Name: patterning of embryonic stem cells using the bio flip chip
Uploaded: 2007-10-01T13:21:11
Description: Watch this Scientific Journal Video about Patterning of Embryonic Stem Cells Using the Bio Flip Chip at JoVE.com

Bu çalışma, Ulusal Sağlık Enstitüsü, hibe EB007278 tarafından desteklenen oldu.

BFC yapma BFCs 4 &quot;Si ana gofret üzerinde kalıp polidimetilsiloksan (PDMS) tarafından yapılır. Bir ana gofret yapma <ol><li> 130 30 dakika Si gofret dehydrating başlayın ° C </li><li> Bir spin-lak gofret koyun, sırasıyla, 40-30 mm özelliği yükseklikleri verim s için 30 300 dev / s 3000-4000 rpm rampası, gofret üzerine SU8-2050 (Microchem) dökün ve spin. </li><li> Iplik sonra, 65 ° C ocak gofret, hemen 95 ° C &#39;de 5-6 dak...

<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>

Burada gösterilen protokol ve video desen fare embriyonik kök hücreleri (mESCs) BFC 1 kullanarak tarif olmasına rağmen, BFCs desen ilgi pratikte herhangi bir hücre tipi için kullanılabilir . Biri yapmak gerekebilir tek değişiklik, mikro oyukların boyutunu değiştirmek için. Bizim tecrübelerimize göre en iyi sonucu verir desen tek hücre başka bir hücre tipi, kuyu çapı ve unattached hücre çapı 10 mikrondan daha büyük eşit yükseklikte BFC kullanmak. BFCs protokolünde önemli değişiklikler olmada...

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<td>PDMS</td>
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<td>3/4" binder clips</td>
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patterning of embryonic stem cells using the bio flip chip

Difüze sinyalizasyonu ve hücre-hücre teması (juxtacrine sinyalizasyon) oluşan hücre-hücre etkileşimleri, tümör büyümesini, kök hücre farklılaşması ve kök hücre kendini yenileme gibi çok sayıda biyolojik süreçlerde önemli. Yöntemler bir dizi in vitro hücre sinyal modüle var. Difüze sinyal modüle toplam miktarın bir yöntem kültür sırasında hücre ekim yoğunluğu değişebilir. Hücre tohumlama, bu gerçek, hücre-hücre aralığı ve hücre-hücre teması miktarı önemli farklılıklar sonuçları ve yerel çevre reçete rasgele doğası nedeniyle. Modüle hücre sinyal iletimi için daha özel bir yaklaşım özel sinyalleme proteinleri yıkmak için moleküler inhibitörleri ya da genetik yaklaşımlar kullanmak, ancak bu yöntemlerin her ikisinin en iyi moleküllerin az sayıda işlemek için uygundur. Burada, bir substrat üzerindeki istenilen noktada farklı hücre koyarak hücrelerden oluşan bir küme yerel çevre modüle modüle hücre-hücre sinyalleşme için yeni bir yaklaşım göstermektedir. Bu yöntem, hücreler arasındaki yerel yayılabilir ve juxtacrine sinyalizasyon kontrol etmek için birbirini tamamlayıcı bir şekilde sağlar. Bizim metodumuz Bio Flip-Chip (BFC) kullanımı, tek bir hücrenin veya hücre az sayıda tutmak için mikro oyukların, her büyüklükte yüzlerce-binlerce içeren bir microfabricated silikon çip yapar. Biz sadece dizi kuyu üzerine pipetleme ve dizi kapalı boş hücreleri yıkama hücreleri ile yonga yükleyin. Hücreleri desenlendirme bakımı, kuyuların ve yüzey üzerine dökülür sayede çip, sonra bir yüzey üzerine çevrilmiş. Hücreleri bağlı sonra, çip (veya sol) silinebilir. Bu yaklaşım, hücre desenlendirme benzersiz olduğunu: 1) böylece hücreleri çoğalırlar ve geçiş için izin, yüzey kimyası değiştirmez;, doku kültürü polistren, cam, matrigel de dahil olmak üzere, herhangi bir yüzey üzerine desenlendirme sağlar 2) ve Hatta besleyici hücre tabakaları ve 3) Bu adaların içine yerleştirilen hücreler ile ekstraselüler matriks adalar oluşturmaya olanak sağlayarak, geleneksel microcontact baskı ile uyumludur. Bu video, BFC kullanarak, doku kültürü polistiren üzerine fare embriyonik kök hücreleri desenlendirme göstermektedir.

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

Biz, bir substrat üzerindeki istenilen noktada hücre yerleştirmek için basit bir yöntem ortaya koymaktadır. Bu yöntem desen hücreleri yüzeye hücreleri ile dolu mikro oyukların içeren bir silikon çip saygısız. Bu yöntem yayılabilir ve juxtacrine hücreleri arasındaki sinyal modüle etmek için yeni bir yol sağlar.

Bio Flip-Chip kullanarak Embriyonik Kök Hücreler, Desenlendirme

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

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.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

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-Lenfosit Yakalama HIV için Disposable mikroakışkan Chip

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

Bio-Rad Total RNA Kiti Kullanarak Neuroprecursor Hücreler RNA Ekstraksiyon

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.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

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.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

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.

Asma Bırak Yöntemiyle Fare Embriyonik Kök (MES) Hücreler in vitro Farklılaşma

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.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

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.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

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.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

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