Name: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces (Video) | JoVE Journal
Uploaded: 2014-09-26T14:00:00.000+00:00
Duration: 8 min 50 s
Description: Discover how to optimize polymer coated surfaces for long-term culture of stem cell derived human hepatocytes using polyurethane synthesis and spin coating techniques. This method improves cell adhesion and metabolic function.

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5 minutes

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="Polyester synthesis diagram; includes chemical structures, reaction conditions, catalyst usage." fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Scheme 1">
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>
	<li>Apply heat treatment to the monomers 1,6-hexanediol, di(ethylene glycol) and neopentyl glycol at 40 °C for 48 hr in a vacuum oven to remove any residual water. Allow to cold down to RT under vacuum.</li>
	<li>Add 22 mmol of each monomer and adipic acid (55 mmol) to a two-necked round bottom flask equipped with a stirred bar and connected to a Dean-Stark apparatus.</li>
	<li>Place the whole assembly under a vacuum and gently heat the glassware at 40 °C for 6 hr, in order to avoid any moisture absorption during the addition of the chemical into the flask.</li>
	<li>Add via syringe, drop by drop, 0.0055 mol of the catalyst titanium (IV) butoxide.</li>
	<li>Stir the reaction mixture at 180 °C, under an N2 atmosphere for 24 hr, and collect residual water in the Dean-Stark trap. Allow the product to cool to RT.</li>
</ol>
2. Synthesis of PU134

<img alt="Polyurethane synthesis diagram; chemical reaction process with catalyst and chain extender details." fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Scheme 2">
Scheme 2: Synthesis of PU134. Schematic representation of the synthesis of polyurethane 134. PU134 was prepared by the reaction of 1.0 equiv of a PHNGAD with 2.0 equiv of a 4,4′-Methylenebis(phenyl isocyanate), followed by the addition of 1.0 equiv of a 1,4-butanediol chain extender.

