이 연구는 UCI (AR 및 SP)에서 학부 연구 기회 프로그램에서 보조금 캘리포니아 알츠하이머 질병 이니셔티브없이 건강 부여의 국립 연구소의 국가에서 보조금에 의해 지원되었다. HD38466, 그리고 알츠하이머 병 연구 센터는 부여하지 않습니다. AG16573 (JB)

1. 두피 조직 블록의 Cryopreservation 재료 준비 <ol><li> 멸균 물 (1시 9분)에서 10X 재고 솔루션에서 diluting에 의해 1X 행크의 버퍼 소금 솔루션 (HBSS)을 준비합니다. 4oC에 저장 HBSS. </li><li> HBSS (1시 9분)에 DMSO를 diluting 10 % DMSO 냉동 매체를 준비합니다. 냉장 냉동 때까지 매체 cryopreservation 전에 신선했고 4oC에 저장해야합니다. </li><li> 강철 면도날은 조직의 체계적인 돼지에 사용됩니다. 사용하기 전에 면도날이 2 시간을위한 70 %의 에탄올에 잠수하여 소독을합니다. 즉시 조직 처리하기 전에 멸균 물 3 회와 면도날을 씻어. 그것이 산화하는 경향이므로, 시간의 과도한 금액에 대한 멸균 물의 면도칼을 떠나 피하십시오. </li><li> Nalgene 냉동 컨테이너는 cryovials (2.5 ML)를로드하고 살균 생물 학적 캐비닛으로 가지고있다. 냉장 냉동 중간 1 ML 각 유리병에 추가됩니다. 냉동 컨테이너는 다음 적어도 2 시간에 대한 4oC에 배치됩니다. </li></ol> , 청소 초핑 및 냉동 <ol><li> 냉동으로 뇌 조직은 meningeal 막과 혈관의 청소입니다. 얼음 팩 상단에서 작업하는 동안주의 깊게 조직에서 파편을 제거하는 살균 바늘 도움말을 사용하십시오. 청소 조직은 차가운 HBSS로 가볍게 씻어서 자르고을위한 새로운 배양 접시로 전송해야합니다. </li><li> 얼음 팩 위에 작업, 빠른 속도로 약 1mm 3 개의 블록으로 조직을 잘라하는 소독 면도날을 사용합니다. 더 균일한 블록 크기의 결과, 자르고 절차보다 나은 제어에 시간 결과를 청소 조직의 작은 부분 작업. </li><li> 천천히 50 ML 원뿔 튜브 HBSS 50 ML에 다진 조직을 추가합니다. 부드럽게 남아있는 조직 블록을 수집하기 위해 HBSS와 페트리 접시를 씻어. 조직 블록이 느슨하게 포장 조직 블록 펠렛을 만들고, 튜브의 바닥에 내려 수 있습니다. </li><li> 조직이 정착되는 동안, 생물 안전 캐비닛에 이전에 차게 냉동 컨테이너와 cryovials 가져. 조직을 추가하는 과정을 촉진하는 모든 cryovials을 모자를 벗다. </li><li> 모든 조직 블록 정착 후, 부드럽게 펠렛 위의 미디어의 매우 얇은 레이어를 떠나, 초과 HBSS를 대기음. 그것은 조직 블록이 서로 준수 원인이되므로 원심 분리는 크게 냉동 효율이 떨어지면서, 낙담합니다. </li><li> 천천히 넓은 orifices (멸균 가위로 끝부분을 절단)에 피펫 팁을 사용하여 느슨한 조직 블록 펠렛의 맨 아래에 200 μL를 수집합니다. 한 cryovial로 자료를 전송하고 펠렛의 맨 아래에서 다음 다시 수집 조직에 이동합니다. 이것은 전체 조직 할당 절차 냉동 컨테이너 당 3-4 분 이상하지 않는 것이 중요합니다. 이것은 조직이 동결되기 전에 시간은 짧은 기간 동안 DMSO에 노출되는 보장합니다. 유리병에 조직을 전송 후, 최소 4 시간을위한 A - 80oC 냉동실에 냉동 컨테이너를 놓습니다. 또는 냉동 컨테이너는 -80 OC에서 하룻밤 남아있을 수 있습니다. 남아있는 냉동 컨테이너에 대해이 과정을 반복합니다. </li><li> 냉동 컨테이너 (S)에서 장기 저장을위한 액체 질소 탱크에 cryobox과 장소에 cryovials를 전송합니다. </li></ol> 2. 