这项工作是由美国国立卫生研究院授予EB007278支持。

制作一个BFC BFCs超过4个“四主晶圆成型聚二甲基硅氧烷（PDMS）是由。 使主晶片<ol><li>开始脱水硅片为30分钟，在130 ° C。 </li><li>晶圆放置在一个旋转涂布机，倒到晶圆SU8 - 2050（Microchem），坡道从3000-4000转300转/秒，以及30秒的旋转产生功能高度40-30毫米，分别。 </li><li>纺纱后，放置在65℃烤盘晶圆，立即提升温度至95 ° C为5-6分钟，然后斜下来到65 ° C。 </li><li>公开的晶圆到200兆焦耳/厘米2，在接触使用暗视野面罩对准的紫外线剂量。 </li><li>将晶圆上65℃烤盘，立即提升温度至95 ° C为4-5分钟，然后斜下来到65 ° C。 </li><li>发展在PM醋酸和异丙醇晶圆。 </li><li> Silanize晶圆为30分钟，使用六甲基二硅氧烷（信越MicroSi），或（Tridecafluoro - 1 ,1,2,2 - tetrahydrooctyl）- 1 -三氯硅烷（t2492 -公斤，开普敦大学专科，布里斯托尔，PA），以防止PDMS从坚持“四主晶圆。 </li></ol>成型芯片<ol><li> PDMS（道康宁，Sylgard 184）10:1的比例混合，倒在主硅晶圆每片晶圆（约15克），并让它一夜之间在室温下固化。 </li><li>剥离固化硅晶片上的PDMS和使用刀片切割出每个芯片。 </li><li>债券每个芯片的一个1x1厘米的切割，易于处理的显微镜幻灯片。 </li></ol>准备使用的BFC <ol><li>浸泡在PBS BFC过夜，以防止媒体在实验过程中吸收到的PDMS。 </li><li>干与Kimwipe（Kimtech科学）的芯片。 </li><li>将下降7.5％牛血清白蛋白（BSA），（〜200毫升）的BFC图案的表面，然后刮去，用枪头驱散BSA和删除任何气泡从水井。 </li><li> BFC表面上保留至少0.5小时的牛血清白蛋白。理想的情况下搅拌BSA每15分钟，以防止结痂。 </li><li>为了适应在一个35x10毫米的TCPS菜BFC（猎鹰，35-3001），切一个TCPS盘中途轮辋上下，¾“长尾夹（欧迪办公）适合在菜RIM钳芯片菜一起。 </li><li>剪下从一个硅橡胶垫片板材垫片（框形外缘20&#39;20毫米，15&#39;15毫米内缘，厚度为250-500毫米）（硅特产）或任何其他硅，并将其应用的菜用小镊子。 </li></ol>图案与BFC细胞<ol><li>消毒1分钟的紫外线光下的菜，垫片，BSA的涂层芯片，和2个长尾夹。 </li><li>吸BSA和BFC用200 mL PBS冲洗两次。 </li><li>添加明胶削减TCPS菜，如果需要为您的细胞系。否则，湿与一些媒体的TCPS表面，然后将它应用到芯片（见下文）。这可防止气泡形成在会议厅内。 </li><li>吸气的PBS后，加入200 mL的细胞溶液（〜1 × 10 6细胞/ mL）的BFC表面上。让细胞为5-10分钟解决。 </li><li>在〜15 °角倾斜的BFC一个角落，慢慢地用200毫升吸管吸管的细胞溶液。然后，将BFC单位，并添加100毫升的PBS或空白媒体到对面的BFC角落。以这种方式冲洗BFC一个额外的2-4倍，在必要时，清除所有细胞间以及区域。 </li><li>吸取200毫升到芯片表面的媒体。吸明胶/媒体的TCPS。然后反转预湿的菜和BFC起来，慢慢推入菜。 </li><li>应用粘结剂剪辑，两个腔两侧密封。从长尾夹取出金属耙子，使菜保持水平翻转过来。 </li><li>最后，快速翻转以上设置，同时避免任何不必要的移动，并放置在孵化器。 </li></ol>

