我们要感谢史密斯菲尔德农场捐赠了完整的猪肺组织。我们也想感谢胡扬博士，唐克里斯蒂娜博士和VCU整形外科部允许我们使用他们的设备。水凝胶和组织样品进行SEM准备在解剖学和神经生物学显微镜基金的VCU部支持的，一部分是由NIH-NINDS中心核心格兰特5 P30 NS047463的资金，部分通过资金形式NIH-NCI癌症中心支援津贴P30 CA016059。在VCU纳米技术核心表征基金（NCC）的SEM图像。这项工作是由美国国家科学基金会，CMMI 1351162资助。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 解 </td><td> 无菌过滤器 </td><td> 说明 </td></tr><tr><td> DIH 2 O </td><td>是</td><td> DIH 2 O;无菌过滤</td></tr><tr><td> 0.1％的Triton X-100溶液</td><td>是</td><td>在通风柜100微升的Triton-X 100溶液加至100毫升DIH 2 O和搅拌至溶解; 无菌过滤器。 </td></tr><tr><td> 2％的脱氧胆解决方案</td><td>是</td><td>在通风橱中添加2克脱氧胆酸钠溶液 每100毫升DIH 2 O和搅拌至溶解;无菌过滤器</td></tr><tr><td> 1M NaCl的</td><td>是</td><td> 58.44克氯化钠加入1升DIH 2 O的;搅拌至溶解; 无菌过滤器</td></tr><tr><td> DNA酶的解决方案</td><td>是</td><td>添加12000单位DNase的1升DIH 2 O;添加0.156克硫酸镁 （无水），和0.222克CaCl 2;搅拌至溶解;无菌过滤器</td></tr><tr><td> PBS </td><td>是</td><td>结合27.2克的 Na 2 HPO 4·7H 2 O（二钠七水合物） 80克氯化钠和2g氯化钾用10L DIH 2 O;搅拌至溶解;调节pH值至7.4;无菌过滤器</td></tr></tbody></table> 表1: 所需的组织脱细胞的解决方案。使上述对脱细胞过程的解决方案。保存在4℃。约将需要一个猪肺2.5升。 1.水凝胶形成<ol><li> 猪肺脱细胞（改编自引用9,10） <ol><li>苦练内功， 整块正常猪肺，从屠宰场，与心脏和VASCulature完好无损。 </li><li>用组织剪或手术刀解剖远离心脏，血管切割尽可能靠近心脏，血管其余将用于灌注后面的步骤中使用。 </li><li>小心解剖距离围绕气管，支气管，和使用解剖刀或剪刀脉管结缔组织。 注:确定最佳肺通过选择肺不通过胸膜大切口或穿刺和大量肺不张或血管闭塞可能妨碍脱细胞过程的有效性以用于程序的其余部分。 </li><li>切去次优肺留下附着到气管尽可能多支气管（心脏，结缔组织和除去肺的凸起可设置的）。应该有一个肺保持附着到气管和血管。 注意:如果需要保留的部分组织学。 9 </li><li>关闭用夹子固定或缝合至P断开支气管r事件多余的回流。 </li></ol><ol start="6"><li>直到需要（ 表1）在4℃准备脱细胞的解决方案和冷藏。 </li><li>使用插管以适合肺动脉，通过两个肺动脉和气管灌注肺组织用DI水3次手泵。第一血管灌注每次。与周围的1L到脉管和1.5升进气管每个灌注开始，但是作为细胞碎片除去更多的液体可通过该系统来灌注。 </li><li>灌注两个脉管和气管用Triton X-100溶液。 </li><li>淹没肺组织中的Triton X-100解决方案，在4℃下24小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>既灌注血管和气管与脱氧胆酸的解决方案。 </li><li>浸没在脱氧胆酸盐的组织切片，在4℃下24小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>灌注两个脉管和气管用NaCl溶液。 </li><li>淹没在过滤的NaCl溶液用于组织在4℃1小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>既灌注血管和气管用DNase解决方案。 </li><li>淹没在过滤的DNA酶溶液中的组织在4℃1小时。 </li><li>灌注两个脉管和气管5次，用PBS。 </li><li>解剖走明显的软骨组织，气管和传导气道，只留下呼吸的区域（主要是周边地区）的所有直径管2mm或更大（主要围绕肺门和肺的中间部分找到）。 </li><li>解剖组织成1英寸的部分或更小。的组织的取向并不重要此步骤。 </li><li>在50ml锥形管中倒出多余的液体和地方组织。冷冻在-80℃下的组织。 注:保留部分组织学，以确保去除细胞和细胞debri的S，如果需要的话。我们使用H＆E，α半乳糖苷酶的PicoGreen，羟脯氨酸，茚三酮，阿新蓝，SDS-PAGE和质谱表征。 9 </li></ol></li><li> 隆处理 <ol><li>拆除含脱细胞肺组织冷冻管盖。 </li><li>将滤纸在管口并用橡皮筋固定。 注:管材与内容仍然应该被冻结，否则置于-80℃直至冻结。 </li><li>冻干组织，直到所有多余的液体是根据制造商的方向走了，用冷冻干燥机。储存在-80°C，直到准备磨。 </li><li>才开始加液氮冷冻研磨机冷却绝缘和内部元件的工作条件。 </li><li>从磨筒中取出磁磨棒并加入组织覆盖底部。 </li><li>更换磨棒，并添加松散的组织。该厂酒吧还是应该自由移动。 </li><li>关闭工厂管和PLACE在冷冻研磨机。 </li><li>填写液氮最大填充线。 </li><li>冷冻粉碎机所有组织成细粉（约5分钟，〜600分钟嵌塞速率-1）时 ，在用不锈钢冲击的聚碳酸酯圆筒以及根据制造商的说明使用冷冻粉碎机不锈钢端塞。储存在-80°C，直到准备使用。 </li></ol></li><li> 微多孔凝胶形成（8毫克/毫升）（改编自引用9,11） <ol><li>添加1％（重量/体积）的脱细胞的粉末和0.1％的（重量/体积）胃蛋白酶的0.01M HCl中，在恒定搅拌下（应能看到流在液体的顶层），在室温下，为48小时。 注:粉末带有静电，因此添加盐酸的粉末，得到机会洗过量离管壁之后。 </li><li>消化48小时后，将溶液和试剂在冰上5分钟。 </li><li>使用冷冻的10％（体积/体积）0.1M NaOH中，一个ð11.11％（体积/体积）的10倍的PBS（以使整个溶液为1x浓度），使消化的蛋白溶液，以7.4生理pH。 注意:溶液现在可以储存在4℃下长达一周。执行如果需要使用流变9凝胶动力学。 </li></ol></li></ol> 2.交联水凝胶，以提高机械强度<ol><li>通过京尼平粉末的所需量溶解在10％DMSO中制备的1％，0.1％和0.01％w / v的京尼平交联溶液的解决方案。 </li><li>涡的解决方案，每15分钟1小时。 </li><li>加京尼平溶液以覆盖先前已经组装在部分1.3.4 ECM凝胶（100μL用于96孔板的每个孔）。 </li><li>离开每个ECM凝胶在37℃下交联24小时。 </li><li>冲洗的ECM凝胶用PBS 3次，直到洗涤液不再蓝色。然后将交联的水凝胶将准备用于进一步表征和CELL文化研究。 </li></ol> 3.细胞培养与微孔凝胶<ol><li> 二维涂层 <ol><li>从1.3.3使用溶液中，添加20微升的预凝胶溶液到96孔非治疗的组织培养板的每个孔中。 </li><li>在4℃下冷藏过夜，使蛋白质吸附于板。 </li><li>吸预凝胶溶液，用PBS冲洗。 </li><li>传代细胞9。 </li><li>加10,000个细胞/ cm 2至孔中。 </li><li>增加媒体每孔100μl，并在37℃下孵育。 </li></ol></li><li> 三维细胞培养9 <ol><li>从1.3.3采用溶液，悬浮沉淀的细胞，以100万个细胞/ ml的预凝胶溶液中的浓度。 </li><li>迅速分配16微升预凝胶细胞悬液到96孔平板的每个孔中，以形成一个5〜50微米厚的水凝胶为三维细胞培养物。 </li><li>孵育在37℃，30分的凝胶形成。 </li><li>加入100微升的媒体到形成水凝胶的顶部，并在37℃孵育。 </li></ol></li><li> 在变刚度凝胶的三维细胞培养 <ol><li>从1.3.3使用溶液，吸管100微升的预凝胶溶液加入到96孔板的每个孔中。 </li><li>孵育在37℃下30分钟进行凝胶形成。 注意:交联从第2部分的步骤可在此使用。 </li><li>传代细胞9。 </li><li>加10,000个细胞/ cm 2至孔中。 </li><li>增加媒体每孔100μl，并在37℃下孵育。 </li></ol></li></ol>

