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

इस काम NIH अनुदान EB007278 द्वारा समर्थित किया गया था.

एक BFC बनाना BFCs एक 4 &quot;सी मास्टर वफ़र पर ढलाई polydimethylsiloxane (PDMS) द्वारा किए गए हैं. एक मास्टर वफ़र करना <ol><li> 30 मिनट के लिए 130 सी वेफर्स dehydrating द्वारा प्रारंभ डिग्री सेल्सियस </li><li> एक स्पिन coater पर ?...

<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>

हालांकि प्रोटोकॉल और वीडियो यहाँ दिखाया पैटर्न माउस भ्रूणीय स्टेम कोशिकाओं (mESCs) BFC एक का उपयोग कर का वर्णन करने के लिए, BFCs व्यावहारिक रूप से ब्याज की किसी भी कोशिका प्रकार के पैटर्न के लिए इस्तेमाल किया जा सक?...

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<td>PDMS</td>
<td>&nbsp;</td>
<td>Dw Corning</td>
<td>Sylgard 184</td>
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<td>Silicon master mould</td>
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<td>35-3001</td>
<td>35 mm</td>
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<td>3/4" binder clips</td>
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<td>PBS</td>
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<td>hexamethyldisiloxane</td>
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patterning of embryonic stem cells using the bio flip chip

सेल सेल diffusible संकेतन और सेल सेल संपर्क (juxtacrine संकेतन) से मिलकर बातचीत ट्यूमर के विकास, स्टेम सेल भेदभाव, और स्टेम सेल आत्म नवीकरण जैसे कई जैविक प्रक्रियाओं में महत्वपूर्ण हैं. तरीकों का एक नंबर वर्तमान में इन विट्रो में सेल संकेतन मिलाना मौजूद है. Modulating diffusible संकेतन की कुल राशि का एक तरीका संस्कृति के दौरान सेल बोने घनत्व भिन्न है. सेल बोने, वास्तविक रिक्ति सेल सेल और सेल सेल संपर्क की राशि में काफी भिन्नता में इस परिणाम, और स्थानीय पर्यावरण लिख नहीं कर सकते के यादृच्छिक प्रकृति के कारण. Modulating संकेतन सेल के लिए एक अधिक विशिष्ट दृष्टिकोण आणविक inhibitors या आनुवंशिक दृष्टिकोण का उपयोग करने के लिए नीचे विशिष्ट संकेत प्रोटीन दस्तक है, लेकिन इन दोनों तरीकों में से सबसे अच्छा अणुओं के छोटे संख्या से छेड़छाड़ करने के लिए अनुकूल हैं. यहाँ, हम modulating संकेतन सेल सेल है कि एक सब्सट्रेट पर वांछित स्थानों पर कोशिकाओं के विभिन्न संख्या रखकर कोशिकाओं का एक समूह के स्थानीय पर्यावरण modulates एक नया दृष्टिकोण प्रदर्शित करता है. यह विधि एक पूरक कोशिकाओं के बीच स्थानीय diffusible और juxtacrine संकेतन नियंत्रण प्रदान करता है. हमारे विधि जैव फ्लिप चिप (BFC) का उपयोग, एक microfabricated सिलिकॉन चिप microwells, प्रत्येक आकार के सैकड़ों हजारों युक्त या तो एक एकल कक्ष या कक्षों की छोटी संख्या पकड़ बनाता है. हम बस उन्हें कुओं की सरणी पर pipetting और सरणी बंद उतार कोशिकाओं धोने के द्वारा कोशिकाओं के साथ चिप लोड. चिप तो एक सब्सट्रेट पर रूप से फ़्लिप किया है, जिससे कोशिकाओं कुओं और सब्सट्रेट बाहर गिर, उनके patterning को बनाए रखने. बाद कोशिकाओं संलग्न है, चिप (या पर बाईं) हटाया जा सकता है. सेल patterning के लिए इस दृष्टिकोण में अद्वितीय है कि: 1) सब्सट्रेट के रसायन शास्त्र को बदल नहीं करता है किसी भी सब्सट्रेट पर इस तरह कोशिकाओं को पैदा करना और की ओर पलायन की अनुमति, 2) patterning की अनुमति देता है ऊतक संस्कृति polystyrene, कांच, matrigel सहित, और यहां तक ​​कि सेल परतों फीडर, और 3) पारंपरिक microcontact मुद्रण के साथ संगत है, उन द्वीपों के अंदर रखा कोशिकाओं के साथ बाह्य मैट्रिक्स द्वीपों के सृजन की अनुमति है. इस वीडियो में, हम माउस भ्रूण स्टेम कोशिकाओं ऊतक संस्कृति polystyrene पर BFC का उपयोग patterning प्रदर्शित करता है.

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

हम एक सब्सट्रेट पर वांछित स्थानों पर कोशिकाओं रखने के लिए एक सरल तरीका प्रदर्शित करता है. यह विधि एक सिलिकॉन सब्सट्रेट कोशिकाओं के साथ भरा microwells युक्त चिप flipping द्वारा कोशिकाओं के पैटर्न. इस पद्धति का एक नया करने के लिए कोशिकाओं के बीच diffusible और juxtacrine संकेतन मिलाना प्रदान करता है.

जैव फ्लिप चिप का प्रयोग भ्रूण स्टेम कोशिकाओं के patterning

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

मानव भ्रूण स्टेम कोशिकाओं (ते) के प्रचार - प्रसार

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.

Murine भ्रूण स्टेम कोशिकाओं से hematopoietic स्टेम सेल की व्युत्पत्ति

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

+ टी lymphocyte कैद CD4 एचआईवी के लिए एक डिस्पोजेबल microfluidic चिप का उपयोग

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

Neuroprecursor जैव रेड कुल शाही सेना किट का उपयोग कर कक्ष से शाही सेना निकालना

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.

मानव भ्रूण स्टेम कोशिकाओं के Immunosurgery द्वारा व्युत्पत्ति

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.

मानव भ्रूण स्टेम कोशिकाओं से chromatin 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.

माउस भ्रूणीय स्टेम (एमईएस) हैंगिंग ड्रॉप विधि का उपयोग कोशिकाओं की इन विट्रो भेदभाव में

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.

संस्कृति और मानव भ्रूण स्टेम कोशिकाओं के रखरखाव

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.

मानव भ्रूण स्टेम कोशिकाओं को ठंड और विगलन

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.

अलगाव और माउस भ्रूणीय कीटाणु कक्ष की व्युत्पत्ति

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