Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

뇌 기증 등록을 기부하는 그들의 의도 등록자에 의한 설명서가 포함되어 있습니다. 법이 허용 조직 검색에 대한 부검 권한은 친척의 다음에 의해 제공됩니다. 수집 부검 표본을 사용하여 연구 연구는 건강 보험 양도 및 책임에 관한 법률 (HIPAA) 규정을 준수하기 위해 제도적 검토위원회 (IRB)에 의해 검토된다. 참고 : Leptomeninges 샘플은 뇌 해부 동안 뇌 해부학자 또는 신경 병리학에 의해 수집되고, 25 ~ 30 ML의 수막 성장 미디어를 포함하는 50 ML 원뿔 튜브에 저장됩니다. 샘플은 샘플 준비 될 때까지 4 ℃에서 저장된다. 세포의 생존 시간의 사후 감소로 처리가 가능한 한 빨리 수행되어야한다. 1 세트 업 Leptomeninges 해부를 시작하기 전에 참고 : - 1.3 바이오 안전성 캐비닛 내부에서 수행 할 수 있습니다 1.1 단계를 반복합니다. <ol><li> 375 mL의 둘 베코 변형 이글 매체를 조합하여 수막 성장 배지를 준비 태아 소 혈청 100 ㎖, 5 ㎖의 10 mM 비 필수 아미노산, 5 ㎖의 200 mM의 L- 알라 닐 -L- 글루타민, 5 ㎖의 100 mM의 피루브산 나트륨 5 ㎖ 10,000 U / ㎖ 페니실린 / 스트렙토 마이신 및 250 μg의 5 ㎖ / ㎖의 암포 테리 신 B, 500㎖ 필터 유닛이다. 필터와 혼합한다. 최대 4 주 동안 용지를 사용하십시오. </li><li> 해부를 시작하기 전에 바이오 안전성 캐비닛에있는 모든 재료를 넣 배 배양 접시, 배 6 웰 플레이트, 배 메스, 15 ~ 20 멸균 15 mm 커버 전표, 피펫을 흡입 인산염 완충 생리 식염수 (PBS), 혈청 학적 피펫. </li><li> 6 웰 플레이트의 각 웰에 멸균 멸균 0.1 % 젤라틴 용액 1 ㎖를 추가한다. 30 ~ 60 분 동안 따로 접시를 설정합니다. 배 6 웰 플레이트를 준비합니다. </li></ol> Leptomeninges 샘플 2. 준비 참고 : - 2.3 바이오 안전성 캐비닛 내부에서 수행 할 수 있습니다 2.1 단계를 반복합니다. <ol><li> 에 PBS의 약 10 mL를 넣고멸균 10 cm 조직 배양 접시는 접시에 수막 샘플을 배치합니다. 포셉 사용하여 부드럽게 혈액을 제거하기 위해 PBS와 leptomeninges을 씻는다. </li><li> 신선한 PBS의 10 ㎖를 포함하는 새로운 10 cm 조직 배양 접시에 수막을 전송하고 세척을 계속합니다. 가능한 한 많은 혈액을 제거 할 필요 반복합니다. </li><li> 약 6cm X 6cm 큰 조각으로 조직 leptomeninges 절단이 메스를 사용. </li><li> 수평 층류 후드의 해부 현미경으로 배양 접시 및 전송에 놓고 덮개. </li></ol> Leptomeninges 조직 3. 해부 주 - 3.3 해부 현미경을 사용하여 수평 층류 후드 내부에서 수행되어야 3.1 단계. <ol><li> 적어도 20 ~ 25 3mm X 3mm 조각을 생성 할 수있을만큼 leptomeninges 조각 (단계 2.3)에 영역에서 혈관을 제거합니다. 한 메스과 장소에 leptomeninges을 잡고 다른과, 부드러운 얕은 긁어 모 사용기는 혈관을 분리합니다. </li><li> 준비된 지역에서 조직의 작은 사각형을 잘라. 첫 번째 큰 조각을 한 다음 작은 섹션으로 사람들을 슬라이스하여 단계적 과정에서 잘라. </li><li> 요동과 조직을 절단하는 대신에 조직과 다른 메스를 개최 한 메스를 사용합니다. 조직의 일관성으로 인해, 서로에 대해 메스 슬라이드하는 것이 곤란하다. 이것은 따로 조직을 찢어 및 비정형 가장자리에 발생합니다. </li><li> 20 ~ 25 3mm X 3mm 조각이 될 때까지 반 조각을 절단 계속합니다. </li></ol> 조직 배양 플레이트 상에 해부 수막 조각 4. 전송 주의 : 단계 4는 바이오 캐비닛 내부에 수행되어야한다. <ol><li> 6 웰 플레이트에서 (단계 1.3에서) 대기음 젤라틴. 각 웰에 수막 성장 미디어의 1 ML을 추가합니다. 멸균 집게를 사용하여 각 웰에 3-4 생검 조각을 배치합니다. </li></ol> M을 통해 커버 전표 5. 배치eninges 조각 주 - 5.2 해부 현미경을 사용하여 수평 층류 후드 내부에서 수행되어야 5.1 단계. <ol><li> 물론 당 1-2 멸균 15 mm 원형 된 커버와 조각을 커버. </li><li> 조각이 판의 하단을 터치되도록 집게로 단단히 누르십시오. 이 웰의 바닥에 조직 조각을 부착 할 수있다. </li><li> 37 ° C, 5 % CO 2 배양기로 6 웰 플레이트를 놓습니다. </li></ol> 6. 세포 배양 유지 보수 참고 : - 6.3 바이오 안전성 캐비닛 내부에서 수행 할 수 있습니다 6.1 단계를 반복합니다. 