우리의 작품은 스위스 국립 과학 재단 (SNF), 시스템 생물학 (SystemsX.ch), 근육질 영양 장애 협회 미국 (MDA), 협회 테크 프랑세즈 contre 레 Myopathies (AFM), 미국 Mitochondrial 질병 재단에서 스위스의 이니셔티브에 의해 지원됩니다 (UMDF), Gebert-RUF 재단 희귀 질병 프로그램 (GRF), 근육 질환 (SSEM / FSRMM), 스위스 생활 &quot;Jubiläumsstiftung FÜR Volksgesundheit 깨물어 medizinische Forschung&quot;, 로슈 학술 진흥 재단과 대학의 연구에 대한 스위스의 학회 바젤의.

1. 기본 인간 근육 세포 배양의 준비 <ol><li> explantation - 다시 explantation 기법 넷에 따르면 인간의 근육 세포 배양을 마련한다. 첫째, biopsies에서가 아닌 근육 조직을 제거합니다. 그런 다음, 섬유아 세포가 explants에서 나타날 수있는 플라즈마의 coagulum에 1mm 3 근육 explants를 포함. </li><li> 임베디드 explants는 플라즈마-젤라틴 코팅 접시에 옮겨주고 (MEM 10 % 태아 소 혈청 [FBS], 1 % 페니실린 / 스트렙토 마이신, 10 μg / ML 인슐린, 25 %의 중간 199, 함께 보완 정상적인 성장 매체로 성장하게됩니다 10 NG / ML 인간 표피 성장 인자 [EGF], 12.5 NG / ML 인간 fibroblast의 성장 인자 단일층의 [FGF]). 이러한 조건 하에서 explants 반발 세포는 근육 세포입니다. 이러한 세포는 explants이 제거된 후에도 단일층 성장 나갈 것입니다. </li><li> 문화 aneurally 정상적인 성장 매체의 0.1 % 젤라틴 코팅 접시에있는 세포. 세포가 될 수 있습니다aneurally 배양해하고, 필요에 따라 증폭. 그것은 공동 문화권을 수행하기 위해 3와 8 사이 통로에서 근육 세포를 사용하는 것이 좋습니다. 그것은 젊은 myotubes에 신경을 분포시키다 것이 중요하다,이 시점 이후, 공동 문화는 실패의 위험이 있습니다. 또한, myotubes 너무 크고 너무 두꺼운가 없습니다. </li><li> innervation이 계획되어 이틀 전, 플레이트 정상적인 성장 매체에 요리 당 500,000 세포 밀도에서 35mm 조직 문화 요리 (또는 6 잘 접시)에서 근육 세포. 그 농도에서 세포 그 이후는 다음날 융합을 시작할 적절한 confluency 및 후속 innervation 날에입니다. 이러한 조건을 사용하여 myotubes는 최적의 innervation시키고 그럼으로써 이후 섬유 수축에 맞는 단계에 있습니다. </li><li> 다음 날, 25 %의 중간 199 5 % FBS, 10 μg / ML 인슐린과 페니실린 / 스트렙토 마이신의 1 %로 보충 MEM 구성된 융합 미디어로 정상 성장 매체를 교체하십시오. </li></ol> 2. Isolati<html> 쥐 배아 척수의 Explants의에 척수의 explants를 분리하기위한 모든 절차는 10% 태아 소 혈청 (FBS)을 포함하는 행크의 균형 소금 솔루션 (HBSS, Gibco / Invitrogen 심판 14,170)에, 쌍안 현미경으로 수행됩니다. <ol><li> 이것은 쥐의 배아가 다른 개발 단계로 ED 13 층과 14 층 사이에 있다고하는 것은 전체 과정을 위태롭게 할 것이 매우 중요합니다. CO 2와 임신 여성을 희생하고 HBSS 매체 전체 배아 체인을 수집합니다. 각각의 배아를 분리하고 안락사를 위해 목을 벨. </li><li> 한 조각의 신체의 나머지 부분에서 배아 척수를 해부하며 주위의 근육과 결합 조직을 제거하십시오. 기능성 innervation을 보장하기 위해 척수에 연결된 지느러미 루트 ganglions (DRGs)를 유지하는 데 매우주의하십시오. </li><li> 각 explant는 최소한 2 DRG (1mm 3 explants) 그것에 첨부를 포함하는 방식으로 transversally 각 척수를 썰어. </li></ol><html> 배아 쥐 척수의 Explants과 인간 근육 세포 ove_title &quot;&gt; 3. Innervation <ol><li> 단일층에 매체의 벌금 레이어를두고, 근육 세포 배양에 융합 매체를 제거합니다. </li><li> 근육 세포 층, 35 밀리미터 접시 당 다섯 균등하게 게재 explants에 신중 explants를 놓습니다. </li><li> 매우 느리게 추가, 각 explant에 현명, 퓨전 미디어를 놓습니다. 그것은 explants 먼저 준수하고 세포와 접촉을하도록 너무 많은 살아남기 위해 근육 세포에 대한 매체를 추가할 필요가 있지만, 아니다. 매체가 제대로 (너무 강하게 또는 너무 많은 매체)에 추가되지 않은 경우 explants는 역부족되며 근육 세포를 신경을 분포시키다되지 않습니다. </li><li> explants가 준수하기 시작하면, 35 밀리미터 접시 당 2 ML로 융합 미디어를 위로하고, 인큐베이터에 다시주의 깊게 요리를하다. </li></ol> 4. Heterologous 신경 근육의 공동 문화의 유지 보수 <ol><li> 일주일에 두 번 융합 미디어를 변경합니다. </li></ol>_title &quot;&gt; 5. 대표 결과 즉시 이일로 innervation 후, 그것은 각 explant에서 신흥 및 버튼과 같은 구조 (그림 1)로 표시 근육 세포와 연락처를 확립 neurites을 볼 수 있습니다. 이르면 innervation 후 4-6일로서, 몇 가지 개별 섬유 계약에 시작됩니다. 수축이 단순히 일주일에 두 번 배지를 변경하여 유지, 여러 달 동안 유지됩니다. 약 2 주가 지난 후, 척수의 explant 자체는 썩어빠진지만 innervation이 보존되어있다. 이것은 정상입니다. 신경 구성 요소의 벌금 레이어 근육 세포 레이어의 표면에 유지됩니다. 이 신경 네트워크는 모터 장치의 기능을 유지하는 데 충분합니다. explants이 innervation 후 매체를 하루에 떠있다면, 그들은 준수하지 않습니다. 근육 세포 계층이 때 발생충분히 합류, 또는 경우 매체 아니라도 대략 추가됩니다. 때로 explant을 준수합니다 그런데 neurites가 등장할 것입니다. 이것은 가능성이 그것에 첨부 DRG의 부족 때문입니다. 물론이 경우, innervation을 설정할 수 없습니다. explants 여전히 쉽게 분리 수 있으므로, 첫째 주에 매우 신중하게 요리를 조작. 융합 미디어를 교환하려면, 셀 부대를 피하기 위해 최소한의 속도로 액체 드롭 현명한를 추가합니다. <img alt="그림 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> 그림 1. 척수 explants - 근육 세포 일 ~ 15 일 1에서 공동의 문화.

