This work was funded by the National Science Foundation (CBET 0845754, AHH).

1.准备海藻酸钠光盘的形成<ol><li>制备4％（重量/体积）在1×Dulbecco氏磷酸盐缓冲盐水藻酸盐溶液没有在37℃水浴中加入氯化钙或氯化镁和地点。藻酸盐溶液的浓度可以不同，但​​1 - 4％（重量/体积）藻酸盐溶液推荐使用。 </li><li>混合以1:1的比率的藻酸盐溶液:1用温细胞培养基基为所需的细胞类型（ 例如 ，Dulbecco氏改良的Eagle培养基）。现在藻酸盐的浓度为原始浓度的一半， 即 ，2％（重量/体积）。用无菌0.2微米的尼龙注射器过滤器消毒海藻酸钠/媒体解决方案。 </li><li>制备无菌102 1mM氯化钙二水合物的浴在无菌水中有足够的溶液淹没模具（〜200 - 300毫升）中。 </li><li>收集无菌"凝胶形成模子"（直径6毫米×3毫米高的圆柱形孔中3在铝基板×3，看<strong&gt;图1）和端板（底:一种3在铝板×3，上图:在铝板×3 1 1.5）和如下制备模具: <ol><li>切割厚滤纸（吸墨纸滤纸）和细胞微筛膜（10微米孔径），以在顶部和底部端板的尺寸，并将其放置在氯化钙浴中直到饱和的，大约为1分钟。如果需要的话，灭菌通过高压釜或紫外光的厚滤纸和微筛膜为在使用前30分钟。 </li><li>设置在模具构建体的一半（下半部分）。 <ol><li>放置以下项顶上彼此按下列顺序，并使用无菌刮刀平滑:大板（3×3英寸），厚滤纸，然后在微筛膜。 </li><li>倒置并按模具到纸巾的叠层，以确保过量氯化钙溶液的损失。 </li><li>放置"凝胶形成模子"与圆柱孔对C的顶部ELL微筛膜，并轻轻两侧使用左侧和右侧长尾夹固定在一起的模具。确保（在×3 1.5）留下足够的空间顶部端板后完全覆盖井。 </li></ol></li><li>设置在模具构建体的第二半（上半部分）。 <ol><li>将以下物品之上彼此以下顺序和平滑:小终板，厚滤纸，微筛膜。 </li><li>倒置并按下此半模具的上纸巾的叠层，以确保过量氯化钙溶液的损失。不要拧紧这一半使用长尾夹此时的模具。 </li></ol></li></ol></li><li>培养细胞根据制造商的说明建议。收获所需的细胞分层海藻酸钠水凝胶嵌入。推荐的细胞密度为1 - 2×10 6个细胞/ ml，但可以根据所期望的实验而变化。 注意:使用此方法用于与不同类型的细胞层，确保文化两类细胞并行的层次感。间充质干细胞（MSCs）接种1×10 6个细胞的细胞密度/ ml的将被用作用于以下方案的例子。细胞数量和盘片号码可以按需要被缩放为实验。 <ol><li> 5000细胞/ cm 2在10毫升基础生长培养基（Dulbecco氏改良的Eagle培养基，10％胎牛血清，2％L-谷氨酰胺，1 -在3000的细胞密度嵌入，种子间质干细胞前一到两周％非必需氨基酸，1％青霉素/链霉素）中的T-75烧瓶中。 </li><li>移除用过的培养基，每2 - 3天用10 ml基础生长培养基替换直至细胞是80 - 90％汇合。 </li><li>收获细胞，取出用过的培养基，和吸管3毫升1×Dulbecco氏磷酸盐缓冲盐水的不加氯化钙或氯化镁冲洗细胞。删除此解决方案，吸管3毫升0.25％的尝试PSIN-EDTA到在T-75瓶中生长的细胞，并在37℃下孵育5分钟。后细胞已经从粘附表面抬起，添加6 - 8 ml基础生长培养基。 </li><li>离心机的MSC在600 xg离心在室温下5分钟，吸出上清液，并在1重悬 - 5 ml基础生长培养基。使用计数使用设备指令的血球细胞。 </li><li>从总细胞悬浮除去1×10 6个细胞，使用相同的条件下离心，并吸出上清液。 </li></ol></li></ol> 2.细胞海藻晶种光盘的形成<ol><li>重悬用1ml无菌藻酸盐/介质溶液的细胞沉淀，以达到1×10 6个细胞/ ml的细胞密度。细胞 - 海藻酸钠混合物将是均匀混浊的外观，当细胞的适当混合。 </li><li>吸移管130微升细胞 - 藻酸盐混合物的成六个直径6毫米×3毫米高孔在模具的下半部分构造滴加以免产生任何气泡。轻微凸弯月形应该是每个边缘上方可见很好。 </li><li>使用无菌刮刀仔细平滑模具构建体的上半部分和翻面，从而使细胞微筛膜是在井的顶部。将模具构造上的孔的顶部，确保以覆盖含有细胞和完全藻酸盐混合物的孔中。 </li><li>解除装载模具构建体和，同时用力压下的中心，粘合剂夹剩下的两个边（顶部和底部）来固定模具构建体的顶部和底部两半。细胞 - 藻酸盐溶液应牢固坐落在此时的孔中。 </li><li>沉浸在102毫氯化钙浴中的固定模结构，确保整个构建体淹没。孵育在室温下细胞培养罩90分钟。 </li><li>在的90分钟结束时，请从102毫米氯化钙模具结构浴和地点纸巾在细胞培养罩堆栈上。除去所有四个长尾夹和模具结构的两半分开。形成在井中水凝胶不应该有任何气泡，并应完全填满孔中。 </li><li>用锅铲，仔细追查含有水凝胶仔细放松和楔形出来的水凝胶孔的边缘。从模具构建体除去水凝胶后，丢弃水凝胶直接进入1X Dulbecco氏磷酸盐缓冲盐水的用氯化钙和氯化镁的浴中。完全覆盖凝胶洗掉过量氯化钙溶液进行1 - 5分钟。 </li><li>转移的水凝胶成期望的盘基底的生长培养基溶液（ 例如 ，6孔板），该完全覆盖凝胶。孵育在37℃下在细胞培养孵化器至少1小时，5％CO 2继续成层步骤之前。 注意:含有细胞的水凝胶，可以保持在此时无限期细胞培养培养箱直到与另一细胞群体的凝胶的分层是期望的，提供该媒体的变化，每2完成 - 3天。 </li></ol> 3.