커버 슬립이 이동하고 수막 조각을 제거하지 말아야으로 치료와 배양 접시를 처리합니다. <ol><li> 문화 2 일 후, 각 웰에 신선한 수막 성장 미디어의 추가 1 mL를 넣어. </li><li> 7 일에, 신중하게 미디어를 대기음하고 각 웰에 신선한 미디어의 2 mL를 넣고. </li><li> 9 일 후, 성장 배지를 매일 계속 변화문화까지 다른 일 합류하게된다. </li></ol> (7)과 Passaging 참고 : 모든 단계는 바이오 안전성 캐비닛 내부에서 수행 할 수 있습니다. 수막 아세포는 수막 조직편에서 마이그레이션 큰 배양 접시에 수막 섬유 아세포를 배양 용기의 가장자리를 향해 성장 확장 시작되면. <ol><li> 15 ~ 30 분 동안 1 % 젤라틴과 계대 코트 배양 접시를 시작하기 전에. </li><li> 대기음 6 잘 접시에서 미디어 및 2 mL의 PBS로 세척 세포. </li><li> 각 웰에 트립신 / EDTA의 1 mL를 넣고 5 ~ 10 분 동안 37 ° C에서 품어. 또한, 커버 슬립 또는이 단계의 수막 조직을 제거 할 필요가 없다. </li><li> 셀은 원형이며, 분리 배지 2 mL를 첨가하기 시작하면. </li><li> 피펫 세포까지 완전히 모든 대단히 짧은 시간을 방해 큰 배양 접시에 세포 솔루션을 전송하려면 아래로. </li><li> 1 내지 3 : 1의 비율을 사용하여 4 세 6 웰 접시에서 세포를 결합 할한 T75의 배양 접시에. </li><li> 수막 섬유 아세포 세포를 T75 배양 플라스크에서 컨 플루 언트되면, 세포를 Trypsinize, 1 × 106 세포 / mL의 바이알에서 동결 배지에서 cryopreserve. 합류 수막 섬유 아세포와 함께 한 T75 문화 플라스크에 대한 5-7000000 세포가 포함되어 있습니다. </li></ol> 면역 염색에 의한 수막 섬유 아세포의 8 특성 주 : 절차를 시작하기 전에, PBS (블로킹 완충액)에서 4 % 파라 포름 알데히드 용액을, 5 % 정상 염소 혈청을 제조 0.3 % 트리톤 X-100의 PBS (permeabilization 버퍼), 일차 및 이차 항체 용액, 및 1 μg의 중 / ㎖의 훽스트 33342 10 ㎎ / ㎖의 재고에서 PBS에 희석. <ol><li> 8 잘 챔버 슬라이드 씨 15,000 30,000 수막 섬유 아세포는 0.1 % 젤라틴으로 코팅. </li><li> 세포가 부착 된 경우 24 시간 후, 실온에서 10 분 동안 웰 당 100 μL 4 % 파라 포름 알데히드로 세포를 고정한다. 1X PBS로 우물 3 회 반복한다. </li><li> 실온에서 5 분 동안 0.3 % 트리톤 X-100 150 μL와 세포 Permeabilize 하시려면. 1X PBS로 우물 3 회 반복한다. </li><li> 실온에서 1 시간 동안 차단 용액 200 μL (PBS + 5 % 정상 염소 혈청)을 추가한다. </li><li> 차단 솔루션을 기음과 솔루션을 차단에 희석 원하는 차 항체의 150 μL를 추가합니다. 이중 염색의 경우, 다음과 같이 얼음에 별도의 원심 분리기 튜브의 일차 항체의 작업 희석을 준비 : <ol><li> 안티 피브로넥틴 + 1 (300) 희석 : 솔루션을 차단하는 안티 네 스틴의 200 희석 (1)을 준비합니다. 안티 SERPINH1 + 1 (250) 희석 : 솔루션을 차단하는 안티 TUJ1 1000 희석 1을 준비합니다. (1) 준비 : 안티 - SERPINH1 250 희석 + 1 : 솔루션을 차단하는 안티 SOX2 100 희석 </li></ol></li><li> 밤새 4 ° C에서 품어. </li><li> 1X PBS로 우물 3 회 반복한다. </li><li> 작업 한 준비 : 이차 항체는 항 - 토끼를 염소 라벨 형광 400 희석 (녹색, 예 / 엠 2 </s최대&gt; 519분의 495 ㎚)과 염소 항 - 마우스 (빨강, 예 / 엠 2 617분의 590 ㎚) 차단 솔루션과 각 웰에 100 μL를 추가합니다. 실온에서 어두운 곳에서 1 시간 동안 배양. 그런 다음 1X PBS로 한번 우물을 씻는다. </li><li> 각 솔루션은 물론 3 분 동안 어둠 속에서 부화 / mL의 훽스트 1 μg의 100 μL를 33342 (예 / 엠 2 461분의 358 nm의 청색)를 추가합니다. 웰의 최종 1X PBS 세척에두고 1X PBS로 웰을 3 회 세척하고, 알루미늄 호일로 슬라이드 커버. </li><li> 형광 현미경으로 세포를 분석 할 수 있습니다. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은 뇌 기증과 관련하여 수집 된 인간 사후의 leptomeninges에서 수막 섬유 아세포 문화를 유도하는 간단하고 강력한 프로토콜을 설명합니다. 사후 인간의 물질로 세포 배양을 유도하는 프로토콜의 거의 설명이 있습니다. 두 연구는 피부 유래 섬유 아세포를 배양 7, 8, 9, 한 연구는 경질 샘플 (10)을 설?...