<ol>
	<li>Lee, J., Cuddihy, M., Kotov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Three-Dimensional Cell Culture Matrices: State of the Art. 14, (2008).</li><li>Haycock, J. W. <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 Cell Culture. , (2011).</li><li>Walker, J. M., Ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Epithelial cell culture protocols. , (2012).</li><li>Badylak, S. F., Freytes, D. O., Gilbert, T. W. <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+as+a+biological+scaffold+material:+Structure+and+function.">Extracellular matrix as a biological scaffold material: Structure and function.</a> Acta Biomater. 5 (1), 1-13 (2009).</li><li>Koo, H. -. J., Lim, K. -. H., Jung, H. -. J., Park, E. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+evaluation+of+gardenia+extract,+geniposide+and+genipin.">Anti-inflammatory evaluation of gardenia extract, geniposide and genipin.</a> J. Ethnopharmacol. 103 (3), 496-500 (2006).</li><li>Jeffords, M. E., Wu, J., Shah, M., Hong, Y., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Material+Properties+of+Cardiac+Matrix+Hydrogels+to+Induce+Endothelial+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells.</a> ACS Appl. Mater. Interfaces. 7 (20), 11053-11061 (2015).</li><li>Suckow, M. A., Voytik-Harbin, S. L., Terril, L. A., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bone+regeneration+using+porcine+small+intestinal+submucosa.">Enhanced bone regeneration using porcine small intestinal submucosa.</a> J. Invest. Surg. 12 (5), 277-287 (1998).</li><li>Brown, B. N., Badylak, S. F. <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+as+an+inductive+scaffold+for+functional+tissue+reconstruction.">Extracellular matrix as an inductive scaffold for functional tissue reconstruction.</a> Transl. Res. J. Lab. Clin. Med. 163 (4), 268-285 (2014).</li><li>Pouliot, R. 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=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> J.Biomed.Mater.Res.A. , (2016).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., 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=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+initiation+and+growth+of+tumor+cell+lines,+cancer+stem+cells+and+biopsy+material+in+mice+using+basement+membrane+matrix+protein+(Cultrex+or+Matrigel)+co-injection.">Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.</a> Nat Protoc. 7 (6), 1138-1144 (2012).</li><li>Zhang, W. -. 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=The+reconstruction+of+lung+alveolus-like+structure+in+collagen-matrigel/microcapsules+scaffolds+in+vitro.">The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro.</a> J. Cell. Mol. Med. 15 (9), 1878-1886 (2011).</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 (9), 866-876 (2016).</li><li>Matsuda, 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=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., 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=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. D., 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=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