<ol>
	<li>Delaporte, C., Dautreaux, B., Fardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myotube+differentiation+in+vitro+in+different+culture+conditions.">Human myotube differentiation in vitro in different culture conditions.</a> Biol. Cell. 57, 17-22 (1986).</li><li>Kobayashi, T., Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+cultured+in+monolayer+and+cocultured+with+fetal+rat+spinal+cord:+importance+of+dorsal+root+ganglia+for+achieving+successful+functional+innervation.">Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.</a> J. Neurosci. 7, 3131-3141 (1987).</li><li>Braun, S., Croizat, B., Lagrange, M. C., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+muscular+abnormalities+in+culture+in+spinal+muscular+atrophy.">Constitutive muscular abnormalities in culture in spinal muscular atrophy.</a> Lancet. 345, 694-695 (1995).</li><li>Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+program+for+investigating+adult+human+skeletal+muscle+grown+aneurally+in+tissue+culture.">A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.</a> Neurology. 25, 58-67 (1975).</li><li>Guettier-Sigrist, S., Coupin, G., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+required+to+efficiently+innervate+human+muscle+cells+in+vitro.">Cell types required to efficiently innervate human muscle cells in vitro.</a> Exp. Cell Res. 259, 204-212 (2000).</li><li>Lefebvre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+spinal+muscular+atrophy-determining+gene.">Identification and characterization of a spinal muscular atrophy-determining gene.</a> Cell. 80, 155-165 (1995).</li><li>Schrank, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+survival+motor+neuron+gene,+a+candidate+gene+for+human+spinal+muscular+atrophy,+leads+to+massive+cell+death+in+early+mouse+embryos.">Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 9920-9925 (1997).</li><li>Hsieh-Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+spinal+muscular+atrophy.">A mouse model for spinal muscular atrophy.</a> Nat. Genet. 24, 66-70 (2000).</li><li>Monani, U. 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+human+centromeric+survival+motor+neuron+gene+(SMN2)+rescues+embryonic+lethality+in+Smn(-/-)+mice+and+results+in+a+mouse+with+spinal+muscular+atrophy.">The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.</a> Hum. Mol. Genet. 9, 333-339 (2000).</li><li>Sleigh, J. N., Gillingwater, T. H., Talbot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mouse+models+to+understanding+the+pathogenesis+of+spinal+muscular+atrophy.">The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy.</a> Dis. Model. Mech. 4, 457-467 (2011).</li><li>Durbeej, M., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies+involving+the+dystrophin-glycoprotein+complex:+an+overview+of+current+mouse+models.">Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models.</a> 12, 349-361 (2002).</li><li>Schnabel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Standard+model.">Neuroscience: Standard model.</a> Nature. 454, 682-685 (2008).</li><li>Benatar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+treatment+trials+in+the+SOD1+mouse+and+in+human+ALS.">Lost in translation: treatment trials in the SOD1 mouse and in human ALS.</a> Neurobiol. Dis. 26, 1-13 (2007).</li><li>Dorchies, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+innervation+and+differentiation+of+X-linked+myotubular+myopathy+muscle+cells+in+a+nerve-muscle+coculture+system.">Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.</a> Neuromuscul. Disord. 11, 736-746 (2001).</li><li>McFerrin, J., Engel, W. K., Askanas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+innervation+of+cultured+human+muscle+overexpressing+betaAPP+experimentally+and+genetically:+relevance+to+inclusion-body+myopathies.">Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies.</a> Neuroreport. 9, 3201-3205 (1998).</li></ol>

