이 연구는 중국 국립 자연과학 재단(81673676)과 동관과학기술국(2019622101002)의 지원을 받았습니다. 저자는 메리 로즈 설리반 박사 (수술조교수, 하버드 의과 대학) 기술 컨설팅에 감사드립니다.

모든 실험 프로토콜과 동물 절차는 국가 연구 위원회의 윤리적 원칙 지침을 준수했다.
1. 재료 의 준비
<ol><li>실험을 수행하기 전에 자동 복제를 사용하여 모든 기기및 ddH2O를 멸균합니다. 기기에는 수술 용 가위, 안과 가위, 집게, 숟가락 핵 칸막이, 유리 접시 (직경 60-100mm) 및 유리 차단기를 포함하지만 이에 국한되지 않습니다.</li>
	<li>다음과 같이 Krebs 용액을 준비(표 1): 사용하기 전에 ddH2O와 함께 모든 화학 물질을 용해하십시오. CaCI2를 별도로 녹이고 침전물을 피하기 위해 교반하는 동안 혼합 용액에 천천히 추가합니다.</li>
	<li>10% 태아 소 혈청 (FBS) 및 1 % 항생제 / 항진균 (100x)F12 미디어로 구성된 린스 미디어를 준비하십시오. F12 미디어의 44.5 mL에 FBS 5mL 및 항생제/항마이코틱의 0.5 mL를 추가합니다.</li>
	<li>2% B-27, 1% FBS, 1% L-글루타민, 1% 항생제/항마이코틱(100x), 그리고 0.1% glia 유래 신경영양인(GDNF)을 포함하는 신경물질 A를 포함하는 신경 세포 A를 준비합니다. B-27의 200 μL, FBS 100 μL, L-글루타민 100 μL, 항생제/항진균제 100μL(100x), GDNF(10 μg/mL) 10μL를 신경분질 A 미디어의 9.5mL에 추가합니다. 참고: B-27, L-글루타민 및 GDNF의 신선도를 보장하기 위해 사용 후 1주일 이내에 뉴런 미디어 A를 준비합니다.</li>
	<li>뉴런 미디어 A의 준비와 동일한 절차를 사용 하 여 신경 미디어 B를 준비 하지만 FBS를 추가 하지 않고.</li>
	<li>소화용액 10 mg의 콜라게나아제 타입 II, 소 세럼 알부민 6mg, 산소 안정크렙스 용액 10mL에서 항생제/항진균(100x)의 200μL를 용해하여 소화용액 1을 준비한다.</li>
	<li>행크의 균형 잡힌 소금 용액 4mL로 0.25% 트립신 1mL을 희석하여 소화 용액 2를 준비합니다.</li>
	<li>코팅 된 커버립접시를 준비합니다.<ol>
<li>유리 커버립을 48웰 배양판에 넣을 때 라미나르 플로우 벤치에 집게를 사용하십시오.</li>
		<li>폴리-D-리신을 ddH2O로 희석하여 폴리-D-lysine 재고로 0.1 mg/mL농도로 희석합니다. 재고를 -20°C에 보관하고 사용하기 전에 해동합니다.</li>
		<li>각 커버슬립 위에 폴리-D-리신 스톡 40μL을 추가하고 실온에서 10분 동안 용액을 배양합니다. 참고: 기저 영역이 다른 플레이트의 경우 농도를 4 μg/cm2로조정합니다. 예를 들어, 24웰 플레이트로부터 1개의 우물의 기저면적이 2cm2인경우, 폴리-D-리신 재고의 80 μL을 잘 전달해야 한다. 각 cm2에는 폴리 D-리신 4μg가 포함됩니다.</li>
		<li>48웰 플레이트에서 폴리-D-리신을 제거하고, ddH2O 세 번으로 커버립을 헹구세요.</li>
		<li>플레이트가 무수성인지 확인하기 위해 최소 30분 동안 라미나르 플로우 후드로 플레이트를 건조시다.</li>
		<li>라미닌 코팅 전에 4°C에서 플레이트를 1일 동안 보관하십시오. 플레이트를 -20°C로 저장하는 것이 장기 보관에 바람직하다.</li>
		<li>4 °C에서 라미닌을 해동하십시오. 라미닌 육수로 50 μg/mL의 농도로 ddH2O로 라미닌을 희석시하십시오. 재고를 -20°C로 저장하고 사용하기 전에 4°C에서 해동하십시오.</li>
		<li>파이펫을 사용하여 희석된 라미닌 100μL을 각 커버슬립의 맨 위로 옮기고 라미닌을 4°C에서 1h로 배양한다. 참고: 기저 영역이 다른 플레이트의 경우 농도를 5 μg/cm2로조정합니다. 예를 들어, 24웰 플레이트로부터 하나의 우물의 기저 면적이 2cm2인경우, 희석된 라미닌200μL을 우물에 옮기는다. 각 cm2에는 라미닌 5μg가 포함됩니다.</li>
		<li>라미닌 용액을 제거하고 커버립을 ddH2O로 한 번 헹구는 다. 긁어방지를 위해 커버슬립 가장자리에서 이 작업을 수행합니다. 참고: 코팅된 커버립은 최대 2주 동안 4°C로 보관할 수 있습니다.</li>
	</ol>
</li>
</ol>2. 방광 수확
<ol><li>5주 된 스프라그-Dawley 쥐를 구하십시오.</li>
	<li>안정적인 산소 상태와 pH 수준에 도달하기 위해 얼음 욕조에서 적어도 30 분 동안 크렙스 용액에 카보겐 (95 % 산소, 5 % CO2)을주입합니다.</li>
