这项工作是由美国国立卫生授予NIH / NICHD的HD051998（CAM）的机构的支持。

1。胶原珠的制备<ol><li>在此之前的3 - D细胞培养加载EVTs，需要准备的Cytodex - 3微载体珠: <ol><li>称取适量的实验所需的Cytodex - 3珠。该协议是适应农村信用社的10毫升船只，其中珠0.05克需要。对于一个50毫升农村信用社的船只，相应的规模。在一个50毫升的高压灭菌的锥形管，混合磷酸12mL贝科250毫克Cytodex - 3珠缓冲液（DPBS）。这一数额是足够5个农村信用社的船只。 </li></ol></li><li>确保有足够的量是锥形管中，反应釜过程将导致蒸发。松散帽管和高压灭菌10分钟，在110 ° C。 </li><li>拆下的反应釜周期完成锥管和冷却C​​ytodex - 3珠的解决方案。 </li><li>锥形管冷却至室温后，使用无菌技术，使总体积为12.5mL 1X的DP学士学位。 </li><li>第储存在室温下准备Cytodex - 3珠。允许准备珠膨胀和冷却到室温，使用前。 Cytodex - 3珠子上面的准备，将提供五个10毫升农村信用社的船只。我们观察Cytodex - 3珠在室温下长期储存会导致性能的降低，胶原蛋白会破坏后约一个月。 Cytodex - 3珠的尺寸范围从133-215微米。 </li></ol> 2。媒体准备<ol><li>优化GTSF - 2媒体（改编来自22个 ），其中40％的MEM ALPHA组成，加上补充和60％的L - 15 Leibovitz的媒体（改编22日 ）农村信用社准备1L。首先，准备在纪念阿尔法辅以总量400ML: 21.2mM碳酸氢钠 0.06％，蛋白胨 0.7mm的果糖 1.4mm的半乳糖 5.6mM葡萄糖 1％的HEPES 1％L -谷氨酰胺 0.5％ 10％F学士</li><li>为了准备其原液 ​​，溶于5毫升无菌酸化H 2冰醋酸（约0.05mL），除了准备Ø 。旋流解散，请用45mL无菌水。 </li><li>称取中等600毫升足够的L - 15 Leibovitz的粉末溶解在组织文化品位H 2 O </li><li>使细胞培养液中的L - 15 Leibovitz的培养基总体积至1L。过滤，消毒，并保存于4 ° C黑暗环境中。添加1％青霉素，链霉素单独使用每个中等等分。 </li><li> GTSF - 2媒体以外的许多媒体配方已成功地在农村信用社进行测试。个别实验室必须自己决定，正在执行的试验介质是最佳。 </li></ol> 3。细胞和珠孵化<ol><li>传播EVTs〜80％汇合，trypsinize和计数用公认的细胞培养方法。 </li><li>暂停在温暖的4ML 1 × 10 6 EVTs版媒体。 </li><li>轻轻混合编写的Cytodex - 3珠。使用无菌技术，消除了使用广泛的一角，10ml血清移液管中，并转移到一个未使用的15ML的锥形管的准备珠2.5毫升。请注意，有可能是一个小的损失珠由于它们附着于移液器。 </li><li>允许Cytodex - 3珠，收于管底。经过沉淀，使用移液器删除，而不会干扰Cytodex - 3珠的DPBS的顶层。 </li><li>混合准备Cytodex - 3珠准备EVTs媒体1 × 10 6。 </li><li>细胞珠的混合物在室温下孵育30分钟。定期轻轻混匀。为进一步孵育30分钟，在37 ° C和5％CO 2 。定期轻轻混匀。 </li></ol> 4。载入的RWV <ol><li>在层流柜，从包装中取出一个10ML农村信用社和地方稳定在无菌6孔培养板。请参考图1为标记的RC图政务司司长。 </li><li>删除从港口大塞。 </li><li>把孵化细胞珠的混合物总量与温暖媒体10ML。 </li><li>通过大港口，装载到农村信用社的细胞/珠混合物。农村信用社应倾斜45 °角（大港口最多）加载时，以帮助消除潜在的气泡。防止填充船只缓慢而稳定地积聚的正压力。 </li><li>更换大型港口塞。 </li><li>取出3毫升注射器的活塞，并将其放置到小港口的空注射器。添加到每个注射器媒体1 - 3ml的。慢慢打开两个阀门。更换注射器轻轻注射器活塞。添加从一个注射器，直到所有的气泡从内室中删除的媒体。 </li><li>拿起农村信用社，并同时旋转它在你的面前检查气泡，轻轻敲击一边。