우리는 공개 할게 없다.

근육 세포 배양은 보통 여러 셀 및 셀 타입의 연결의 중요성을 요점을 되풀이하지 않기 때문에 정상과 병리 학적 맥락에서 근육 세포 기능을 연구하는 생리학의 체외 도구, myologists에 대한 높은 관심이다. 근육 세포에 모터 뉴런을 정화의 첨가 Schwann 세포의 존재는 5 개의 효율적이 될 innervation 필요하고 관련 프로토콜이 여기에 설명된 (3D + 특수 유통) topologically하지 않았습니다이기...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> HBSS </td><td> Gibco / Invitrogen </td><td> 14,170 </td></tr><tr><td> MEM </td><td> Gibco / Invitrogen </td><td> 31,095 </td></tr><tr><td> 중형 199 </td><td> Gibco / Invitrogen </td><td> 31,153 </td></tr><tr><td> 태아 소 혈청 </td><td> 태아 복제 Perbio </td><td> SH30066.03 </td></tr><tr><td> 인슐린 </td><td> 시그마 </td><td> I9278 </td></tr><tr><td> 인간 EGF </td><td> 시그마 </td><td> E9644 </td></tr><tr><td> 인간 FGF </td><td> 시GMA </td><td> F0291 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 용액 </td><td> Gibco </td><td> 15,140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

