The Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canadian Institutes of Health Research (CIHR) have provided funding for this work. The authors would like to acknowledge Dr. Amin Rizkalla for the use of his electrospraying system, the Nanofabrication Facility at Western University for use of SEM imaging equipment, the Mount Brydges Abattoir for the provision of porcine tissue samples, and Drs. Aaron Grant, Brian Evans, and Robert Richards for their clinical collaborations in support of this research.

1.脱细胞组织处理<ol><li>脱细胞和冰冻干燥<ol><li>以下建立的协议的感兴趣组织脱细胞化（多个）。 注:支架在目前的研究已准备按照公开的脱细胞协议人类DAT 28，猪DDT 8，和猪DLV 32。市售的不溶性胶原，也可用于制造泡沫和微载体，例如胶原蛋白从牛腱，其已经成功地用于在10的浓度范围源 - 50毫克/毫升。其它不溶性胶原源可以被利用，但可能需要优化以识别的浓度范围内，这将产生稳定的支架。 </li><li>转移水合脱细胞化组织或纯化的胶原到50mL离心管中与钳，并添加足够的双蒸水（DDH 2 O）以浸没所述组织，叔的35毫升OA最大总体积。 </li><li>过夜冷冻样品在-80℃下，有冻随后冻干期间最大限度地为升华的表面积期间水平定位在所述离心管中。 </li><li>取下离心管盖和冷冻样本转移到冻干瓶中。 </li><li> 72小时或直至完全干燥 - 使用实验室冷冻干燥器48冻干样品。 注:干燥时间可以为不同的冻干机和脱细胞组织而变化。 </li><li>精细地剁碎冻干ECM成小块-使用锋利的外科剪刀（〜1 2 立方毫米）。 </li><li>存放在干燥器中碎ECM直到准备进行进一步的处理。 注:建议将切碎ECM可以立即使用，以防止对环境吸潮。 </li></ol></li><li>低温研磨（可选泡沫制造） <ol><li>填有25毫升不锈钢研磨室用于laboratoRY球磨机系统剁碎冻干ECM​​并添加两个10毫米的不锈钢研磨球。 </li><li>关闭和完全浸没在液氮中所装载的研磨室3分钟。 </li><li>磨机以30Hz（1800 rpm）的3分钟的冷冻样本。 </li><li>直到ECM磨成细粉重复步骤1.2.2和1.2.3。 注意:次这些步骤应重复可以ECM来源之间变化的数量。例如，DAT通常需要3个铣削循环，以产生细粉。 </li><li>转移使用scoopula到玻璃小瓶中的粉末，紧密地密封，并在干燥器中储存。 </li></ol></li><li> ECM悬浮液的制备<ol><li>称出250毫克或者剁碎（为泡沫）或低温研磨的（对于泡沫或微载体）的ECM并将其转移至15mL离心管中。分别指到步骤1.1.6和1.2节的制备切碎的ECM和低温研磨ECM的，。 注意:此协议被设计以制备50mg / mL的ECM suspen锡永，它被推荐用于人类DAT，猪DDT和猪DLV。这取决于特定的ECM源上，也可以在更高或更低的起始浓度来产生均匀的悬浮液。 </li><li>添加的0.22摩尔的NaH 2 PO 4（pH为5.4）缓冲液中至离心管中，并标记在管中的液位5毫升。 </li><li>通过加入7.5毫克α淀粉酶的1毫升0.22摩尔的NaH 2 PO 4（pH为5.4）缓冲液制备的α淀粉酶储液。添加α淀粉酶原液的100μL（0.75毫克α淀粉酶; 0.3％w / w的干的组织）到样品中。添加0.22摩尔的NaH 2 PO 4，得到10 mL的最终体积。 </li><li>以300rpm在室温下72个小时连续地搅拌该悬浮液。 </li><li>离心悬浮液在1500×g离心10分钟。小心收集并弃去上清液而不扰乱消化ECM颗粒。重悬在10毫升的5％的NaCl的沉淀材料在DDH 2 O稀释</li><li>总共两次漂洗，用5％的NaCl在DDH 2 O重复步骤1.3.5 </li><li>弃上清，重悬沉淀材料在10毫升的DDH 2 O的</li><li>连续搅拌该悬浮液以300rpm在室温下10分钟。 </li><li>离心悬浮液在1500×g离心10分钟。小心收集并弃去上清液而不扰乱消化ECM颗粒。 </li><li>添加0.2M的乙酸的5毫升标记在步骤1.3.2制成。 </li><li>连续搅拌该悬浮液以120rpm O / N在37℃。 </li><li>在均质化使用配备有锯齿10mm宽的探针，直到没有可见的碎片残留的手持均化器10秒的时间间隔在室温下将悬浮液ECM。放置在悬浮液的冷水均化间隔之间的烧杯以防止过热。 注意:根据所述ECM源上，在0.2M的乙酸进一步稀释，可能需要以获得均匀的悬浮液。 Ë所述ECM悬浮液xcessive冷却会导致粘度的增加可与有效均化干扰。如果粘度增加注意，可将样品复温至37℃，并进行进一步的处理。 </li><li>储存于4℃下之前泡沫或微载体制造长达一个月。 </li></ol></li></ol> 2. ECM-源性泡沫制造<ol><li>在37℃连续搅拌孵育从步骤1.3.13的ECM悬浮液（切碎或低温研磨）以120rpm，直到悬浮液是温暖的。 </li><li>稀释在0.2M乙酸的ECM酸性悬浮液至所需浓度。 注意:根据所述ECM来源的，稳定的泡沫体可以在10的范围内制备 - 100毫克/毫升，用15 - 50毫克/毫升，作为DAT，DDT和DLV的建议范围。通常，在较高浓度下制造的泡沫将稍微少多孔但在培养物中更稳定和更容易处理。 </li><li>使用3毫升注射器瓦特第i个18号针以防止在ECM悬浮气泡形成，填充与ECM悬浮液所需的模具。为了制造DAT，DDT，和DLV泡沫，分配400μL的35毫克/毫升悬浮液ECM到48孔细胞培养物处理的板。 注意:所选择的模具的形状和ECM悬浮液的体积，将决定所得到的泡沫的几何形状。 </li><li>覆盖，并通过将它们放置在一个-20℃或-80℃冰箱中过夜冷冻的模具。 注:冻结的温度可能会影响泡沫的孔隙率。当样品在-20℃下冷冻相比，-80℃下，由于较大的冰晶33的形成预计更大的孔。