如果有任何气泡，你必须让他们的商会，气泡会干扰形成ØF细胞聚集和引进的剪切力。旋转的农村信用社，直到气泡下的小口，取出泡沫。然后轻轻地往下推对面注射器，强行进入的端口和出室气泡。 </li><li>关闭一个侧面端口。其他注射器活塞轻轻推下来的船只，防止形成气泡引入少量的正压。关闭第二个阀门。 </li><li>转子装到农村信用社。在37℃CO 2培养箱，在19rpm开始旋转。 </li></ol> 5。更改媒体<ol><li>更改媒体隔日的前三天，然后以后每天。 </li><li>关闭转子和消除农村信用社。拉活塞，创造出一些吸，去除注射器从每个小口，放在农村信用社的角度，使珠解决大型港口对面。 </li><li>珠后一切都解决了，打开一个小阀门允许媒体流农村信用社，并成为一个废物容器。在这种方式中删除媒体的2 / 3。可以肯定的，不打扰珠，不放弃任何聚合。 </li><li>关闭小阀门，并打开大港口。加回农村信用社通过大型港口的媒体。更换大型港口塞。 </li><li>重复步骤4.6 - 4.9。 </li><li>由于总量增加的大小，你需要增加转速，保持他们在任何时候都处于悬浮状态，在一个小的循环模式的理想在最佳的速度。一般来说，每次喂奶后增加0.3-0.7 RPM之间的速度，一旦聚合开始日益明显。 </li></ol> 6。收集传播细胞<ol><li>农村信用社从转子取出。从每个小口取出注射器和地方对农村信用社的角度，使珠子可能定居的大型港口对面。 </li><li>珠后一切都解决了，打开一个小阀门，并允许媒体都流出来了农村信用社和成一个废物容器。在这种方式中删除媒体的1 / 3。 </li><li>关闭小阀门，并打开大港口。轻轻摇动船只驱散聚集成溶液。空的液体混合物进入无菌50ml的锥形管。允许收集聚集在锥形管，使用前上清彻底清洗的培养容器，最大限度地聚集恢复解决。聚合，可立即用于下游分析，如流式细胞仪检测，入侵检测，免疫和其他。 </li></ol> 7。代表性的成果生长在Cytodex - 3珠农村信用社的EVT样细胞（SGHPL - 4滋养细胞系）的一个例子是如图2所示。 EVT -样细胞株显示预测远离主要集群扩展和附加到邻近的集群。许多珠子完全覆盖与传播细胞。一旦从农村信用社和解放军泰德外基质，EVT般的3 - D细胞生长大举入侵和/或迁移（图3）。 RT - PCR检测数据证实了在3 - D聚集，而不是传统的细胞培养单层（图4），基质金属蛋白酶的表达增加。有趣的是，不与入侵相关的基因也上调农村信用社（见表 1），并可能在划定侵略滋养细胞免疫的相互作用等方面的援助。 <img alt="图1" src="/files/ftp_upload/3367/3367fig1.jpg" /> 图1。卡通描绘的旋转细胞培养系统（农村信用社）。 <img alt="图2" src="/files/ftp_upload/3367/3367fig2.jpg" /> 图2。期后5天在农村信用社的增长代表总的对比显微照片。箭头指示侵入细胞和*表示Cytodex - 3微载体珠。 <imgALT ="图3"SRC ="/ files/ftp_upload/3367/3367fig3.jpg"/&gt; 图3。农村信用社生长聚合，嵌入在纤维蛋白凝胶。通过纤维蛋白凝胶的入侵（一）24小时后的纤维蛋白嵌入的早期观察，（二）继续通过48小时。箭头指示侵入细胞和*表示Cytodex - 3微载体珠（改编许可，从编号7）。 <img alt="图4" src="/files/ftp_upload/3367/3367fig4.jpg" /> 图4明胶酶谱的比较单层和农村信用社。传播SGHPL - 4的EVT样细胞。亲和活性形式的MMP - 2和MMP - 9分泌的农村信用社生长的滋养细胞（改编许可编号7）。 <img alt="表1" src="/files/ftp_upload/3367/3367table1.jpg" /> 表1。SGHPL - 4细胞生长在农村信用社（改编许可编号7）芯片结果的摘要。 <ol class="lalpha"><li&gt;折叠诱导的3 - D总量计算如下:正常化的3 - D基因值/归monlayer基因值。 </li><li>表示低于检测限制在单层的基因。 </li></ol>