	<li>자궁 경부 탈구를 통해 안락사 후, 살균을 위해 30 에탄올에 75 %의 쥐를 담그십시오.</li>
	<li>소독 수술 수건에 쥐를 놓고 복부를 노출. 복강을 열고 가위와 집게 세트로 방광을 드러냅니다.</li>
	<li>방광을 부드럽게 들어 올리고 방광목에서 방광을 다른 가위와 집게로 잘라 교차 오염을 방지합니다. 방광을 감기 산소 안정 크렙스 솔루션에 빠르게 배치하여 세포 생존을 향상시킵니다. 참고: 방광이 제거되면 다음 작업을 신속하게 수행하여 뉴런 생존 가능성을 개선하십시오.</li>
	<li>크렙스 용액을 세 개의 유리 요리와 유리 차단기에 추가합니다.</li>
	<li>프리쿨링을 위해 얼음 욕조에 크렙스 용액으로 유리 접시와 유리 차단기를 준비합니다.</li>
	<li>혼동을 방지하기 위해 해당 번호 1-3로 이러한 컨테이너를 표시합니다.</li>
	<li>각 유리 접시에 집게와 숟가락 핵 분배기와 함께 페어링합니다.</li>
	<li>유리 접시 1에서 안과 가위로 방광을 열고 집게와 숟가락 핵 칸막이로 펴놓습니다.</li>
	<li>방광을 유리 차단기 1에 헹신 다음 유리 접시 2에 놓습니다.</li>
	<li>유리 접시 2의 집게와 안과 가위를 사용하여 조직 표면에 부착 된 지방을 제거하십시오.</li>
	<li>방광을 유리 차단기 2에 헹신 다음 유리 접시 3에 놓습니다.</li>
	<li>집게와 숟가락 핵 분배기를 유리 접시 3에 사용하여 방광을 부드럽게 긁어 외인성 부착물을 제거합니다.</li>
	<li>방광을 유리 차단기 3에서 헹구고, 방광을 14mL의 콜드 크렙스 용액으로 15mL 원심분리기 튜브로 옮기고, 356 x g 및 4°C에서 1분 동안 샘플을 회전시합니다.</li>
	<li>오염을 줄이기 위해 크렙스 용액을 사용하여 두 개의 다른 튜브에서 이전 단계를 두 번 반복합니다.</li>
</ol>3. 2단계 방광 소화
<ol><li>원심분리기 관에서 1mL의 소화용액 1을 포함하는 2mL 바이알로 방광을 전달한다. 안과 가위를 사용하여 방광을 용액의 작은 조각 (1mm 보다 작은)으로 자른다.</li>
	<li>방광 용액을 멸균 세포 배양 접시(직경 100mm)에 9mL의 소화용액 1과 섞는다. 5% CO2,37°C 및 200 rpm 미만의 1시간 동안 흔들리는 인큐베이터에서 1단계 소화를 수행합니다.</li>
	<li>1단계 소화 후, 원심분리기는 용액을 4°C에서 356 x g에서 8분 동안 356 x g로 한다.</li>
	<li>예열을 위해 37°C 수조에 소화용액 2를 놓습니다.</li>
	<li>원심 분리 후, 소화 용액 1을 포함하는 상체를 제거하고 세포 퇴적물을 수확한다. 일부 남은 액체는 허용됩니다. 용액의 완전한 제거는 세포 손실을 촉진할 수 있다.</li>
	<li>15mL 원심분리기 튜브에 따뜻한 소화용액 2와 세포 침전물을 섞고 37°C 수조에서 소화하면서 혼합물을 흔들어 5분 동안 섞는다. 단계 2 소화의 7 분 이상하지 마십시오 또는 뉴런은 멸망한다.</li>
	<li>소화 후, 즉시 차가운 헹기 매체의 10 mL와 혼합물에 트립신을 비활성화합니다. 참고: 0°C-4°C에서 다음 단계를 수행합니다. 얼음 목욕은 그러한 상태를 제공 할 수 있습니다.</li>
	<li>원심분리 후 세포 퇴적물을 4°C에서 356 x g에서 8분 동안 수확하십시오. 남은 트립신이 세포 성장에 유해하기 때문에 가능한 한 많은 미디어를 제거하십시오.</li>
	<li>3mL의 뉴런 미디어로 퇴적물을 부드럽게 재중단합니다. 높은 생존율을 위해 세포를 포함하는 용액에서 기포가 생성되지 않았는지 확인합니다.</li>
	<li>70 μm 세포 스트레이너를 통해 혼합물 매체를 50mL 원심분리기 튜브로 필터링합니다.</li>
	<li>30 분 동안 얼음 목욕에 30 rpm에 셰이커에 여과물을 유지합니다. 이 단계는 필요하지 않지만 권장됩니다.</li>
	<li>4°C에서 356 x g에서 원심분리로 세포를 수거하고, 1mL의 뉴런 미디어 A에서 세포 펠릿을 부드럽게 재보냅니다.</li>
	<li>준비된 48웰 플레이트의 각 웰에 500 μL의 셀 혼합물을 넣습니다.</li>
	<li>37°C 및 5% CO2에서인큐베이터내의 배양 세포.</li>
	<li>모든 미디어를 1h의 뉴런 미디어 B로 교체하여 혈청없는 문화를 제공합니다.</li>
	<li>3일마다 뉴런 미디어 B의 절반을 변경합니다. 참고: 뉴런은 5-7일 간의 배양 후 면역 세포화학 실험을 위한 준비가 되어 있습니다.</li>
</ol>