制造具有均匀的多孔结构的泡沫，确保模具不与导电表面，以防止定向冷却接触。 </li><li>将包含冷冻样本到冻干机烧瓶中的模具。冻干机瓶连接到实验室及其他实验室ORY冷冻干燥器系统和干燥24个小时。 注:重要的是，样品保持冻干之前冻结。 </li><li>存储冻干泡沫在干燥器中备用。 </li></ol> 通过电喷3. ECM来源的微载体制备注:建立电喷雾的概述示于图2。 <ol><li>与在100rpm O / N连续搅拌孵育从步骤1.3.13的低温研磨ECM悬浮液在37℃下。 注:低温研磨ECM被用于制造ECM来源的微载体，以确保在该悬浮液更均匀，并防止电喷射期间堵塞。 </li><li>负载3毫升ECM悬浮到3mL的路厄氏锁注射器的并附加有翼输注设置在注射器的孔中。 注:针规的范围，可以使用识别出选择可影响所得到的微载体的尺寸和形状。为了fabricatE中的DAT，DDT，以及微载体DLV，使用25号针。 </li><li>安全注射泵内的注射器。紧固针到铁架台，并在4:1的距离针尖垂直定位 - 6厘米从低形式杜瓦瓶的顶部（250 - 500推荐毫升大小范围）。 </li><li>附加弹簧夹电极到针的尖端并把它连接到所述高电压电源的正极端子。 </li><li>折叠铝箔（12×5厘米）的条带在真空瓶的边缘。 </li><li>附加一个第二弹簧夹电极箔的外边缘，并将其连接到电源的接地源极端子。 </li><li>用液氮杜瓦瓶填以从顶部大约1厘米，从而使3/4箔片被淹没。 注意:保持充满液氮的杜瓦瓶是很重要的。液氮的表面必须在电喷雾PROCES不大于从烧瓶的顶部2厘米少秒。液氮的连续再填充是必需的电喷射期间维持恒定的水平。 </li><li>设定注射器泵以30毫升/小时的输注速率。 注:改变输注速率可以改变微载体的形状和直径，但是推荐的范围是25 - 35毫升/小时。虽然它是高度地依赖于悬浮液的粘度，低于25毫升/小时的流速可能导致较大的微载体的产生。在非常低的流量，微载体的大小和形状可能变得不那么均匀。在输注速率高于35毫升/小时， 通过电喷雾产生的液滴的均匀性可能被破坏，从而导致非球形的微载体具有更宽的粒度分布34。 </li><li>应用20千伏的电压并打开注射泵启动电喷雾。 注:电压可以变化，以调节所述微载体的尺寸和趋向于对直径比infusio产生更大的影响氮肥用量。推荐的电压范围为15 - 25千伏。在微载体直径的减小预期的增加电压35。 </li><li>一旦输注完成后，小心地倒出从杜瓦过量液氮，留下悬浮在约25mL液体氮的微载体，以确保它们保持冷冻。 </li><li>立即以平滑的动作转移浇注用液氮到50mL离心管中，微载体。收集剩余的杜瓦用scoopula冰冻微载体，并将其添加到离心管中。 注意:根据所述杜瓦瓶的大小，在液氮中的微载体可以是最初注入中型容器中，然后立即转移到50毫升离心管中。 </li><li>覆盖包含在与在冻干制剂小孔穿孔铝箔液氮微载体的离心管中。 </li><li>将覆盖离心机管放入冻干器烧瓶中。立即连接到烧瓶冻干机和干燥样品O / N。 注:这是保持悬浮在液态氮，以确保他们没有这一步之前解冻微载体非常重要，因为融化可能导致崩溃和/或聚集。使用上述的电喷雾条件中的35毫克/毫升的浓度制成DAT，DDT和DLV微载体典型地将具有一种水合直径范围350之间 - 在大小为500μm。尺寸分布可以根据所述ECM来源的不同而不同。 </li><li>存储冻干微载体在干燥器中备用。 </li></ol><img alt="图2" src="/files/ftp_upload/55436/55436fig2.jpg" /> 图2.概述微载体制造中使用的电喷雾装置的。 一个 :图片示出密钥电喷雾设备，包括注射泵的布置和高压电源，以及相对于液氮的杜瓦针的定位。 B:电喷射原理图，包括用于电压，输注速率和距离在建议范围。 <a href="http://ecsource.jove.com/files/ftp_upload/55436/55436fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.制备泡沫和微载体用于细胞培养的<ol><li>补液<ol><li>转移用钳子冻干泡沫到含有过量的无水乙醇（〜99.9％）的50mL离心管中（约5:1的比例的乙醇，以泡沫）。类似地，重悬用于收集原始离心管内的过量的无水乙醇的冻干微载体。使用血清移液管，过滤器通过具有限定目尺寸不锈钢筛微载体进入一个新的50毫升离心管中，以去除任何聚集体并选择所需的尺寸范围。 注意:如果泡沫TCPS板内制成，它们可以直接这些血管内再水化。 </li><li>孵育在4℃下4个小时或直至泡沫或微载体具有重力沉降至离心管的底部。执行使用无菌的试剂和适当的无菌技术在层流罩的所有后续步骤。 </li><li>使用血清移液管除去无水乙醇，并添加过量（5:1的比例）的95％乙醇，用无菌磷酸盐缓冲盐水（PBS）稀释至支架。孵育在4℃下4个小时或直至ECM来源的支架已经沉没到补液容器的底部。 </li><li>通过乙醇系列（90，85，80，75，70，50，25和0％，用无菌PBS稀释）逐渐再水合支架。在每一步，在孵育4℃，直到支架具有改变溶液之前沉到容器的底部。脱气下光真空支架如果支架内形成气泡是OBServed。 </li><li>更换无菌PBS额外冲洗两次以去除残留的乙醇。 </li><li>储存在100％无菌PBS在4℃下，直到准备用于细胞培养。以下补液2个星期 - 使用1内的支架。 </li></ol></li><li>用于细胞接种制备<ol><li>上播种前一天，转移使用镊子的泡沫体成细胞培养物处理的孔板或transwell插入并添加足够的细胞培养基（基于感兴趣的细胞类型选择），以完全浸没的支架（ 例如，2.5毫升/支架中12孔平板）。类似地，允许微载体沉降重力离心管内，小心使用血清移液管，而不会干扰所述微载体移除PBS，并添加细胞培养基以达到5:介质的至微载体1的比例。 注:对于接种人类的ASC，利用Dulbecco改良的Eagle氏培养基（DMEM）:Ham氏F12营养混合物补充有10％胎牛血清（FBS）和1％青霉素 - 链霉素。 </li><li>总共一个漂洗的更换细胞培养基。 </li><li>平衡在细胞培养基中的支架过夜，在37℃。该支架将准备细胞接种的第二天。 注:泡沫可以静态地接种在细胞培养插入物或组织培养孔板29，或使用实验室摇动器36动态地接种。根据不同的ECM来源，加工方法和悬浮液浓度上，动态播种可提高初始细胞附着，增殖和浸润。微载体可以动态地使用旋涂器培养系统11接种。 </li></ol></li></ol>