海藻酸钠光盘的分层<ol><li>在1小时培养的最后半小时，收集和退火凝胶准备无菌模具及解决方案。 <ol><li>通过以x具有孔的一半"凝胶形成模具"的高度的（直径6毫米×1.5毫米高孔中3×3在铝板）的模具紧固到一个端板（3准备了"切割模具"在铝板3）用的左，右两侧的粘合剂片段。 </li><li>通过紧固的模具为3的直径比"凝胶形成模具"较大毫米x中准备了"退火模具"（9毫米直径×3毫米高孔中3×3在铝盘），一个端板（3在铝板3）用的左，右两侧的粘合剂片段。 </li><li>制备的溶液中的100mM柠檬酸钠/ 30毫摩尔EDTA（柠檬酸钠二水合物，乙二胺四乙酸四钠盐二水合物）在无菌水中。 </li></ol></li><li>除去所需的细胞群的凝胶（在这个例子中，两个圆盘具有的hMSCs）从在盘媒体和放置到"切割模具"井。每个凝胶应紧贴入孔与模具上方突出的凝胶的一半。 </li><li>使用手术刀，片沿着所述模具的表面的凝胶（这将减少一半的水凝胶）。翻转凝胶的上半部分，将其放入一个敞开的模具很好。凝胶的一半还应该紧贴到模具，但是现在两个半凝胶应与切内表面可见模具的高度。与第二凝胶重复。 注:警告:只有内切表面会形成多层光盘。使用的外表面将导致半部分离。它被怀疑从转印到凝胶表面防止微筛膜纹理退火过程的成功。 </li><li>放置在孔的上方的一块干细胞微筛膜，确保微筛膜是在与所有的凝胶半部的接触，进行退火处理，并覆盖它们完全。放置在微筛膜顶部厚滤纸，确保完全覆盖凝胶。 </li><li>直到它被饱和吸取的100mM柠檬酸钠/ 30毫摩尔EDTA（柠檬酸钠二水合物，乙二胺四乙酸四钠盐二水合物）的到厚滤纸的溶液。约750微升足以用于四口井。 </li><li>孵育凝胶在室温下1分钟。然后，取出细胞微筛膜厚滤纸丢弃它们。除去粘结剂剪辑和打开模具。 </li><li>对于每个退火凝胶，水凝胶的一种柠檬酸钠/ EDTA处理的一半转移到准备"退火模子"用切面朝上。这将是退火常数的二分之一构作。 </li><li>使用刮刀，升降机和放置柠檬酸第二钠/ EDTA处理的半凝胶（可以含有不同的细胞类型）已经在"退火模具"凝胶上，翻转这个第二半凝胶，使得切割表面是在与凝胶的切割表面接触已经处于"退火模具"。 </li><li>按用抹刀两个凝胶之间消除任何气泡两种凝胶轻轻放下。另外，根据需要，以确保它们是直接在彼此的顶部重新定位凝胶。 </li><li>轻轻提起"退火模具"和淹没它在102毫氯化钙浴中30分钟。切勿用端板的井。 </li><li> 30分钟的孵育后，去掉"退火模子"，并将其放置到纸巾。去除粘合剂的剪辑和模具从端板分开。 </li><li>用刮刀收集退火凝胶并将它们放入一个1X贝科的磷酸盐缓冲液与氯化钙和Magnesium氯化物浴洗凝胶。接着，转移退火水凝胶到细胞培养基中的所需盘（ 例如 ，6孔板）进行培养。 </li></ol>