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Gastrointestinal and Liver Physiology. 301 (3), 498-507 (2011).</li><li>Le Berre-Scoul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enteric+neuron-glia+coculture+system+reveals+the+role+of+glia+in+neuronal+development.">A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.</a> The Journal of Physiology. 595 (2), 583-598 (2017).</li><li>Hayashi, H., Yamada, M., Kumai, J., Takagi, N., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+laminin-111-derived+peptide-chitosan+matrices+in+a+primary+culture+of+rat+cortical+neurons.">Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.</a> Archives of Biochemistry and Biophysics. 648, 53-59 (2018).</li><li>Bottenstein, J. E., Sato, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+rat+neuroblastoma+cell+line+in+serum-free+supplemented+medium.">Growth of a rat neuroblastoma cell line in serum-free supplemented medium.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (1), 514-517 (1979).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Survival+of+Hippocampal+Neurons+in+B27-Supplemented+Neurobasalm,+a+New+Serum-free+Medium+Combination.">Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasalm, a New Serum-free Medium Combination.</a> Journal of Neuroscience Research. 35 (5), 567-576 (1993).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1 (5), 2406-2415 (2006).</li><li>Georgas, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+illustrated+anatomical+ontology+of+the+developing+mouse+lower+urogenital+tract.">An illustrated anatomical ontology of the developing mouse lower urogenital tract.</a> Development. 142 (10), 1893-1908 (2015).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single-cell+RNA-seq+artifacts:+a+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li></ol>