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一般来说，从脱细胞组织来源的生物支架可以相比于基于二维TCPS合成的支架或标准培养模型更接近在天然细胞微环境的ECM的复杂的三维组合物和结构。如先前所讨论的，细胞-ECM相互作用是介导无论是在培养和在本体1中细胞行为是至关重要的。认识到ECM的生物化学，生物物理和生物力学特性是唯一的每个组织，有越来越多的证据，以支持生物材料组织工程的设计应用特定的组织方式，以及在更多的发展的理论基础生理学相关的文化模型， 在体外实验中 20。利用脱细胞组织为起始原料，我们的方法可以将更多的cust内的组织特异性细胞外基质的复合组合物omizable脚手架格式。而将导致天然ECM超微结构的损失的机械和酶处理步骤，与DAT先前的研究已经表明，所述脂肪来源的ECM的启发作用在这些支架格式是保守的，这表明所述生物支架组合物是关键介质的细胞功能11,29。以相比于完整脱细胞组织使用ECM来源的泡沫和微载体细胞培养基板A显著优点是，它们是更均匀，从而可以提高在细胞分布和细胞 - 细胞/细胞-ECM相互作用的均匀性。 这里描述的方法可以用于产生用于在细胞培养和组织工程应用中使用组织特异性的生物支架的宽阵列。例如，除了DAT，DDT和DLV，我们的实验室已成功地应用了这些技术来生成3D POROU中泡沫使用脱细胞的骨，软骨，髓核和纤维环，以及从牛腱衍生市售的不溶性胶原。来自体外的角度来看，这些生物支架可以作为一个基础，更高保真度的3D培养模型用于研究细胞生物学，生理学或疾病病理学38，如在高通量药物筛选平台39，或者用于干作为启发矩阵的生物活性底物细胞分化40,41。在25浓度制造DAT，DDT和DLV泡沫- 50 mg / mL的是在体外培养物（试验到28天）的长期稳定。此外，所有三种类型的微载体可以支持动态条件下的细胞附着和增殖在低剪切搅拌培养系统（10 - 15）离心至少2周。对于体内应用，生物相容的和biodegradaBLE ECM衍生的泡沫和微有希望作为现成的现成产品来刺激建设性的组织重构和再生11,29。此外，细胞粘附性的支架可作为细胞治疗递送系统42，43。作为一个例子，示出了泡沫DAT当与同种异基因的ASC接种和免疫活性大鼠模型29皮下植入促进血管生成和脂肪生成。相对于完整DAT，更加高度处理的DAT的泡沫降解更加迅速，随着体积减少50％3周注意，因为他们变得由12周宿主组织整合，和几乎完全吸收。然而，该泡沫体也诱导了更有效的血管生成反应，这表明酶消化ECM具有独特的促再生的效果。类似地，ECM来源的微载体可以用作体外&lt;/ em&gt;的动态培养系统内的细胞培养基材和可注射细胞递送载体11，30，44。更具体地，微载体的小直径和大的表面积可以使细胞大量的在小体积的递送，同时提供了基质，其可以有助于支持细胞活力和在注射30的部位增加细胞保留。在任何活的系统在使用之前，关键是要确保源ECM基本上没有抗原性的细胞组分和/或潜在的细胞毒性试剂脱细胞可能触发一个负宿主应答7。 该蛋白水解酶胃蛋白酶在ECM来源的水凝胶15的制备常用。胃蛋白酶是一种非特异的蛋白酶，将消化的胶原蛋白和其它ECM蛋白诠释o小碎片45。而从胃蛋白酶消化ECM制造水凝胶已被报道具有细胞-指令的影响，限制是这些材料往往是非常弱的机械46。在我们的微载体的DAT的初始发展，我们利用一种复合方法，其中胃蛋白酶消化的DAT与藻酸盐混合并滴加到CaCl 2的，以形成球形珠30。将珠随后的光交联和用柠檬酸钠萃取藻酸盐。除了用于化学交联的要求，关键限制是，使用这种方法制造的微载体具有低于900的尺寸范围稳定性差- 950微米30。代替胃蛋白酶，这里提出的方法使用的ECM的更温和的消化与糖酵解酶α淀粉酶，其被假定为从telope裂解碳水化合物基团胶原的ptide区域，由此在乙酸37增加溶解度。