Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Summary

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

Abstract

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.

Introduction

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 perform....

Protocol

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 aci....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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