저자는 큰 이해 상충을 선언하지 않습니다.

플레이트 준비 면역형광 또는 칼슘 이미징 실험을 위한 6-, 12 또는 48웰 배양 판에서 유리 커버립을 사용하는 것은 경제적이고 시료 절약 적인 동작입니다. 세포는 1 차적인 세포 배양의 준비 도중 커버립 없이 접시에서 잘 자랍니다. 따라서, 커버립은 서양 블롯 또는 폴리머라제 연쇄 반응과 같은 실험에서 분배할 수 있다. 또한, 코팅은 커버립의 유무에 관계없이 세포를 도금하기 전...

하부 요로에는 두 가지 주요 기능이 있습니다: 주기적인 소변 저장 및 배뇨1. 하부 요로 신경계(LUTNS)는 이러한 기능을 제어하고 많은 신경병증에 섬세하고 민감하며, 이는 선천적(포르피리아), 획득(라임병), 질병 상태(당뇨병 세포질), 약물 유도(출혈성 방광염), 수술 발생(복부 절제술 절제술, 4, 외상성 척추 상해, 4, 외상성 척추 상해, 4, 외상성 척추 손상, 4, 외상성 척추 손상, 3) 및 상해(외상성 척추 손상, 3) 및 상해(외상성 척추 손상, 5, 외상성 척추 손상,3) 생리적/병리학 연구에서 생체 내 및 체외 실험도 똑같이 중요합니다. LUTNS에 대한 생체 내 연구는 장기, 세포 및 분자 수준에서 한동안 수행되었지만, 오줌 방광에서 1 차 적인 뉴런에 대한 체외 연구는 거의 존재하지 않는8,9. 본 연구는 제한적이지만, 우리는 다른 연구자들이 그것을 개선할 수 있도록 이 지역에 있는 연구를 개척하기를 희망합니다. 이러한 방식으로, 이러한 공동 배양은 방광 뉴런 기능 장애와 같은 표현형에 있는 생리적 기능 장애의 세포 이해로 이끌어 낼 수 있습니다.
근육 세포의 명확한 방향성을 가진 장내 근육과 는 대조적으로, 방광의 근육은 조직되지 않습니다10. 따라서 방광의 외부 층을 벗겨내는 대신, 이 방법은 방광 전체를 소화하여 작동의 어려움을 줄이고 높은 세포 생존율에 대한 전처리 시간을 단축할 것을 제안합니다.
이 방법에 따라, 우리는 뉴런 과 다른 세포의 혼합 배양을 얻을 수 있습니다. 그들의 존재는 생체 내환경(11)을모방하기 때문에 다른 세포는 필수 불가결하다. 또한, 이러한 세포는 배지에서 사용할 수 없는 물질을 제공한다.
이 방법은 소화를 위한 두 단계를 포함합니다. 먼저, 콜라게나아제 형 II는 콜라겐을 가수분해하는 데 사용되며, 트립신이 이어서 조직을세포(10)로해리한다. 이런 식으로, 방광 조직은 단 하나 세포로 분산되고 그 때 상대적으로 독립적으로 성장합니다. 뉴런의 문화가 성숙할 때, 뉴런은 화상 진찰 또는 기능적인 소사에 사용될 수 있습니다.

