这项工作是由NHMRC项目资助＃568969（PSS）和医学院，新南威尔士大学，干细胞的倡议。（KSS）支持。

下面所有的程序进行了II级生物安全柜内部使用无菌技术。在下表中列出所用的试剂和设备。 1。 1.1％的海藻酸钠的制备（W / V） <ol><li>加入纯化的海藻酸钠0.275克（高葡萄糖醛酸含量≥60％，粘度&gt; 200 MPa级小号，内毒素≤100 EU / G）在无菌的50毫升管，并添加25毫升无菌0.1％的明胶溶液，提前准备（0.5 gelatin/500毫升的Milli-Q H 2 O和由蒸压溶解）。 </li><li>涡管约30秒，部分溶于海藻酸钠粉，然后将其放置在10 XG过夜轨道混频器管（室温）。 </li><li>新增的9％无菌氯化钠（4.5克）NaCl/50毫升的Milli-Q H 2 O 2.778毫升至25毫升海藻酸钠溶液。涡管约30秒，随后5分钟离心95 XG。 </li><li>存储海藻酸钠溶液在4°，C为短期存储（1-2月），或在-20°C长期存放（约一年）。 </li></ol> 2。制备氯化钙沉淀浴<ol><li>在1公升的Milli-Q H 2 O溶解14.7克氯化钙2·2H 2 O和2.38肝素钠Ğ </li><li>调整pH值至7.4。 </li><li>使用0.22微米的过滤消毒的解决方案。 </li><li>可存放在室温（6-12个月）降水浴。 </li></ol> 3。解压缩解决方案的准备<ol><li>在500毫升杜尔贝科磷酸盐缓冲液（D - PBS），0.5M EDTA的添加50毫升和5毫升1M肝素钠。 </li><li>消毒用0.22微米的过滤器或釜，在121°C间为20分钟。 </li><li>储存在室温（6-12个月）的解压缩解决方案。 </li></ol> 4。制备血浆置换（SR）中等<ol><li>封装之前，提前准备为德SR介质scribed表在特定的试剂和设备。 </li></ol> 5。 ROCK抑制剂的制备（Y 27632） <ol><li> Y型27632粉稀释在0.1％的人血清白蛋白（HAS）的D-PBS使一个5毫米的解决方案。 </li></ol> 6。人类胚胎干细胞制备的封装<ol><li>治疗前与补充两个小时，在37℃（避光）岩石抑制剂（RI）与10微米的培养媒体的细胞。 </li><li>国际扶轮治疗后，的D-PBS洗细胞两次，并打跑的细胞培养板，10分钟accutase酶在37°C </li><li>用吸管或细胞刮刀轻轻刮去细胞，并收集了15毫升管。抵消accutase SR 1:1的比例中等。 </li><li>要准备一个单细胞悬液，使用40微米过滤器过滤瓦解细胞和收集在一个新的50 mL离心管。 </li><li>计算解决方案在使用血球细胞的总数。 </李&gt; <li>细胞悬液，离心95 XG为5分钟和精心弃上清事后。预热的0.9％氯化钠冲洗细胞。 95 XG和离心5分钟，弃上清。 </li></ol> 7。单元封装<ol><li>准备1毫升注射器连接到14G×2“静脉导管软塑料管，这是用来吸装上注射泵，如在图1所示的细胞悬液。 </li><li>悬浮预热的海藻酸钠溶液的细胞密度在125万细胞/毫升海藻酸钠。用注射器轻轻混匀，避免产生气泡。 </li><li>成立珠发生器，注射泵和空气流量计，如在图1所示。特定的试剂和设备表中列出了这些设备的更多细节。 </li><li>到注射器抽吸细胞，静脉导管丢弃的塑料管和注射器连接到封装机。确保有10厘米的缝隙<html>之间最终的封装机和集合点​​，如在图1所示。 </li><li>细胞封装，通过设置在20毫升/小时，8 L / min的空气流速和压力为100 kPa（胶囊的大小可以通过改变空气流量变化）注射泵。 </li><li>收集到包裹细胞培养皿充满20毫升预热的7分，以稳定胶囊降水浴（100×15毫米）。 </li><li>包裹细胞转移到50 mL离心管填充20毫升0.9％氯化钠温柔的愿望。 </li><li>允许胶囊定居到试管底部，然后轻轻地弃上清。重复洗涤过程中，用0.9％氯化钠。 </li><li>包裹细胞悬浮预热的培养辅以RI（10μM），转移到培养瓶中，并在37°C / 5％CO 2孵育介质。 </li></ol> 8。封装的人类胚胎干细胞分化为多巴胺神经元ve_content“&gt;封装的人类胚胎干细胞分化前3天的国际扶轮治疗。 <ol><li>鼠标种子的基质细胞系，PA6的细胞密度1.0×10 4每平方厘米 0.1％明胶涂层的T75瓶，条件尼龙细胞中多巴胺神经分化培养基的分化前24小时（材料表）。 </li><li>胶囊转移至50 mL离心管中，并允许他们定居到试管底部。 </li><li>弃上清，PA6的细胞调节DA神经分化培养基中悬浮粒。 </li><li>文化与PA6的28天与第4天的媒体的变化和隔日（9×10 6每7.5×10 5 PA6的细胞的人类胚胎干细胞）细胞单层封装后人类胚胎干细胞（中等每次只改变的一半）。 </li><li>经过3周的文化与PA6细胞，补充的DA神经分化培养基100毫微克/毫升SHH和100毫微克/毫升FGF8a余下的一周。 </李&gt; </ol> 9。解封的封装人类胚胎干细胞<ol><li>到15 mL离心管吸胶囊，并允许他们定居在试管底部。然后小心弃上清。 </li><li>胶囊的D-PBS洗两次。让胶囊收于试管底部，然后取出上清液。 </li><li>添加5毫升胶囊解封的解决方案。彻底混合暂停之前，通过吸入孵化室温度为4-5分钟。 </li><li>离心3分钟95 XG的解封细胞，弃上清。 </li><li>洗颗粒细胞的D-PBS离心3分钟，95 XG。重复。 </li><li>解封的细胞可以进一步培养的单层或用于下游分析。 </li></ol> 10。代表结果海藻酸钠微胶囊的直径为400-500微米。胶囊内的细胞数量是估算通过计算细胞总数除以每运行胶囊总数。因此，估计约5.0×10 4个细胞胶囊。由此，我们推测细胞胶囊可以包含的最大数量为约1.0×10 5。封装的人类胚胎干细胞的生存能力是&gt; 80％（图2）使用羧基双乙酸钠succinimidly酯（CFDA）/碘化丙啶（PI）的检测来确定。我们已经优化了人类胚胎干细胞封装的条件下，通过减少海藻酸钠的浓度从2.2％下调至1.1％，并通过改变降水浴氯化钡氯化钙。从这些条件，我们发现，只有1.1％海藻酸钙包裹细胞可以存活，增殖并形成胚在体外 1。进一步优化的条件下，培养媒体和RI的影响，Y 27632进行了调查。图2和3的数据表明，国际扶轮preventeð分离，诱导细胞凋亡，并保持细胞活力，促进集群形成1,6。此外，封装的人类胚胎干细胞培养HFF - CM +注册机的存活率显着高于没有注册机补充的其他组高，但是这是没有显着不同封装的人类胚胎干细胞培养的SR +注册机。同样，细胞增殖的BrdU检测单细胞发展成集群（图3）从25％提高到75％。凋亡TUNEL检测显示，在SR培养基培养的微囊单细胞凋亡（数据未显示），而HFF-CM检索集群大多TUNEL法负。在一定程度上，与碱性成纤维细胞生长因子补充HFF - CM也促进了中的Y-27632的情况下生存和封装的胚胎干细胞的增殖。然而，与Y-27632（2小时）治疗前和后封装（额外4天）明显增强活力，增殖和集群封装的人类胚胎干细胞的形成1.1％海藻酸钙微胶囊。 以前，我们表明，封装人类胚胎干细胞可以成功地分化明确的内胚层1。在这里，我们研究应用细胞封装，三维封装的人类胚胎干细胞分化成多巴胺神经元的平台。人类胚胎干细胞，形成胚体（EB），胶囊直接分化后2-3天的文化所描述的条件下解封表明一个渐进的神经元的形态（图4）超过90％的存活率。基因表达分析表明下调的多能干细胞的标记，OCT4标记neuroprogenitor，PAX6的多巴胺神经元标记，TH，而分化后7天（图5A）上调。免疫荧光染色显示分化的人类胚胎干细胞是在7天的PAX6的阳性（&gt; 80％），但的OCT4负。进一步分化的人类胚胎干细胞显示，经过21天的TH阳性（&gt; 90％）神经元（图5B）。 Western blot分析也表明上调TH的表达（图5C）从第14天，而PAX6的表达下调后21天。相比之下，细胞培养的二维（2D）的环境下，类似的条件下（如RI为2小时和RI治疗后治疗前3天前分化）保持了PAX6的阳性细胞的比例很高（&gt; 80 ％），整个分化时期不区分封装（图5A和C）提供的3D环境中的TH阳性细胞（&lt;60％）的有效。

