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 epithelial cells. BMs serve an important role in regulating cell function, such as cell polarity and migration, in health and disease.¹ The composition, stiffness, architecture, and porosity of BMs varies across organ systems to facilitate distinct physiological functions. For example, BM pores are critical for maintaining cell-cell communication, soluble molecule diffusion, and for migration of immune cells during inflammation or bacteria during infection. In the airways, pores span the full thickness of the BM, with diameters ranging from 0.75 to 3.86 µm.²

The thin nature of the BM ensures that although cell types are physically separated from one another, intercellular communication via paracrine- and contact-mediated signaling is preserved. Thus, to study human disease in vitro, researchers have relied on porous permeable support inserts to culture cellular bilayers.³ These models have been critical for understanding the cellular communication that plays a role in health and disease.^3,4,5,6,7 Permeable support inserts satisfy the basic requirements for understanding how cell-cell signaling regulates physiological processes, such as leukocyte recruitment and bacterial infiltration; however, the inserts have significant limitations and fail to mimic a human BM. Permeable support inserts lack both mechanical and biochemical tunability, and the simplistic porous structure does not mimic the fibrous structure that creates the irregular pores typical of BMs. Therefore, there is a growing need for tunable systems that can recreate the native BM properties that influence cellular processes.

Polymer-based substrates are ideal candidates for the development of biomimetic BMs to study cellular bilayers in a context that more closely mimics the in vivo environment.^8,9,10,11,12 Polymers are mechanically tunable and can be chemically modified to incorporate biomimetic peptide fragments.^11,12,13 The bioinert polymer polyethylene glycol (PEG) can be used to construct biomimetic BMs, and recent work has detailed the synthesis of mechanically tunable PEG arginine-glycine-aspartic acid (RGD) gels with porous networks that support cell growth and inflammatory cell chemotaxis.¹⁴ Although published PEG-based substrates provided a more realistic model of a human ECM than permeable supports, many of these models are extremely thick, with a depth of roughly 775 µm that limits the ability to create bilayer cultures with cell-cell contacts.¹⁴

Here, we present a protocol for the creation of a PEG polymer-based BM mimic that overcomes many of the limitations of current cell bilayer culture technologies. We have developed a templating method that incorporates zinc oxide, an extensively used material for the manufacture of microcrystalline production, into the polymer during synthesis and crosslinking, which is subsequently and selectively removed from the resulting bulk polymer. This process generates a random porous network, mimicking the tortuous and interconnected pore network of human BMs. Further, the porosity can be altered by changing the size and shape of the zinc oxide microcrystals via modification of the reaction stoichiometry during needle production. The technique developed here creates an ultrathin hydrogel that mimics the thickness of human BM. Lastly, the mechanics, the porosity, and the biochemical composition of these BM-like constructs can easily be altered to generate a microenvironment that is most similar to that seen in vivo.

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.
5. Add 150 mL NaOH solution (a white precipitate will form briefly and then disappear as the two solutions continue to mix). Stir for 2 h uncovered.
6. Heat the flask to 55 - 60 °C and continue stirring for 24 h.
7. Turn off the heat and allow solution to cool to room temperature while stirring.
8. Filter the solution on a Büchner funnel with an 11 µm pore size and allow to dry overnight, uncovered. When the filter paper is no longer wet from the poured solution and a white powder has formed over the funnel holes, the particles are fully dried.
9. Collect ZnO needles and prepare for scanning electron microscopy (SEM) to confirm the needle-like morphology. Briefly, mount carbon tape onto a pin stub and use a metal spatula to smear the ZnO needles onto the carbon tape. Sputter coat with 8 mm iridium at a density of 22.4 g/cm³ and acquire images at 10 kV.

