Name: ultrathin porated elastic hydrogels as a biomimetic basement membrane for dual cell culture
Uploaded: 2017-12-26T13:00:00
Description: Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

The authors would like to thank Prof. Paul Van Tassel and Prof. Chinedum Osuji for their thoughtful conversations and materials science expertise. Funding for this work was provided by the Dubinsky New Initiative Award and National Institutes of Health NIBIB BRPR01 EB16629-01A1.

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
<ol>
	<li>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.</li>
	<li>Prepare 150 mL of 1 M NaOH by adding 6 g of NaOH to 150 mL of water.</li>
	<li>Set up a mineral oil bath on a hot plate with stirrer, and submerge a 500-mL round bottom flask into the oi...

<ol>
	<li>Domogatskaya, A., Rodin, S., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+laminins.">Functional diversity of laminins.</a> Annu Rev Cell Dev Biol. 28, 523-553 (2012).</li><li>Howat, W. J., Holmes, J. A., Holgate, S. T., Lackie, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+pores+in+human+bronchial+epithelium:+a+conduit+for+infiltrating+cells.">Basement membrane pores in human bronchial epithelium: a conduit for infiltrating cells.</a> Am J Pathol. 158, 673-680 (2001).</li><li>Lauridsen, H. M., Pober, J. S., Gonzalez, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+composite+model+of+the+human+postcapillary+venule+for+investigation+of+microvascular+leukocyte+recruitment.">A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment.</a> FASEB J. 28, 1166-1180 (2014).</li><li>Mul, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+migration+of+neutrophils+across+monolayers+of+endothelial+and+epithelial+cells.">Sequential migration of neutrophils across monolayers of endothelial and epithelial cells.</a> J Leukoc Biol. 68, 529-537 (2000).</li><li>Hermanns, M. I., Unger, R. E., Kehe, K., Peters, K., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+epithelial+cell+lines+in+coculture+with+human+pulmonary+microvascular+endothelial+cells:+development+of+an+alveolo-capillary+barrier+in+vitro.">Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro.</a> Lab Invest. 84, 736-752 (2004).</li><li>Birkness, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+tissue+culture+bilayer+model+to+examine+early+events+in+Mycobacterium+tuberculosis+infection.">An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection.</a> Infect Immun. 67, 653-658 (1999).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+alveolar+epithelial+cells+attenuate+pulmonary+microvascular+endothelial+cell+permeability+under+septic+conditions.">Human alveolar epithelial cells attenuate pulmonary microvascular endothelial cell permeability under septic conditions.</a> PLoS One. 8, 55311 (2013).</li><li>Pellowe, A. S., Gonzalez, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+biomimicry+for+the+creation+of+investigational+and+therapeutic+devices.">Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.</a> Wiley Interdiscip Rev Nanomed Nanobiotechnol. 8, 5-22 (2016).</li><li>Peyton, S. R., Raub, C. B., Keschrumrus, V. P., Putnam, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+poly(ethylene+glycol)+hydrogels+to+investigate+the+impact+of+ECM+chemistry+and+mechanics+on+smooth+muscle+cells.">The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells.</a> Biomaterials. 27, 4881-4893 (2006).</li><li>West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-patterned+hydrogels:+Customized+cell+microenvironments.">Protein-patterned hydrogels: Customized cell microenvironments.</a> Nat Mater. 10, 727-729 (2011).</li><li>DeLong, S. A., Gobin, A. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+immobilization+of+RGDS+on+hydrogel+surfaces+to+direct+cell+alignment+and+migration.">Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration.</a> J Control Release. 109, 139-148 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Taite, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+hydrogel+substrates:+probing+leukocyte+receptor-ligand+interactions+in+parallel+plate+flow+chamber+studies.">Bioactive hydrogel substrates: probing leukocyte receptor-ligand interactions in parallel plate flow chamber studies.</a> Ann Biomed Eng. 34, 1705-1711 (2006).</li><li>Lauridsen, H. M., Walker, B. J., Gonzalez, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically-+and+mechanically-tunable+porated+polyethylene+glycol+gels+for+leukocyte+integrin+independent+and+dependent+chemotaxis.">Chemically- and mechanically-tunable porated polyethylene glycol gels for leukocyte integrin independent and dependent chemotaxis.</a> Technology. 02, 133-143 (2014).</li><li>DeLong, S. A., Moon, J. J., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+gradients+of+bFGF+on+hydrogel+scaffolds+for+directed+cell+migration.">Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration.</a> Biomaterials. 26, 3227-3234 (2005).</li><li>Lauridsen, H. M., Gonzalez, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic,+ultrathin+and+elastic+hydrogels+regulate+human+neutrophil+extravasation+across+endothelial-pericyte+bilayers.">Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.</a> PLOS one. 12, 0171386-0171405 (2017).</li><li>Peters, E. B., Christoforou, N., Leong, K. W., Truskey, G. A., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(Ethylene+Glycol+Hydrogel+Scaffolds+Containing+Cell-Adhesive+and+Protease-Sensitive+Peptides+Support+Microvessel+Formation+by+Endothelial+Progenitor+Cells.">Poly(Ethylene Glycol Hydrogel Scaffolds Containing Cell-Adhesive and Protease-Sensitive Peptides Support Microvessel Formation by Endothelial Progenitor Cells.</a> Cellular and Molecular Bioengineering. 9, 38-54 (2016).</li><li>Schwartz, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+strategy+for+mimicking+the+extracellular+matrix+provides+new+insight+about+tumor+cell+migration.">A synthetic strategy for mimicking the extracellular matrix provides new insight about tumor cell migration.</a> Integr Biol (Camb). 2, 32-40 (2010).</li><li>Booth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+normal+and+fibrotic+human+lung+matrices+as+a+culture+system+for+in+vitro+investigation.">Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.</a> Am J Respir Crit Care Med. 186, 866-876 (2012).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes:+structure,+assembly+and+role+in+tumour+angiogenesis.">Basement membranes: structure, assembly and role in tumour angiogenesis.</a> Nat Rev Cancer. 3, 422-433 (2003).</li><li>Roudsari, L. C., Keffs, S. E., Witt, A. S., Gill, B. J., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Poly(ethylene+glycol)-based+Tumor+Angiogenesis+Model+to+Study+the+Influence+of+Vascular+Cells+on+Lung+Tumor+Cell+Behavior.">A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior.</a> Scientific Reports. 6, 1-15 (2016).</li><li>Bermudez, L. E., Sangari, F. J., Kolonoski, P., Petrofsky, M., Goodman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+the+translocation+of+Mycobacterium+tuberculosis+across+a+bilayer+of+epithelial+and+endothelial+cells+as+a+model+of+the+alveolar+wall+is+a+consequence+of+transport+within+mononuclear+phagocytes+and+invasion+of+alveolar+epithelial+cells.">The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells.</a> Infect Immun. 70, 140-146 (2002).</li></ol>