<ol>
	<li>Mix one equivalent of the polyol PHNGAD (Mn ~1,800 Da, 3.2 mmol) with two equivalents of 4’4-Methylenebis(phenyl isocyanate) (6.4 mmol) in anhydrous N,N-Dimethylformamide (12 ml).</li>
	<li>Stir the reaction mixture at 70 °C, under an N2 atmosphere.</li>
	<li>Add via syringe, drop by drop the catalyst titanium (IV) butoxide (0.8% wt).</li>
	<li>After 1 hr, add one equivalent of the chain extender 1,4-butanediol (3.2 mmol). Increase the temperature at 90 °C and stir for 24 hr under an N2 atmosphere.</li>
	<li>Following the reaction, collect the polyurethane by precipitation by adding diethyl ether (hexane or water could also be used) drop wise into the reaction solution until the precipitation occurs.</li>
	<li>Centrifuge the solution at 5,300 x g for 5 min.</li>
	<li>Decant the supernatant and dry off at 40 °C in a vacuum oven until the solvent evaporates. 
	Note: In order to ensure that the final product of the reaction possess the correct parameters regarding to the molecular weight distribution, the functional groups of the polymer, and the melting and glass transition temperatures, various analytical techniques and methods can be used, such as gel permeation chromatography or FITR spectroscopy.</li>
</ol>
3. Preparation of PU134 Solutions
<ol>
	<li>Weigh 200 mg of PU134 into a glass bottle.</li>
	<li>Dilute PU134 to a final concentration of 2% in a number of solvents: chloroform, a combination of chloroform and toluene in 1:1 ratio, tetrahydrofuran, and combination of tetrahydrofuran and dicloromethane in 1:1 ratio. 
	Note: The election of the right solvent varies depending on the polymer to use. Different solvents possess different boiling points that can affect the solubilization of the polymer.</li>
	<li>Shake the solution vigorously for 20 min at RT using a shaker at a speed of 200 mot/min, until the solution becomes homogeneous and no precipitate is observed.</li>
</ol>
4. Coating of Glass Slides with PU134
<ol>
	<li>Place a round 15 mm2 coverslip on the spin coater.</li>
	<li>Apply 50 μl of the PU134 solution to each using a pipette. Adjust the volume of the PU134 solution accordingly for the required coverslip size keeping the volume to surface ratio proportional.</li>
	<li>Spin each coverslip for 7 sec at 23 x g.</li>
	<li>Air dry coverslip at RT for at least 24 hr before sterilization.</li>
</ol>
5. Irradiation of Coverslip
<ol>
	<li>Gamma-irradiate polymer coated coverslip by applying a dose of 10 Grays using a laboratory irradiator for 12 min.</li>
	<li>UV- irradiate polymer coated coverslip using a 30 W, UV bulb for 16 min each side.</li>
	<li>Place the polymer coated coverslip in a suitable tissue culture plate according to the slide size.</li>
</ol>
6. Scanning Electron Microscopy
<ol>
	<li>Gold coat polymer coverslip by sputtering for 200 sec in an atmosphere of 5 x 10-1 millibars of pressure.</li>
	<li>Capture the micrographs of the polymer coated coverslip using a scanning electron microscope at an accelerating voltage of 20 kV in secondary electron imaging mode.</li>
</ol>
7. Atomic Force Microscopy Observations
<ol>
	<li>Scan an area of 20 x 20 μm of the polymer surface.</li>
