Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

Summary

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Abstract

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Introduction

Extracellular matrices (ECM) make up the protein scaffolds that support cell attachment and serve as barriers between distinct cell types and are an essential component of complex tissues and organs. In contrast to interstitial connective tissue, the basement membrane (BM) is a specialized type of ECM that acts as a barrier to divide tissue compartments from one another. BMs are approximately 100 µm thick, and therefore allow for direct and indirect communication between cells on either side. Two common examples of BMs are vascular BMs, found in the microvascular wall between pericytes and endothelial cells, and airway BMs that are found between endothelial and e....

Protocol

Please read Material Safety Data Sheet (MSDS) of all materials prior to use and use safety precautions at all times.

1. Synthesis of Zinc Oxide Needles

1. Prepare 250 mL of a 0.04 M Zn(NO₃)₂*6H₂O solution by adding 2.9749 g of zinc nitrate to 250 mL of water.
2. Prepare 150 mL of 1 M NaOH by adding 6 g of NaOH to 150 mL of water.
3. Set up a mineral oil bath on a hot plate with stirrer, and submerge a 500-mL round bottom flask into the oil bath at room temperature.
4. Add 250 mL of Zn(NO₃)₂*6H₂O to the flask and begin stirring the reaction.
....

Results

PEG-RGD hydrogels were formed by sandwiching the polymer solution between two sacrificial zinc oxide layers and creating pore templates with zinc oxide needles. Sacrificial zinc oxide components were then removed with hydrochloric acid, generating ultrathin PEG hydrogels with continuous pores (Figure 1). The morphology of zinc oxide needles was confirmed by scanning electron microscopy (SEM), and the average length and width were determined to be 3.92 ±&.......

Discussion

The protocol detailed here has allowed us to create a tunable PEG hydrogel to serve as a biomimetic BM scaffold. Specifically, by varying PEG molecular weights, peptide conjugation strategies, and zinc oxide microcrystalline structures or concentrations, the elastic modulus, biochemical properties, and porous structure of the hydrogels can be modified, respectively. The ultrathin PEG scaffold features a higher pore density and a smaller pore diameter that is more mimetic of the features found in in vivo basement.......

Materials

Name	Company	Catalog Number	Comments
1M Hydrogel Chloride (HCl)	EMD	HX0603-75 2.5L	Sterile. Use in fume hood with eye protection and gloves.
1X PBS	Gibco	14040-133 500 mL	None
Zinc Nitrate Hexahydrate (Zn(NO3)2•6H2O)	Sigma-Aldrich	228737-500g	Use with eye protection and gloves.
Sodium Hydroxide (NaOH)	Macron Chemicals	278408-500g	Use with eye protection and gloves.
Zinc Acetate Dihydrate ((CH3O2)2Zn2+•2H2O)	Fisher Scientific	AC45180010 1 kg	Use with eye protection and gloves.
Methanol (CH3OH)	J.T. Baker	9070-05 4L	Use in fume hood with eye protection and gloves.
VWR Life Science Seradigm Premium Grade FBS	VWR	97068-085	Sterile filter. 5 mL FBS in 45 mL PBS
Mineral oil	CVS	PLD-B280B	None
Round bottom flask	ChemGlass		N/A
Thermometer			N/A
Stir bar			N/A
Plain precleaned microscope slides 3"x1"x1" mm thick	Thermo Scientific	420-004T	Spray with ethanol and let dry prior to use.
Glass pasteur pipets			N/A
1 mL rubber bulbs			N/A
Plastic 100 mm petri dishes			N/A
Sterile forceps			N/A
Silicone isolators			0.8 mm thick
Polydimethylsiloxane (PDMS) punches			N/A
Glass bottles			N/A
6 well plates	Cellstar	657 160	N/A
Filter Paper	Whatman	8519	N/A
Stirrer-hot plate	VWR Dya-Dual	12620-970	Use with eye protection and gloves.
2,2-Dimethoxy-2-phenylacetophenone (C6H5COC(OCH3)2C6H5	Sigma-Aldrich	24650-42-8	Use with eye protection and gloves.
1-Vinyl-2-pyrrolidone (C6H9NO)	Aldrich		Use with eye protection and gloves.
Polyethylene Glycol 10,000 (H(OCH2CH2)10,000OH)	Fluka	81280-1kg	Use with eye protection and gloves.
RGDS	Life Tein	180190	Use with eye protection and gloves.
Blak-Ray long wave UV lamp	UVP	Model B 100AP	N/A
Eppendorf tubes	USA Scientific	1615-5500	N/A