뇌종양 치료에 대한 프레스톤 A. 웰스 주니어 센터,이 작품은 뇌 종양 연구 플로리다 센터에서 보조금에 의해 지원되었다.

1. 기본 GBM 조직의 수집 <ol><li> glioblastoma mutliforme (GBM) 종양은 암 진단과 수술을받은 환자로부터 얻은 것입니다. </li><li> 준비는 신경 외과 팀하셔야합니다. 신경 외과는 신경 줄기 세포 (NSC) 10-15%의 항생제 (Peniciline / Streptomycine)와 보충 기저 매체를 포함하는 적절한 크기의 튜브에 resected의 GBM 종양 추가됩니다. 차가운 매체 실험실로 수술실에 제공해야합니다. 또는 감기에 HEPES - 버퍼 최소 필수 배지 (앙) 또는 인산염은 항생제의 높은 농도와 염분 (PBS)는 또한 이러한 목적으로 사용할 수 있습니다 버퍼. </li><li> resected 종양 조직은 얼음에있는 실험실로 전달, 그리고 후드를 배치됩니다. </li></ol> 2. 단일 셀 서스펜션에 차 종양의 분해 <ol><li> 원래 중간 / PBS를 초과은 팔콘 튜브에서 제거되고 샘플 5-10m로 2-3 번 씻어입니다PBS / NSC 기저 매체의 전 혈액과 이물질을 제거합니다. PBS / 매체가 제거되고 GBM 종양 조직은 배양 접시에 배치됩니다. </li><li> 조직은 작은 조각으로 잘라 trypsinization 프로세스의 면적을 증가 작은 조각에 번호 10 메스 블레이드와 다진 있습니다. 닦지는 종양의 크기에 따라 1~3분 소요될 수 있습니다. </li><li> 다진 조직은 사전 예열 % 0.05 37 ° C의 물을 욕조에 10-15분에 대한 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 3 - 5ml에 trypsinized 있습니다. 전자 피펫은 15ml 팔콘 튜브에 작은 종양의 조각과 트립신를 전송하는 데 사용됩니다. </li><li> 대두 트립신 억제제의 동등한 볼륨은 잠복기 후 효소 트립신의 반응을 중지에 추가됩니다. </li><li> 트립신의는 불활 성화를 여러 번 다운 서스펜션을 pipetting과에 의해 보장됩니다. 그런 다음 중지를위한 5 분 800rpm (110g)에 centrifuging에 의해 아래 pelleted 있습니다. </li><li> 뜨는가 삭제되고 조직 조각은 무균 NSC 바의 1ml에 resuspended 아르샐 매체. 대단히 짧은 부드럽게 부드러운 하얀 단일 세포 현탁액을 얻을 때까지 (3-7 회) 위아래 pipetting 및하여 dissociated 수 있습니다. pipetting 단계의 수는 직접 다진 조직의 입자의 크기에 따라 달라집니다. 그것이 세포 죽음과 구체 형성의 감소가 발생 하듯이 장기와 활발한 기계적 분리는 피해야한다. </li><li> 취소 dissociated 조각과 파편을 제거하려면, 기저 매체 10-15 ML은 튜브에 추가되고 세포 현탁액은 50ml 튜브로 40 마이크론 셀 스트레이너를 통해 필터링됩니다. </li><li> 필터링 정지 5 분에 대한 800rpm (110g)에 centrifuged입니다. 뜨는는 나중에 삭제됩니다. </li><li> Pelletted 전지는 다음 셀 카운트를위한 완벽한 NSC 매체의 1 2ml에 resuspended 있습니다. </li></ol> 3. 셀 카운트 및 도금 <ol><li> 세포 현탁액의 10 μL은 1ml eppendorf 튜브에 0.04 % Trypan 블루 90 μL에 추가됩니다. 참고 : 다른 적절한 세포를 거치도록tions도 사용할 수 있습니다. </li><li> 정지를 섞어 아래 피펫합니다. 