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>

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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><tbody><tr><td>Alginic Acid sodium salt</td><td>Sigma Aldrich</td><td>A1112</td><td>Solution made in wt% using DPBS (-/-)</td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (-/-)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (+/+)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11965</td><td>Example. Use desired medium type</td></tr><tr><td>Syringe Filters (0.02 &amp;#956;m Nylon)</td><td>FisherBrand</td><td>0979C</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Fisher Bioreagents</td><td>BP510</td><td>Prepare solution in sterile water</td></tr><tr><td>Criterion Blotter Filter Paper</td><td>Biorad</td><td>1704085</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Cell Microsieve Membrane (10 &amp;#956;m pore size)</td><td>Biodesign Inc of New York</td><td>N10R</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Sodium citrate dihydrate</td><td>FisherScience</td><td>S93364</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td><td>FisherBioreagent</td><td>BP121</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco Life Technologies&amp;nbsp;</td><td>26140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Penicilin/Streptomycin (10,000 U/ml)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>15140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>25030081</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Non-essential Amino Acids (100x)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>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

Universidad de Cartagena UDEC

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

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

发育生物学

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

医学

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, ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

微凝胶-细胞外基质复合材料支持人类神经结构的嵌入式3D打印

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...