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>
	<li>Apply heat treatment to the monomers 1,6-hexanediol, di(ethylene glycol) and neopentyl glycol at 40 &#176;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 &#176;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 &#176;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="Scheme 2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" />
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&#8242;-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&#8217;4-Methylenebis(phenyl isocyanate) (6.4 mmol) in anhydrous N,N-Dimethylformamide (12 ml).</li>
	<li>Stir the reaction mixture at 70 &#176;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 &#176;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 &#176;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&#160;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 &#956;l 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 &#956;m 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" /> 
	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" /> 
	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 &#8220;L&#8221;. 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&#8217;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 &#176;C.</li>
	<li>Collect supernatants from cells and media, carry out the assay as per manufacturer&#8217;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 &#176;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 &#176;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 &#176;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>

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</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 differentiation approach, stabilizes the hepatocyte phenotype and improves cell function when compared with matrigel 15.
Handling of existing biological matrices, commonly used in culturing and differentiating stem cells such as vitronectin 22, laminin 521 23 or matrigel, is effective for short term maintenance. In contrast, PU134 coated surfaces provide a superior substrate for hepatocyte culture. Moreover, the optimization of surface topography and polymer sterilization have led to further improvements in cell performance, a key factor in delivering human liver models at scale with dependable performance.
The nature of the chain extender and phenyl isocyanate represents one of the critical stages of the process; as elimination or replacement of either component would significantly impair the polymer surface and therefore the cellular attachment and functional activity of stem cell derived hepatocytes. The composition of the polymer affects physical parameters such as elasticity and wettability 24, which has impacts the capacity of the polymer to absorb key extracellular matrix proteins involved in hepatocyte function. In addition, variations in the catalyst, reaction time and temperature affect the molecular weight distribution of the polymer.
The selection of the solvent to solubilize the polymer represents another critical point in the obtaining of an optimized polymer coated surface. We have observed that the nature of the solvent affects the topography of the coated surface, which has direct implications in the function of the cells 25-31. In addition, the spinning time and speed are also crucial in obtaining this uniform and homogeneous coating. Another step to take in consideration is the sterilization procedure applied on the polymer coated surface, as the activity of the cells can be influenced by the sterilization treatment used. This may be due to the presence of sensitive area(s) within the structure of the polymer to different sterilization procedures 32-34.
Current pluripotent stem cell based cell models possess phenotypic and functional limitations. We believe that this optimized polymer coated surface represents an advance in the field, circumventing some of the limitations associated with the use of biological substrates. Furthermore, the possibility of using the polymer within 3D matrices to support the attachment of different cellular types found in the tissue of interest, may further improve cell fidelity.
In conclusion, the use of synthetic materials is becoming increasingly important in the delivery of human soma from pluripotent stem cells. We have developed an optimized surface and coating procedure that is robust and reliably delivers functional human hepatocytes with significant implications for cell based modeling and regenerative medicine.