해동 및 냉동 두피 조직 블록의 Culturing 재료 준비 <ol><li> 하루 전에 해동 조직 샘플을, 폴리 - L - 라이신 코팅 배양 접시 / coverslips 준비가되어 있습니다. 일반적으로 대뇌 피질의 뉴런에 우리는 500 μg / ML 또는 1mg / ML로 코팅 요리를 준비합니다. 완전히 접시의 바닥을 감추기에 충분 할만큼 폴리 - L - 라이신 솔루션을 (borate 버퍼에서 만든)을 추가합니다. 유리 coverslips이 필요한 경우, 그들은 완전히 폴리 - L - 라이신 솔루션에 가라앉아 있는지 확인합니다. 최소한 12 시간을위한 부화. 사용하기 전에 철저한 멸균 물의 자유로운 양의 3 배, 5 분 각 요리를 씻어. </li><li> 따뜻한 HBSS은 해동 조직뿐만 아니라 세포 suspensions로 조직을 떼어 놓다을 청소하는 데 사용됩니다. 필요한 HBSS의 볼륨 해동하는 조직의 양에 따라 달라질 수 있지만, 일반적으로 1 cryovial은 HBSS의 50 ML에 희석하고 HBSS 10 ML에 dissociated 것입니다. </li><li> Dulbecco의 수정된 이글 매체와 10% 철분 보충 소 송아지 혈청 (EBS)를, 1 % antimycotic / 항생제 (AA) 혼합 (DMEM (++))가 37 ° C 물 목욕에 배치해야합니다. </li><li> 도금 매체 (NB (+++))는 사용하기 전에 신선한하여야한다. 이 매체는 1퍼센트 항생제 / antimycotic 플러스 B27와 N2의 보충을 추가하여 준비가되어 있습니다. </li></ol> 참고 : 십자가 (+)로 추가 미디어 이름은 미디어의 기본 조성에 첨가제의 포함을 나타냅니다. 이 문서에서는 DMEM (+ +) 나타냅니다 DMEM 플러스 10% EBS 플러스 1퍼센트 AA, 반면 NB (+++) 상징 NB PL우리 1퍼센트 AA 플러스 B27 플러스 N2. 해동 및 Culturing <ol><li> 액체 질소 탱크에서 cryovials를 제거하고 급속 해동 37 °에만 작은 얼음 펠렛 때까지 물을 욕조에 C는 유리 내부에 관찰 내용입니다. </li><li> 부드럽게 원뿔 관에 따뜻한 HBSS의 50mL에 병을 콘텐츠를 전송할 수 있습니다. 부드럽게 cryovial 항아리에 남아있는 조직 블록을 이동시키다하는 넓은 구멍 팁을 사용합니다. 관에게 3-4 회를 반전하고 조직이 서서히 바닥에 수집 수 있습니다. 조직 빠르게 정착해야하지만 블록이 너무 작은 경우에는이 권장됩니다 원심 분리 (~ 200 X g)를 발생 가벼운하지 않을 수 있습니다. </li><li> 초과 HBSS를 대기음. </li><li> 조직 펠렛 및 0.25 %의 트립신과 DNAase 50 μL 300 μL에 따뜻한 HBSS 10 ML을 추가합니다. 궤도 통에 다음 5 분, 장소에 대해 37 ° C 물 욕조에 품어는 ° C 추가 5 분 80 RPM 37로 설정합니다. </li><li> 이 기간 이후, 생물 안전 캐비닛에 조직 블록을 가지고 있으며 흐린 세포 현탁액을 형성 때까지 신중하게 조직 블록을 선동하기 위해 10 ML의 피펫을 사용합니다. 따뜻한 DMEM 10 ML을 추가하여 트립신을 비활성화 (+ +) 세포 현탁액 수 있습니다. </li><li> 5 분 1,200 X g에서 dissociated 세포를 원심 분리기. 뜨는을 기음과 폐기, 따뜻한 DMEM 10 ML에있는 세포 펠렛을 resuspend (+ +)과 hemocytometer를 사용하여 휴대폰 번호를 계량. </li><li> 폴리 - L - 라이신 코팅 접시 플레이트 세포. 일반적으로 60 mm 요리가 1x10 6 전지와 도금 것입니다. </li><li> 세포 조직 문화 인큐베이터에서 약 1 시간을위한 요리 / coverslips에 연결할 수 있습니다. 