우리는이 작품의 지원 CIRM 감사하고 싶습니다. 셀 라인은 넉넉한 마운트시나이 병원, 토론토, 온타리오에서 박사 Andras 나지에서 기증되었다.

1. Shrinky - 딩크 몰드 만들기 <ol><li> 좋은 정의 프린터를 사용 shrinky - 딩크 시트에 원하는 패턴을 인쇄합니다. </li><li> 163에서 베이크 shrinky - 딩크 시트 ° C 약 10 분, 또는 때까지 대해 전적으로 축소하고 정기적인 모양을 인수하는 데. </li><li> shrinky - 딩크의 금형이 냉각되면, 이소프로판올 욕조에서 잠수함 그것은 전체 표면은 거의 덮여 때까지. </li><li> 신중하게, 금형을 통해 몇 가지 아세톤을 스프레이하고 컨테이너 몇 번 흔들. 아세톤의 초과를 씻고 shrinky - 곰팡이가 깨끗해 보여요 때까지이 단계 몇 번 반복해야 더 이소프로판올 추가합니다. </li><li> 남아있는 유기 용매 씻어 10 분 정도 증류수에 담가 곰팡이. </li><li> 에어 shrinky - 곰팡이 청소. 163에서 5 분 동안 그것을 다시 열 ° C. 이것은 남아있는 용매를 증발 압축 잉크 및됩니다. </li></ol> 2. PDMS microwells 만들기 <ol><li> 10시 1분 PDMS / 치료 에이전트 혼합물을 준비하고, 몇 분 동안 적극적으로 선동. </li><li> 작은 페트리 접시에 넣어 shrinky - 딩크 금형. 그것은 금형 표면 2분의 1에 대한 cm에 도달할 때까지 PDMS 혼합물을 붓고. </li><li> PDMS 혼합물에서 모든 거품을 제거하기 위해 진공 종 아래 플레이스 요리. </li><li> 70 오븐에 넣어 요리 ° C, 하룻밤. </li><li> 금형에서 고체 PDMS를 차단하고 단지 압력을 적용하여 유리 슬라이드에 바인딩합니다. </li><li> 그것이 PDMS 사이 incrusted 잉크 잔류물을 가지고 있기 때문에, 첫번째 microwell 칩 폐기하십시오. </li><li> 잉크 무료이며 더 정의된 모양을 가지고 두 번째 칩을 생산하기 위해이 절차를 반복합니다. </li><li> 청소는 microwell 칩 70 % 에탄올 솔루션을 사용합니다. 그것을 소독하기 위해 10 분 동안 자외선 광원 아래로 놓으십시오. </li></ol> 3. microwells에서 트래핑 세포 <ol><li> 세포를 카운트하고 원하는 농도 (당신이 싶은 얼마나 많은 우물의 초기 세포에 따라)에 문화 미디어에서 그들을 희석. 예를 들어, 잘 당 약 10-15 세포 (평균 = 11, SD = 5.4, 로딩 속도 = 93%)하기 위해서, 우리는 8 농도 × 10 4 세포 / ML를 사용합니다. 17 × 10 4 세포 / ML의 농도에 대해서는 안정적으로 잘 당 25의 사이 35 세포를 (평균 = 27.17857 SD는 = 7.7, 로딩 속도 = 100 %)받을 수 있습니다. </li><li> 조심스럽게 확정 PDMS 기반을 포함하는 50 ML의 원심 분리기 튜브의 microwell 칩 장소. </li><li> 세포 솔루션의 2JDP ML에 대한 추가합니다. </li><li> 5 760 RPM 분만에 4 ° C.위한 원심 분리기 </li><li> 초과 솔루션 아웃 Pipet 조심스럽게 PBS 1X 솔루션 microwell 씻는다. </li><li> 원심 분리기 튜브의 칩을하면서 조심하고, 작은 페트리 접시에 microwell를 배치합니다. </li><li> 1 X PBS 솔루션을 사용하여 셀 초과 씻으십시오. </li><li> 물론 당 세포의 의도 번호를 확인할 수 거꾸로 현미경 microwell 칩을 놓으십시오. </li><li> 부화 microwell 표준 조건에서 세포를 포함하는. <img src="/files/ftp_upload/old/692_2008_7_21_1/692_Figure-01.png" alt="그림 - 01" width="350" height="183" /></li></ol> 4. 세포 배양 <ol><li> 연구실에 정상 EB 프로토콜을 따르십시오. </li><li> 천천히 챔버의 측면에서 매체를 변경, microwell에있는 세포를 방해하지 마십시오. </li></ol>

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li><li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines:+Formation+of+visceral+yolk+sac,+blood+islands+and+myocardium.">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li><li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li><li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies.">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li><li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+differentiation+of+stem+cells.">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li><li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP/BMPR/Smad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media.">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li><li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li></ol>

우리는 microfabricated 웰스 (Shrinky - Dinks에서 성형)에 세포의 미리 정의된 숫자를로드하고 embryoid 몸 개발을 위해 그들을 유지하기 위해, 간단한 신속하고 확장 가능한 문화 방식을 개발했습니다. 마지막으로, 이러한 전지는 더 이상 분석 및 실험을 위해 쉽게 액세스할 수 있습니다. 이 방법은 실험실 의무이며, 우리가 석판술의 필요성을 사전에 제거하다 때문에 아무 전용 장비를 필요하지 않습니다. 우리는 embryoid ?...

