Este estudo foi apoiado pela Fundação Nacional de Ciência Natural da China (Grant nº 81673676) e pelo Dongguan Science and Technology Bureau (Grant nº 2019622101002). Os autores agradecem à Dra.

Todos os protocolos experimentais e procedimentos animais cumpriram as diretrizes do princípio ético do Conselho Nacional de Pesquisa.
1. Preparação de materiais
<ol><li>Esterilize todos os instrumentos e ddH2O usando uma autoclave antes de realizar o experimento. Os instrumentos incluem, mas não se limitam a tesouras cirúrgicas, tesoura oftálmica, fórceps, divisor de núcleos de colheres, pratos de vidro (60-100 mm de diâmetro) e quebra-vidros.</li>
	<li>Prepare a solução Krebs da seguinte forma(Tabela 1): Dissolva todos os produtos químicos juntamente com ddH2O antes de usar. Dissolva o CaCI2 separadamente e adicione lentamente na solução mista enquanto mexe para evitar sedimentos.</li>
	<li>Prepare a mídia Rinse, que consiste em mídia F12 com 10% de soro bovino fetal (FBS) e 1% antibiótico/antimíctico (100x). Adicione 5 mL de FBS e 0,5 mL de antibiótico/antimíctico a 44,5 mL de mídia F12.</li>
	<li>Prepare a mídia de neurônioS A, que compreende a mídia neurobásica A com 2% B-27, 1% FBS, 1% L-glutamina, 1% antibiótico/antimíctico (100x) e fator neurotrófico derivado de glia de 0,1% (GDNF). Adicione 200 μL de B-27, 100 μL de FBS, 100 μL de L-glutamina, 100 μL de antibiótico/antimíctico (100x) e 10 μL de GDNF (10 μg/mL) a 9,5 mL de mídia neurobásal A. NOTA: Prepare a mídia de neurônios A dentro de uma semana de uso para garantir o frescor de B-27, L-glutamina e GDNF.</li>
	<li>Prepare a mídia de neurônio B usando o mesmo procedimento que a preparação da mídia de neurônios A, mas sem adicionar FBS.</li>
	<li>Prepare a solução de digestão 1 dissolvendo 20 mg de colagenase tipo II, 6 mg de albumina de soro bovino e 200 μL de antibiótico/antimíctico (100x) em 10 mL de solução krebs estável em oxigênio.</li>
	<li>Prepare a solução de digestão 2 diluindo 1 mL de 0,25% de trippsina com 4 mL da solução de sal balanceada da Hank.</li>
	<li>Prepare um prato com tampas revestidas.<ol>
<li>Use fórceps em um banco de fluxo laminar ao colocar tampas de vidro em uma placa de cultura de 48 poços.</li>
		<li>Diluir a poli-D-lysina com ddH2O a uma concentração de 0,1 mg/mL como estoque de poli-D-lysina. Armazene o estoque a -20 °C e descongele antes de usar.</li>
		<li>Adicione 40 μL de material de poli-D-lisina em cima de cada deslizamento de cobertura, e incubar a solução em temperatura ambiente por 10 minutos. NOTA: Para diferentes placas com diferentes áreas basais, ajuste a concentração para 4 μg/cm2. Por exemplo, se a área basal de um poço de uma placa de 24 poços é de 2 cm2,então devemos transferir 80 μL de estoque de poli-D-lysina em um poço. Cada cm2 conteria 4 μg de poli-D-lysine.</li>
		<li>Remova a poli-d-lisina na placa de 48 poços e enxágue os deslizamentos com ddH2O três vezes.</li>
		<li>Seque a placa em um capô de fluxo laminar por pelo menos 30 minutos para garantir que a placa esteja anidro.</li>
		<li>Armazene a placa a 4 °C antes do revestimento de laminina durante no máximo 1 dia. Armazenar a placa a −20 °C é preferível para armazenamento a longo prazo.</li>
		<li>Descongele laminina a 4 °C. Diluir laminina com ddH2O a uma concentração de 50 μg/mL como estoque de laminina. Armazene o estoque a -20 °C e descongele a 4 °C antes de usar.</li>
		<li>Use uma pipeta para transferir 100 μL de laminina diluída para o topo de cada deslizamento de cobertura e incubar laminina a 4 °C por 1h. NOTA: Para diferentes placas com diferentes áreas basais, ajuste a concentração para 5 μg/cm2. Por exemplo, se a área basal de um poço de uma placa de 24 poços for de 2 cm2, então transfira 200 μL de laminina diluída em um poço. Cada cm2 conteria 5 μg de laminina.</li>
		<li>Remova a solução de laminina e enxágue as tampas com ddH2O uma vez. Realize esta operação na borda do deslizamento para evitar raspagem. NOTA: As tampas revestidas podem ser armazenadas a 4 °C durante 2 semanas no máximo.</li>
	</ol>
</li>
</ol>2. Colheita da bexiga
<ol><li>Obter ratos Sprague-Dawley de cinco semanas de idade.</li>
	<li>Infundir carbogen (95% oxigênio, 5% CO2) na solução Krebs por pelo menos 30 minutos em um banho de gelo para atingir um estado estável de oxigênio e nível de pH.</li>
	<li>Após a eutanásia por deslocamento cervical, mergulhe os ratos em 75% de etanol para 30 s para esterilização.</li>