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我们什么都没有透露。

利用小鼠胚胎干细胞和胚胎干细胞的一些研究已经证明，生物材料和组织工程2,3三维培养系统的好处。我们作为一个合适的3D平台研究钡海藻酸钠相比，人类胚胎干细胞的增殖和分化，自人类胚胎干细胞表现出显着高于可行性封装比钡海藻酸钠海藻酸钙海藻酸钙微胶囊。这种文化系统还允许一个高密度细胞培养和交流的营养和氧气穿过细胞膜7。胶囊内自发分化先前已观察到的，它...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 笔记 </td></tr><tr><td>海藻酸钠（Pronova向上MVG） </td><td> NovoMatrix </td><td> 4200101 </td><td>高葡萄糖醛酸含量≥60％，粘度&gt; 200 MPa级小号，内毒素&lt;100欧/克</td></tr><tr><td>明胶</td><td> Sigma-Aldrich公司</td><td> G1890-100G </td><td></td></tr><tr><td> 0.9％的NaCl </td><td>百特医疗用品</td><td> AHF7123 </td><td></td></tr><tr><td> J1的珠发电机类型</td><td>南钢工程公司</td><td> SPA - 0447 </td><td></td></tr><tr><td>移相多注射泵</td><td>新时代热泵系统公司</td><td> NE-1000型</td><td></td></tr><tr><td> EZI流医疗流量计</td><td>吹牛系统</td><td> G0149 </td><td></td></tr><tr><td> Y型27632 </td><td>默克公司</td><td> 688000 </td><td></td></tr><tr><td>人血清白蛋白</td><td> Sigma-Aldrich公司</td><td> A4327-1G </td><td></td></tr><tr><td> accutase </td><td> Millipore公司</td><td> SCR005 </td><td></td></tr><tr><td> 14G x 2“静脉导管</td><td> Terumo公司</td><td> SR-OX1451C的</td><td></td></tr><tr><td>淘汰赛-DMEM培养液</td><td> Invitrogen公司</td><td> 10829-018 </td><td>对于SR介质（基底） </td></tr><tr><td> GlutaMAX我</td><td> Invitrogen公司</td><td> 35050-061 </td><td>对于SR培养基（2毫米） </td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td>对于SR中等（20％） </td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15070063 </td><td>对于SR培养基（2.5 U / ml的） </td&gt; </tr><tr><td>胰岛素转硒区（ITS） </td><td> Invitrogen公司</td><td> 41400045 </td><td>对于SR培养基（1X） </td></tr><tr><td> β-巯基乙醇</td><td> Invitrogen公司</td><td> 21985-023 </td><td>对于SR中型（0.1毫米） </td></tr><tr><td>的MEM NEAA解决方案</td><td> Invitrogen公司</td><td> 11140-050 </td><td>对于SR中等（5毫米） </td></tr><tr><td>格拉斯哥最低基本中等</td><td> Invitrogen公司</td><td> 11710035 </td><td>对于多巴胺神经分化培养基（基底） </td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td>对于多巴胺神经分化培养基（10％） </td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td><td> 11360070 </td><td>对于多巴胺神经分化培养基（1毫米） </td></tr><tr><td>的MEM NEAA解决方案</td><td> Invitrogen公司</td><td> 11140-050 </td><td>对于伤残列印eural分化培养基（0.1毫米） </td></tr><tr><td> β-巯基乙醇</td><td> Invitrogen公司</td><td> 21985-023 </td><td>对于多巴胺神经分化培养基（0.1毫米） </td></tr><tr><td>刺猬（Shh） </td><td> R＆D系统</td><td> 1314-SH-025/CF </td><td> DA神经分化（100毫微克/毫升） </td></tr><tr><td>成纤维细胞生长因子8A（FGF8a） </td><td> R＆D系统</td><td> 4745-8-050 </td><td> DA神经分化（100毫微克/毫升） </td></tr></tbody></table>