뇌경막, 지주막과 피아 이잖아요 : 수막은 뇌를 보호하는 세 개의 막으로 구성되어 있습니다. 최근에, 또한 수막 뇌 발달 한 뇌 항상성에 중요한 역할을한다는 것을 인식하고있다. 수막은 중간 엽 및 신경 능선 유래 세포로부터 유래하고 흥미로운 시험 관내 및 생체 내 2, 3, 4에서의 이식 후 뉴런을 야기 할 수있는 수막에 존재하는 전구 세포를 보였다. 들은 도파민 뉴런 (5)에 배아 줄기 세포의 분화를 위해 간질 세포 유래 유도부 활성을 갖는 수막으로 배양 성공적, 급전 층으로서 사용되어왔다. 또한 leptomeninges 직접 허혈성 조건 하에서 6 뉴런, 아스트로 사이트, 올리고 덴드로 사이트와로 분화 할 수있는 잠재력을 가지고있다. 이 프로토콜의 경우, 인간의 사후 수막 샘플을 총칭하여 지주막과 피아 이잖아요에서 수집 leptomeninges라고, 연구 목적으로 인간의 두뇌 기부의 일환으로 조달된다. 뇌의 해부 죽음의 24 시간 내에서 수행되며,이 프로토콜 여기에 도시 된 바와 같이 leptomeninges 샘플은 다음 6-8 시간 내에서 추가 처리를 위해 저온 성장 배지에 배치된다. 이 프로토콜은 환자의 기본 leptomeninges 세포 배양의 개발에 대한 해부와 인간의 수막 샘플의 준비에 대해 설명합니다. 조직은 약 3mm X 3mm 사각형의 25 ~ 30 조각으로 절단됩니다. 세 가지 각 6 웰에 배치됩니다 젤라틴 코팅 잘 라운드 커버 글라스로 누르고. 뇌막 해부은 약 25 ~ 35 분 소요됩니다. 이 문화의 주요 과제는 뇌 조달, 운송 및 박리 등의 불임은 일반적으로 무균 조건 하에서 수행하지 않는 것입니다. 티herefore, 페니실린, 스트렙토 마이신 및 암포 테리 신 B의 칵테일 배양 배지를 보충하고 별도로 배양 조직 조각을 멀티 웰 접시를 사용하는 것이 중요하다. 수막 섬유 아세포의 부산물은 일반적으로 첫 주 내에서 발생합니다. 세포가 합류 할 때까지 미디어 매 2 ~ 3 일 변화 및 세포 효소 적으로 계대된다. 수막 섬유 아 세포는 냉동 보존 매체에 ㎖ / 바이알 당 100 만 세포의 냉동 보관한다. 이 프로토콜로, 20-30000000 수막 섬유 아 세포는 냉동 보존에 대한 6-8주에서 유도 될 수있다. 이러한 수막 섬유 아세포의 다운 스트림 응용 프로그램은 질병 연구, 직접 신경 세포 분화 또는 질병 메커니즘의 이해 및 약물 개발을위한 leptomeninges에서 유도 만능 줄기 세포의 유도를위한 차 문화입니다.

