Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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.

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Protokół

1. Synthesis of PHNGAD (Poly[1,6-hexanodiol/neopentyl glycol/di(ethylene glycol)-alt-adipic acid]diol)

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.

1. 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.
2. 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.
3. 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.
4. Add via syringe, drop by drop, 0.0055 mol of the catalyst titanium (IV) butoxide.
5. 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.

2. Synthesis of PU134

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.

1. 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).
2. Stir the reaction mixture at 70 °C, under an N₂ atmosphere.
3. Add via syringe, drop by drop the catalyst titanium (IV) butoxide (0.8% wt).
4. 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.
5. 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.
6. Centrifuge the solution at 5,300 x g for 5 min.
7. 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.

3. Preparation of PU134 Solutions

1. Weigh 200 mg of PU134 into a glass bottle.
2. 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.
3. 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.

4. Coating of Glass Slides with PU134

1. Place a round 15 mm² coverslip on the spin coater.
2. 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.
3. Spin each coverslip for 7 sec at 23 x g.
4. Air dry coverslip at RT for at least 24 hr before sterilization.

5. Irradiation of Coverslip

1. Gamma-irradiate polymer coated coverslip by applying a dose of 10 Grays using a laboratory irradiator for 12 min.
2. UV- irradiate polymer coated coverslip using a 30 W, UV bulb for 16 min each side.
3. Place the polymer coated coverslip in a suitable tissue culture plate according to the slide size.

6. Scanning Electron Microscopy

1. Gold coat polymer coverslip by sputtering for 200 sec in an atmosphere of 5 x 10^-1 millibars of pressure.
2. 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.

7. Atomic Force Microscopy Observations

1. Scan an area of 20 x 20 μm of the polymer surface.
2. Set up a scan rate from 1.32 Hz to 1.60 Hz.
3. Set up a resolution of 512 x 512 pixels in the scanned region.
4. 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:
   
   Where Zi is the current Z value, and N is the number of points within the given area.
5. 7.5 Calculate the deviation or mean surface roughness (Ra) of the image using,
   
   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.

8. Cell Culture and Differentiation

1. Culture and differentiate the human embryonic stem cell line (hESC) H9 as described in Hay ¹⁷.
2. 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.

9. Cytochrome p450 Functional Assay

1. Measure CYP3A activity following the manufacturer’s instructions.
2. 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.
3. Collect supernatants from cells and media, carry out the assay as per manufacturer’s instructions.
4. Measure the level of CYP activity and normalized relative to the surface area (cm²).

10. Immunostaining

1. Wash hESC derived hepatocytes with PBS, for 1 min, repeat two times.
2. Add ice cold 100% methanol to fix cells, place in -20 °C for 10 min.
3. Wash cells with PBS for 5 min and repeat twice.
4. Incubate cells with PBS/T (0.1% tween)/10% BSA for 1 hr at RT.
5. 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.
6. Wash cells with PBS/T (0.1% tween)/1% BSA for 5 min and repeat three times.
7. 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.
8. Wash cells with PBS for 5 min and repeat three times.
9. 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.

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Wyniki

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...

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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips
Shaker	Edmun Bühler	KS-15
Irradiator	CIS Biointernational	IBL 637
Spin coater	Specialty Coating System	P-6708
Scanning Electron Microscope	Philips	XL30CPSEM
Atomic Force Microscope	DimensionV Nanoscope, VEECO
p4-GLO CYP3A4	Promega	V8902
UV bulb	ESCO
NanoScope analysis software	VEECO		version 1.20
Fluorescence microscope	Olympus	TH45200	Use Volocity 4 Software
Tissue culture plates	Corning, UK	3527
glass slides	Scientific Laboratory Supplies	MIC3308
Diethylene glycol	Sigma–Aldrich	93171
1,6-hexanediol	Sigma–Aldrich	240117
Neopentyl glycol	Sigma–Aldrich	408255
Adipic acid	Sigma–Aldrich	9582
anhydrous N,N-Dimethylformamide	Sigma–Aldrich	227056
Diethyl ether	Sigma–Aldrich	676845
titanium (IV) butoxide	Sigma–Aldrich	244112
1,4-butanediol	Sigma–Aldrich	493732
Vacuum oven	Thermoscientific
4,4’-Methylenebis(phenyl isocyanate)	Sigma–Aldrich	101688
Tetrahydrofurane	Sigma–Aldrich	401757
Sputter coater	Bal-Tec SCD 050
Inmunostaining
Phosphate buffer saline (-MgCl2, -CaCl2)	Gibco	10010031	Store at room temperature
PBST, PBS made up with 0.1% TWEEN 20	Scientific Laboratory Supplies Ltd	EC607
Methanol	Scientific Laboratory Supplies Ltd	CHE5010
Bovine Serum Albumin	Sigma-Aldrich, UK	A7906
MOWIOL 488 DAPI	Calbiochem	475904	Made up in Tris HCl and glycerol as per manufacturers instructions
Cell culture and Functional assay
CYP3A activity pGLO kit	Promega	V8902
Hepatozyme	Gibco	17705021
TryLE express	Life Technologies	12604013