We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

참고 : 동물 실험은 실험 동물의 관리 및 사용을위한 건강 가이드의 국립 연구소에 따라 수행 하였다. 의학의 동북 대학 의과 대학의 동물 실험에 대한위원회는 여기에 기술 된 실험 절차를 승인했다. 1. 마취와 개복술 <ol style="margin-left: 40px;"><li> 혈액 수집의 경우, 특정 병원균이없는 수컷 흰쥐를 사용합니다. 물을 제공하는 동안 적어도 18 시간 동안 쥐를 빨리. 혈액을 수집하기 위해 연령 (550-650그램) 6-8 개월에 은퇴 한 남성 개종 쥐를 사용합니다. </li><li> 10 분 동안 2.0-3.5 L / min으로 마취를 소개하는 마취 장치에 연결 상자에, 흡입 마취제 2.5-4.0 % 이소 플루 란 (isoflurane)을, 흐름. 마취를 소개하는 상자에 쥐를 전송합니다. <ol><li> 집게와 발에 자극에 대한 반응의 손실을 확인하여 마취를 확인합니다. 쥐 위스콘신의 코를 커버깊게 채혈 동안 래트를 마취 마취 장치에 접속 마스크를 토륨. 농도 및 유량은 각각 2.5-4.0 %에서 유지 2.0-3.5 L / 분됩니다. 참고 : 배아이 시약 전시 이소 플루 란 (22) 마취 쥐에서 얻은 혈청에서 배양 된 배아에 비해 배아 발달의 지연으로 마취 쥐에서 수집 된 혈청에서 배양하기 때문에 할로 세인은 흡입 마취에 사용해서는 안된다. </li></ol></li><li> 모피의 오염을 방지하기 위해, 마취 된 쥐의 복부에 70 % 에탄올을 주입하여 피부를 소독. 포셉 한 쌍의 하부 영역에서 소독 된 피부와 복부 벽을 선택하고 내부 장기를 노출하는 큰 가위로 가슴 지역으로 동시에 이러한 레이어를 잘라. 포셉 한 쌍의 복강 외부의 창자를 반영합니다. </li><li> 와 함께 두 다리쪽으로 피부와 복부 벽을 잘라큰 가위의 쌍은 더 뒤 복부를 노출합니다. 포셉 한 쌍의 복강 외부에 여분의 지방을 반영한다. </li><li> 포셉 두 쌍을 사용하여 중간 선에서 내장 지방을 선택하고 복부 대동맥을 노출 평행 방향으로 조심스럽게 지방을 분할. 쉽게 복부 대동맥의 분기점을 식별 할 수있는 길이 방향으로 내장 지방을 분할하려면이 절차를 반복합니다. 또, 대동맥 박동성이며,이 특성을 이용하여 인접 대정맥으로부터 구별 될 수있다. </li><li> 포셉 두 쌍의 신중 복부 대동맥 주위에 지방과 분기를 들고 복부 대동맥에 지방을 분리합니다. </li></ol> 2. 혈액 수집 <ol style="margin-left: 40px;"><li> 조심스럽게 아래 방향으로 경사와 대동맥의 분기점, 즉시 두개골 20 ML의 주사기에 연결된 21 G 바늘의 끝 부분을 삽입합니다. 하나와 주사기를 잡고손과 남성 쥐의 혈액 15 ㎖를 수집하기 위해 천천히 주사기를 잡아 당깁니다. </li><li> 주사기에서 바늘을 제거하고, 두 개의 얼음처럼 차가운 10-ML 멸균 테스트 튜브 (그림 1A)에 혈액을 붓는다. 원심은 혈액 응고의 형성을 지연 할 때까지 얼음에 튜브를 유지합니다. 한 번에 여러 개의 튜브를 원심 분리하여 원심 분리 시간을 줄일 수 있습니다. </li><li> 개흉술 및 심장이나 잘린 절단에 의해 마취 된 쥐를 안락사. </li></ol> 3. 쥐 혈청 준비 <ol style="margin-left: 40px;"><li> 상부층과 하부층 (도 1C)에 혈액을 분리하기 위해 1200 XG 실온 (RT)에서 5 분 동안 수집 된 혈액을 원심 분리기. </li><li> 섬유소의 응고를 해결하기 위해 1.5 ~ 2 시간 동안 4 ° C에서 튜브를 유지합니다. </li><li> 곡선 포셉 한 쌍을 사용하여 상위 계층에있는 섬유소의 응고를 선택하고, 혈청을 분리하는 섬유소 응고를 짠다. 그런 다음 할 일에 직면 곡선 집게와 섬유소 응고를 눌러두 개의 레이어 (그림 1D) 사이의 인터페이스 근처 wnwards. </li><li> 또한 섬유소의 응고를 분리하는 단계 3.3를 반복합니다. </li><li> 1,200 XG 및 RT (그림 1E)에서 5 분 동안 테스트 튜브를 원심 분리기. </li><li> 조심스럽게 층류 공기 흐름 캐비닛에 멸균 피펫을 사용하여 15 ㎖의 멸균 튜브에 혈청을 전송합니다. </li><li> 남아있는 붉은 세포를 제거하기 위해 1,200 XG와 RT에서 5 분 동안 수집 된 쥐의 혈청을 원심 분리기. </li><li> 튜브 (그림 1 층)의 하단에 잔류 붉은 세포에 의한 오염을 방지하기 위해 멸균 피펫을 사용하여 관을 소독 새로운 15 ㎖의주의 혈청 해변. </li><li> 보체계를 실활 30 분 동안 56 ℃에서 수욕 혈청을 배양한다. </li><li> 층류 공기 흐름 캐비닛에 RT로 쥐의 혈청 튜브를 냉각. 혈청 개별 멸균 튜브에 10 ml로 분주합니다. 사용 (그림 1H)까지 -20 ° C에서 튜브를 저장합니다. </li></ol>