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...

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 &#181;m 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 &#181;m.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 &#181;m 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:737px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1M Hydrogel Chloride (HCl)</td>
			<td style="width:117px;">EMD</td>
			<td style="width:119px;">HX0603-75 2.5L</td>
			<td style="width:167px;">Sterile. Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">1X PBS</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:119px;">14040-133 500 mL</td>
			<td>None</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">Zinc Nitrate Hexahydrate (Zn(NO3)2&#8226;6H2O)</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">228737-500g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Sodium Hydroxide (NaOH)</td>
			<td style="width:117px;">Macron Chemicals</td>
			<td style="width:119px;">278408-500g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:335px;">Zinc Acetate Dihydrate ((CH3O2)2Zn2+&#8226;2H2O)</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">AC45180010 1 kg</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">Methanol (CH3OH)</td>
			<td style="width:117px;">J.T. Baker</td>
			<td style="width:119px;">9070-05 4L</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">VWR Life Science Seradigm Premium Grade FBS</td>
			<td style="width:117px;">VWR</td>
			<td style="width:119px;">97068-085</td>
			<td>Sterile filter. 5 mL FBS in 45 mL PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Mineral oil</td>
			<td style="width:117px;">CVS&#160;</td>
			<td style="width:119px;">PLD-B280B</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Round bottom flask</td>
			<td style="width:117px;">ChemGlass</td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Thermometer</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stir bar</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Plain precleaned microscope slides 3&#34;x1&#34;x1&#34; mm thick</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:119px;">420-004T</td>
			<td>Spray with ethanol and let dry prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass pasteur pipets</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1 mL rubber bulbs</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plastic 100 mm petri dishes</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sterile forceps</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Silicone isolators</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>0.8 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Polydimethylsiloxane (PDMS) punches</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass bottles</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6 well plates</td>
			<td style="width:117px;">Cellstar</td>
			<td style="width:119px;">657 160</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Filter Paper</td>
			<td style="width:117px;">Whatman</td>
			<td align="right" style="width:119px;">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stirrer-hot plate</td>
			<td style="width:117px;">VWR Dya-Dual</td>
			<td style="width:119px;">12620-970</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:335px;">2,2-Dimethoxy-2-phenylacetophenone (C6H5COC(OCH3)2C6H5</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">24650-42-8</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-Vinyl-2-pyrrolidone (C6H9NO)</td>
			<td style="width:117px;">Aldrich</td>
			<td style="width:119px;"></td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:335px;">Polyethylene Glycol 10,000 (H(OCH2CH2)10,000OH)</td>
			<td style="width:117px;">Fluka</td>
			<td style="width:119px;">81280-1kg</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">RGDS</td>
			<td style="width:117px;">Life Tein</td>
			<td align="right" style="width:119px;">180190</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Blak-Ray long wave UV lamp</td>
			<td style="width:117px;">UVP</td>
			<td style="width:119px;">Model B 100AP</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Eppendorf tubes</td>
			<td style="width:117px;">USA Scientific</td>
			<td style="width:119px;">1615-5500</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

ultrathin porated elastic hydrogels as a biomimetic basement membrane for dual cell culture

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.

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 &#177;&...

Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

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

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.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