作者什么都没有透露。

之一的生物学的积分方面是自组织分子成执行特定任务层次结构。 13在实验室中，自组装取决于许多因素，如盐浓度，pH和消化时间。如图所示，自组织的水凝胶形式时溶解蛋白质返回到生理温度。形成的水凝胶是能够促进在体外的细胞附着和增殖的。 从ECM生物物理信号的细胞应答不能使用的聚合物进行研究。生物物理信号组织特异性细胞应答不能使用其他组织进行研究。再生研究人员和医生认为用脱细胞支架提高组织再生和伤口愈合。慢性肺病患者需要治疗的改进，以增加器官的可用性和降低免疫原性。器官特异性，细胞ECM互动必须是研究进一步器官生物学。 使用ECM作为用于肺细胞的细胞支架材料已经显示出一致的结果。 14,15从患病车型使用肺片鼓励疾病续期16干细胞或祖细胞，在远端肺中发现，17,18和需要特殊利基防止形成畸胎瘤。使用组织特异性的ECM作为递送媒介物，祖细胞的再生效果可以增强。该ECM水凝胶建立一个更适合的环境来评估细胞外基质细胞反应。 大量的研究机构存在看着僵硬，因为它涉及到细胞信号传导。我们的京尼平交联的方法允许细胞刚度信令以及细胞-ECM组件交互。而水凝胶的刚度可通过ECM量和蛋白质的比率被操纵，使用京尼平的允许有些独立的ECM含量的水凝胶的刚度变化。调整根亿品浓度可允许的机械性能的精确调整到最接近自然的肺组织6。使用来自外周肺水凝胶在本研究中获得的京尼平数据，一个15倍的增加（从5 Pa至75帕）被记录在案。加入1％京尼平外周肺水凝胶或增加0.01％，至全肺水凝胶（估计在此基础上，用同样的方法去细胞活检正常大鼠肺组织的流变数据，我们就可以实现近似的外周肺组织硬度（数据未显示） ）。使用周围型肺癌ECM由肺实质与我们先前公布的全肺ECM水凝胶的结果相比，7取得了相当柔软的凝胶。因此除了京尼平，选择肺组织的特定区域，以decellularize可以显着地影响刚性。 有改进这种方法，该方法可以包括在perfusi常用的附加步骤的机会在整个肺组织的脱细胞。此方法已被用于在正常的肺组织，并可能需要对患病的肺的脱细胞修饰。我们还没有研究特定疾病状态这一过程。我们得到我们从屠宰场运冰隔夜肺部。这种方法可能会限制我们的过程中因肺不张或血管闭塞的疗效;然而，我们很少有以除去尚未彻底脱细胞肺癌的领域。当获得新的肺组织，我们会建议让所有让肺部，以防止组织处理任何停顿前所需的解决方案。先前的研究已经发现，使用过乙酸作为最终杀菌工序到矩阵生产导致浸润免疫细胞，19潜在地改善去细胞的组织的proregenerative效果的再生M1巨噬细胞的表型。其他的研究已经进入使用其他清洁剂来完成这个过程，但那些APPEAR以成为实验室特定的喜好与最终的脱细胞同样的影响。脱细胞过程和消化步骤可以根据研究人员的特定应用进行优化。例如改变凝胶消化时间或中和方法可以改变它的蛋白质组合物，机械性能，或凝胶化动力学。 替代这里所讨论的方法可以在复杂程度取决于所需的实验的结果而改变。例如，如果目标是保持基底膜完好尽可能，用压力限制控制灌注泵可降低屏障的损坏。此外，如果一个人可用，以保持双肺完整体积较大的品种实验室设置中，脱细胞处理可提高疗效。相反，手磨用研钵和研杵技术可应用于冻干组织为不具有cryomill谁实验室。我们希望一方面折磨g至产生较大的片段，限制可用于酸消化的表面积，以便在消化时间可能需要在此情况下被改变。 替代品并不限定于脱细胞的步骤和形成水凝胶的。戊二醛已被用来作为水凝胶的交联剂，提供一种可能的替代方案增加用于模型系统中的生物凝胶的机械强度。其它模型系统包括完整脱细胞肺作为体外三维培养系统，或更常见的看着肺片作为模型三维体外的支架。 20我们已经提到，来自与聚合物支架，以及单一组分支架工作的限制，但这些仍尽可能替代这里提出的步骤。此外，还有一些其他方法来确定脱细胞的功效（如DNA分离），凝胶化（可以使用浊度测定法），和细胞附着一个第二扩散（如采用CCK8的）。我们发现，这里所描述的方法是绰绰有余表征凝胶用作模型的三维支架。 我们在这里描述的肺ECM水凝胶为研究细胞ECM互动的有效平台。该预凝胶溶液具有作为聚合物药物载体的涂层的潜力，在交货间质干细胞的协助，或由本身的药物递送平台。这里提出的协议提供了用于创建从肺ECM来源一个多功能水凝胶的基本步骤。

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

在这里，我们提出了体外肺细胞培养建立的多组分细胞培养水凝胶的方法。用健康途中从猪，大鼠，或小鼠集团肺组织开始，组织灌注并在随后的化学洗涤剂浸没以除去细胞碎片。处理之前和之后的组织的组织学比较证实除去双链DNA和α-半乳糖苷酶染色的95％以上表明大部分细胞碎片除去。脱细胞后，组织是冻干然后cryomilled成粉末。基质粉末在酸性胃蛋白酶消化溶液消化48小时，然后中和形成的预凝胶溶液中。该预凝胶溶液的凝胶化可通过温育在37℃下被诱导，并且可以立即使用以下中和或储存在4℃下长达两周。涂层可以用在未处理板c中的预凝胶溶液来形成埃尔附件。细胞可以悬浮在之前自组装，实现了3D培养的预凝胶，电镀形成的凝胶从该细胞可以通过在支架迁移，或镀在涂层的表面上。改变的策略提出可影响凝胶化温度，强度，或蛋白质片段的大小。以外的水凝胶的形成，所述水凝胶的刚度可使用京尼平可以增加。

