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 ...

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Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...