The overall goal of this protocol is to describe a polyethylene glycol and zinc oxide templating method to create a porous, ultra-thin, biomimetic basement membrane that supports dual cell culture, and has tuneable mechanical and chemical properties. The main advantage of this technique is that we can create hydrogels that are ultra-thin, porated, mechanically and biochemically tuneable. They allow support of either single or dual cell culture.This method can help answer key questions in the tissue engineering field, including how dual cell cultures react to, and interact with, microenvironments that mimic human basement membranes. The implications of this technique extend toward a wide variety of biological systems and tissues, ultimately allowing more biomimetic research with enhanced translation for human disease. Add 250 milliliters of zinc nitrate to a flask and begin stirring the reaction.Then, add 150 milliliters of sodium hydroxide solution. A white precipitate will form briefly and then disappear as the two solutions continue to mix. Stir the mixture for two hours uncovered.Heat the flask to 55 to 60 degrees celsius and continue stirring for 24 hours. Turn off the heat and allow solution to cool to room temperature while stirring. Filter the solution though a Buchner funnel with an 11 micron pore size and allow it 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. After checking the needle leg morphology of zinc oxide needles, as described in the text protocol, clean three inch by one inch plain microscope slides with 70 percent ethanol and a disposable wipe. Hold the glass slide with tweezers and use a glass pipet with a pipet bulb to coat the slides with five drops of zinc acetate forming a thin layer dispersed on the slide.Allow excess solution to drip back into the stock solution. Then, place the slide into a preheated Petri dish on a hot plate with the zinc acetate coated side facing up for 15 minutes. Remove the slide with tweezers and allow it to cool to room temperature.The slide will appear to be coated with white streaks. Remove any excess zinc that is remaining on the slides, using a disposable wipe to remove permanent white streaks. The surface should be uniform.Suspend 20 milligrams of zinc oxide needles in 270 microliters of ten percent FBS/PBS solution. Vortex to mix for approximately 15 seconds. Briefly spin the solution in a bench top mini centrifuge to bring any zinc oxide aggregates to the base of the tube.Following centrifugation, collect 250 microliters from the top of the zinc oxide solution to ensure that aggregates remain at the bottom of the tube. Combine the collected zinc oxide solution with PEG diacrylate and PEG-RGD to create a PEG solution with approximately 2.5 millimolar RGD. Then vortex to mix for approximately 15 seconds.Next, add two microliters of acetophenone and vinyl pyrrolidone photoinitiator and vortex briefly to mix. Add 20 microliters of the polymer solution along the center of the zinc oxide coated slides. Slowly lower a second slide on top, with the zinc oxide coating facing down so that the PEG solution is between two zinc oxide coated layers.Try to prevent the formation of any air bubbles. Move the slides laterally along their axis to allow any bubbles to escape and to spread the solution into a thin layer. Create a slight overhang with the top slide to ensure that the slides can be pulled apart in their later steps.Finally, cross link the slides under a 365 nanometer UV lamp for 15 minutes. Apply pressure to the overhanging slide and manually pull the two slides apart at a slow pace in order to avoid cracking the slides. Allow the gels to dry for at least five minutes or overnight.Once the gels are dry, place the slide in a sterile 25 milliliter glass Petri dish and gently pour the one molar HCL solution onto the slide using only enough to cover the slide. 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. Rinse the gel by gently pouring approximately 25 milliliters of 1x PBS into the Petri dish until the slide and gel are submerged. Carefully pour off PBS into a waste container.Then, gently pour approximately 25 milliliters of 1x PBS into the Petri dish until the gel is floating in the solution. Use tweezers to carefully slide the silicone isolator under the gel and lift the gel onto it. Transfer the isolator and the gel into a six well dish filled with sterile 1x PBS.Prepare A549 cells as detailed in the text protocol. While cells are spinning down, add a small drop of complete Dulbecco's Modified Eagle Medium to each well of a six well plate. Use tweezers to transfer the silicone isolators with the gels, into a new six well plate such that the gel is resting on the drop of media.Now that the gels are spread into a thin layer, check to ensure that there are no large holes visible to the eye. Add the resuspended cell suspension drop-wise to the center of the gel, trying to maintain a meniscus in the punched out area of the silicone membrane. After allowing the cells to adhere for four hours, add 0.5 milliliters of Dulbecco's Modified Eagle Complete Medium to the well.Incubate overnight at 37 degrees Celsius to allow for complete adhesion. The following day, prepare huvecs for cell seeding as described in the text protocol. Then, add a small drop of Complete Medium 199 to each well in a new six well plate.Place a new silicone isolator in the well with a drop of media in the center of the silicone. Using tweezers, carefully flip the silicone isolators supporting the peg gel with A459 cells onto the isolator in the new six well plate. Add the huvec suspension to the center of the flipped gel drop-wise to maintain a meniscus on the gel within the punched out area of the silicone membrane.After allowing the cells to adhere for two hours, gently add two milliliters of M199 Complete Media to each well. Use a manual pipet to carefully remove the media from the gels, and add approximately 500 microliters of four percent PFA to the center of the gel where the cells are seeded. After the PFA sits for 30 minutes at room temperature, carefully remove the PFA and add approximately 500 microliters of two percent BSA in PBS to each gel.Following one hour incubation at room temperature, carefully remove the BSA. Add 500 microliters of prepared primary antibodies at a dilution of one to 100 in two percent BSA in PBS to each gel. Apply the secondary antibodies and phalloidin as detailed in the text protocol.Then, using tweezers and silicone isolators, transfer one gel to a glass slide. Mount the gel using a DAPI mounting medium. Finally, seal the samples with nail polish and acquire the images.Endothelial cells are able to form confluent monolayers on PEG hydrogels as demonstrated by fluorescent staining for PECAM-1 in red and DAPI in blue. Similarly, fluorescent staining for A549 in red demonstrates that epithelial cells also form confluent monolayers. Furthermore, endothelial cells can form intact cadherin junctions on PEG hydrogels as demonstrated by fluorescent staining for VE-cadherin in green and DAPI in blue.Staining for E-cadherin in green reveals the epithelial cells also form intact cadherin junctions. Following this technique, other methods like migration assays or permeability assays can be performed to understand how dual cell cultures and different microenvironments maintain or attenuate barrier function. After watching this video, you should have a good understanding on how to create ultra-thin PEG gels and modulate their porosity in addition to their mechanical and chemical properties in order to mimic the basement membrane.This technique will pave the way for researchers in the fields of biomaterials and biomedical engineering to explore and decouple mechanical, biochemical, and cell-mediated pathways that contribute to human disease. Though the system is widely applicable to biological applications, it can be applied to any system where cell-cell signaling and mechanical or biochemical extracellular cues can play a role.

Research

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Bioengineering

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