使用这种方法，我们已经产生从正常猪，大鼠和小鼠的肺（ 图1）的水凝胶。处理肺提供一种估计分别5毫克，40毫克，和10g的ECM粉末。该过程的概要示于图2。在此过程中关键的可视化包括:清洗后脱氧胆肺的白色外观;该预凝胶形成后，溶液应是不透明的，如果保存在4℃的溶液应几个月出现均匀。我们已经包括了基本的故障排除表，以帮助确定在整个过程中（ 表2）错误源。胶凝后，水凝胶可立即使用或储存供日后使用。流变测试证实，该胶凝的超过90％，在第一分钟内达到37℃（ 图3）后发生。形成的水凝胶是在PBS中长时间，但细胞附着和proliferatio稳定n可以改变（ 图4）。 虽然该协议用于猪肺写的，空间的局限限制了我们脱细胞猪肺时只用一个肺。我们已经使用了相同的方法稍作修改decellularize其他物种两肺。对于大鼠和小鼠，肺部可原封不动和心脏附着，但结缔组织仍然应该用解剖刀或剪刀除去。我们保持心脏完整，并附上大鼠和小鼠，并通过右心室，而不是肺动脉灌注的解决方案。气管灌注仍然是相同的;然而，套管可以在气管为了便于较小物种访问的缝合。 我们增加了多种类型的细胞，骨髓间充质干细胞，小鼠9间充质干细胞，上皮细胞株（A549s），人原代微血管内切thelial细胞（HpuVECs），以及内和肺的ECM凝胶的表面上的人脐静脉上皮细胞（HUVEC）。我们没有遇到就形成凝胶的异种性质的问题。我们以前的工作已经表明在猪水凝胶在α半乳糖苷酶一显著降低。 9预凝胶溶液也可以被用作涂层在4℃下，以改善细胞的附着。当添加到肺ECM包被组织培养板的预凝胶解决方案提高HpuVEC附着和增殖。包被的孔与非处理的孔（ 图5）相比，演示了显著改善细胞附着。 添加京尼平增加水凝胶交联。该增加的交联可以提供更生理学相关的刚度，而减少ECM的降解。在降解的降低，虽然较小的体外 ，可能会发生因之间增加粘结蛋白质和ECM重塑因此更抗性细胞。我们用在流变仪频率扫描来测量交联的水凝胶的机械性能。水凝胶的刚度被发现直接相关京尼平浓度中位数和最相关的浓度为0.01％（ 图6，未示出天然组织）。 MTT测定法来定量上的交联水凝胶的细胞增殖和存活。细胞增殖是在交联的水凝胶（ 图7）更高。这支持来自其他增加的刚度增加的细胞增殖。 12变京尼平交联的时间或温度可能会导致不同的交联度。 <img alt="图1" src="/files/ftp_upload/55094/55094fig1.jpg" /> 图1:肺的照片，我们有脱细胞。从（A）小鼠肺图片decellulari后采样与心脏取出，后的Triton X-100冲洗掉，（B）的大鼠（C）的猪，初始漂洗之前;它们分别收率〜5毫克，40毫克，和10克隆的ECM。 （D）小鼠肺和完整心脏，脱细胞处理后，用套管仍缝合到位。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55094/55094fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55094/55094fig2.jpg" /> 图2:方法概述。这是产生从肺水凝胶的方法的概述。整块猪肺发生脱细胞离开该由ECM的蛋白质只有白色不透明或半透明的组织开始。然后除去任何剩余的水，并增加表面面积，以优化酸消化后的组织是溶解。交流的中和ID和增加盐生理水平创建预凝胶溶液准备凝胶化。一旦溶液形成，在37℃的水凝胶，表征发生用凝胶流变仪和扫描电子显微镜。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55094/55094fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55094/55094fig3.jpg" /> 图3:代表流变数据根据温度斜坡凝胶。后的初始振幅扫描，以确定线性粘弹范围内，温度斜坡来测定凝胶化动力学。数据表示为平均值+/-标准偏差。 N = 4。 <img alt="图4" src="/files/ftp_upload/55094/55094fig4.jpg" /> 图4:长期稳定性。一个IM储存在PBS中在室温下在6个月的猪的肺的ECM凝胶的年龄。 <img alt="图5" src="/files/ftp_upload/55094/55094fig5.jpg" /> 图5:HpuVECs的代谢测定。从生长在非治疗的组织培养板人类初级微血管内皮细胞（HpuVECs）MTT增殖数据。从1.3.3预凝胶溶液加入到板，冷藏过夜，然后漂洗，以形成预凝胶肺的ECM包被的板。相比，非涂覆HpuVECs显示对ECM包被的板更高的MTT活性。* P &lt;0.05与对照相比。数据为均值±标准差N = 3每组。 <img alt="图6" src="/files/ftp_upload/55094/55094fig6.jpg" /> 图6:流变数据以增强交联。流变数据，京尼平交联。与GENI的浓度增加了刚度增加引脚在1％京尼平高原。数据被归一化，以显示与京尼平交联一倍。 <img alt="图7" src="/files/ftp_upload/55094/55094fig7.jpg" /> 图7;在京尼平交联凝胶细胞增殖。