	<li>Set up a scan rate from 1.32 Hz to 1.60 Hz.</li>
	<li>Set up a resolution of 512 x 512 pixels in the scanned region.</li>
	<li>Calculate the root mean square (RMS or Rq) of the coating by using the average of height deviations taken from the mean image data plane, expressed as: 
	<img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" alt="Surface roughness, Rq equation formula, showing root mean square height calculation." data-altupdatedat="1787630408000"> 
	Where Zi is the current Z value, and N is the number of points within the given area.</li>
	<li>7.5 Calculate the deviation or mean surface roughness (Ra) of the image using, 
	<img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" alt="Equation for arithmetic average roughness, Ra=∫|Z(x)|dx, formula used in surface analysis." data-altupdatedat="1787630408000"> 
	Where Z (x) is the function that describes the surface profile analyzed in terms of height (Z) and position (x) of the sample over the evaluation length “L”. Ra represents the mean value of the surface relative to the center plane.</li>
</ol>
8. Cell Culture and Differentiation
<ol>
	<li>Culture and differentiate the human embryonic stem cell line (hESC) H9 as described in Hay 17.</li>
	<li>Detach cells using a dissociation reagent and replate them onto PU134 coated slides at Day 9 of the differentiation process, in the presence of a serum free medium as describe in Szkolnicka 18,19. 
	Note: The use of an enzymatic cell dissociation is preferred to physical cell detachment.</li>
</ol>
9. Cytochrome p450 Functional Assay
<ol>
	<li>Measure CYP3A activity following the manufacturer’s instructions.</li>
	<li>Incubate hESC derived hepatocytes at Day 13 or Day 19, and media without cells - as a negative control, with the appropriate substrate for 5 hr at 37 °C.</li>
	<li>Collect supernatants from cells and media, carry out the assay as per manufacturer’s instructions.</li>
	<li>Measure the level of CYP activity and normalized relative to the surface area (cm2).</li>
</ol>
10. Immunostaining
<ol>
	<li>Wash hESC derived hepatocytes with PBS, for 1 min, repeat two times.</li>
	<li>Add ice cold 100% methanol to fix cells, place in -20 °C for 10 min.</li>
	<li>Wash cells with PBS for 5 min and repeat twice.</li>
	<li>Incubate cells with PBS/T (0.1% tween)/10% BSA for 1 hr at RT.</li>
	<li>Aspirate the PBST solution and add the appropriate primary antibody diluted in PBS/T (0.1% tween)/1% BSA, incubate at 4 °C with gentle agitation O/N.</li>
	<li>Wash cells with PBS/T (0.1% tween)/1% BSA for 5 min and repeat three times.</li>
	<li>Dilute the appropriate antibody in PBS/T (0.1% tween)/1% BSA, add to the cells and incubate in the dark at RT for 1 hr with gentle agitation.</li>
	<li>Wash cells with PBS for 5 min and repeat three times.</li>
	<li>Add MOWIOL 488 (containing DAPI 1:1,000) to each well, add a coverslip gently to reduce number of air bubbles. Store fixed cells at 4 °C in the dark. Observe staining using a microscope with appropriate filter and fluorescent lamp. The optimized primary and secondary antibodies are listed in Table 1.</li>
</ol>