그것은 효과적인 첨부 파일이 관찰 때까지 한 판의 모든 10 분 첨부 파일의 진행 상황을 모니터링하는 것이 좋습니다. 그런 다음, 부드럽게 신선한 DMEM (++).으로 미디어를 대체 </li><li> 24 시간 후, DMEM를 교체 (+ +) 따뜻한 NB (+++).과 중간 부분의 변화 (50 %)는 신선한 NB (+++).있는 모든 오일 수행됩니다 건강한 문화는 일반적으로 24 시간 후에 분화와 프로세스의 성장의 흔적을 보여줍니다. 이러한 조건에서 모든 4-5일 일부 매체 변경을 수행, 문화가 최대 4-6주 시간의 연장 기간 동안 유지하실 수 있습니다. </li><li> 의 연결을 전구체 세포 (NPCs)의 생성을 위해, 유사한 프로토콜은 원심 분리 후, EBS없는 DMEM 사용되는 예외와 함께 사용됩니다. 세포 neuropheres 형성을 허용하는 B27과 EGF (20 NG / ML)로 보충 DMEM/F12 (1시 3분)에 T - 25 flasks에 도금입니다. NPCs을 차별화하는 데, neurospheres는 laminin (10 UG / ML) DMEM/F12 (1시 3분) N2와 보충 매체 코팅 coverslips에 도금입니다. </li></ol> 3. 대표 결과 건강한 문화 오일 이후의 해동 후 크게 성장과 분화를 표시하고 보통 10 일 (그림 1)에 의해 성장에 안정합니다. 이러한 문화의 Immuncocytochemical 얼룩은 수많은 astrocytes (그림 2A)를 공개하고 인간과 쥐의 모두 기본의 연결을 문화 뉴런 (그림 2B). 이 프로토콜은 또한 자유 부동 neurospheres (그림 3A), 차별 조건, 고품질의 혼합 문화 (그림 3B, C)의 결과에 따라.로서 NPCs의 생성에 적합합니다 <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="그림 1" /> 그림 1. 냉동 인간의 대뇌 피질 조직의 세포 문화. 냉동 인간의 대뇌 피질 조직 블록 해동 및 도금 폴리 - 리신 코팅 coverslips과 10 일간 재배하고 있습니다. 으로 5 일째 셀 확장은 분명하고 일 10 confluency이 이루어진다. 스케일 바 : 100 μm의. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="그림 2" /> 그림 2. 셀 문화 Immunocytochemical 얼룩이. (A) 문화가 glial 마커 glial fibrillary 산성 단백질 (GFAP, 굵은 화살표)와 스테인드되었습니다. (B) 뉴런은의 연결 마커 베타 tubulin III (시까지, 얇은 화살표)를 발견했다. (C) 인간과 쥐의 문화 두 문화에서 10 일 후에 풍부한 glia과 뉴런을 보여줍니다. 차 항체 : 마우스 방지 GFAP (1:1000)와 토끼 안티 때까지 (1:1000). 차 항체 : 안티 - 마우스 알렉사 594 (1:500)와 안티 - 토끼 알렉사 488 (1:500). 핵의 얼룩 : Hoestch 파란색 (1:1000). 스케일 바 : 50 μm의. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="그림 3" /> 그림 3. neurospheres의 생성. (A) 냉동 조직 위에서 설명한 및 neurospheres로 전파할 수로 처리되었습니다. (B) Neurospheres가 떨어진 neurosphere에서 마이 그 레이션하는 세포의 결과, 차별 조건 하에서 유리 coverslips에 도금 및 10 일 동안 성장했다. (C) 뉴런과 astrocytes 모두 차별 neurosphere의 확장 주변에 존재한다. 원자력 counterstaining, 그림 2에서와 같이 사용되는 기본 및 보조 항체.