这种方法使可用于产生纯ECM来源的泡沫和微载体，而不需要化学交联或其它添加剂高度聚合的胶原的分离。这些生物支架是通过物理相互作用和氢键保存完好的胶原原纤维，类似于在天然ECM微环境中的胶原之间稳定。 该泡沫体是一种高度灵活的平台，可在宽范围内根据所选择的特定的几何形状的模具来制造。用于细胞培养研究中，泡沫体可直接在TCPS孔板施放，以形成涂层或不同厚度的3D支架。制造具有非常均匀的表面三维泡沫，则建议定制模具设计，可以在两侧用塑料或玻璃载玻片进行密封。或者切碎或低温研磨ECM可被用于合成泡沫。一般情况下，我们已经发现，低温研磨泡沫往往是宏观柔软，在低浓度31有多个被破坏的超微结构，36。根据不同的组织源上，另外的机械加工步骤可能会导致在ECM组合物可能影响细胞功能的改变。例如，在我们以前的工作，在碎DLV泡沫检测层粘连蛋白，但不能进行低温研磨DLV泡沫31。与此相反，I型胶原，IV型胶原，层粘连蛋白和纤连蛋白在两个切碎进行检测，并低温研磨DAT泡沫36。除了机械加工步骤中，泡沫的孔隙率和孔径可通过改变ECM悬浮液浓度和冷冻温度47被调谐到一定程度。一般来说，较低浓度的泡沫（〜10 - 15毫克/毫升）是定性更加多孔的，但是可以迅速地收缩并且具有差的稳定性在长期培养31,36。类似地，较慢的冷冻速率，典型地通过较高的冷冻温度来实现，可以导致在泡沫由于制造29过程中形成的冰晶体的尺寸更大的孔。所有这些参数可以影响细胞间的相互作用与材料，包括附件，浸润和重塑。例如，与较高的ECM浓度制造在泡沫细胞的生长可被限制到的表面区域，特别是与切碎ECM来源和静态培养条件下36。 对于微载体，其可被调谐的关键参数是ECM悬浮液浓度，针规，和施加电压，以较高的浓度典型地产生是长期动态培养下更稳定的微载体。继电喷雾的开始时，ECM suspens离子液滴应该迅速落入烧瓶的中心，朝着铝箔集电体的方向。为了防止聚集，所述珠子接触液氮箔片之前，它是重要的。针和液氮的表面之间的距离可以调整，以满足这些要求。需要注意的是优化可以根据每个特定的ECM来源的特性是需要的，特别是在选择将产生稳定的生物支架的浓度范围是重要的。另一个关键因素是用于生成起始原料，因为这会降低ECM或残留试剂（ 例如表面活性剂）的存在可以得到的泡沫体和微载体的稳定性产生负面影响脱细胞的方法脱细胞协议。如果挑战遇到了生物支架的稳定性，可以包括使用更渐进的过程补液进行调查选项，增加ECM停赛ension浓度，并探索与剁碎ECM低温研磨。如果所有这些选项不能解决问题，可能有必要探索其他脱细胞协议或ECM来源。 为了确保支架生产过程中的重复性，必须特别注意在协议的某些步骤服用。当低温研磨将脱细胞化组织中，则建议铣削在干燥环境中，以减少颗粒聚集的可能性冻干后立即由于湿气从环境吸收进行的。在微载体制造中，有人建议将悬浮液以小批量电喷雾，用3mL的最大体积，以避免与样品冷却问题，可能导致针的堵塞。此外，至关重要的是，微载体是不允许的电喷雾处理后解冻。为了保持其球面几何学和机械稳定性，在microcarriERS应该从液氮中被收集，以液体充满氮气的容器运送，并立即冷冻干燥。最后，对于泡沫和微载体都，这是至关重要的补液步骤历时多天的缓慢地进行。快速补液可以导致在宏观和/或微观尺度结构崩溃。此外，必须慢慢地发生再水合，以防止支架内的小气泡，这可能需要在光下真空的时间脱气一个显著量的形成。 总之，在本文提出的方法可用于制造由纯的，非化学交联的ECM的组织特异性泡沫和微载体的多样性阵列。用于生物研究的优点是，所述生物支架是易于处理和诸如以下的技术组织学，免疫组织化学，或基因和蛋白质表达分析进行分析时，可以类似地处理组织。此外，ECM来源的骨架可以是酶促降解来提取种子的细胞群或可直接用作生物可降解的和生物相容的细胞递送载体。总体而言，这一灵活的平台技术，拥有众多的应用，包括用于研究细胞功能的3D细胞培养的研究非常实用，如细胞扩增的基材，并作为促再生的生物支架。