alginate microcapsule as a 3d platform for propagation and differentiation of human embryonic stem cells (hesc) to different lineages

人类胚胎干细胞（胚胎干细胞）正在成为一个有吸引力的替代细胞替代治疗源，扩大在文化，因为它们可以无限期地在身体的任何类型的细胞分化。不同类型的生物材料也被用于干细胞培养提供了一个微环境，模仿的干细胞小生境1-3。后者是重要的促进提供一个三维（3D）环境中，如封装4细胞与细胞间的相互作用，细胞增殖，分化成特定的谱系，以及组织机构。 2三维文化的半渗透膜的范围内对细胞封装的原则，涉及诱捕的活细胞。这些膜允许交换横跨膜的营养物质，氧气和刺激，而从主机比胶囊孔径大的抗体和免疫细胞被排除。在这里，我们预文化派的做法和三维使用海藻酸钠微囊微环境中的人类胚胎干细胞多巴胺神经元分化。我们已经修改了培养条件，以加强封装的人类胚胎干细胞的可行性。我们先前已经表明，除了显着增强的3D平台盘绕线圈P160-Rho相关激酶（ROCK）抑制剂Y-27632和人类的胎儿成纤维细胞调节血清更换介质（HFF-CM）封装的人类胚胎干细胞的可行性在细胞中表达明确的内胚层标记基因1。我们现在已经传播到人类胚胎干细胞高效分化的多巴胺神经元用于这个3D平台。多巴胺神经细胞分化的最后阶段后，蛋白和基因表达分析表明酪氨酸羟化酶（TH）的表达增加，多巴胺神经元的标志，&gt; 2周后的100倍。我们假设，我们的3D平台，使用海藻酸钠微囊可能是有用的，以研究细胞增殖和定向分化人类胚胎干细胞，以不同的谱系。这个3D系统还允许从人类胚胎干细胞饲养层细胞的分离，并在分化过程中，也有可能在将来移植免疫隔离。

Watch this Scientific Journal Video about Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages at JoVE.com

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

我们已经优化了微胶囊技术作为一种有效的繁殖和胚胎干细胞分化为内胚层和多巴胺（DA）神经元的3D平台1。它还提供了一个机会，从宿​​主细胞移植过程中的免疫隔离。这个平台可以适应其他类型的细胞。

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 ...

alginate-microcapsule-as-3d-platform-for-propagation-differentiation

Research

JoVE Journal

Bioengineering

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

16.2K 次观看.  The University of New South Wales. 我们已经优化了微胶囊技术作为一种有效的繁殖和胚胎干细胞分化为内胚层和多巴胺（DA）神经元的3D平台1。它还提供了一个机会，从宿​​主细胞移植过程中的免疫隔离。这个平台可以适应其他类型的细胞。

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

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 ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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 ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

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.

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...