	<li>Coloque ratos em uma toalha cirúrgica esterilizada e exponha seu abdômen. Abra a cavidade abdominal e revele a bexiga com um conjunto de tesouras e fórceps.</li>
	<li>Levante a bexiga suavemente e corte a bexiga do pescoço da bexiga com outro conjunto de tesouras e fórceps para evitar contaminação cruzada. Coloque a bexiga rapidamente em solução krebs estável em oxigênio frio para melhorar a sobrevivência celular. NOTA: Uma vez que a bexiga é removida, realize as seguintes operações rapidamente para melhorar a perspectiva de sobrevivência dos neurônios.</li>
	<li>Adicione a solução Krebs em três pratos de vidro e quebra-vidros.</li>
	<li>Prepare pratos de vidro e quebra-vidros com solução Krebs em um banho de gelo para pré-fabricação.</li>
	<li>Marque esses recipientes com números 1-3 correspondentemente para evitar confusão.</li>
	<li>Emparelhe cada prato de vidro com fórceps e um divisor de núcleos colheres.</li>
	<li>No prato de vidro 1, abra a bexiga com uma tesoura oftálmica, e desdobre-a com fórceps e um divisor de núcleos de colheres.</li>
	<li>Enxágüe a bexiga no quebra-vidro 1 e coloque-a no prato de vidro 2.</li>
	<li>Elimine a gordura aderente na superfície do tecido usando as fórceps e a tesoura oftálmica no prato de vidro 2.</li>
	<li>Enxágüe a bexiga no quebra-vidro 2 e coloque-a no prato de vidro 3.</li>
	<li>Raspe suavemente a bexiga usando as fórceps e o divisor do núcleo das colheres no prato de vidro 3 para remover os acessórios exógenos.</li>
	<li>Enxágüe a bexiga no disjuntor de vidro 3, transfira a bexiga para um tubo de centrífugas de 15 mL com 14 mL de solução krebs fria, e gire a amostra por 1 min a 356 x g e 4 °C.</li>
	<li>Repita a etapa anterior duas vezes em dois outros tubos com solução Krebs para reduzir a contaminação.</li>
</ol>3. Digestão da bexiga de dois passos
<ol><li>Transfira a bexiga do tubo centrífuga para um frasco de 2 mL contendo 1 mL de solução de digestão 1. Use uma tesoura oftálmica para cortar a bexiga em pequenos pedaços (menores que 1 mm) na solução.</li>
	<li>Misture a solução da bexiga com 9 mL de solução de digestão 1 em um prato de cultura celular estéril (100 mm de diâmetro). Realize a digestão passo 1 em uma incubadora de agitação por 1h abaixo de 5% de CO2, 37 °C e 200 rpm.</li>
	<li>Após a digestão do passo 1, centrifugar a solução a 356 x g a 4 °C por 8 min.</li>
	<li>Coloque a solução de digestão 2 em um banho de água de 37 °C para pré-aquecimento.</li>
	<li>Após a centrifugação, remova o sobrenante que contém a solução de digestão 1, e colmeia o sedimento celular. Algum líquido restante é permitido. A remoção completa da solução pode promover a perda celular.</li>
	<li>Misture sedimentos celulares com solução de digestão quente 2 em um tubo de centrífugas de 15 mL e agite a mistura enquanto digere em um banho de água de 37 °C por 5 min. Não exceda 7 minutos de digestão do passo 2 ou os neurônios perecerão.</li>
	<li>Após a digestão, desative imediatamente a trippsina na mistura com 10 mL de mídia de lavagem a frio. NOTA: Realize as seguintes etapas em 0 °C–4 °C. Um banho de gelo poderia fornecer tal condição.</li>
	<li>Colher o sedimento celular após centrifugação a 356 x g a 4 °C por 8 min. Remova o máximo de mídia possível porque a trippsina restante é prejudicial ao crescimento celular.</li>
	<li>Sedimento resuspend com 3 mL de mídia de neurônios suavemente. Certifique-se de que as bolhas de ar não sejam geradas na solução que contém células para uma alta taxa de sobrevivência.</li>
	<li>Filtre a mídia da mistura através de um coador de células de 70 μm em um tubo centrífuga de 50 mL.</li>
	<li>Mantenha o filtrado em um shaker a 30 rpm em um banho de gelo por 30 minutos. Esta etapa não é necessária, mas recomendada.</li>
	<li>Coletar células por centrifugação a 356 x g a 4 °C por 8 min, e resuspensar suavemente as pelotas celulares em 1 mL de mídia neurônio A.</li>
	<li>Adicione 500 μL de mistura celular em cada poço da placa de 48 poços preparada.</li>
	<li>Células de cultura em uma incubadora a 37 °C e 5% DE CO2.</li>
	<li>Substitua toda a mídia por mídia de neurônios B em 1h para fornecer uma cultura livre de soro.</li>
	<li>Troque metade da mídia de neurônios B a cada 3 dias. NOTA: Os neurônios estão prontos para experimentos imunocitômicos após 5-7 dias de cultura.</li>
</ol>