<ol>
	<li>Sophia Fox, A. J., Bedi, A., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Basic+Science+of+Articular+Cartilage.">The Basic Science of Articular Cartilage.</a> Sports Health. 1 (6), 461-468 (2009).</li><li>Neidlinger-Wilke, C., 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=Mechanical+loading+of+the+intervertebral+disc:+from+the+macroscopic+to+the+cellular+level.">Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level.</a> Eur. Spine J. 23 (3), 333-343 (2013).</li><li>Klein, T. 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=Tissue+engineering+of+stratified+articular+cartilage+from+chondrocyte+subpopulations.">Tissue engineering of stratified articular cartilage from chondrocyte subpopulations.</a> Osteoarth. Cartilage. 11 (8), 595-602 (2003).</li><li>Han, E., 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=Scaffold-free+Grafts+for+Articular+Cartilage+Defects.">Scaffold-free Grafts for Articular Cartilage Defects.</a> Clin. Orthop. Relat. R. 466 (8), 1912-1920 (2008).</li><li>Ng, K. W., Ateshian, G. A., Hung, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zonal+chondrocytes+seeded+in+a+layered+agarose+hydrogel+create+engineered+cartilage+with+depth-dependent+cellular+and+mechanical+inhomogeneity.">Zonal chondrocytes seeded in a layered agarose hydrogel create engineered cartilage with depth-dependent cellular and mechanical inhomogeneity.</a> Tissue Eng. A. 15 (9), 2315-2324 (2009).</li><li>Ng, K. 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=A+layered+agarose+approach+to+fabricate+depth-dependent+inhomogeneity+in+chondrocyte-seeded+constructs.">A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-seeded constructs.</a> J. Orthop. Res. 23 (1), 134-141 (2005).</li><li>Nguyen, L. H., Kudva, A. K., Saxena, N. S., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+articular+cartilage+with+spatially-varying+matrix+composition+and+mechanical+properties+from+a+single+stem+cell+population+using+a+multi-layered+hydrogel.">Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel.</a> Biomaterials. 32 (29), 6946-6952 (2011).</li><li>Leone, G., 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=Continuous+multilayered+composite+hydrogel+as+osteochondral+substitute.">Continuous multilayered composite hydrogel as osteochondral substitute.</a> J. Biomed. Mater. Res. A. 103 (8), 2521-2530 (2015).</li><li>Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+catalyze+cartilage+formation+by+neonatal+articular+chondrocytes+in+3D+biomimetic+hydrogels.">Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels.</a> Sci. Rep. 3, (2013).</li><li>Vigf&#250;sd&#242;ttir, &. #. 1. 9. 3. ;. T., Pasrija, C., Thakore, P. I., Schmidt, R. B., Hsieh, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Pericellular+Matrix+in+Mesenchymal+Stem+Cell+Deformation+during+Chondrogenic+Differentiation.">Role of Pericellular Matrix in Mesenchymal Stem Cell Deformation during Chondrogenic Differentiation.</a> Cell. Mol. Bioeng. 3 (4), 387-397 (2010).</li><li>Lee, C. S. D., Gleghorn, J. P., Won Choi, N., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+layered+chondrocyte-seeded+alginate+hydrogel+scaffolds.">Integration of layered chondrocyte-seeded alginate hydrogel scaffolds.</a> Biomaterials. 28 (19), 2987-2993 (2007).</li><li>Gleghorn, J. P., Lee, C. S. D., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85 (3), 611-618 (2008).</li><li>Gharravi, A. M., Orazizadeh, M., Ansari-Asl, K., Banoni, S., Izadi, S., Hashemitabar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Fabrication+of+Anatomical+Bioreactor+Systems+Containing+Alginate+Scaffolds+for+Cartilage+Tissue+Engineering.">Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering.</a> Avicenna J. Med. Biotechnol. 4 (2), 65-74 (2012).</li><li>Xu, 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=Chondrogenic+Differentiation+of+Human+Mesenchymal+Stem+Cells+in+Three-Dimensional+Alginate+Gels.">Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Three-Dimensional Alginate Gels.</a> Tissue Eng. A. 14 (5), 667-680 (2008).</li><li>Yeatts, A. B., Gordon, C. N., Fisher, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+an+aggregated+alginate+construct+in+a+tubular+perfusion+system.">Formation of an aggregated alginate construct in a tubular perfusion system.</a> Tissue Eng. C, Methods. 17 (12), 1171-1178 (2011).</li></ol>