<ol><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMOylation+of+HNF4%CE%B1+regulates+protein+stability+and+hepatocyte+function%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">SUMOylation of HNF4α 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/pubmed?term=((The+influence+of+hydrogel+modulus+on+the+proliferation+and+differentiation+of+encapsulated+neural+stem+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Neural+Progenitor+Cells+Grown+on+Hydrogel+Surfaces+Respond+to+the+Product+of+the+Transgene+of+Encapsulated+Genetically+Engineered+Fibroblasts%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+matrix+composition+of+3D+constructs+on+embryonic+stem+cell+differentiation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Hyaluronic+acid+hydrogel+as+Nogo-66+receptor+antibody+delivery+system+for+the+repairing+of+injured+rat+brain%3A+in+vitro%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">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/pubmed?term=((Release+of+angiogenic+growth+factors+from+cells+encapsulated+in+alginate+beads+with+bioactive+glass%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Differentiation+of+human+embryonic+stem+cells+into+hepatocytes+in+2D+and+3D+culture+systems+in+vitro%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Directed+osteogenic+differentiation+of+human+mesenchymal+stem%2Fprecursor+cells+on+silicate+substituted+calcium+phosphate%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">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/pubmed?term=((Deciphering+cellular+morphology+and+biocompatibility+using+polymer+microarrays%5BTitle%5D)+AND+%22Biomedical+Materials%22%5BJournal%5D)">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/pubmed?term=((Differentiation+of+cardiosphere-derived+cells+into+a+mature+cardiac+lineage+using+biodegradable+poly%28N-isopropylacrylamide%29+hydrogels%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((A+microarray+approach+to+the+identification+of+polyurethanes+for+the+isolation+of+human+skeletal+progenitor+cells+and+augmentation+of+skeletal+cell+growth%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Strategies+for+cell+manipulation+and+skeletal+tissue+engineering+using+high-throughput+polymer+blend+formulation+and+microarray+techniques%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((A+thermoresponsive+and+chemically+defined+hydrogel+for+long-term+culture+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Developing+high-fidelity+hepatotoxicity+models+from+pluripotent+stem+cells%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((Unbiased+screening+of+polymer+libraries+to+define+novel+substrates+for+functional+hepatocytes+with+inducible+drug+metabolism%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">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/pubmed?term=((Maintaining+hepatic+stem+cell+gene+expression+on+biological+and+synthetic+substrata%5BTitle%5D)+AND+%22BioResearch+Open+Access%22%5BJournal%5D)">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/pubmed?term=((Highly+efficient+differentiation+of+hESCs+to+functional+hepatic+endoderm+requires+ActivinA+and+Wnt3a+signaling%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">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/pubmed?term=((Pluripotent+Stem+Cell%E2%80%93Derived+Hepatocytes%3A+Potential+and+Challenges+in+Pharmacology%5BTitle%5D)+AND+%22Annu+Rev+Pharmecol+Toxicol%22%5BJournal%5D)">Pluripotent Stem Cell–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/pubmed?term=((Accurate+prediction+of+drug-induced+liver+injury+using+stem+cell-derived+populations%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((Robust+Generation+of+Hepatocyte-like+Cells+from+Human+Embryonic+Stem+Cell+Populations%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Culture+of+organized+cell+communities%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">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/pubmed?term=((Recombinant+Vitronectin+Is+a+Functionally+Defined+Substrate+That+Supports+Human+Embryonic+Stem+Cell+Self-Renewal+via+%CE%B1V%CE%B25+Integrin%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via αVβ5 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/pubmed?term=((Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521%2FE-cadherin+matrix+in+defined+and+xeno-free+environment%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((High-Throughput+Evaluation+of+the+Wettability+of+Polymer+Libraries%5BTitle%5D)+AND+%22Macromolecular+Rapid+Communication%22%5BJournal%5D)">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/pubmed?term=((Cell+Sensing+and+Response+to+Micro-+and+Nanostructured+Surfaces+Produced+by+Chemical+and+Topographic+Patterning%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">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/pubmed?term=((Epithelial+contact+guidance+on+well-defined+micro-+and+nanostructured+substrates%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">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/pubmed?term=((Focal+adhesion+interactions+with+topographical+structures%3A+a+novel+method+for+immuno-SEM+labelling+of+focal+adhesions+in+S-phase+cells%5BTitle%5D)+AND+%22Journal+of+Microscopy%22%5BJournal%5D)">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/pubmed?term=((Nano-+and+microscale+holes+modulate+cell-substrate+adhesion%2C+cytoskeletal+organization%2C+and+-beta1+integrin+localization+in+SV40+human+corneal+epithelial+cells%5BTitle%5D)+AND+%22IEEE+Transactions+on+Nanobioscience%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+substratum+topography+on+osteoblast+adhesion+mediated+signal+transduction+and+phosphorylation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Matrix+elasticity+directs+stem+cell+lineage+specification%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Myogenic+Induction+of+Aligned+Mesenchymal+Stem+Cell+Sheets+by+Culture+on+Thermally+Responsive+Electrospun+Nanofibers%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">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/pubmed?term=((UV+and+gamma+irradiation+effects+on+surface+properties+of+polyurethane+derivate+from+castor+oil%5BTitle%5D)+AND+%22Pol%C3%ADmeros%22%5BJournal%5D)">UV and gamma irradiation effects on surface properties of polyurethane derivate from castor oil.</a> Polímeros. 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/pubmed?term=((Effect+of+UV+radiation+on+the+semi-interpenetrating+polymer+networks+based+on+polyurethane+and+epoxy+maleate+of+bisphenol+A%5BTitle%5D)+AND+%22Journal+of+Photochemistry+and+Photobilogy+A%3A+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Blistering+and+degradation+of+polyurethane+coatings+under+different+accelerated+weathering+tests%5BTitle%5D)+AND+%22Polymer+Degradation+and+Stability%22%5BJournal%5D)">Blistering and degradation of polyurethane coatings under different accelerated weathering tests.</a> Polymer Degradation and Stability. 77 (1), 103-109 (2002).</li>
</ol>

