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

作者希望感谢 Prof. 的 Prof. Chinedum Osuji 的谈话和材料科学的专业知识。这项工作的经费由 Dubinsky 新倡议奖和国家卫生研究院 NIBIB BRPR01 EB16629-01A1 提供。

在使用之前请阅读所有材料的材料安全数据表 (MSDS), 并在任何时候使用安全预防措施。
1. 氧化锌针的合成
<ol>
	<li>准备250毫升的0.04 米锌 (没有3)2* 6H2O 解决方案, 加入2.9749 克的硝酸锌到250毫升的水。</li>
	<li>将6克氢氧化钠加到150毫升的水中, 制备150毫升的1米氢氧化钠。</li>
	<li>在带有搅拌器的热板上设置矿物油浴, 并在室温下将500毫升圆底烧瓶浸入油?...

<ol>
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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>

这里详述的协议允许我们创建一个可调谐的 PEG 水凝胶作为仿生 BM 支架。具体而言, 通过改变 PEG 分子量、肽共轭策略和氧化锌微晶结构或浓度, 可以分别修改水凝胶的弹性模量、生物化学性质和多孔结构。超薄的 PEG 支架具有更高的孔隙密度和更小的孔径, 更模拟了在体内基底膜中发现的特征, 与标准的渗透性支持模型 (表 2) 相比。此外, 通过这种方法, 我们实现了一个平均厚度?...

细胞外基质 (ECM) 构成了支持细胞附着的蛋白质支架, 在不同细胞类型之间充当屏障, 是复杂组织和器官的重要组成部分。与间质结缔组织不同, 基底膜 (BM) 是一种特殊类型的 ECM, 它是一种屏障, 可以将组织间隔分开。BMs 大约有100µm 厚, 因此可以在两边的细胞之间进行直接和间接的交流。两个常见的例子的 bms 是血管 bms, 发现在微血管壁之间的周和内皮细胞, 和气道 bms, 发现之间的内皮细胞和上皮组织。BMs 在调节细胞功能, 如细胞极性和迁移, 在健康和疾病中起着重要的作用。1 BMs 的组成、刚度、结构和孔隙度在不同的器官系统间变化, 以促进不同的生理功能。例如, BM 毛孔对于维持细胞间的通讯、可溶性分子的扩散, 以及在感染期间炎症或细菌迁移的免疫细胞是至关重要的。在气道中, 孔隙横跨 BM 的全厚度, 直径范围从0.75 到3.86 µm.2
BM 的稀薄的本质保证, 虽然细胞类型是物理上分开的, 通过分泌-和接触介导的信令的胞间通信被保留。因此, 研究人类疾病在体外,研究人员依靠多孔渗透支持插入到培养细胞双层。3这些模型对于了解在健康和疾病中发挥作用的蜂窝通信非常重要。3,4,5,6,7渗透性支持插入满足了了解细胞信号如何调节生理过程的基本要求, 如白细胞的招募和细菌浸润;然而, 这些插入物有很大的局限性, 无法模仿人类的 BM. 渗透性支持插入缺乏机械和生化调谐, 并且简单化的多孔结构不模仿造成不规则的毛孔的纤维结构典型的 BMs。因此, 越来越需要可调谐的系统, 可以重现影响细胞过程的自然 BM 属性。
聚合物基板是发展仿生 BMs 研究细胞双层的理想候选对象, 它在更接近于体内环境的背景下进行。8,9,10,11,12聚合物是机械可调谐的, 可以进行化学修饰, 以加入仿生肽片段.11,12,13 bioinert 聚合物聚乙二醇 (peg) 可用于构建仿生 BMs, 最近的工作详细介绍了可用于支持细胞生长的多孔网络的机械可调谐 PEG 精氨酸-甘氨酸 (RGD) 凝胶的合成和炎症细胞趋化。14虽然发布的 PEG-based 基板提供了比可渗透支持更逼真的人类 ECM 模型, 但这些模型中的许多都非常厚, 有大约775µm 的深度, 从而限制了用细胞细胞创建双层培养物的能力。联系.14
在这里, 我们提出了一个协议, 建立一个 PEG 聚合物 BM 模仿, 克服了目前的细胞双层培养技术的许多限制。我们已经开发了一种模板化方法, 它将氧化锌作为一种用于制造微晶生产的广泛使用的材料, 在合成和交联过程中加入到聚合物中, 随后被选择性地从产生的散装聚合物。这个过程产生一个随机的多孔网络, 模仿人类 BMs 的曲折和互联的孔隙网络。此外, 通过改变针生产过程中的反应化学计量, 可以通过更改氧化锌微的大小和形状来改变孔隙率。在这里开发的技术创建了一个超薄的水凝胶, 模仿人类 bm 的厚度. 最后, 这些 bm 结构的力学、孔隙率和生物化学组成很容易被改变, 产生微环境, 这是最类似于在体内看到的。

<table border="0" cellpadding="0" cellspacing="0" style="width:737px;" width="738">
	<colgroup>
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		<col />
		<col />
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	</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

基底膜是细胞双层的一个重要组成部分, 它的刚度、组成、结构和孔隙度都不同。体外内皮上皮双层的研究传统上依赖于允许双层培养的渗透性支持模型, 但渗透性支持在复制人类基底膜多样性的能力上是有限的。相比之下, 需要化学合成的水凝胶模型是高度可调谐的, 允许通过加入仿生肽或蛋白质来修改材料的硬度和生物化学成分。然而, 传统的水凝胶模型是有限的功能, 因为他们缺乏孔隙的细胞接触和功能的体外迁移研究。此外, 由于传统的水凝胶的厚度, 合并毛孔, 跨越整个厚度的水凝胶一直是挑战。在本研究中, 我们使用聚乙二醇 (PEG) 水凝胶和一种新颖的氧化锌模板化方法来解决仿生水凝胶以前的缺点。因此, 我们提出了一个超薄, 基底膜状水凝胶, 允许融合细胞双层的文化在一个可定制的脚手架, 具有可变的孔隙结构, 机械性能和生物化学成分。

RGD 水凝胶是在两个牺牲氧化锌层之间夹聚合物溶液形成的, 用氧化锌针创建孔隙模板。然后用盐酸除去牺牲的氧化锌组分, 生成具有连续孔隙的超薄 PEG 水凝胶 (图 1)。用扫描电镜 (SEM) 对氧化锌针的形貌进行了验证, 平均长度和宽度分别确定为3.92 ±0.089 µm 和0.43 ±0.02 µm (图 2A-2B)。在除去氧化锌针头后, 用 SEM 对水凝?...

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

目前的双层培养模式不允许功能性的体外研究, 模仿体内环境。使用聚乙二醇和氧化锌模板化方法, 该协议描述了一个超薄仿生基底膜的发展, 具有可调谐的刚度, 孔隙率和生物化学组成, 密切模仿在体内细胞外基质。

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

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

JoVE Journal

Bioengineering

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

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

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

科研

生物工程

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

三维仿生技术：新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

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

RGD功能化水凝胶作为治疗应用的工具合成

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

用于细胞培养应用光介导的形成和构图的水凝胶

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

可调水凝胶由肺细胞外基质的三维细胞培养

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

水凝胶的合成与防污性能作为膜的净水

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

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

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

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

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

用Janus碱纳米管和纤维素制造用于干细胞粘附的仿生纳米矩阵

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

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

用于细胞和药物输送应用的可注射超分子-纳米粒子水凝胶

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...