细胞外基质（ECM）是由蛋白质，糖蛋白和多糖1的复杂的三维网络。一度被视为一个主要的结构框架，它现在公认的是，ECM结合的生物活性分子的多样性阵列与重要的功能性作用2。细胞-ECM相互作用可以直接细胞存活，粘附，迁移，增殖和分化3。而ECM大分子的主要类别通常是公跨组织和物种中是保守，每个组织具有独特的基质组合物和体系结构4。总体而言，组织特异性的ECM提供一种有益的微环境，从亚细胞到组织/器官秤5介导的功能。 由于ECM在介导细胞功能的重要作用，已经在德日益增长的兴趣在组织工程和再生医学中的应用ECM衍生的生物支架的velopment。特别地，去细胞化的方法已经被广泛探索作为从广泛范围的组织用作组织再生和细胞递送5，6，7的支架材料的获得ECM的一种手段。脱细胞通常涉及一系列的机械，化学和/或生物处理针对去除细胞和细胞组分的级，而理想地产生最小的改变到ECM 8的三维结构和组成。通过测量文献，各种脱细胞协议可以被识别为在本体7几乎所有组织中。 虽然去细胞的组织，可直接使用作为可植入支架或三维细胞培养基材，细胞浸润可在组织被限制具有致密结构的ECM 9。此外，在ECM中的天然异质性可能会导致在脱细胞基质，这可能潜在地影响的细胞反应10内细胞附着和分布的可变性。总体而言，虽然有希望用于一些应用中，它们的完整形式施加脱细胞组织提供在调谐脚手架特性方面包括形状，孔隙率，和刚度有限的通用性，以及输送的用于体内应用的模式。 为了克服这些限制，许多研究小组正在施加进一步的处理方法来生成使用脱细胞组织为母材的定制支架格式。在最简单的形式，这可能涉及低温研磨将脱细胞化组织来产生可注射的组织特异性的ECM颗粒11。这些ECM颗粒可被掺入作为细胞instru与其他生物材料复合支架，如原位交联的水凝胶12，13，14莫如组件。除了机械加工，脱细胞组织也可进行酶消化的蛋白水解和/或糖酵解酶以制造ECM来源的水凝胶，泡沫，微载体，和涂层15，16，17，以及以合成为3D打印bioinks 18。 除了组织工程应用，ECM衍生的生物支架保持在体外模型保真度更高的生物学研究产生巨大的潜力。有一个显著需要开发更好地概括了原生细胞微环境19的3D细胞培养系统。大多数体外 Ç迄今为止ELL培养研究是在组织培养聚苯乙烯（TCPS），其具有与活组织20内找到的生物复杂和动态的细胞环境几乎没有相关性进行的。同时方便对这些简化刚性2D衬底的受控环境中研究细胞相互作用，培养细胞改变细胞附着和形态，以及二者的细胞-细胞和细胞-ECM的相互作用的21，22。二维观察到TCPS蜂窝适应可影响调节多种细胞功能，包括存活，增殖，迁移和分化，提高在建模2D研究的相关性的问题， 在体内系统23细胞内信号通路。人们已经日益认识到细胞行为可以在2D很大变化与 3D系统24，以及生化和Bi与ECM omechanical信令是细胞功能25的关键介质。许多研究小组已经尝试通过与ECM组分如胶原，层粘连蛋白和纤连蛋白涂布TCPS克服建立2D系统的局限性。虽然这些策略可以改善细胞附着并可能改变细胞的反应，这些车型仍然受到其2D的配置，并不模拟天然ECM 26，27的复杂空间组织或生物化学有限公司。 我们的生物工程实验室一直有意ECM衍生的生物支架的发展为三维细胞培养和组织工程应用的基材。特别是，我们已经开创了使用脱细胞的脂肪组织（DAT）作为脚手架平台脂肪再生28。此外，我们已经建立了合成使用D- 3D微载体和多孔泡沫方法在与蛋白水解酶胃蛋白酶或糖酵解酶消化的α淀粉酶29，30，31。值得注意的是，我们已经在所有的这些支架格式，所述脂肪来源的ECM提供了用于在培养的人类脂肪来源的干细胞/基质细胞（ASCs）的成脂分化的诱导微证明。最近，我们扩展我们的制造方法，以生成从脱细胞左心室（DLV）α淀粉酶消化的猪三维多孔泡沫（脱细胞的方法改编自Wainwright的等人 32），并显示它们用于诱导早期心肌提供一个支持平台在人类心包脂肪衍生的ASC 31标记表达。 本文详细描述了用于合成纯粹衍生的非化学交联的三维多孔泡沫和微载体的方法的从α淀粉酶消化的ECM用作体外细胞培养基材的生物复杂的3D和用作生物材料用于组织再生。在理论上，可以使用含有高分子量的胶原ECM的任何来源作为这些技术的起始材料。为了证明这种方法的灵活性，该方法已被应用到产生的组织特异性的生物支架使用人DAT，脱细胞猪真皮组织（DDT）8，和猪DLV作为代表例。 图1提供了对ECM来源的泡沫和微载体制造过程的可视概况。 <img alt="图1" src="/files/ftp_upload/55436/55436fig1.jpg" /> 图1.组织特异性ECM来源的泡沫和微载体的生产方法的概述。 1.脱细胞组织中，制备按照既定减速lularization协议，可用于组织特异性ECM来源的生物支架制造。宏观图像示水合人类DAT的，猪DDT（如在2010年弗林28中所述制备）（如在Reing，JE， 等人描述的方法制备。2010 8），和猪DLV（如在Wainwright的等人描述的方法制备。2010 32 ），因为这可以用作起始材料的来源ECM代表性实例。标尺代表3厘米。 2.脱细胞组织被冷冻干燥，然后机械地3.切碎。标尺代表1厘米。 4.将切碎的ECM然后可以低温研磨，这是可选的泡沫制造中，但需要对微载体的合成。比例尺代表为3mm。 5.切碎或低温研磨ECM然后用α淀粉酶消化，并均化以形成均匀悬浮液ECM。比例尺代表1厘米。 <str翁&gt; 6a）中对于泡沫制造中，ECM的悬浮液转移到用户定义的模具中，冷冻，并冻干以产生具有良好限定的几何形状的多孔3D支架。比例尺代表1厘米。 图6b）对于微载体制造中，低温研磨ECM悬浮液被电喷雾到产生离散的微载体的球形。比例尺表示2毫米。 7.泡沫和微载体然后可以逐渐再水化并接种有细胞。代表性图像显示人的ASC（活细胞=绿色）接种到DAT泡沫（左）和DAT微（右）。标尺代表100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55436/55436fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>BioShop</td>
			<td>ACE222.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Alligator clip leads</td>
			<td>VWR</td>
			<td>&#160;470149-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-101</td>
			<td></td>
		</tr>
		<tr>
			<td>a-amylase&#160;</td>
			<td>Sigma</td>
			<td>101074694</td>
			<td>from Aspergillus aryzae</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>CPA225D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td>75004251</td>
			<td>With swinging bucket rotor for 15 and 50 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL)</td>
			<td>Sarstedt</td>
			<td>62.554.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>Sarstedt</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from bovine achilles tendon (insoluble)</td>
			<td>Sigma</td>
			<td>C9879&#160;</td>
			<td>Or similar insoluble collagen source; Can be used as an alternative to decellularized tissues to fabricate the foams and microcarriers&#160;</td>
		</tr>
		<tr>
			<td>Cryomilling system</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td>MM 400</td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Fisher Scientific</td>
			<td>8624426</td>
			<td>For lyophilized ECM and bioscaffold storage</td>
		</tr>
		<tr>
			<td>DMEM: F12 Hams</td>
			<td>Sigma</td>
			<td>D6421</td>
			<td>Used for proliferation media</td>
		</tr>
		<tr>
			<td>Dewar flask</td>
			<td>Fisher Scientific</td>
			<td>10-196-6</td>
			<td>Low form; volume range of 250 - 500 mL</td>
		</tr>
		<tr>