D.C.H. is CSO, Director, founder and a shareholder in FibromEd Products Ltd. M.B. and J.P.I. are founder shareholders in FibromEd Products Ltd.

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><tbody><tr><td colspan="4">&lt;strong&gt;Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips&lt;/strong&gt;</td></tr><tr><td>Shaker</td><td>Edmun B&amp;uuml;hler</td><td>KS-15</td><td></td></tr><tr><td>Irradiator</td><td>CIS Biointernational</td><td>IBL 637&amp;nbsp;</td><td></td></tr><tr><td>Spin coater</td><td>Specialty Coating System</td><td>P-6708</td><td></td></tr><tr><td>Scanning Electron Microscope&amp;nbsp;</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&amp;nbsp;</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&amp;ndash;Aldrich</td><td>93171</td><td></td></tr><tr><td>1,6-hexanediol</td><td>Sigma&amp;ndash;Aldrich</td><td>240117</td><td></td></tr><tr><td>Neopentyl glycol</td><td>Sigma&amp;ndash;Aldrich</td><td>408255</td><td></td></tr><tr><td>Adipic acid</td><td>Sigma&amp;ndash;Aldrich</td><td>9582</td><td></td></tr><tr><td>anhydrous &lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma&amp;ndash;Aldrich</td><td>227056</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma&amp;ndash;Aldrich</td><td>676845</td><td></td></tr><tr><td>titanium (IV) butoxide&amp;nbsp;</td><td>Sigma&amp;ndash;Aldrich</td><td>244112</td><td></td></tr><tr><td>1,4-butanediol&amp;nbsp;</td><td>Sigma&amp;ndash;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&amp;rsquo;-Methylenebis(phenyl isocyanate)</td><td>Sigma&amp;ndash;Aldrich</td><td>101688</td><td></td></tr><tr><td>Tetrahydrofurane</td><td>Sigma&amp;ndash;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 colspan="4">&lt;strong&gt;Inmunostaining&lt;/strong&gt;</td></tr><tr><td>Phosphate buffer saline (-MgCl&lt;sub&gt;2&lt;/sub&gt;, -CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Gibco</td><td>10010031</td><td>Store at room temperature</td></tr><tr><td>PBST, PBS made up with 0.1% TWEEN 20&amp;nbsp;</td><td>&amp;nbsp; Scientific Laboratory Supplies Ltd</td><td>EC607&amp;nbsp;</td><td></td></tr><tr><td>Methanol</td><td>&amp;nbsp; 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 colspan="4">&lt;strong&gt;Cell culture and Functional assay&lt;/strong&gt;</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>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/12604013&quot;&gt;12604013&lt;/a&gt;</td><td></td></tr></tbody></table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

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...

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 ...