2. Addition of Sacrificial Zinc Layer on Microscope Slides for HCl Induced Release of PEG

1. Prepare zinc acetate solution by dissolving 1.756 g of zinc acetate in 200 mL of methanol.
2. Clean 3" x 1" plain microscope slides with 70% ethanol and a disposable wipe. Allow to air dry for 10 min.
3. Place a 150-mm glass Petri dish onto a hot plate and preheat to 150 - 160 °C.
4. Using a glass pipet with a pipet bulb, hold the glass slides with tweezers and coat slides by applying 5 drops of zinc acetate, forming a thin layer dispersed on the slide. Allow excess solution to drip back into stock solution.
5. Place the slide in the pre-heated Petri dish (150 - 160 °C) with the zinc acetate coated side facing up. Leave on the hotplate for 15 min.
6. Remove the slide with tweezers and allow it to cool to room temperature. The slide will appear to be coated with white streaks.
7. Remove any excess zinc that is remaining on the slides using a disposable wipe to remove prominent white streaks. The surface should be uniform.
8. Expose the the prepared slides to UV light in a biosafety hood for at least 1 h to ensure sterility.
   Caution: UV light is harmful to eyes and exposed skin. Avoid direct exposure to eyes or skin and turn off electrical supply when not using.

3. Preparation of Silicone Isolators

1. Cut silicone sheet into squares less than 1" x 1" (isolators must be able to fit in a 6-well dish).
2. Punch 8 - 12 mm holes in the center of the squares with a biopsy punch.
3. Autoclave silicone isolators for 20 min at 121.0 °C and 1.12 kg/cm.

4. Preparation of PEG solution and PEG Gel Synthesis

Note: Functionalization and polymerization of PEG has been extensively explored and detailed previously by our lab and others.^{8,10,11,13,14,15,16,17}

1. Combine the RGD cell adhesive peptide with acryloyl-PEG-NHS in 50 mM sodium bicarbonate (pH 8.5) at a 1:1 molar ratio to enable functionalization of the amine terminus of the peptide with the acrylate moiety, a biochemical reaction that has been previously characterized to yield greater than 85% efficiency.¹⁷
2. Prepare photo-initiator by mixing 2,2-Dimethoxy-2-phenylacetophenone with 1-Vinyl-2-pyrrolidone at a concentration of 300 mg/mL.
3. In separate 1.5 mL tubes, weigh out the following: 20 mg of ZnO needles, 12.5 mg of PEG-DA, and 2.5 mg of PEG-RGD.
4. Suspend 20 mg of ZnO needles in 270 µL of 10% FBS/PBS solution; vortex to mix (approximately 15 s).
5. Briefly (<1 s) spin the solution in a benchtop mini centrifuge to bring any ZnO aggregates to the base of the tube.
6. Collect 250 µL from the top of the ZnO solution, to ensure that aggregates remain at the bottom of the tube. Combine with PEG-DA and PEG-RGD to create a PEG solution with approximately 2.5 mM RGD. Vortex to mix (approximately 15 s).
7. Add 2 µL of acetophenone/n-vinyl pyrrolidone photo-initiator and vortex briefly to mix (approximately 5 s).
8. Add 20 µL of the polymer solution along the center of the ZnO coated slides.
9. Slowly lower a second slide on top, with the ZnO coating face down (the PEG solution should be between two ZnO coated layers). Try to prevent the formation of any air bubbles. Move the slides laterally along the major axis to allow any bubbles to escape and to spread the solution to a thin layer. Cover the majority of the slide with the polymer solution. Create a slight overhang with the top slide to ensure that the slides can be pulled apart in later steps.
10. Crosslink the slides under a 365 nm UV lamp (approximately 10 mW/cm²) for 15 min.

5. Release of PEG Gels from Glass Slides

1. Apply pressure to the overhanging slide and manually pull the two slides apart at a slow pace in order avoid cracking the slides. Allow the gels to dry for at least 5 min, or overnight.
2. Place the slide in a sterile 25 mm glass Petri dish and gently pour the 1 M HCl solution onto the slide, using only enough to cover the slide (approximately 10 - 20 mL). Gently rock the Petri dish; the gel should begin to lift off the slide as the HCl dissolves the sacrificial zinc coating and needles. Once the gel is free from the slide, pour the HCl back into the stock solution.
3. Rinse the gel by gently pouring approximately 25 mL of 1x PBS into the Petri dish until the slide and gel are submerged. Carefully pour off PBS into a waste container.
4. Gently pour approximately 25 mL of 1x PBS into the Petri dish until the gel is floating in the solution.
5. Using tweezers, carefully slide the silicone isolator under the gel and lift the gel onto it.
6. Transfer the isolator and the gel into a 6-well dish filled with sterile 1x PBS.
7. Repeat this process until all of the gels have been removed from the slides and are soaking in PBS. Expose the prepared gels to UV light in a biosafety hood for at least 1 h to ensure sterility.