<ol>
	<li>Fowler, C. J., Griffiths, D., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+micturition.">The neural control of micturition.</a> Nature Reviews Neuroscience. 9 (6), 453-466 (2008).</li><li>Golbidi, S., Laher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+diabetes+mellitus.">Bladder dysfunction in diabetes mellitus.</a> Frontiers in Pharmacology. 1, 136 (2010).</li><li>Iwasawa, E., 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=Long-term+Effects+of+Intravenous+Cyclophosphamide+in+Combination+with+Mesna+Provided+Intravenously+and+via+Bladder+Perfusion+in+a+Patient+with+Severe+Multifocal+Motor+Neuropathy.">Long-term Effects of Intravenous Cyclophosphamide in Combination with Mesna Provided Intravenously and via Bladder Perfusion in a Patient with Severe Multifocal Motor Neuropathy.</a> Internal Medicine. 56 (14), 1893-1896 (2017).</li><li>Lange, M. M., van de Velde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+and+sexual+dysfunction+after+rectal+cancer+treatment.">Urinary and sexual dysfunction after rectal cancer treatment.</a> Nature Reviews. Urology. 8 (1), 51-57 (2011).</li><li>Lin, C. S., 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=Nerve+function+and+dysfunction+in+acute+intermittent+porphyria.">Nerve function and dysfunction in acute intermittent porphyria.</a> Brain. 131, 2510-2519 (2008).</li><li>Rantell, A., 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=What+is+the+utility+of+urodynamics,+including+ambulatory,+and+24+h+monitoring,+in+predicting+upper+urinary+tract+damage+in+neuro-urological+patients+and+other+lower+urinary+tract+dysfunction?+ICI-RS+2017.">What is the utility of urodynamics, including ambulatory, and 24 h monitoring, in predicting upper urinary tract damage in neuro-urological patients and other lower urinary tract dysfunction? ICI-RS 2017.</a> Neurourology and Urodynamics. 37, 25-31 (2018).</li><li>Halperin, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Lyme+neuroborreliosis.">Diagnosis and management of Lyme neuroborreliosis.</a> Expert Review of Anti-Infective Therapy. 16 (1), 5-11 (2018).</li><li>Walter, 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=Reliability+of+supraspinal+correlates+to+lower+urinary+tract+stimulation+in+healthy+participants+-+A+fMRI+study.">Reliability of supraspinal correlates to lower urinary tract stimulation in healthy participants - A fMRI study.</a> Neuroimage. 191, 481-492 (2019).</li><li>Leitner, L., 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+infusion-drainage+device+to+assess+lower+urinary+tract+function+in+neuro-imaging.">A novel infusion-drainage device to assess lower urinary tract function in neuro-imaging.</a> BJU International. 119 (2), 305-316 (2017).</li><li>Smith, T. H., Ngwainmbi, J., Grider, J. R., Dewey, W. L., Akbarali, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in-vitro+preparation+of+isolated+enteric+neurons+and+glia+from+the+myenteric+plexus+of+the+adult+mouse.">An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse.</a> Journal of Visualized Experiments. (78), e50688 (2013).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Roppongi, R. T., Champagne-Jorgensen, K. P., Siddiqui, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Density+Primary+Hippocampal+Neuron+Culture.">Low-Density Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (122), e55000 (2017).</li><li>Arms, L., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptides+in+lower+urinary+tract+function.">Neuropeptides in lower urinary tract function.</a> Handbook of Experimental Pharmacology. (202), 395-423 (2011).</li><li>Moriyama, Y., Hiasa, M., Sakamoto, S., Omote, H., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+nucleotide+transporter+(VNUT):+appearance+of+an+actress+on+the+stage+of+purinergic+signaling.">Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.</a> Purinergic Signal. 13 (3), 387-404 (2017).</li><li>Chaudhury, A., He, X. D., Goyal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+plays+a+key+role+in+nitrergic+neurotransmission+by+transporting+nNOS&#945;+to+enteric+varicosity+membrane.">Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOS&#945; to enteric varicosity membrane.</a> American journal of physiology. 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>

Os autores não declaram nenhum grande conflito de interesses.