			<td>Double distilled water</td>
			<td></td>
			<td></td>
			<td>From Barnstead GenPure xCAD Water Purification System</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Wisent</td>
			<td>311425125</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM&#160;</td>
			<td></td>
			<td></td>
			<td>Isolated from human adipose tissue, porcine dermis or porcine myocardium, as described in Flynn et al. 2010, Reing et al. 2010, and Wainwright et al. 2010 (ref # 28, 8, 32)</td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Greenfield Specialty Alcohols Inc.</td>
			<td>P016EAAN</td>
			<td>Absolute</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>80150</td>
			<td>Used for proliferation media</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>VWR&#160;</td>
			<td>37-501-32</td>
			<td>For transferring the foams</td>
		</tr>
		<tr>
			<td>Freezer (-20 &#176;C)</td>
			<td>VWR</td>
			<td>97043-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer ( -80 &#176;C)</td>
			<td>Thermo Scientific</td>
			<td>EXF40086A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Fisher Scientific</td>
			<td>03-339-26D</td>
			<td>To store lyophilized cryomilled ECM</td>
		</tr>
		<tr>
			<td>Hand held homogenizer&#160;</td>
			<td>Fisher Scientific</td>
			<td>14-359-251</td>
			<td>Speed: 8000 - 30,000 RPM</td>
		</tr>
		<tr>
			<td>Homogenizer accessories: saw tooth bottom generator probes</td>
			<td>Fisher Scientific</td>
			<td>14-261-17</td>
			<td>10 X 95 mm</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>For electrospraying</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7750021</td>
			<td>FreeZone4.5</td>
		</tr>
		<tr>
			<td>Milling chamber</td>
			<td>Retsch</td>
			<td>02.462.0213</td>
			<td>25 mL volume recommended</td>
		</tr>
		<tr>
			<td>Milling balls</td>
			<td>VWR</td>
			<td>16003-606</td>
			<td>10 mm diameter, stainless steel recommended</td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>VWR</td>
			<td>C ABD305185</td>
			<td>For dispensing ECM suspension into moulds</td>
		</tr>
		<tr>
			<td>Orbital incubator shaker&#160;</td>
			<td>SciLogex</td>
			<td>832010089999</td>
			<td>Temperature controlled (37 &#176;C)</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Used for proliferation media</td>
		</tr>
		<tr>
			<td>Pipet-Aid XP</td>
			<td>Mandel Scientific</td>
			<td>DRU-4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort stand</td>
			<td>VWR</td>
			<td>470019-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort stand clamp</td>
			<td>VWR</td>
			<td>21573-606</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety-Lok Syringe</td>
			<td>BD</td>
			<td>309606</td>
			<td>3 mL luer lock syringe for microcarrier fabrication and dispensing ECM suspension</td>
		</tr>
		<tr>
			<td>Serological pipettes (10 mL )</td>
			<td>Sarstedt&#160;</td>
			<td>86.1254.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (25 mL)</td>
			<td>Sarstedt&#160;</td>
			<td>86.1685.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>BioShop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic&#160;</td>
			<td>BioShop</td>
			<td>10049-21-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoopula</td>
			<td>VWR</td>
			<td>89259-968</td>
			<td>For collecting microcarriers</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>VWR</td>
			<td>82027-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>VWR</td>
			<td>10117-490</td>
			<td>Microprocessor controlled</td>
		</tr>
		<tr>
			<td>High voltage power supply</td>
			<td>Gamma High Voltage Research</td>
			<td>ES30P-5W/DDPM</td>
			<td>Capable of recommended 15 - 25 kV voltage range</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Fisher Scientific</td>
			<td>12565321</td>
			<td>For use as molds during foam fabrication; Other sizes or user-defined molds can also be selected</td>
		</tr>
		<tr>
			<td>Winged infusion set</td>
			<td>Terumo</td>
			<td>22258092</td>
			<td>30 cm tubing length, 25 G 3/4 &#34; recommended; Other needle gauges can be used and may influence microcarrier diameter</td>
		</tr>
	</tbody>
</table>