6. Seeding Cell Bilayers

1. Prepare A549 cells for seeding. Briefly, rinse a 75 cm² flask with 5 mL of 1x PBS, add 3 mL of 0.25% Trypsin-EDTA and let sit for 2 - 3 min at 37 °C.
   1. Mechanically agitate the flask, quench the reaction with 3 mL Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin-streptomycin, and collect into a 15-mL conical flask. Rinse with an additional 4 mL of complete Dulbecco's Modified Eagle Media and collect into the same conical. Spin cells at 475 x g for 6 min.
2. While cells are spinning down, add a small drop of complete Dulbecco's Modified Eagle Medium to each well of a 6-well plate. Use tweezers to transfer the silicone isolators with the gels into the new 6-well plate, such that the gel is resting on the drop of media. Now that gels are spread into a thin layer, check to ensure that there are no large holes visible to the eye. This should be done immediately prior to cell seeding to avoid drying and cracking of the gels.
3. Resuspend cells in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin-streptomycin at a concentration of 6 x 10⁵ cells/mL. Add the cell suspension drop wise to the center of the gel, trying to maintain a meniscus in the punched out area of the silicone membrane. Allow cells to adhere for 4 h.
4. After 4 h, add 0.5 mL of Dulbecco's Modified Eagle Complete Medium to the well and incubate overnight at 37 °C to allow for complete adhesion.
5. The following day, prepare HUVECs for cell seeding.
   1. Rinse a 75 cm² flask with 5 mL of 1X PBS, add 3 mL of 0.25% Trypsin-EDTA and let sit for 2 - 3 min at 37 °C.
   2. Mechanically agitate the flask, quench the reaction with 3 mL M199 media with 20% fetal bovine serum, 1% penicillin-streptomycin, and 1% growth supplement, and collect into a 15-mL conical.
   3. Rinse with 4 mL of M199 complete media and collect into the same conical. Spin cells at 475 x g for 6 min.
6. While cells are spinning down, add a small drop of complete medium 199 to each well in a new 6-well plate. Place a new silicone isolator in the well with the drop of media in the center of the silicone.
7. Using tweezers, carefully flip the silicone isolator supporting the PEG gel with A549 cells onto the isolator in the new 6-well plate.
8. Resuspend HUVECs at a concentration of 6 x 10⁵ cells/mL and add the cell suspension to the center of the flipped gel drop wise, trying to maintain a meniscus on the gel within the punched out area of the silicone membrane. Allow cells to adhere for 2 h.
9. After 2 h, gently add 2 mL of M199 complete media to each well.

7. Immunofluorescence

1. Carefully remove media from gels with a manual pipette and add approximately 500 µL of 4% paraformaldehyde (PFA) to the center of the gel where the cells are seeded. Let sit for 30 min at room temperature.
   ​Caution: PFA is a toxic chemical; wear protection and ensure handling is done in a biological safety cabinet.
2. Carefully remove the PFA and add approximately 500 µL of 2% BSA in PBS to each gel. Let sit for 1 h at room temperature.
3. Carefully remove BSA. Prepare primary antibodies at a dilution of 1:100 in 2% BSA in PBS, and add 500 µL to each gel. Let sit for 1 h at room temperature. Rinse gently with 500 µL of 1x PBS.
   1. Repeat the same process with the secondary antibodies. Use following antibodies: 1) anti-human CD144 (VE-Cadherin) clone 16B1; 2) anti-human CD324 (E-Cadherin) clone 6714; 3) anti-human CD31 (PECAM-1) clone C-20; 4) anti-A549; 5) anti-mouse FITC; 6) anti-goat Alexa Fluor 647. To visualize F-actin, add 500 µL of Phalloidin (10 µg/mL in PBS) for 20 min.
4. Carefully remove secondary antibodies or phalloidin and add 500 µL of DAPI (0.1 µg/mL) for 20 min. Carefully remove DAPI solution and gently add 1.5 mL of 1x PBS to each well so that gels are in suspension.
   1. Using tweezers and silicone isolators, transfer one gel to a glass slide. Do this for each gel immediately before imaging, and replace gels into PBS solution after imaging.
   2. Alternatively, mount gels using a DAPI mounting medium. Seal well the samples with nail polish, acquire images within 2 days to prevent drying and cracking of the gel. Refer to Table 1 for troubleshooting.

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