The overall goal of this procedure is to optimize polymer coated surfaces to preserve the long-term stable culture of stem cell derived human hepatocytes. This is accomplished by first synthesizing polyurethane 1 3 4 or PU 1 3 4. The second step is to choose the right solvent to solubilize PU 1 34.Next, the spin coating process with the PU 1 3 4 on the glass cover slip is optimized. The final step is optimization of the sterilization procedure of the polymer P 1 34 coated glass cover slips ultimately scanning electron microscopy, atomic force microscopy and drug metabolism. Assays are used to show the influence of the PU 1 34 polymer coating in the function of stem cell derived hepatocytes.The main advantage of this technique over existing methods like the use of al Metrices in culture in maintaining URI potent stem cells that have hepatocytes, is that these synthetic materials can be fine tuned for purpose and a s scale to quality assured standards. In addition, they display batch two bus consistency. The implications of this technique extend toward therapy of liver diseases because it represents an example of how the surface of a synthetic polymer can be optimized to preserve cell phenotype.Demonstrating the procedure will be Dr.Jeffrey Walton, a postdoc from my laboratory Prior to starting this procedure. Apply heat treatment to the mono's one six hexane dial di ethylene glycol and neo PenTile glycol at 40 degrees Celsius for 48 hours in a vacuum oven. To remove any residual water, add 22 millimoles of each monomer and 55 millimoles of a dpic acid to a two necked round bottom flask connected to a dean stark apparatus and equipped with a magnetic stir bar.Following this place the whole assembly under a vacuum and gently heat the glassware at 40 degrees Celsius for six hours in order to avoid any moisture absorption during the addition of the chemical to the flask. After removing the assembly from the oven and placing the system under nitrogen, add 0.0055 moles of the catalyst titanium four, but oxide dropwise by a syringe to the flask. Stir the reaction mixture at 180 degrees Celsius for 24 hours, collecting residual water in the dean stark trap.Allow the product to cool to room temperature. Next mix 3.2 millimole, or one equivalent of the polyol P-H-N-G-A-G with 6.4 milli mole or two equivalents of four four methyl and a bisphenol isocyanate. In 12 milliliters of anhydrous DML to a two necked round bottom flask connected to a dean stark apparatus and equipped with a magnetic star bar.Stir the reaction mixture at 70 degrees Celsius under a nitrogen atmosphere. Then add the catalyst titanium four oxide dropwise via syringe to a final concentration of 0.8%After one hour, add one equivalent of the chain extender one four butane dial to give copolymer product. Increase the temperature to 90 degrees Celsius and starve for 24 hours under a nitrogen atmosphere.Once the reaction mixture has called to room temperature a dal ether dropwise to the reaction mixture until precipitation of the polyurethane product is observed. After transferring the reaction mixture to a centrifuge tube centrifuge the solution at 5, 300 times G for five minutes. After decanting, the supinate dry off at room temperature until the solvent evaporates.Operates next weigh 200 milligrams of PU 1 34 into a glass bottle. Dilute the sample to a final concentration of 2%in chloroform or chloroform and toluene in a one-to-one ratio or tetra roran or a combination of tetra roran anti chloro methane. In a one-to-one ratio shake the solution vigorously for 20 minutes at room temperature using a shaker at a speed of 200 motions per minute until the solution becomes homogeneous and no precipitate is observed.When finished, place around 15 millimeter square glass cover slip on the spin coter. Apply 50 microliters of the PU one through four solution to the cover slip using a pipette spin each cover slip for seven seconds at 23 times G.Then air dry the cover slip at room temperature for at least 24 hours before sterilization. At this point, gamma irradiate each polymer coated cover slip by applying a dose of 10 grays using a laboratory radiator for 12 minutes.Alternatively, UV irradiate each polymer coated cover slip using a 30 watt UV bulb for 16 minutes on each side. When finished, place the polymer coated cover slip in a suitable tissue culture plate according to the cover slip size. After culturing and differentiating the human embryonic stem cell line detached cells using dissociation reagent and replay them onto the PU 1 34 coated cover slips at day nine of the differentiation process in the presence of serum free medium scanning electron microscopy.Images of the different coated glass cover slips show that the coating with toluene or chloroform was not homogenous, demonstrating an uneven coating with PE 1 34 precipitation. In contrast, tetrahedran allowed the spin coating of a much more uniform surface. Additionally, atomic force microscopy analysis of the different polymer coatings shows a 40%reduction in the roughness of P 1 34 solubilized in tetra hydro furin in comparison to other solvents demonstrating a more uniform PU 1 34 coating a biological application hepatic endoderm differentiation was induced and at day nine, hepato blasts like cells were apparent with the majority of cells expressing cytokeratin 19 alpha feta protein and hepatocyte nuclear factor for alpha and low levels of albumin as a measure of metabolic function.Cytochrome P four 50 function was assessed four days post replating onto different polymer coated surfaces. A twofold increase in metabolic activity in cells replated on tetra hydro fure mpu 1 3 4 coated surfaces was observed. These results demonstrate that human embryonic stem cell derived hepatocyte metabolic activity is improved with a more uniform P 1 34 coating face Contrast imaging showed no gross differences post UV or gamma irradiation, which was further confirmed by scanning electron microscopy analysis.Human embryonic stem cell derived hepatocytes replated on gamma radiated PU 1 34 coated glass cover slips displayed a threefold increase in CYP three a activity over cells replated on UV radiated PU 1 34 coated glass cover slips. These observations demonstrate the gamma radiation is the optimal sterilization technique. While attempting this procedure, it's important to remember to check the homogeneity of the coating between different spin coating CLOs, And don't forget that working with chemical solvents instrumentation can be extremely dangerous.Applications such as wearing a safety goggles should always be taken while performing this procedure.

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Research

JoVE Journal

Chemistry

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Discover how to optimize polymer coated surfaces for long-term culture of stem cell derived human hepatocytes using polyurethane synthesis and spin coating techniques. This method improves cell adhesion and metabolic function.

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

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