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No conflicts of interest are declared.

WEC 실험 결과의 재현성이 높은 품질의 IC 쥐의 혈청과 정확한 배아 해부 기술 3의 사용에 의존하고 있습니다. 금식은 혈액에있는 포도당의 농도를 표준화 할 필요가 있기 때문에 혈액을 수집하기 전에 금식의 기간을 단축하지 마십시오. 호르몬 수준은 발정주기에 따른 여성 동물에서 변화하기 때문에 암컷 쥐 혈청의 제조에 사용되지 않아야하고, 에스트라 디올과 같은 호르몬 변화는 배아...

모델 동물의 다양한 분자 및 세포 수준에서 발달 메커니즘을 조사하기 위해 발달 생물학에 사용됩니다. 예를 들어, 양서류와 조류 종은 널리 이러한 태아가 어머니의 외부 개발하기 때문에 태아의 직접 조작에 적합한 클래식 모델 동물로 사용되어왔다. 이 동물과는 달리, 포유류의 태아는 엄마의 자궁에서 성장하고, 이후 단계에서의 성장은 자궁의 기능에 결정적으로 의존한다. 따라서, 직접적 초기 단계에서 마우스 및 래트에서 것과 포유류 배아를 조작하는 통상적 어렵다. 1960 년대에, 데니스 새로운 지속적인 산소 공급 및 열 제어 1 WEC 장치를 사용하여 포유류의 전체 배아 문화 (WEC) 기술을 설립했다. WEC에서, 마우스 및 쥐의 배아는 생체, (즉, 자궁의 이상으로) 증가 할 수 있습니다. WEC 기술은 종종 다양한 Chemic의를 추가하여 기형에 사용되었지만AL은 배양액에 화합물이 기술은 또한 포유 동물 2-4에서 고유 발달 메카니즘을 조사하기 위해 다양한 개발 생물학 연구에서 사용되었다. 예를 들어, WEC는 형광 염료 4.0, 세포 이식 (6) 및 리포 펙션 (7) 및 일렉트로 8-13 통한 유전자 도입을 이용하여 야생형과 돌연변이 등의 배아 세포 라벨링 등의 다른 기술과 결합된다. 최근 자궁 조작에 나중 단계에서 쥐의 배아에서 발달 과정을 분석하는 데 사용되었으며, 일렉트로 기술 14 ~ 16과 결합되어 있습니다. 그러나, 이들 기술은 초기 단계 배아에 DNA 용액의 정확한 로컬 주입을 달성 어려움으로 인해 postimplantation 및 midgestation 배아의 조작에 적합하지 않다. 비록 초기 배아 (에 초음파 유도 세포 이식 및 바이러스 벡터를 주입 즉,, E8.5-E-9.5 마우스의) 자궁 내 이전에 17, 18, ​​우수한 기술은 높은 성공률로이 실험을 수행하는 데 필요한보고되었습니다. 따라서, 높은 접근성 생체와 WEC는 마우스와 쥐 배아의 조작에 대한 장점이 있습니다. 수컷 쥐에서 준비 즉시 원심 분리 (IC) 쥐의 혈청은 종종 WEC 매체에 사용됩니다. 배아 (쥐 마우스 나 E10.0에서 E8.0 이전 즉,) postimplantation 단계에서 배양하면, 합성 중간 IC 쥐 혈청의 혼합물은 종종 WEC 19을위한 매체로 사용됩니다. 더 현재 대안 미디어가 할 수 없기 때문에 그러나, 중반 임신에서 문화 배아에 (즉., E8.0 12.5 마우스 배아 또는 쥐의 배아에서 E10.0-E14.5에서), 100 %의 혈청 매체로 사용한다 2 일 이상 체외에서 정상적으로 성장하는 배아. 고품질 쥐 혈청의 제조는WEC 실험에서 재현성을 달성하기위한 중요한 단계. 연기를 원심 분리에 비해, IC는 적혈구의 대부분이 이미 피브린 응괴로부터 분리 되었기 때문에 혈청 피브린 응괴를 짜내 수집 될 때 용혈을 줄이는 이점이있다. 용혈성 쥐의 혈청은 래트 및 마우스 배아의 정상적인 성장을 지원하는 데 실패 할 때, 즉시 원심 분리하여 혈청을 준비 지연 원심 분리를 사용하여 제조하는 것이 바람직하다. 우리의 프로토콜이 다른 프로토콜에 비해 두 단계를 추가로 포함 18 ~ 20 (예., 이전에 첫 번째 원심 분리에 얼음에 수집 된 혈액을 저장하고 처음으로 원심 분리 한 후 4 ° C에서 2 시간 동안 수집 된 혈액 샘플을 유지). 전반 단계는 혈병의 형성을 지연시킬 수 있고, 후자의 단계는 락 스퀴즈 피브린 응괴의 응고를 촉진시킨다. 따라서, 우리의 프로토콜은 초보자를 사용할 수 있습니다. 그러나, 재생 채혈하고 혈청단순히 프로토콜 책을 참조하여 정확하게 추출은 19 ~ 21 매우 어렵습니다. 이 비디오 문서에서는, 우리는 수컷 쥐와 원심 분리하여 혈청을 추출의 복부 대동맥에서 채혈을 포함 최적의 IC 쥐 혈청의 준비를위한 우리의 표준 프로토콜을 보여줍니다.