KO DMEM : 40.8ml, 81.6ml 15 % KSR : 7.5ml, 15ml 1X 펜 - Strep : 0.5 ML, 1ml 2mM L - 글루타민; (100ml 50ml 대비 총 미디어 :) mESC 미디어 (맥클로스키 실험실 프로토콜)에 대한 0.5 ML, 1ml NEAA : 0.5 ML, 1ml 평범한 : 100ul, 200ul BMP - 4 (10ng/ml) : 50ul, 100ul 희석 BME :. 50ul, 100ul (PBS와 주사기 필터 5ml에 살균 필터 BME의 35ul 추가 소독 삭제 이주 후에. 솔루션에 최종 농도는 0.1 MM입니다)

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

Embryoid 단체 (EB)는 배아 줄기 세포 집합체입니다. 이러한 집합체를 만드는 가장 일반적인 방법은 매달려 드롭 방식, 잘 접시에 세포의 임의의 숫자를 pipetting의 힘드는 접근이다. 서로의 가까이에 강제 줄기 세포 사이의 상호 작용은 EBS의 생성을 촉진. 우물의 각 미디어가 수동으로 매일 교환해야했기 때문에, 이러한 방식은 수동으로 집약적이다. 동일한 조건에서 양식 때 또한, 셀 셀, 셀 용해 계수 상호 작용, 산도, 그리고 산소의 가용성을 포함한 환경 매개 변수 EB 크기의 기능을 수 있기 때문에, 전통적인 매달려 방울에서 얻은 세포 집단도 크게 다를 수 있습니다. 최근 연구는 실제로 집계를 형성하는 세포의 초기 숫자가 줄기 세포 분화에 중요한 영향을 미칠 수있는 것으로 나타났습니다. 우리는 microfabricated 우물로 세포의 미리 정의된 숫자를로드하고 embryoid 몸 개발을 위해 그들을 유지하기 위해, 간단한 신속하고 확장 가능한 문화 방식을 개발했습니다. 마지막으로, 이러한 전지는 더 이상 분석 및 실험을 위해 쉽게 액세스할 수 있습니다. 이 방법은 실험실 의무이며 전용 장비를 필요하지 않습니다. 우리는 붉은 형광 마우스 세포 라인을 (129S6B6 - F1)를 사용 embryoid 시체를 작성하여이 방법을 보여줍니다.

Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

우리는 microfabricated 우물로 세포의 미리 정의된 숫자를로드하고 embryoid 몸 개발을 위해 그들을 유지하기 위해 간단하고 빠른 방법을 보여줍니다.

방울을 공중 Shrinky - 딩크 : Embryoid 기관 폼 문화하는 간단한 방법

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

shrinky-dink-hanging-drops-simple-way-to-form-culture-embryoid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K 조회수.  University of California Merced - UC Merced. 우리는 microfabricated 우물로 세포의 미리 정의된 숫자를로드하고 embryoid 몸 개발을 위해 그들을 유지하기 위해 간단하고 빠른 방법을 보여줍니다.

비디오: Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

연구

생체공학

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 ...

생물학

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

의 준비 Drosophila</em공중 드롭 프로토콜을 사용하여 라이브 영상을위한> 태아

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

인간 배아 줄기 세포에서 뇌 오르가노이드를 생성하기위한 정적 자기 지향 방법

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

발생학

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

광학 핀셋을 사용하여 감금된 세포 전 역학의 직접 힘 측정

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Hanging Drop Method: A Technique to Generate 3D Melanoma Spheroids

Source: M&#252;ller, I. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/57500"> A 3D Organotypic Melanoma Spheroid Skin Model</a>. J. Vis. Exp. (2018)
This video describes the technique of generating three-dimensional spheroids via the hanging drop method using melanoma cells. The generated in vitro&#160;three-dimensional (3D) organotypic melanoma spheroid model&#160;can portray the&#160;in vivo&#160;architecture of malignant melanoma and may give insights into intra-tumoral interactions.&#160;

행잉 드롭 방법(Hanging Drop Method): 3D 흑색종 스페로이드를 생성하는 기술

Source: M&#252;ller, I. et al.,  A 3D Organotypic Melanoma Spheroid Skin Model. J. Vis. Exp. (2018)
This video describes the technique of generating ...

JoVE Encyclopedia of Experiments

암 연구학

Skin Cancer

Assays and techniques

Developing Neuron Balls Using a Hanging Drop Culture Technique

Source: Parvin, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/59893">Presynapse Formation Assay Using Presynapse Organizer Beads and &#34;Neuron Ball&#34; Culture.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the process of generating neuron balls using a hanging drop culture technique. Within the hanging drops, cells move towards the bottom, self-assembled into three-dimensional structures forming neuron balls, which can be used for neurobiological studies.