fabrication of extracellular matrix-derived foams and microcarriers as tissue-specific cell culture and delivery platforms

细胞的功能是通过与胞外基质（ECM），其具有复杂的组织特异性的组成和结构的相互作用介导的。本文的重点是用于制造ECM来源的多孔泡沫体和微载体用作体外细胞培养物模型或作为促再生支架和细胞递送系统用于组织工程和再生医学的先进的三维生物相关底物的方法。使用脱细胞组织或纯化的不溶性胶原作为原料，该技术可以适用于合成具有可定制的几何形状的组织特异性的生物支架的宽阵列。该方法涉及的机械加工和温和酶消化以产生用于通过控制冷冻和冷冻干燥过程来制造三维泡沫或微载体的ECM悬浮液。这些纯ECM来源的支架是高度多孔的，但稳定的，而不需要CHEMIC人交联剂或其他添加剂可以细胞功能产生负面影响。该支架属性可以被调谐以通过改变因素在一定程度上，如ECM悬浮液浓度，机械加工的方法，或合成条件。一般地，支架是稳健的和易于处理，并且可以被处理为组织大多数标准生物测定法，提供一种多用途的且用户友好的三维细胞培养平台，该平台上模拟天然ECM组合物。总体而言，用于制造定制的ECM来源的泡沫材料和这些微载体直接的方法可以是感兴趣的两个生物学家和生物医学工程师作为体外和体内应用的组织特异性细胞-指令平台。

