate beads with bioactive glass.</a> Biomaterials. 26 (19), 4171-4179 (2005).</li><li>Baharvand, H., Hashemi, S. M., Kazemi Ashtiani, S., Farrokhi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+hepatocytes+in+2D+and+3D+culture+systems+in+vitro.">Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro.</a> The International Journal of Developmental Biology. 50 (7), 645-652 (2006).</li><li>Cameron, K., Travers, P., Chander, C., Buckland, T., Campion, C., Noble, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+osteogenic+differentiation+of+human+mesenchymal+stem/precursor+cells+on+silicate+substituted+calcium+phosphate.">Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.</a> Journal of Biomedical Materials Research Part A. 101 (1), 13-22 (2013).</li><li>Pernagallo, S., Unciti-Broceta, A., Diaz-Mochon, J. J., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+cellular+morphology+and+biocompatibility+using+polymer+microarrays.">Deciphering cellular morphology and biocompatibility using polymer microarrays.</a> Biomedical Materials. 3 (3), 034112 (2008).</li><li>Li, Z., Guo, X., Matsushita, S., Guan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+cardiosphere-derived+cells+into+a+mature+cardiac+lineage+using+biodegradable+poly(N-isopropylacrylamide)+hydrogels.">Differentiation of cardiosphere-derived cells into a mature cardiac lineage using biodegradable poly(N-isopropylacrylamide) hydrogels.</a> Biomaterials. 32 (12), 3220-3232 (2011).</li><li>Tare, R. S., Khan, F., Tourniaire, G., Morgan, S. M., Bradley, M., Oreffo, R. O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microarray+approach+to+the+identification+of+polyurethanes+for+the+isolation+of+human+skeletal+progenitor+cells+and+augmentation+of+skeletal+cell+growth.">A microarray approach to the identification of polyurethanes for the isolation of human skeletal progenitor cells and augmentation of skeletal cell growth.</a> Biomaterials. 30 (6), 1045-1055 (2009).</li><li>Khan, F., Tare, R. S., Kanczler, J. M., Oreffo, R. O. C., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cell+manipulation+and+skeletal+tissue+engineering+using+high-throughput+polymer+blend+formulation+and+microarray+techniques.">Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques.</a> Biomaterials. 31 (8), 2216-2228 (2010).</li><li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermoresponsive+and+chemically+defined+hydrogel+for+long-term+culture+of+human+embryonic+stem+cells.">A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells.</a> Nature Communications. 4 (1335), (2013).</li><li>Medine, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+high-fidelity+hepatotoxicity+models+from+pluripotent+stem+cells.">Developing high-fidelity hepatotoxicity models from pluripotent stem cells.</a> Stem Cells Translational Medicine. 2 (7), 505-509 (2013).</li><li>Hay, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+screening+of+polymer+libraries+to+define+novel+substrates+for+functional+hepatocytes+with+inducible+drug+metabolism.">Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism.</a> Stem Cell Research. 6 (2), 92-102 (2011).</li><li>Lucendo-Villarin, B., Khan, F., Pernagallo, S., Bradley, M., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+hepatic+stem+cell+gene+expression+on+biological+and+synthetic+substrata.">Maintaining hepatic stem cell gene expression on biological and synthetic substrata.</a> BioResearch Open Access. 1 (1), 50-53 (2012).</li><li>Hay, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+differentiation+of+hESCs+to+functional+hepatic+endoderm+requires+ActivinA+and+Wnt3a+signaling.">Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling.</a> Proceedings of the National Academy of Sciences. 105 (34), 12301-12306 (2008).</li><li>Szkolnicka, D., Zhou, W., Lucendo-Villarin, B., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+Stem+Cell&#8211;Derived+Hepatocytes:+Potential+and+Challenges+in+Pharmacology.">Pluripotent Stem Cell&#8211;Derived Hepatocytes: Potential and Challenges in Pharmacology.</a> Annu Rev Pharmecol Toxicol. 53, 147-149 (2013).</li><li>Szkolnicka, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+prediction+of+drug-induced+liver+injury+using+stem+cell-derived+populations.">Accurate prediction of drug-induced liver injury using stem cell-derived populations.</a> Stem Cells Translational Medicine. 3 (2), 141-148 (2014).</li><li>Medine, C. N., Lucendo-Villarin, B., Zhou, W., West, C. C., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Generation+of+Hepatocyte-like+Cells+from+Human+Embryonic+Stem+Cell+Populations.">Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations.</a> Journal of Visualized Experiments. (56), (2011).</li><li>Freed, L. E., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+organized+cell+communities.">Culture of organized cell communities.</a> Advanced Drug Delivery Reviews. 33 (1-2), 15-30 (1998).</li><li>Braam, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Vitronectin+Is+a+Functionally+Defined+Substrate+That+Supports+Human+Embryonic+Stem+Cell+Self-Renewal+via+&#945;V&#946;5+Integrin.">Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via &#945;V&#946;5 Integrin.</a> Stem Cells. 26 (9), 2257-2265 (2008).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521/E-cadherin+matrix+in+defined+and+xeno-free+environment.">Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.</a> Nature Communications. 5 (3195), (2014).</li><li>Thaburet, J. -. F. O., Mizomoto, H., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Evaluation+of+the+Wettability+of+Polymer+Libraries.">High-Throughput Evaluation of the Wettability of Polymer Libraries.</a> Macromolecular Rapid Communication. 25 (1), 336-370 (2003).</li><li>Lim, J. Y., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Sensing+and+Response+to+Micro-+and+Nanostructured+Surfaces+Produced+by+Chemical+and+Topographic+Patterning.">Cell Sensing and Response to Micro- and Nanostructured Surfaces Produced by Chemical and Topographic Patterning.</a> Tissue Engineering. 13 (8), 1879-1891 (2007).</li><li>Teixeira, A. I., Abrams, G. A., Bertics, P. J., Murphy, C. J., Nealey, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+contact+guidance+on+well-defined+micro-+and+nanostructured+substrates.">Epithelial contact guidance on well-defined micro- and nanostructured substrates.</a> Journal of Cell Science. 116 (10), 1881-1892 (2003).</li><li>Biggs, M. J. P., Richards, R. G., Wilkinson, C. D. W., Dalby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+interactions+with+topographical+structures:+a+novel+method+for+immuno-SEM+labelling+of+focal+adhesions+in+S-phase+cells.">Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells.</a> Journal of Microscopy. 231 (1), 28-37 (2008).</li><li>Karuri, N. W., Porri, T. J., Albrecht, R. M., Murphy, C. J., Nealey, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-+and+microscale+holes+modulate+cell-substrate+adhesion,+cytoskeletal+organization,+and+-beta1+integrin+localization+in+SV40+human+corneal+epithelial+cells.">Nano- and microscale holes modulate cell-substrate adhesion, cytoskeletal organization, and -beta1 integrin localization in SV40 human corneal epithelial cells.</a> IEEE Transactions on Nanobioscience. 5 (4), 273-280 (2006).</li><li>Hamilton, D. W., Brunette, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+substratum+topography+on+osteoblast+adhesion+mediated+signal+transduction+and+phosphorylation.">The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation.</a> Biomaterials. 28 (1), 1806-1819 (2007).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Dang, J. M., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+Induction+of+Aligned+Mesenchymal+Stem+Cell+Sheets+by+Culture+on+Thermally+Responsive+Electrospun+Nanofibers.">Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers.</a> Advanced Materials. 19 (19), 2775-2779 (2007).</li><li>Azevedo, E. C., Nascimento, E. M., Chierice, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+and+gamma+irradiation+effects+on+surface+properties+of+polyurethane+derivate+from+castor+oil.">UV and gamma irradiation effects on surface properties of polyurethane derivate from castor oil.</a> Pol&#237;meros. 23 (3), 305-311 (2013).</li><li>Rosu, L., Cascaval, C. N., Ciobanu, C., Rosu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV+radiation+on+the+semi-interpenetrating+polymer+networks+based+on+polyurethane+and+epoxy+maleate+of+bisphenol+A.">Effect of UV radiation on the semi-interpenetrating polymer networks based on polyurethane and epoxy maleate of bisphenol A.</a> Journal of Photochemistry and Photobilogy A: Chemistry. 169 (2), 177-185 (2005).</li><li>Yang, X. F., Tallman, D. E., Bierwagen, G. P., Croll, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blistering+and+degradation+of+polyurethane+coatings+under+different+accelerated+weathering+tests.">Blistering and degradation of polyurethane coatings under different accelerated weathering tests.</a> Polymer Degradation and Stability. 77 (1), 103-109 (2002).</li></ol>