对于A549s标准化MTT数据对京尼平交联水凝胶。增加的细胞增殖看出具有增加的刚度。 P &lt;0.05与对照相比。数据为均值±标准差。 N = 3每组。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 问题 </td><td> 问题 </td><td> 解析度 </td></tr><tr><td>红色/粉红色脱细胞后保留</td><td>脱细胞不力</td><td>新肺部重新开始</td></tr><tr><td></td><td></td><td>删除不完全脱细胞领域&lt;/ TD&gt; </tr><tr><td></td><td>血管闭塞</td><td>新肺部重新开始</td></tr><tr><td></td><td></td><td>删除不完全脱细胞领域</td></tr><tr><td>显著细胞碎片保留</td><td>脱细胞不力</td><td>新肺部重新开始</td></tr><tr><td></td><td></td><td>删除不完全脱细胞领域</td></tr><tr><td></td><td>血管闭塞</td><td>新肺部重新开始</td></tr><tr><td></td><td></td><td>删除不完全脱细胞领域</td></tr><tr><td></td><td>肺不张</td><td>新肺部重新开始</td></tr><tr><td></td><td></td><td>删除不完全脱细胞领域</td></tr><tr><td>膜的破坏组织样本中发现</td><td>灌注过程中血管的超压</td><td>减少灌注压清洁剂</td></tr><tr><td>大颗粒物仍是研磨后</td><td>在管太多的冻干材料</td><td>在研磨缸减少材料</td></tr><tr><td></td><td>不研磨足够长</td><td>增加球磨时间</td></tr><tr><td>预凝胶液不胶凝</td><td>溶液的pH值过高</td><td>使pH值7.4 </td></tr><tr><td></td><td></td><td>首先鲜粉预凝胶液</td></tr><tr><td></td><td>溶液的pH值过低</td><td>使pH值7.4 </td></tr><tr><td></td><td></td><td>首先鲜粉预凝胶液</td></tr><tr><td></td><td> ECM在消化</td><td>首先鲜粉预凝胶液</td></tr><tr><td>细胞不存活</td><td>盐酸消化后引入微生物</td><td>用鲜粉重新开始，并使用无菌过滤解决方案</td></tr><tr><td></td><td> ØpH值FF </td><td>重新开始预凝胶液</td></tr><tr><td></td><td></td><td>使pH值7.4 </td></tr><tr><td></td><td>细胞破裂</td><td>修正了预凝胶液盐含量</td></tr></tbody></table> 表2:故障排除指南，以确定潜在的误差源。

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

这是创建从肺的细胞外基质的三维细胞培养物的支架的方法。完好肺被加工成可支持细胞以三维生长的水凝胶。

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 ...

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.5K 次观看.  Virginia Commonwealth University. 这是创建从肺的细胞外基质的三维细胞培养物的支架的方法。完好肺被加工成可支持细胞以三维生长的水凝胶。 

视频: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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 particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

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 ...

科研

生物工程

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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.

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.

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 ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...