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>For rat anesthesia.</td></tr><tr><td>Large scissors</td><td>Napox</td><td>B-7H</td><td>Stelized scissors for cutting the skin and muscle of rats.&amp;nbsp;</td></tr><tr><td>Curved forceps</td><td>Napox</td><td>A-3-2</td><td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Charles Rivers Laboratories</td><td></td><td>Retired male rats from colony in the lab or purchased retired male rats.</td></tr><tr><td>Syringe (20 ml)</td><td>TERUMO</td><td>SS-29ESZ</td><td></td></tr><tr><td>Needle (21 G x 5/8&quot;)</td><td>TERUMO</td><td>NN-2116R</td><td></td></tr><tr><td>Sterile test (spitz) tube (10 ml)</td><td>ASIAKIZAI</td><td>1101C000B-10</td><td>For collection of boold</td></tr><tr><td>Sterile disposable pipette</td><td>Eiken Chemical</td><td>CD2000 No.4</td><td></td></tr><tr><td>Sterilie disposable tube (15 ml)&amp;nbsp;</td><td>Falcon</td><td>2196</td><td></td></tr></tbody></table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

도 1은 혈액 세포 및 혈청의 분리에 개시된 방법의 대표적인 결과를 나타낸다. 우리는 일반적으로 은퇴 한 남성 쥐 (그림 1A)에서 혈액 15 ㎖를 얻을 수 있습니다. 원심 분리에 의해 수집 된 혈액 혈청과 화살표로 표시되는 피브린 응고, 및 혈액 세포 (도 1c)를 포함하는 하부층을 포함하는 상부 층으로 분리 될 수있다. 중요한 사실은, 용혈성 혈액 샘플은 혈청, ?...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

포유류의 전체 배아 문화에 적합 쥐 혈청의 제조

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.8K 조회수.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

비디오: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

공동 문화 시스템에 신경 연결의 형성을 평가하기위한 Optogenetic 접근

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

연구

발생학

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

혈청이없는 단층 문화 마우스 배아 줄기 세포의 신경 분화

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

초기 Postimplantation 마우스 배아로 세포 나 DNA를 정확하게 현지화 전송하는 방법

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

쥐 희소 돌기 아교 세포 전구 문화의 준비 및 Oligodendrogenesis의 정량화 이중 적외선 형광 스캔을 사용하여

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

쥐 배아 Hemogenic 내피 세포의 분리

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

신경과학

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

신경 세포와 Glia 개발 연구 랫 두뇌 집계 문화의 준비

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

생물학

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

엑스 우테로 사전 배후에서 고급 유기발생에 이르는 마우스 배아의 배양

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...