세포 / trypan 파랑 혼합 10 μL은 세포 밀도를 계산하기 위해 hemocytometer로 전송됩니다. </li><li> 세포의 0.2 % 헤파린 (2μg/ml)의 20ng/ml EGF, 10ng/ml bFGF와 1μl/ml와 보충 완료 NSC 매체 (9시 1분 비율 NSC 기저 매체와 NSC 확산 보완의 혼합물)에 도금 아르 적절한 조직 문화 선박. 중간의 5, 20, 40 ML는 각각, T25, T80과 T175 flasks에 사용됩니다. 항생제는 오염의 기회를 감소 1:100의 농도에서 매체에 추가할 수 있습니다. </li><li> 플라스크는 37 ° C와 5% CO 2 배양기 세트에 배치됩니다. </li></ol> 4. Passaging와 GBM 파생 분야의 확장 : <ol><li> neurospheres 직경 150-200 μm의의 평균 크기에 도달하면 문화 subculture 준비가되었습니다. 각 플라스크의 내용은 적절한 크기의 살균 tiss에서 제거 및 위치UE 문화 튜브, 그리고 실온에서 5 분 800 RPM (110g)에 centrifuged. </li><li> 뜨는이 제거되고 펠렛은 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 1 ML에 resuspended 있습니다. </li><li> 최적의 trypsinization을 달성하기 위해, 세포 현탁액은 37 ° C 2-3 분 물 목욕에 incubated입니다. 트립신 활동을 중지하려면 대두 트립신 억제제의 동등한 볼륨 세포 현탁액에 추가되고 세포 현탁액은 부드럽게 위아래로 pipetted하고 있습니다. </li><li> 세포 현탁액은 5 분 800 RPM (110g)에 centrifuged입니다. 그런 다음, 표면에 뜨는이 제거되고 세포는 NSC 매체의 1 ML에 resuspended 있습니다. </li><li> 앞에서 설명한대로 셀 개수가 수행됩니다. </li><li> 세포는 성장 인자와 같은 이전 부분에 설명된 적절한 크기의 조직 문화 선박의 도금과 보충 완료 NSC 매체에 5x10 4 셀 / ML의 농도에 도금하고 있습니다. </li><li> 습기에 ° C 37 incubated 때 보조 neurospheres는 70~10일에 형성5% CO 2 ified 인큐베이터. </li></ol> 5. 대표 결과 : GBM 종양 조직에서 수확한 단일 세포를 도금 후, 종양 줄기와 같은 세포가 세포 분열 따위에 의해 번식과 (비디오와 그림 1 참조) 3-4일에 몇 가지 세포로 구성된 세포의 작은 클러스터를 생성합니다. 이러한 클러스터의 성장에 따라, 그들은 150-200 마이크론 양식 (비디오 및 그림 2 참조)의 평균 직경 7~8일하여 수 있도록 좀 더 원형 모양, 적절한 단계 밝은 분야를 취득. 높은 배율에서 건강 분야는 일반적으로 자신의 주변에서 microspikes를 보여줍니다. 문화 개시는 문화의 binging에 비 dissociated 대단히 짧은 시간의 존재로 인해 진정한 분야로 착각해서는 안됩니다 이후 1~2일의 클러스터와 같은 큰 범위를 가졌어요. 기본 종양 영역의 문화에 파편의 양은 조직의 초기 소스에 따라 다릅니다 그리고 그것은 모든 주위의 뇌 조직을 포함할지 여부. 적절한 조직 준비 기법매체의 충분한 양의 효소와 기계적 분리, 그리고 샘플의 후속 필터를 포함하는 것은 문화에 덜 파편이 발생할 수 있습니다. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="그림 1" /> 그림 1. 사일 도금 후에 기본 GBM의 구체 문화. 종양 줄기 같은 세포가 세포 분열 따위에 의해 번식하고 3~4일에서 세포의 작은 클러스터를 생성합니다. 원본 확대, 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="그림 2" /> 그림 2. 팔일 도금 후 항로 일 GBM의 영역 문화를. 원본 확대, 20x.