Hanging Drop Culture 기법을 이용한 뉴런 볼(Neuron Ball) 개발

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

A Technique to Optimize Cerebral Organoid Culture Using Lateral Soft Light Illumination

Source: Chen, B. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63989">Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination.</a>&#160;J. Vis. Exp.&#160;(2022).
This video showcases the utilization of a lateral soft light illumination technique to improve the naked-eye visualization of embryoid bodies, thereby facilitating their manipulation and transfer during cerebral organoid culture. It elucidates the setup of soft light illumination, methods for media changes and embryoid body transfer, as well as the procedure for inducing neural differentiation in embryoid bodies for cerebral organoid development.

측면 소프트 라이트 조명을 사용하여 대뇌 오가노이드 배양을 최적화하는 기술

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of the soft light ...

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Aspirate the medium with a pipette, and wash the cells once with DPBS. Then, add 500 microliters of cell detachment solution, or 0.5 millimolar EDTA, to dissociate the colonies, and incubate at 37 degrees Celsius for 3 to 5 minutes. Next, add 1 milliliter of fresh E8 media to each well, and pipette gently to detach all cells.
Transfer the entire cell suspension into a 15-millimeter centrifuge tube, and add another 1 milliliter of fresh E8 media. Then, spin down the cells at 290 g for 3 minutes at room temperature.
After discarding the supernatant, resuspend the cell pellet in 1 milliliter of E6 media, supplemented with 50 micromolar ROCK inhibitor, and count the cells using a hemocytometer.
Prepare a cell suspension of the desired seating density in E6 media, supplemented with the ROCK inhibitor. Then, add 150 microliters of the cell suspension into each well of a 96-well ultra-low attachment plate.
Next, centrifuge the plate at 290 g for 3 minutes at room temperature to force-aggregate the cells. Then, incubate the plate at 37 degrees Celsius in a humidified atmosphere with 5% carbon dioxide for embryoid body formation.
After 24 hours of incubation, transfer the plate to the hood, and carefully aspirate 120 microliters of the media using a pipette, taking care not to aspirate the embryoid body by lowering the pipette tip too far into the well.
Next, add 150 microliters of fresh E6 media, supplemented with 2 micromolar XAV939 and the SMAD inhibitors to each well. Transfer the plate back to the incubator, and replace the medium daily with freshly prepared E6, supplemented with the inhibitors.
At day seven of incubation, check whether all the embryoid bodies have reached a diameter of 550 to 600 micrometers and display a smooth, clear edge. At this stage, they are ready to be embedded in an extracellular matrix.
To embed the organoids, prepare dimpled embedding sheets by placing a 4-inch long thermoplastic sealing film sheet on an empty P200 box. Then use a 15-milliliter conical tube, or a 500-microliter microcentrifuge tube, and gently press down the film sheet into the holes to make 12, or the desired number of dimples.
Next, spray 70% ethanol on the film sheet, and let it dry inside the hood with the UV light on for at least 30 minutes. Meanwhile, thaw a sufficient quantity of basement membrane matrix on ice. Then, using a wide bore P200 tip, transfer one embryoid body from the 96-well plate to each dimple. Remove as much media as possible with a normal pipette tip, but do not let the embryoid body dry.
Next, with a regular pre-chilled P200 tip, add approximately 30 microliters of undiluted membrane matrix to each organoid, ensuring that the embryoid body is as close as possible to the center of the droplet.
Once all embryoid bodies are embedded in the matrix, place the film sheet in a sterile Petri dish. Then incubate the Petri dish at 37 degrees Celsius for 10 minutes in a humidified atmosphere with 5% carbon dioxide to allow the membrane matrix to solidify.
For organoid differentiation, add 5 milliliters of the prepared differentiation media with B27 without vitamin A to one well of an ultra-low attachment 6-well plate. Pre-warm the plate to 37 degrees Celsius in an incubator. Once the membrane matrix has solidified, transfer the embedded embryoid bodies to the 6-well plate by pushing out the dimples from the back of the film sheet. If necessary, use 1 milliliter of media from the well, and pipette it onto the sheet to detach the droplets from the film sheet.

유도 만능 줄기 세포 유래 대뇌 오가노이드 생성

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Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Begin by setting up the hanging drop cell culture. For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20 microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micropipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren&#39;t too close together to prevent them from merging.
Fill the plate with 5 to 10 milliliters of PBS, and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate, filled with 10 milliliters of differentiation medium. Then, place the plate on an orbital shaker set to low speed in the incubator. After another two days, centrifuge the cells at 200 times g for three minutes, and remove the supernatant.
To induce embryoid body differentiation into neural progenitor cells, resuspend the cells in differentiation medium with 5-micromolar retinoic acid. Incubate the plate for two days. Then, replace the least half of the medium with fresh medium by tilting the plate and pipetting it out.

방울을 공중 Shrinky - 딩크 : Embryoid 기관 폼 문화하는 간단한 방법

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