在目前的研究中，我们使用人DAT，猪DDT，和猪DLV作为代表例说明该技术可被应用以生成使用各种脱细胞组织作为ECM来源的（组织特异性的生物支架制造ECM来源的泡沫和微载体图1）。对于这两种泡沫和微载体制造，胶原蛋白增溶的与α淀粉酶37的糖酵解酶的西原技术被适于产生从所述脱细胞化组织的起始原料，它是用于合成通过控制冷冻和冻干过程的生物支架的粘性ECM悬浮液。 为了制造泡沫，去细胞的组织可以通过任何机械切碎或低温研磨，以增加前，酶消化的表面面积被处理。以下45;淀粉酶处理和均质化，将得到的ECM的悬浮液分配到一个用户定义的模具，然后将其冷冻并冻干。支架可以稳定地贮存在干燥状态下在延长的时间周期。在细胞培养研究在使用前，冻干的支架必须经受受控再水合过程能产生从纯ECM（ 图3A）衍生的多孔和高度水合的均质泡沫。如果泡沫过快复水，迅速肿胀，可能会损坏精致的结构特征，导致完整性和结构倒塌的损失。一般来说，泡沫材料将保持由原始模具以下补液限定的形状并且是稳定的，而不需要化学交联。 图3B 示出了DAT，DDT的代表性扫描电子显微镜（SEM）图像，和DLV的泡沫在35 mg / mL的浓度和-80＆＃冷冻温度与低温研磨ECM制造176℃。 <img alt="图3" src="/files/ftp_upload/55436/55436fig3.jpg" /> 图35毫克/毫升和冷冻的浓度的DAT，DDT，以及DLV泡沫制造成具有低温研磨ECM悬架3.代表性图像在-80℃下。 答 :DAT，DDT，和DLV的宏观视图研磨在以下补液48孔组织培养板中的模具的合成泡沫。标尺代表1厘米。 B:在DAT，DDT，和DLV泡沫的SEM图像示出一个均匀的多孔超微结构。标尺代表100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55436/55436fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的低温研磨ECM悬浮液也可用于产生通过电喷雾技术纯ECM来源的微载体（ 图2 </str翁&gt;）。对于每个ECM来源，悬浮液浓度，针规，输注速率，并且电压可以被调谐以产生离散的微载体球形范围从350 -下面的控制补液（ 图4A）为500μm的直径。虽然微载体可被存储在冻干状态时，建议为易于处理，它们被分配或筛分到所需的尺寸范围以下的乙醇再悬浮。 SEM成像表明微载体超微结构可根据所述去细胞的组织来源（ 图4B）的不同而不同。 <img alt="图4" src="/files/ftp_upload/55436/55436fig4.jpg" /> 图以35毫克/毫升，0.5毫升/分钟的输注速率，和20kV的施加电压的浓度的DAT，DDT，以及微载体DLV制造成具有低温研磨ECM悬架4.代表性图像。 答 :宏观查看OF中的水合DAT，DDT和DLV微载体。比例尺代表4毫米。 B:在DAT，DDT，以及微载体DLV表示超微结构和大小可以根据该ECM源而变化的SEM图像。标尺代表100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55436/55436fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 支架的机械性能是依赖于ECM源上，施加处理的方法（ 即，相对于切碎低温研磨）时，ECM悬浮液浓度，和冷冻温度。一般地，支架是柔软和适形，与杨氏在1的范围内的模量- 5千帕报道的DAT（50 100毫克/毫升）29和DLV泡沫（20 - 50毫克/毫升）31，和&lt;1千帕的微载体。的低温研磨的泡沫通常是较软的第由于其多个被破坏的大自然的碎泡沫。配备有高灵敏度的负载细胞专门的机械试验系统所需的压缩性能的精确的表征。典型地，所述泡沫体和微载体将有由于涉及制造包括酶消化和均化，以及所述支架的非共价交联性质的额外的处理步骤模量比天然脱细胞组织更低。然而，这可以根据所感兴趣的组织变化。例如，在先前的工作中，我们发现，剁碎DAT泡沫塑料在高浓度的ECM制造（100毫克/毫升）也有类似的模量对于天然脂肪组织29。 再水合ECM来源的泡沫和微载体可以与静态或动态培养条件下细胞接种到用于体外细胞培养研究和/ O提供一个电池支持的平台中的 R 体内细胞递送。虽然支架通常支持细胞附着，接种效率将取决于ECM源，所述支架的几何形状和孔隙率，和所述感兴趣的细胞类型上。这样，接种方法和密度将根据用户定义的条件需要优化。对于上述的泡沫，我们推荐的起始细胞浓度在0.25的范围内- 1×10 6个细胞/支架，具有动态播种在轨道摇床上以增强细胞浸润。对于微载体，我们建议在25000个的范围内的初始接种密度- 50,000个细胞的微载体/毫克，与在旋转培养瓶中11动态条件下进行播种。在图1的底部示出的图像表示人类的ASC上的DAT泡沫（左）或DAT微载体接种（右，预先标记与胺-反应性染料和蓝色出现13），通过共聚焦显微可视化作为镜检查使用荧光细胞存活力染色（活细胞=绿色，死细胞=红色;比例尺代表100微米）。共焦显微镜可以被用于可视化接种泡沫的表面上高达2毫米的厚度细胞，但可视化到支架的中央区域是由它们的不透明性质的限制。然而，泡沫体和微载体可以被视为组织和石蜡包埋或冷冻切片用于组织学和免疫组织化学分析以显现细胞分布和标志物表达。

Watch this Scientific Journal Video about Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms at JoVE.com

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

组织特异性细胞外基质（ECM）是细胞功能的关键介质。本文介绍了合成纯ECM衍生的泡沫，并且在培养中稳定，而不需要化学交联在体外细胞培养模型在先进的3D应用程序或为促再生的生物支架的微载体的方法。

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 ...

fabrication-extracellular-matrix-derived-foams-microcarriers-as

Research

JoVE Journal

Bioengineering

13.3K 次观看.  The University of Western Ontario. 组织特异性细胞外基质（ECM）是细胞功能的关键介质。本文介绍了合成纯ECM衍生的泡沫，并且在培养中稳定，而不需要化学交联在体外细胞培养模型在先进的3D应用程序或为促再生的生物支架的微载体的方法。 

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

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 studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

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 tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

胎儿和成人心脏组织外植体作为三维体外研究平台的可比较去角质

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

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 ...