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <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+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

관심 없음 충돌 선언하지 않습니다.

신경 줄기 및 일반 성인과 태아의 뇌 1, 2, 3, 4에서 전구 세포를 분리하고 줄기는 같은 같은 폐 5, 전립선 6, 유방 7 뇌 8, 9로 암 조직에서 세포 neurosphere 분석은 또한 종양되었습니다 자주 선택의 방법으로 사용됩니다. 전직 생체내의 multipotency, 주입시 새로운 종양을 만들 수있는 능력, 그리고 자기 갱신,이 간단하고 재현성 분석을 사용하여 하나는 체세포 줄기 세포와 유사한 특성을 보여주 resected 종양 조직에서 세포의 무기한 번호를 생성할 수 있습니다. 이러한 전지는 휴대 전화의 상호 작용, 그리고 차별화, 마이 그 레이션, 침략과 세포 사망을 포함하여 기본적인 암 세포 생물학을 연구하는 데 사용할 수 있습니다. 또한, 절연 종양 줄기 세포와 같은 방법은 종양의 형태, 진행 및 재발 또한 공부 결국에는 건강에 대한 인사이트를 제공할 수있는 기본 파생 세포 메커니즘을 해명하는 소중한 도구를 제공합니다옵션을 제공합니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 유형 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> NeuroCult NSC 자연적으로 흐르는 것과, 반사적으로 흐르는 중간 (인간) </td><td> 중 </td><td> 세포 기술을 줄기 </td><td> 05,750 </td><td></td></tr><tr><td> NeuroCult NSC 확산 보조 식품 (인간) </td><td> 중간 보충 </td><td> 세포 기술을 줄기 </td><td> 05,753 </td><td></td></tr><tr><td> 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시약 </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * 가상 </td><td> 시약 </td><td> Gibco </td><td> 41500-018 </td><td> 앙 구성 요소 </td></tr><tr><td> * HEPES </td><td> 시약 </td><td> 시그마 </td><td> H4034 </td><td> 앙 구성 요소 </td></tr><tr><td> * 증류수 </td><td> 시약 </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNase I </td><td> 시약 </td><td> 호슈 </td><td> 104,159 </td><td></td></tr><tr><td> ** 대두 트립신 억제제 </td><td> 시약 </td><td> 시그마 </td><td> T6522 </td><td></td></tr><tr><td> 펜 / Strep </td><td> 시약 </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> 번호 10 메스 블레이드 </td><td> 외과 도구 </td><td> BD </td><td> 371,610 </td><td></td></tr><tr><td> 페트리 접시 </td><td> 문화 도자기 </td><td> BD 팔콘 </td><td> 353,003 </td><td></td></tr><tr><td> 작은 집게 </td><td> 외과 도구 </td><td> 파인 과학 도구 </td><td> 11050-10 </td><td></td></tr><tr><td> 셀 스트레이너 </td><td> 체 </td><td> BD 팔콘 </td><td> 352,340 </td><td></td></tr><tr><td> T25 플라스크 </td><td> 문화 도자기 </td><td> Nalge 뉴NC 국제 </td><td> 136,196 </td><td></td></tr><tr><td> T80 플라스크 </td><td> 문화 도자기 </td><td> Nalge Nunc 인터내셔널 </td><td> 178,905 </td><td></td></tr><tr><td> 15 ML 튜브 </td><td> 문화 도자기 </td><td> BD 팔콘 </td><td> 352,096 </td><td></td></tr><tr><td> 50 ML 튜브 </td><td> 문화 도자기 </td><td> BD 팔콘 </td><td> 352,070 </td><td></td></tr><tr><td> EGF </td><td> 성장 인자 </td><td> R &amp; D </td><td> 2028 - EG </td><td></td></tr><tr><td> B - FGF </td><td> 성장 인자 </td><td> R &amp; D </td><td> 3139 - FB </td><td></td></tr><tr><td> 헤파린 </td><td> 성장 인자 </td><td> 시그마 </td><td> H4784 </td><td> PBS의 재구성 </td></tr></tbody></table> * 앙을 1M HEPES의 가상 and160ml의 1x10L 패킷을 혼합하고 증류수를 사용하여 8.75 L로 볼륨을 가지고합니다. 7.4에 대한 최종 PH를 설정D 4 ° C.에서 저장 ** 트립신 억제제 솔루션을 준비하려면 먼저 DNase I 용액 (100 MG DNase이 앙 ​​100 ML에 녹아있는) 10 ML을 만들고 다음 DNase 솔루션에 트립신 억제제의 0.14 g을 추가하고 마지막 앙을 사용하여 1 리터에 볼륨을합니다. -20 ° C의 냉동고에 최종 제품의 aliquots 보관하십시오.