<table border="0" cellpadding="0" cellspacing="0" width="1256">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
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		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

큰 진전 파킨슨 병의 임상 적 특성에 만들어졌다하더라도, 몇몇 연구는 파킨슨 병의 병리학 적 진단이 임상 진단 파킨슨 병의 최대 25 %에서 확인되지 않은 것을보고한다. 따라서, 티슈는 오진의 높은 속도를 가질 수 특발성 파킨슨 병 환자의 임상 진단에서 수집; 전임상 모델이 쓸데 될 수 따라서 같은 조직에서 시험관 연구에서 파킨슨 병을 연구한다. 파킨슨 병의 확인 신경 병리학 적 진단 사후 인간의 leptomeninges를 수집하고 흑질 선조체 세포 손실과 루이 기관이라는 세포 내 단백질 흠을 특징으로 한 임상 관찰 파킨슨 다른 기저 질환 과정 (예를 들어 종양, 동맥 경화)에 의해 발생한 것이 아닌지 확신 할 수 있습니다. 이 프로토콜 presen해부 및 수막 섬유 아세포 문화의 유도에 대한 사후 인간의 leptomeninges의 준비 TS. 이 절차는 견고하고 높은 성공률을 갖는다. 뇌 조달은 일반적으로 무균 조건 하에서 수행되지 않는 문화의 문제는 불임이다. 따라서, 페니실린, 스트렙토 마이신 및 암포 테리 신 B의 칵테일 배양 배지를 보충하는 것이 중요 파킨슨 병 부검 확인 된 경우에서 수막 섬유 아 세포의 유도는 파킨슨 병의 체외 모델링을위한 기반을 제공합니다. 수막 섬유 아세포는 3~9일 샘플 준비 후 나타나는 약 20-30000000 세포 6-8 주 만에 동결 보존 할 수 있습니다. 수막 섬유 아세포 배양 균질하고 세포 피브로넥틴, 수막을 식별하기 위해 일반적으로 사용되는 마커를 표현한다.

leptomeninges 처리 프로토콜이 성공하면이 뇌에 대한 사후 간격의 길이에 따라 달라질 수 있지만, 수막 섬유 아 세포의 생장는 3 구 일 절제술 후 관찰 처음이다. 그림 1은 네 개의 다른 기증자의 수막 섬유 아세포 문화를 보여줍니다. 도 1a는 10 시간 20 분 사후 간격으로 88 세 기증자에서 처리 후 조직 사일 주위에 유리 커버 슬립 (어두운 대각선)와 섬?...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

인간의 사후 뇌에서 leptomeninges 이식편 문화 프로토콜 6-8 주 이내에 피브로넥틴 양성 수막 섬유 아세포를 유도 약 20-30000000 세포를 cryopreserve하기 위해 기술적으로 강력하고 간단한 방법입니다.

사후 인간의 두뇌 기증자로부터 Leptomeninges 이식편 문화의 유도

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

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Neuroscience

8.2K 조회수.  Parkinson’s Institute and Clinical Center.  인간의 사후 뇌에서 leptomeninges 이식편 문화 프로토콜 6-8 주 이내에 피브로넥틴 양성 수막 섬유 아세포를 유도 약 20-30000000 세포를 cryopreserve하기 위해 기술적으로 강력하고 간단한 방법입니다. 

비디오: Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

로던트에서 뉴런과 비 인간 메이트 뇌 조각의 DiOLISTIC 라벨링

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

연구

신경과학

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

포스트 - 나탈 Murine 조직의 공동 문화를 Myelinating 풍부한 Oligodendrocyte 문화와 Oligodendrocyte / 신경 세포의 유도

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

E13 생쥐에서 Glial 제한된 엽 성의 전구 물질의 유도

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

인간 태아 뇌에서 전구 - 파생 Oligodendrocyte 문화 시스템

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

인간 Subcortex의 고해상도 구조 자기 공명 영상<I&gt; 생체</I&gt; 및 사후

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

신선한 인간의 뇌 조직에서 뇌 모세 혈관의 절연

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

사후 인간의 두뇌 기증자로부터 Leptomeninges 이식편 문화의 유도

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