Many of the current methods used to generate hepatocytes from stem cells rely on undefined matrices of animal origin. These substrates can be costly and highly variable, affecting cell function and stability, representing a significant barrier to application. Therefore, we performed a screen for synthetic materials which support the culture of stem cell derived hepatocytes. We have identified, a simple polyurethane (PU134), formed by polymerizing PHNGAD, MDI and an extender, that in combination with a robust hepatocyte d...

Biological materials have been widely used in the maintenance and differentiation of pluripotent stem cells 1. While enabling, these biological substrates often contain a myriad of undefined components. Matrigel is a commonly used substrate for stem cell culture and differentiation. Unfortunately, its variable composition influences cell function and phenotype. Although a variety of alternative, more defined biological matrices have been used 2-7, their animal origin or poor scalability makes them unsuitable candidates for industrial manufacture. Therefore the identification of synthetic alternatives, with defined composition and reliable performance, are key goals in stem cell research.
In an attempt to overcome the limitations of undefined cell culture substrates, interdisciplinary collaborations between chemistry and biology have identified synthetic materials with the capacity to support cell phenotype. Synthetic substrates are scalable, cost effective, and can be manufactured into complex 3D structures, mimicking the in vivo environment. Due to these properties synthetic substrates have been widely used to support and drive differentiation of many cell types 8-10.
Advanced and high throughput assays have facilitated the rapid screening of synthetic materials, from large libraries, and delivered novel materials with flexible properties with wide applications in biomedical research and development 11-13. Utilizing high throughput, polymer micro-array screening technology, we rapidly identified a simple polyurethane (PU134), suitable for maintenance of human stem cell derived hepatocytes. This polymer was found to be superior to animal derived substrates with regard to hepatocyte differentiation and function 14-16. We have subsequently optimized the coating conditions, topography and sterilization process to access effects on polymer performance in stabilizing hepatocyte function and lifespan. This has significant implications with regard to understanding fundamentals of hepatocyte biology for cell based modelling and regenerative medicine applications.
The technology here described represents an example of how the surface of a synthetic polymer can be optimized to preserve cell phenotype. We believe that the combination of this technology with an efficient serum free hepatocyte differentiation protocol has the potential to provide a scalable production of hepatocytes for use in in vitro modelling and regenerative medicine.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Polymer solvent influences the topography of the polymer coated surface
Polyurethane 134 was solubilized in chloroform, either alone or in combination with toluene or tetrahydrofuran or dichloromethane and the glass slides spin-coated with the different formulations. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the physical properties of the polymer coatings (Figure 1). The coating obtained using toluene or chl...

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

Research

JoVE Journal

Chemistry

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...