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

이 작품은 NIH 부여 EB007278에 의해 지원되었다.

BFC 만들기 BFCs는 4 &quot;시 마스터 웨이퍼를 통해 성형 polydimethylsiloxane (PDMS)에 의해 만들어집니다. 마스터 웨이퍼 만들기 <ol><li> 130 30 분시의 웨이퍼를 dehydrating부터 시작 ° C. </li><li> 스핀 - coater에서 웨이퍼를 놓고, 각각 40~30밀리미터의 기능을 높이를 얻을 수 30 s에 대한 3000-4000 RPM 300 RPM / s에서 램프, 웨이퍼에 SU8 - 2050 (Microchem)을 붓고, 그리?...

<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>

여기에 나와있는 프로토콜 및 비디오 패턴 마우스 배아 줄기 세포 (mESCs)에 BFC 1을 사용 설명하지만, BFCs는 패턴의 관심 실질적으로 모든 세포 유형을 사용할 수 있습니다. 하나는해야 수있는 유일한 변화는 microwells의 크기를 변경하는 것입니다. 우리의 경험에서 가장 잘 작동 다른 세포 유형, microwell 직경과 무소속의 세포 직경보다 10 미크론와 같은 높이의 패턴을 하나의 세포로 BFC를 사용할 수 있습니다....

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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>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3001</td>
<td>35 mm</td>
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<td>3/4" binder clips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
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<td>hexamethyldisiloxane</td>
<td>&nbsp;</td>
<td>Shin-Etsu MicroSi</td>
<td>&nbsp;</td>
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<td>New Item</td>
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<td>Microchem</td>
<td>SU8-2050</td>
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<td>PM acetate</td>
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<td>Isopropanol</td>
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<td>BSA</td>
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<td>7.5% Bovine Serum Albumin </td>
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patterning of embryonic stem cells using the bio flip chip

diffusible 신호와 세포 세포 접촉 (juxtacrine 신호)의 구성 세포 - 세포 상호 작용은 종양 성장, 줄기 세포 분화와 줄기 세포 자체 재생과 같은 다양한 생물 학적 과정에 중요합니다. 방법 숫자는 현재 체외에서 세포 신호를 변조하기 위해 존재합니다. diffusible 신호의 modulating 총액의 한 가지 방법은 문화 중에 세포 시딩 밀도를 변경할 수 있습니다. 셀 심는 실제 셀 - 셀 간격 및 셀 셀 문의 금액에 상당한 차이이 결과와 지역 환경을 처방할 수의 무작위 특성으로 인해. modulating 세포 신호에 대한보다 구체적인 접근 방식은 특정 신호 단백질을 노크 분자 억제제 또는 유전자 접근 방법을 사용하는 것입니다하지만, 이러한 방법 모두는 최고의 분자 작은 숫자를 조작하는 데 적합합니다. 여기, 우리는 기판에 원하는 장소에서 세포의 서로 다른 숫자를 배치하여 세포의 클러스터의 로컬 환경을 modulates modulating 세포 세포 신호 전달에 대한 새로운 접근 방식을 보여줍니다. 이 방법은 세포 사이의 지역 diffusible 및 juxtacrine 신호를 제어하는​​ 보완적인 방법을 제공합니다. 우리의 방법은 바이오 플립 칩 (BFC)의 사용, 단일 셀 또는 셀 작은 숫자 중 하나를 잡아 microwells, 각 크기의 수백 - 투 - 수천을 포함하는 microfabricated 실리콘 칩을 수 있습니다. 우리는 단순히 우물의 배열에 그들을 pipetting하고 배열에서 내렸어요 세포를 세척하여 세포와 칩로드합니다. 세포들이 patterning을 유지, 우물과 기판에 빠질 의하여이 칩은 다음, 기판에 뒤집혀 있습니다. 세포가 첨부되면 칩 (또는 왼쪽) 제거할 수 있습니다. 세포 patterning이 방법은 독특한 것을에 : 1) 따라서 세포가 세포 분열 따위에 의해 번식하고 마이 그 레이션 수 있도록 기판의 화학을 변경하지 않고 조직 문화 폴리스티렌, 유리, matrigel 포함한 모든 기판에 patterning이 가능 2), 그리고 심지어 피더 세포 레이어를하며, 3) 그 제도 안에 세포와 세포외 기질 제도의 창조를 허용 전통 microcontact 인쇄와 호환됩니다. 이 비디오에서는, 우리는 BFC를 사용하여 조직 문화의 폴리스티렌에 마우스 배아 줄기 세포의 patterning을 보여줍니다.

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

우리는 기판에 원하는 장소에서 세포를 삽입하기위한 간단한 방법을 보여줍니다. 이 방법은 패턴을 기판에 전지 가득 microwells를 포함하는 실리콘 칩을 틀지하여 세포를. 이 방법은 세포 사이의 diffusible 및 juxtacrine 신호를 조절하는 새로운 방법을 제공합니다.

바이오 플립 칩을 사용하여 배아 줄기 세포의 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

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

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 배아 줄기 세포로부터 조혈 줄기 세포의 유도

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 + HIV에 대한 일회용 Microfluidic 칩을 사용하여 T - 림프구 캡처

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

바이오 - 래드 총 RNA 키트를 사용 Neuroprecursor 셀에서 RNA 추출

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.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

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.

인간 배아 줄기 세포에서 염색질의 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.

공중 드롭 방법을 사용하여 마우스 배아 줄기 (MES) 세포의 체외 차별에

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.

인간 배아 줄기 세포의 문화 및 유지 관리

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.

인간 배아 줄기 세포를 동결 융해

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.

절연 및 마우스 배아 새싹 세포의 유도

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