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我们什么都没有透露。

这里介绍的文化技术提供具有高度侵袭性​​的EVT样细胞的调查。如今，它已被公认，由于抑制细胞反应，化学和分子线索的三个层面（心尖，基底和横向细胞表面 ）10,13在单层发生分化的损失。这种技术反映在子宫内指出入侵的EVT细胞的特点。由于程序模仿传统的单层组织培养的时间动力学，但提供了差异表达的细胞，比较分析可能被雇用。在实验中使用的细胞团可收获，在任?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Cytodex微载体珠</td><td> Sigma - Aldrich公司</td><td> C3275 </td><td></td></tr><tr><td>旋转细胞培养系统（农村信用社） </td><td> Synthecon </td><td>农村信用社- D </td><td>包括转子基地，电源供应器，4个单位一次性农村信用社</td></tr><tr><td>农村信用社一次性单位</td><td> Synthecon </td><td>联系Synthecon </td><td></td></tr><tr><td> 3毫升鲁尔锁提示注射器</td><td>屋宇署</td><td> 309585 </td><td></td></tr><tr><td> 10毫升宽提示血清移液器</td><td>屋宇署</td><td> 357504 </td><td></td></tr><tr><td>纪念阿尔法</td><td> Invitrogen公司</td><td> 12561-072 </td><td></td></tr><tr><td> Leibovitz的L - 15的介质，粉末</td><td> Invitrogen公司</td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O，内毒素</td><td>费舍尔</td><td> MT - 25 - 055 - CM </td><td></td></tr><tr><td>碳酸氢钠</td><td> Sigma - Aldrich公司</td><td> S - 7795 </td><td></td></tr><tr><td>蛋白胨</td><td> Fisher Scientific则</td><td> BP1420 - 100 </td><td></td></tr><tr><td>果糖</td><td> Sigma - Aldrich公司</td><td> F3510 - 100 </td><td></td></tr><tr><td>半乳糖</td><td> Sigma - Aldrich公司</td><td> G5388 - 100 </td><td></td></tr><tr><td>血糖</td><td> Sigma - Aldrich公司</td><td> G7528 - 250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen公司</td><td> 15630-080 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> Invitrogen公司</td><td> 25030 </td><td></td></tr><tr><td>胰岛素转亚硒酸钠（ITS）的</td><td> Sigma - Aldrich公司</td><td> I1884 </td><td></td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10437 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

人类滋养细胞研究领域的艾滋病在认识复杂的环境中建立在胎盘。由于这些研究的性质， 人体内实验是不可能的。一个小学文化，外植体培养和滋养层细胞系的组合，支持我们的绒毛外滋养细胞（EVTs），这是怀孕的成功建立所必需的入侵的子宫壁和子宫螺旋动脉重塑3,4的理解。尽管从这种模型中收集的知识财富，它是接受，使用类似的EVT -细胞株在体外细胞培养模型显示改变细胞特性相比，其在体内的同行5,6时。培养细胞在旋转细胞培养系统（农村信用社）显示的EVT样细胞株的形态，表型和功能特性，更加紧密地模仿u的区别人Tero EVTs，调解入侵（如基质金属蛋白酶（MMPs））和滋养层细胞分化7,8,9的基因表达增加。圣乔治医院的胎盘细胞株- 4（SGHPL 4）（请盖伊惠特利博士和博士朱迪思卡特赖特捐赠）是一个EVT样细胞系，用于在农村信用社的测试。 农村信用社培养容器的设计是基于对器官和组织的原则，即在一个三维（3 - D）环境功能。由于在船只动态的文化条件，包括有关生理剪切条件，细胞生长在三个方面形成聚集基于自然细胞亲和力和分化成器官组织的集会10,11,12。维护流体轨道提供了一个低剪切，低动荡的环境相似，在体内发现的条件。培养细胞沉淀是通过调整旋转反驳加快农村信用社，以确保细胞恒定的自由落体。气体交换发生通过透水疏水膜生物反应器的背面。像他们的父母在体内的组织，能够应对在三个层面（即在其顶端，基底和横向表面），因为他们培养的多孔微载体珠表面上化学和分子梯度力场的细胞生长。像塑料防渗表面的二维膜生长时，细胞被剥夺了这一重要的沟通，在其基底表面。因此，空间限制的环境所施加深刻影响细胞如何从周围的微环境意识和解码信号， 从而暗示13的3 - D环境的重要作用。 我们已经使用了农村信用社工程师7,14,15,16各种人类上皮组织的生物学意义的3 - D模型。事实上，许多以前的报告中有DEMonstrated农村信用社培养细胞可以承担有关生理尚未10,17-21其他机型可能表型。总之，在农村信用社的文化代表了一种简单，重现性好，高通量平台，提供大量分化的细胞，适合各种实验操作。在下面的协议，作为一个例子使用EVTs，我们清楚地说明需要三个维度文化的贴壁细胞在农村信用社的步骤。

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

传统的，二维细胞培养技术，往往导致与分化标志物，细胞因子和生长因子的改变的特点。旋转细胞培养系统的三维细胞培养（农村信用社）重新建立许多因素的表达，绒毛外滋养细胞系。

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 ...

rotating-cell-culture-systems-for-human-cell-culture-human

Research

JoVE Journal

Bioengineering

16.4K 次观看.  Tulane University Medical School. 传统的，二维细胞培养技术，往往导致与分化标志物，细胞因子和生长因子的改变的特点。旋转细胞培养系统的三维细胞培养（农村信用社）重新建立许多因素的表达，绒毛外滋养细胞系。

视频: Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

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

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

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 ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术:协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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 ...