<table><tbody><tr><td>0.25% trypsin</td><td>Gibico</td><td>15050065</td><td>Enzyme digestion</td></tr><tr><td>48-well culture plate</td><td>Corning</td><td>3548</td><td>Coating dish</td></tr><tr><td>antibiotic/antimycotic</td><td>Gibico</td><td>15240062</td><td>Culture media/Rinse media</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein Antibody</td><td>Santa Cruz</td><td>sc-33673</td><td>ICC</td></tr><tr><td>B-27</td><td>Gibico</td><td>17504044</td><td>Culture media</td></tr><tr><td>BSA Fraction V</td><td>Gibico</td><td>332</td><td>Enzyme digestion</td></tr><tr><td>Choline Acetyltransferase Antibody</td><td>Abcam</td><td>ab18736</td><td>ICC</td></tr><tr><td>CO2 Incubator</td><td>Heraeus</td><td>B16UU</td><td>Cells culture</td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>2593923</td><td>Enzyme digestion</td></tr><tr><td>DMEM/F-12</td><td>Gibico</td><td>11330032</td><td>Rinse media</td></tr><tr><td>DYNLL2 Antibody</td><td>Santa Cruz</td><td>sc-13969</td><td>ICC</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibico</td><td>10100147</td><td>Culture media/Rinse media</td></tr><tr><td>Forceps</td><td>Shanghai Jin Zhong Medical Devices</td><td>1383</td><td>10 cm; Sterile operation</td></tr><tr><td>Glass breakers</td><td>Huan Qiu Medical Devices</td><td>1101</td><td>50 ml; Sterile operation</td></tr><tr><td>Glass coverslips</td><td>WHB Scientific</td><td>WHB-48-CS</td><td>Coating dish</td></tr><tr><td>Glass dishes</td><td>Huan Qiu Medical Devices</td><td>1177</td><td>100 mm; Sterile operation</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Hoechst 33342</td><td>BD</td><td>561908</td><td>ICC</td></tr><tr><td>Laminar flow bench</td><td>Su Jie Medical Devices</td><td>CB 1400V</td><td>Sterile operation</td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Coating dish</td></tr><tr><td>L-glutamine</td><td>Gibico</td><td>25030081</td><td>Culture media</td></tr><tr><td>MAP-2 Antibody</td><td>Affinity</td><td>AF5156</td><td>ICC</td></tr><tr><td>Murine GDNF</td><td>Peprotech</td><td>AF45044</td><td>Culture media</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibico</td><td>10888022</td><td>Culture media</td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21010</td><td>12.5 cm; Sterile operation</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000240</td><td>100-1000 ul; Reagent and sample pipetting</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000267</td><td>10-100 ul; Reagent and sample pipetting</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P7280</td><td>Coating dish</td></tr><tr><td>Refrigerated centrifuge</td><td>Ping Fan Instrument</td><td>TGL-16A</td><td>Enzyme digestion</td></tr><tr><td>Shaking incubator</td><td>Haimen Kylin-Bell Lab Instruments</td><td>T8-1</td><td>Enzyme digestion</td></tr><tr><td>SLC17A9 Antibody</td><td>MBL International</td><td>BMP079</td><td>ICC</td></tr><tr><td>Spoons nucleus divider</td><td>Shanghai Jin Zhong Medical Devices</td><td>YZR030</td><td>12 cm; Sterile operation</td></tr><tr><td>Substance P Antibody</td><td>Santa Cruz</td><td>sc-58591</td><td>ICC</td></tr><tr><td>Surgical scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21130</td><td>16 cm; Sterile operation</td></tr><tr><td>Surgical towel</td><td>Fu Kang Medical Devices</td><td>5002</td><td>40 x 50 cm; Sterile operation</td></tr><tr><td>Synapsin-1 Antibody</td><td>CST</td><td>5297T</td><td>ICC</td></tr><tr><td>Tubulin beta Antibody(&amp;beta;-III-tubulin)</td><td>Affinity</td><td>AF7011</td><td>ICC</td></tr></tbody></table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