The authors have nothing to disclose.

在这里，我们描述了用于形成层状藻酸盐水凝胶的光盘用于研究多种细胞群，例如在生理学层状组织，例如软骨的共培养物的协议。层状结构，如所描述的培养平台，可用于检查进行同样的培养环境或在负载下两种不同的细胞群之间的相互影响。 藻酸盐是已被发现是生物相容的，并已成功地用于软骨生物学和组织工程研究12,14的阴离子直链多糖。藻酸盐水凝胶?...

压缩载荷轴承组织如关节软骨或椎间盘组成是在组织都生物力学功能和适当的机械性转导临界异质组织区域。不仅是蜂窝组织和功能在不同的区域不同，但细胞外基质（ECM）是在组合物和组织也变化。例如，关节软骨由具有不同细胞形态，机械功能，和ECM三个主要区域。在他们的ECM导致差承载责任的差异;表面层主要涉及拉伸响应负载，而中间和深区是主要负责为响应于压缩1。同样，在椎间盘，凝胶状髓核由层状纤维环包围，这两个不同的区域中的细胞经历不同类型的生物物理刺激2。在这些类型的组织层内的组织，细胞和细胞外基质的彼此交互的组织经受并响应机械力。 这种多相组织结构的概括保持在组织工程和再生医学的一个挑战，并提供了它们的生物学意义的理解是有限的。有必要培养平台对于一个构建体中分析分层组织以及细胞的不同群体的共培养物。在关节软骨组织工程，支架少层状结构已通过利用带状软骨细胞以沉积不同的ECM来模仿这种组织3,4的不同层的能力构成。然而，层状凝胶结构提供用于研究不同类型的细胞群缺少独立形成一个坚固的组织的能力的相互作用的机会。例如，不同的流行间质干细胞的ulations可分层结构内共培养。这类层状支架已被用于与两个软骨细胞和改进组织工程5分化间质干细胞。不仅可以在不同的细胞群进行共培养在类似凝胶的层，但单一细胞类型也可已操纵以具有变化的刚度或生化内容引发从细胞6,7-不同反应层内培养。 许多不同的生物材料的水凝胶已被用于层软骨组织工程细胞群体，如使用聚乙二醇或聚乙烯醇碱基7-9的那些。然而，藻水凝胶是从中创建层状支架为在共培养研究异质细胞群体的最简单的生物材料中的一个。同时也容易形成琼脂糖凝胶，藻水凝胶具有允许容易是额外的好处从三维构建为单个细胞的分析的细胞olation如前面10所描述。在以前的研究中，已形成在薄片的双层藻酸盐水凝胶和从这些片，切片切片（ 例如 ，使用活检冲）为特定的应用，如用于生化内容或界面剪切性能11,12分析。用于形成薄藻片另一种方法，已与潜在描述为分层成多层，但仍然需要改变，以机械测试13的水凝胶的使用。 在这里，我们提出在细胞共培养不同人群可重复创建双层海藻酸钠水凝胶光盘中使用的方法。这海藻酸钠盘平台具有几个优点。为主，可重现的形状和尺寸小，有利于为嵌入式细胞机械刺激，而不需要活检​​普NCH​​或其它物理变化，以水凝胶对于许多应用。此外，细胞存活率仍然在分层过程高，并且后形成凝胶的凝胶中的两个细胞群的一个明确的分离是没有初始重叠区域可见。

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Alginic Acid sodium salt</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">A1112</td>
			<td style="width:295px;">Solution made in wt% using DPBS (-/-)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (-/-)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (+/+)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11965</td>
			<td>Example. Use desired medium type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Syringe Filters (0.02um Nylon)</td>
			<td style="width:180px;">FisherBrand</td>
			<td style="width:119px;">0979C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Calcium Chloride Dihydrate</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP510</td>
			<td>Prepare solution in sterile water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Criterion Blotter Filter Paper</td>
			<td style="width:180px;">Biorad</td>
			<td style="width:119px;">1704085</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cell Microsieve Membrane (10um pore size)</td>
			<td style="width:180px;">Biodesign Inc of New York</td>
			<td style="width:119px;">N10R</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">0.25% Trypsin-EDTA (1x), phenol red</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25200</td>
			<td>Thaw in 37&#7506;C water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium citrate dihydrate</td>
			<td style="width:180px;">FisherScience</td>
			<td style="width:119px;">S93364</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td>
			<td style="width:180px;">FisherBioreagent</td>
			<td style="width:119px;">BP121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum&#160;</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">26140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicilin/Streptomycin (10000 U/ml)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">15140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-Glutamine (200mM)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25030081</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Non-essential Amino Acids (100x)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11140050</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
	</tbody>
</table>

layered alginate constructs: a platform for co-culture of heterogeneous cell populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

图1描绘了藻酸盐水凝胶的形成和分层。 如图3完成的双层凝胶中所示嵌入这些水凝胶和层状保持为高，并相媲美的散装水凝胶内的人类间充质干细胞的图2。细胞活性）表现出的细胞群的一个完整的初步分离。生存力评估退火后，切片凝胶垂直访问中心，然后染色在退火凝胶活细胞与活细胞追踪器，CMFDA（绿色）和死细胞（红色）乙锭...

Watch this Scientific Journal Video about Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations at JoVE.com

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

layered-alginate-constructs-platform-for-co-culture-heterogeneous

Research

JoVE Journal

Bioengineering

7.8K 次观看.  University of Maryland. Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct. 

视频: Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

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 ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程:建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.