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Cryopreservation는 나중에 사용하기 위해 은행 소중한 뇌 조직 샘플 수있는 기회를 제공하고 있습니다. 우리가 신경 세포 - 풍부한 문화와 냉동 뇌 조직의 연결 블록에서 전구체 세포 모두를 생성하는 간단하지만 효과적인 프로토콜을 설명합니다. 이것은 경제적인 절차는 더 비싼 요금 제어 냉동고를 활용하여 전통적인 cryopreservation 기술의 비용을 방지합니다. 프로토콜은 조직 블록을 동결하는 빠른 아직 효과적인 수단을 제공함으로써 해동 후 가능한의 연결을 문화의 생성을 허용합니다. 전체 냉동 과정은 약간 20 분 정도 걸릴 수 있습니다. 기본의 연결을 문화뿐만 아니라,이 방법을 조직 블록을 사용하는 것은 또한 부유 neurospheres로 성장 NPCs를 생성하기 위해 해동 수 있습니다. 이 점에서 우리 동결 매체 혈청의 부족은 세포가 undifferentiated 상태로 유지되는지 확인합니다. 차별 조건에서 neurospheres는 냉동 조직 쇼 확장과 새로운 조직에서 생성된 neurospheres 매우 비교 차별화 속도에서 생산.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

본 연구에서는, 우리는 성공적으로 cryopreservation과 매우 풍부한 문화의 연결을 생성하는 대뇌 피질의 뇌 조직 블록의 해동을위한 표준 프로토콜을 설명합니다. 이 프로토콜에 사용되는 냉동 매체는 행크의 버퍼 소금 솔루션 (HBSS)에 희석 10% 디메틸 sulfoxide (DMSO)입니다. 대뇌 피질의 조직 단위는 냉동 매체를 포함하는 cryovials로 전송 천천히 -1 속도 제어 냉동 컨테이너 ° C / 분 냉동 있습니다. 포스트 해동 처리와 지속적으로 10 일 이내에 문화의 연결 네트워크의 중요한 확장에 처음 5 일 동안 급속한 성장을 neuritic 전시의 연결 - 풍부한 문화를 생산 냉동 조직 블록의 해리. astrocytic 마커 glial fibrillary 산성 단백질 (GFAP)과의 연결 마커 베타 tubulin 클래스 III와 Immunocytochemical 얼룩은 문화의 뉴런과 astrocytes 높은 숫자를 공개했다. 조직 블록 분리 후 신경 전구체 세포 문화의 생성은 급속도로 차별 조건 뉴런과 astrocytes 다수의 생산 neurospheres을 확장 결과. 이 간단한 cryopreservation 프로토콜은의 연결을 astrocyte와의 연결을 전구체 세포 배양 이후 세대 증가 유연성을 부여 피질 뇌 조직 블록의 급속한, 효율적인, 그리고 저렴한 보존에 대한 수 있습니다.

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

여기, 우리는 효율적인 cryopreservation을위한 방법을 설명하고 매우 풍부한 문화의 연결을 생성하는 대뇌 피질의 뇌 조직 블록의 해동. 이 간단한 프로토콜은의 연결을 astrocyte와의 연결을 전구체 세포 배양 이후 세대 유연성을 제공합니다.

고도의 연결을 강화 문화의 생성을위한 두피 조직 블록의 Cryopreservation

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Research

JoVE Journal

Neuroscience

13.5K 조회수.  University of California, Irvine. 여기, 우리는 효율적인 cryopreservation을위한 방법을 설명하고 매우 풍부한 문화의 연결을 생성하는 대뇌 피질의 뇌 조직 블록의 해동. 이 간단한 프로토콜은의 연결을 astrocyte와의 연결을 전구체 세포 배양 이후 세대 유연성을 제공합니다.

비디오: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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 ...

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 ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

여러인지 기능을 테스트 및 설치류에의 연결 활동을 녹음하기위한 완전 자동화된 고도 다목적 시스템

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

단일 셀의 정밀도와의 연결을 공동 문화의 준비

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

전기 생리학 및 칼슘 이미징에 대한 급성 신경 조직의 장기간 배양

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

미세 전극 배열에서 신경 세포 배양의 자극에 대한 네트워크 반응의 시간 의존적 증가

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

혈액 유래 신경 세포의 GM 무료 생성

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

닭 배아망막에서 콘 세포 상호 작용에 로드를 연구하는 원뿔 농축 배양

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

체외에서 뇌 신경 노화를 모델링하기위한 피질 뇌 오가노이드의 강력하고 재현 가능한 생성

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...