신경 구성 요소의 부재에서, 세포 분화가 제한되며 모터 신경 세포의 자극 1가 누락되어 자연스럽게 있기 때문에 인간의 기본 근육 세포 단일층에 aneurally 드물게 계약을 배양해. 이러한 제한은 교양 근육 세포에있는 많은 신경근육학 질병의 체외 연구를 방해. 중요한 monolayered, 교양 근육 세포의 실험적 제약이 공동 문화권에서 척수의 explants있는 myofibers의 기능 innervation에 의해 극복할 수있다. 여기서는 Askanas이 개발한 방법에 따라 차별화 및 섬유 수축을 완료하는 데 선도적인, 인간의 기본 근육 세포의 효율적이고 적절한 innervation을 달성하는 데 필요한 여러 단계를 보여줍니다. 이렇게하려면, 근육 세포는 여전히 척수의 조각에 부착된 지느러미 루트 신경으로 ED 13.5에서 쥐의 배아의 척수의 explants와 공동 배양해 있습니다. 며칠 후, 근육이 좋bers 계약에 시작하고 결국 근육 세포에 연결되는 척수의 explants에서 돌출 기능 neurites 의해 innervation을 통해 교차 줄무늬가된다. 이 구조는 단순히 문화 매체의 정기 교류에 의해, 많은 달 동안 유지하실 수 있습니다. 그것이 인간의 근육 발달과 innervation의 종합 분석을위한 기능적인 모델을 대표로 귀중한 도구의 응용 프로그램은 다수 있습니다. 사실, 완벽한 드 노보 신경근육학 접합 설치 기본 및 생리적 컨텍스트의 각 단계에서 여러 매개 변수를 쉽게 측정을 수 있도록 문화 접시에 발생합니다. 몇 가지 예제, 게놈 및 / 또는 proteomic 연구를 인용하는 것은 공동의 문화에서 직접 수행할 수 있습니다. 두 구성 요소가 서로 다른 종에서 온 있기 때문에 또한, 사전 및 사후 시냅스 효과는 특별히 별도로, 신경근육학 교차점에서 평가 수쥐, 인간, 각각. 신경 근육의 공동 문화는 또한 근육이나 신경근육학 질병 3 앓고 환자 격리 인간의 근육 세포를 수행할 수 있으며, 따라서 후보 의약품에 대한 선별 도구로 사용할 수 있습니다. 마지막으로, 특별한 장비가 있지만 일반 BSL2 시설은 문화 접시에있는 기능적인 운동 단위를 재현하는 데 필요하지 않습니다. 이 방법은 따라서 정상과 질병 맥락에서, 근육뿐만 아니라 신경근육학 기능의 생리적 및 기계론의 연구 신경근육학 연구 지역 사회 모두에 유용합니다.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

교양 근육 세포 innervated 근육을 요점을 되풀이하는 불충 분한 모델입니다<em&gt; 생체내에서</em&gt;. 기능적인 운동 단위가 복제하실 수 있습니다<em&gt; 체외에서</em&gt; 쥐의 배아 척수의 explants를 사용하여 차별화된 인간의 기본 근육 세포의 innervation에 의해. 이 문서는 척수의 explants와 근육 세포의 공동 문화가 확립되는 방법을 설명합니다.

문화 접시에있는 기능성 모터 단위 : 척수의 Explants와 근육 세포의 공동 문화

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

a-functional-motor-unit-culture-dish-co-culture-spinal-cord-explants

Microsoft Bing

Research

JoVE Journal

Neuroscience

15.2K 조회수.  University of Basel. 교양 근육 세포 innervated 근육을 요점을 되풀이하는 불충 분한 모델입니다 생체내에서. 기능적인 운동 단위가 복제하실 수 있습니다 체외에서 쥐의 배아 척수의 explants를 사용하여 차별화된 인간의 기본 근육 세포의 innervation에 의해. 이 문서는 척수의 explants와 근육 세포의 공동 문화가 확립되는 방법을 설명합니다.

비디오: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

연구

신경과학

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Neurite 가지의 Assays에 대한 해부와 칙 Statoacoustic 신경절과 콜라겐 젤에서 척수의 Explants의 문화

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

닭의 배아 척수 코드 슬라이스 문화 프로토콜

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

미세 유체 공동 문화 장치를 사용하여 치아 세균 신경 분포를 개발 분석

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish의<em&gt; 시츄</em&gt; 척수 척추 감각과 운동 뉴런 전기 생리 녹음 준비

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

마우스에 있는 Laminectomy 및 척수 창 이식

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Sialoglycan의 신진 대사 표지에 의하여 1 차적인 신경 줄기 및 전구 세포의 세포 표면 마커를 확인

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

PiggyBac 벡터를 사용 하 여 기능적 척추 및 두개골 모터 뉴런으로의 인간 유도 만능 줄기 세포 (iPSCs)의 전환

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

쥐와 마우스 두뇌에 신경 줄기 세포의 동맥 내 납품: 대뇌 허혈에 응용 프로그램

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...