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

줄기 세포와 같은 그러한 유방, 폐, 결장, 전립선과 두뇌와 같은 종양에 고립되어있다. 이러한 모든 종양에서 중요한 문제는, 특히 glioblastoma의 mutliforme (GBM)에, 종양 형성, 진행 및 재발 그들의 역할을 조사하기 위해 종양 시작 세포 인구 (들)을 식별하고 격리하는 것입니다. 세포 인구를 시작 이해 종양이 종양에 대한 효과적인 치료 방법을 찾는 데 실마리를 제공할 것입니다. 의 단순 성과 재현성으로 인해 neurosphere 분석 (NSA)이 종양 세포의 많은 고립과 전파에 대한 선택의 방법으로 사용되고 있습니다. 이 프로토콜은 수술 resected 인간 GBM 종양 조직에서 줄기 같은 세포를 분리하고 확장 neurosphere 문화 방법을 보여줍니다. 절차는 초기 화학 소화 및 종양 조직의 기계적 분리, 그리고 이후 NSA 문화에서 발생한 단일 세포 현탁액을 도금이 포함됩니다. 150-2의 7~10일 후, 기본 neurospheres직경 00 μm의 준수 더욱 passaging 및 확장을위한 준비가되어 있습니다.

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

이 비디오 프로토콜은 neurosphere 분석 문화 방법을 사용하여 수술 resected 인간 glioblastoma의 mutliforme (GBM) 종양 조직에서 세포처럼 고립과 줄기의 확장을 보여줍니다.

분리 및 Neurosphere 분석을 사용하여 인간 Glioblastoma의 Multiforme 종양 세포의 확장

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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JoVE Journal

Neuroscience

29.6K 조회수.  University of Florida. 이 비디오 프로토콜은 neurosphere 분석 문화 방법을 사용하여 수술 resected 인간 glioblastoma의 mutliforme (GBM) 종양 조직에서 세포처럼 고립과 줄기의 확장을 보여줍니다.

비디오: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

절연 및 Neurosphere 어세를 사용하여 성인 마우스 신경 줄기 세포의 확장

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

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신경과학

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

인간 Fungiform의 맛 용의자 전지의 분리 및 문화

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

유도 된 신경 세포로 성숙한 인간 두뇌의 혈관 주위 세포에서 파생 된 세포의 리니지 - 프로그래밍

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9를 RARE-LacZ를 셀 기반 리포터 분석을 사용하여 E8.5 배아 및 Neurosphere 문화에서 상대 레티노 산 수준의 정량적 측정

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

미세 주입을 사용하여 인간 헬라 세포에서 스플리세오소말 snRNA 국소화 분석

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

인간 유도 만능 줄기 세포에서 뇌 미세 혈관 내피 세포의 파생, 확장, 냉동 보존 및 특성화

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

성인 얼룩말 물고기를 사용하여 학습 및 메모리 연구에서 인지 평가를위한 연관 학습 도구로 셔틀 박스 분석

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

인간 포피로부터의 일차 슈완 세포, 각질세포 및 섬유아세포의 분리, 배양 및 특성화

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

인간 미세아교세포 유사 세포: 유도만능줄기세포로부터의 분화 및 인간 시냅토좀을 이용한 체외 생세포 식균작용 분석

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...