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

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 chloroform was not homogeneous, demonstrating an uneven coating with PU134 precipitation (Figure 1A). In contrast, tetrahydrofuran was a suitable solvent allowing the spin coating of a much more uniform surface (Figure 1A). Surface topography was measured by AFM using the root mean square (RMS) and the mean roughness (Ra). PU134 dissolved tetrahydrofuran exhibited a 40% reduction in the roughness in comparison to the other solvents (Figure 1B and 1C). These data demonstrate that the use of tetrahydrofuran as a solvent provides a more uniform coating of PU134 for biological application.
Polymer topography has a significant impact on hepatocyte function
hESC can be efficiently differentiated to hepatic endoderm in vitro, exhibiting many of the functions found in the adult liver 17-20. Hepatic endoderm differentiation was induced and at Day 9 hepatoblast-like cells were apparent, with the majority of cells expressing cytokeratin 19 (99% &#177; 1.2), &#945;-fetoprotein (99% &#177; 0.6), and hepatic nuclear factor 4&#945; (97% &#177; 2.6); and low levels of albumin (20% &#177; 1.7) (Figure 2). At this point the cells were detached using TrypLE express and replated onto the different polymer coated surfaces. As a measure of metabolic function, cytochrome P450 function was assessed 4 days post-replating. We observed a 2-fold increase in metabolic activity in cells replated on tetrahydrofuran/PU134 coated surfaces (Figure 3). These results demonstrate that hESC derived hepatocyte metabolic activity is improved with a more uniform coating of PU134, this is consistent with previous approaches 21.
Surface sterilization impacts on the bioactive properties of the polymer
The effect of sterilization on PU134 performance was also investigated. Polymer coated glass slides were exposed to UV or gamma irradiation. Phase contrast imaging of PU134 coated glass slides showed no gross differences post UV or gamma irradiation (Figure 4A). These observations were further confirmed by SEM analysis (Figure 4B). The biological performance of PU134 was examined using hESC derived hepatocytes at 10 days post-replating. hESC derived hepatocytes replated on &#947;-radiated PU134 coated glass slides displayed a 3-fold increase in CYP3A activity over cells replated on UV-radiated PU134 coated glass slides (Figure 4C). These observations demonstrated that gamma-irradiation was the optimal sterilization technique for our purposes.
<img alt="Figure 1" src="/files/ftp_upload/51723/51723fig1highres.jpg" /> 
Figure 1. Optimization of the polyurethane 134 coated surface. (A) Glass slides were coated with a 2% solution of PU134 dissolved in different solvents. Scanning Electron Microscopy (SEM) images of the different coated glass slides show the presence of a homogeneous coated surface and the absence of polymer precipitates when tetrahydrofuran was used compared with toluene, chloroform or mixtures of the solvents (black and white arrows). The images were taken at x1664 magnification and scales bars are shown below the images. (B-C) Atomic Force Microscopy (AFM) was employed to analyze the roughness of the PU134 coated surfaces on selected solvents. Analysis of the roughness parameters RMS (the root mean square) (B) and Ra (the mean roughness) (C) of the different PU134 coated surfaces show that the coating obtained by using tetrahydrofuran as a solvent had the smoothest surface when compared with the rest of the conditions (n=7). Data are expressed as mean &#177; s.d., p&#60;0.05 is denoted *, p&#60;0.01 is denoted **, and p &#60;0.001 is denoted ***, measured by students t-test in comparison to slides coated with PU134 solubilized in tetrahydrofuran. Error bars represent 1 standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51723/51723fig2highres.jpg" /> 
Figure 2. Characterisation of the hESC derived hepatoblasts. Immunocytochemistry showing expression of hepatic markers AFP, CK19, HNF4&#945; and Albumin in hESC (H9) derived hepatic endoderm. Negative controls were performed with corresponding immunoglobulin G (IgG) and representative images are shown. The percentage of positive cells and standard deviation were estimated from at least five random fields of view containing at least 300 cells per view. The images were taken at 20X magnification. Data is expressed as mean &#177; s.d. Error bars represent 1 standard deviation. HNF4&#945;, Hepatocyte nuclear factor 4&#945;; AFP, &#945;-fetoprotein; ALB, Albumin; CK19, Cytokeratin 19, IgG goat, Immunoglobulin G anti-goat, IgG mouse, Immunoglobulin G anti-mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51723/51723fig3highres.jpg" /> 
Figure 3. Functional implications of the topography of the PU134 coated surface on the function of hESC derived hepatocytes. Measurement of the Cytochrome P450 3A activity on hESC-derived hepatocytes maintained for 96 hr on glass slides coated with PU134 dissolved in different solvents. An improvement in the in the cytochrome activity on cells maintained on PU134 coated glass slides solubilized with tetrahydrofuran (n=6). Data are expressed as mean &#177; s.d., p&#60;0.05 is denoted *, p&#60;0.01 is denoted **, and p &#60;0.001 is denoted ***, measured by students t-test in comparison to hESC derived hepatocytes maintained on slides coated with PU134 solubilized on tetrahydrofuran. Error bars represent 1 standard deviation.
<img alt="Figure 4" src="/files/ftp_upload/51723/51723fig4highres.jpg" /> 
Figure 4. Optimizing the sterilization of the coated surfaces. Phase contrast images (A) or SEM images (B) show no gross differences in the PU134 coated surface between &#947;-radiation or UV radiation (UV) treatments. Phase contrast images were taken at 40X magnification and SEM images at 1,664X magnification. (C) Analysis of the cytochrome P450 3A activity on hESC derived hepatocytes maintained for 10 days on PU134 coated glass slides. An increase in the cytochrome P450 3A activity in cells replated on &#947;-radiated PU134 coated glass slides when compared with cells replated on UV radiated PU134 coated glass slides. Units of activity are expressed as relative light units (RLU) ml&#8722;1 cm&#8722;2 of culture surface (n=3). Data are expressed as mean &#177; s.d., p&#60;0.001 is denoted *** measured by students t-test in comparison to hESC derived hepatocytes maintained on PU134 coated glass coverslips sterilized by &#947;-radiation. Error bars represent 1 standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51723/51723fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen</td>
			<td>Type</td>
			<td>Company</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>HNF4&#945;</td>
			<td>Rabbit polyclonal</td>
			<td>Santa Cruz</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Mouse monoclonal</td>
			<td>Sigma</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>AFP</td>
			<td>Mouse monoclonal</td>
			<td>Abcam</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Cytokeratin 19</td>
			<td>Mouse monoclonal</td>
			<td>Dako</td>
			<td>1/50</td>
		</tr>
		<tr>
			<td>IgG</td>
			<td>Mouse monoclonal</td>
			<td>Dako</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit 568 Alexa Flour</td>
			<td>Goat</td>
			<td>Invitrogen</td>
			<td>1/400</td>
		</tr>
		<tr>
			<td>Anti-mouse 488 Alexa Flour</td>
			<td>Rabbit</td>
			<td>Invitrogen</td>
			<td>1/500</td>
		</tr>
	</tbody>
</table>
Table 1. List of optimized primary and secondary antibodies and concentrations used in this study. HNF4&#945;, Hepatocyte nuclear factor 4&#945;; AFP, &#945;-fetoprotein; IgG, Immunoglobulin G.

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 ...

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.2K Views.  University of Edinburgh.  Dit artikel zal zich richten op het ontwikkelen van polymeer gecoate oppervlakken voor de lange termijn, stabiele cultuur van stamcellen verkregen menselijke levercellen. 

Video: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Developmental Biology

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

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Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

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Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

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Biology

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

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Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

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Medicine

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

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Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

A Physiologically-Relevant All-Human 2D+ Hepatic System for Drug Discovery

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Author Spotlight: Developing a Simple and Robust Hepatic Model for Pharmacological and Toxicological Applications 

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

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