<ol>
	<li>Rosenthal, A., Macdonald, A., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Patterning+Chip+for+Controlling+the+Stem+Cell+Microenvironment.">Cell Patterning Chip for Controlling the Stem Cell Microenvironment.</a> Biomaterials. 28, 3208-3216 (2007).</li></ol>

虽然这里显示的协议和视频描述BFCs使用BFC 1模式小鼠胚胎干细胞（mESCs），可用于几乎任何细胞类型的利益格局。唯一的变化可能需要改变大小的微孔。根据我们的经验，使用BFC的另一种类型的细胞，微孔直径和高度大于独立的单元直径10微米等于的单细胞模式效果最好。 BFCs也可以被使用到其他基板，包括其他细胞，格局细胞没有在协议中的重大变化。到现有的细胞层的图案是BFC方法比传统的细胞...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dw Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Silicon master mould</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3001</td>
<td>35 mm</td>
</tr>
<tr>
<td>3/4" binder clips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hexamethyldisiloxane</td>
<td>&nbsp;</td>
<td>Shin-Etsu MicroSi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Microchem</td>
<td>SU8-2050</td>
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<td>PM acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Isopropanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
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patterning of embryonic stem cells using the bio flip chip

在许多生物过程，如肿瘤的生长，干细胞的分化，干细胞的自我更新和扩散信号的细胞 - 细胞接触（juxtacrine信令）组成的细胞间的相互作用是重要的。目前的一些方法存在调节体外细胞信号。扩散信号调制总额的方法之一，是细胞种植密度，在文化的不同而有所差异。由于细胞种植，结果在实际的细胞与细胞间的间距和细胞 - 细胞接触的数量相当大的变化，并不能规定当地环境的随机性质。一个调节细胞信号的更具体的方法是使用分子抑制剂或基因的方法，打倒特定的信号蛋白，但是这两种方法是最适合操纵小分子。在这里，我们展示了一种新的方法来调节细胞 - 细胞信号调制在所需的位置放置在基板上不同的细胞群的细胞当地环境。这种方法提供了一种互补的方式来控制细胞之间的信号的地方可扩散和juxtacrine。我们的方法使使用的生物覆晶（BFC），一个微型硅芯片，含有微孔，每个大小数百到数千持有一个单一的单元格或小的数字。我们只需加载吹打到井阵列和洗衣机关闭阵列卸载细胞与细胞的芯片。该芯片然后翻转到基板上，使细胞的水井和基板上掉下来，维护他们的图案。细胞附着后，芯片可以被删除（或左）。这种细胞图案的方法是独特之处在于：1）不改变基板的化学成分，从而使细胞的增殖和迁移; 2）允许到任何基材上的图案，包括组织培养聚苯乙烯，玻璃，基质胶，和甚至馈线细胞层和3）是与传统的微接触印刷兼容，使创建与这些岛屿内放置的细胞外基质的岛屿。在这段视频中，我们表现出的图案的鼠标到组织培养聚苯乙烯胚胎干细胞BFC。

Watch this Scientific Journal Video about Patterning of Embryonic Stem Cells Using the Bio Flip Chip at JoVE.com

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

我们证明一个简单的方法，放置在基板上的细胞所需的位置。这种方法模式翻转含有一个硅芯片与基板上的细胞充满微孔的细胞。这种方法提供了一种新的方式来调节细胞之间的可传播的juxtacrine信号。

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

patterning-of-embryonic-stem-cells-using-the-bio-flip-chip

Research

JoVE Journal

Biology

9.4K 次观看.  MIT - Massachusetts Institute of Technology. 我们证明一个简单的方法，放置在基板上的细胞所需的位置。这种方法模式翻转含有一个硅芯片与基板上的细胞充满微孔的细胞。这种方法提供了一种新的方式来调节细胞之间的可传播的juxtacrine信号。

视频: Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...