낮은 요로는 두 가지 주요 기능, 즉, 주기적인 소변 저장 및 배뇨; 이러한 기능은 중앙 및 말초 신경 조절을 통해 중재됩니다. 낮은 요로 신경계에 대한 광범위한 연구가 실시되었지만 대부분의 연구는 1 차 문화에 초점을 맞추고 있습니다. 이 프로토콜은 스프라그-Dawley 쥐에서 방광 뉴런과 glia의 격리 및 문화에 대한 방법을 소개합니다. 이 방법에서, 뉴런과 glia는 5-7 일 동안 37 °C, 5 % CO2 인큐베이터에서 배양되었다. 그 결과, 그(것)들은 관련 후속 면역 형광 실험에 적합한 성숙한 모양으로 성장했습니다. 세포는 광학 현미경을 사용하여 형태학적으로 관찰되었다. 뉴런, 시냅스 소포 및 glia는 각각 β-III-tubulin 및 MAP-2, 시냅신-1 및 GFAP 염색에 의해 확인되었다. 한편, 면역세포화학은 콜린 아세틸트랜스퍼라제, DYNLL2 및 SLC17A9와 같은 여러 신경전달물질 관련 단백질에서 수행되었다.

1 차 세포 배양 과정에서, 획득 된 세포는 부착 된 상태 전에 밝고 명확한 경계로 둥글게되었다. 뉴런이 성장함에 따라, 수상자와 축축은 구별되기 시작했다. 문화의 5-7 일 후에, 신경은 화상 진찰 또는 기능 연구 결과에 이상적이었던 긴 돌출을 가진 성숙한 양식에 도달했습니다. 대부분의 불순물 및 세포 파편은 변화하는 매체로 인해 제거될 수 있었지만, 폴리-D-리신 및 라미닌 코팅에 부착된 특?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

이 프로토콜은 추가 세포 실험을 위한 쥐 방광에서 1 차적인 신경세포 및 glia 격리를 위한 반복가능한 프로토콜을 설치하기 위하여 시도합니다.

성인 쥐 요로 방광에서 1 차신경및 glia의 격리 그리고 문화

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K 조회수.  Guangzhou University of Chinese Medicine. 이 프로토콜은 추가 세포 실험을 위한 쥐 방광에서 1 차적인 신경세포 및 glia 격리를 위한 반복가능한 프로토콜을 설치하기 위하여 시도합니다.

비디오: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

뇌실 주위 백질 성인 쥐의 지역 및 인간의 척수에서 신경 줄기 / 전구 세포의 분리

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

연구

발생학

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

신생아 쥐 뇌 조직의 혼합 Glial 세포 배양의 기본 Microglia 격리

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

면역학 및 감염병학

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

신경과학

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

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

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

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

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

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

성인 마우스 척수 조직에서 차 혼합 폐해 문화의 준비

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

신경 전달 물질 방출 공부 하 느 루트 중추 절연 및 주 문화

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

성인 인간 뇌 조직에서 인간 마이크로글리아 획득

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Source: Wang, R. et al., <a href="https://app.jove.com/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

쥐 요로 방광에서 일차 뉴런 및 신경교세포의 분리 및 배양

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

성체 쥐의 척수 뇌실 주위 영역에서 신경 줄기 전구 세포의 격리

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...