Preparação da placa O uso de tampas de vidro em placas de cultura de 6, 12 ou 48 poços para experimentos de imagens imunofluorescentes ou de cálcio é uma operação econômica e poupadora de amostras. As células crescem bem em placas sem tampas durante a preparação das culturas celulares primárias. Portanto, as tampas são dispensáveis em experimentos, como a mancha ocidental ou a reação em cadeia de polimerase. Além disso, o revestimento é um passo necessário antes de emplacamento de c...

O trato urinário inferior tem duas funções principais: armazenamento periódico de urina e micturição1. O sistema nervoso do trato urinário inferior (LUTNS) controla essas funções e é delicado e suscetível a muitas neuropatias, que podem ser inatas (porfiria), adquiridas (doença de Lyme), secundárias aos estados da doença (cistopatia diabética), induzida por drogas (cistite hemorrágica), cirurgia causada (ressecção abdominoperineal) ou lesão causada (lesão medular traumática)2,3,4,5,6,7. Em estudos fisiológicos/patológicos, experimentos in vivo e in vitro são igualmente importantes. Enquanto a pesquisa in vivo em LUTNS tem sido conduzida em níveis de órgãos, celulares e moleculares há algum tempo, a pesquisa in vitro sobre neurônios primários da bexiga urinária é quase inexistente8,9. Embora o presente estudo seja limitado, esperamos ser pioneiros em pesquisas nessa área para que outros pesquisadores possam melhorá-la. Dessa forma, essa cocultura pode levar a uma compreensão celular da disfunção fisiológica em fenótipos, como a disfunção do neurônio da bexiga.
Em contraste com os músculos entéricos com uma direcionalidade clara das células musculares em camadas discretas, os músculos da bexiga são desorganizados10. Portanto, em vez de descascar a camada externa da bexiga, este método propõe digerir toda a bexiga para reduzir a dificuldade de operação e encurtar o tempo de pré-processamento para uma alta taxa de sobrevivência celular.
Seguindo este método, podemos obter uma cultura mista de neurônios e outras células. As outras células são indispensáveis porque sua presença imita um ambiente in vivo11. Além disso, essas células fornecem as substâncias que não estão disponíveis no meio.
Este método envolve dois passos para a digestão. Primeiro, a colagem tipo II é usada para hidrolisar colágeno, seguido de trippsina, para dissociar o tecido nas células10. Dessa forma, os tecidos da bexiga são dispersos em células únicas e, em seguida, crescem relativamente independentes. Quando a cultura dos neurônios amadurece, os neurônios podem ser usados para imagens ou ensaios funcionais.

<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

O trato urinário inferior tem duas funções principais: armazenamento periódico de urina e micturição; essas funções são mediadas através da neuroregulação central e periférica. Embora tenham sido realizadas extensas pesquisas sobre o sistema nervoso do trato urinário inferior, a maioria dos estudos tem se concentrado na cultura primária. Este protocolo introduz um método para o isolamento e cultura dos neurônios da bexiga e da glia dos ratos de Sprague-Dawley. Neste método, os neurônios e a glia foram incubados em uma incubadora de 37 °C, 5% de CO2 por 5-7 dias. Como resultado, eles cresceram em formas maduras adequadas para experimentos de imunofluorescência subsequentes relacionados. As células foram observadas morfologicamente usando um microscópio óptico. Neurônios, vesículas sinápticas e glia foram identificados por β-III-tubulina e MAP-2, Sinapsin-1 e GFAP, respectivamente. Enquanto isso, a imunocitoquímica foi realizada em várias proteínas relacionadas ao neurotransmissor, como acetiltransferase de colina, DYNLL2 e SLC17A9.

No processo de cultura celular primária, as células adquiridas eram redondas com limites brilhantes e claros antes do estado anexo. À medida que os neurônios cresciam, dendritos e axônios começaram a ser distintos. Após 5 a 7 dias de cultura, os neurônios atingiram uma forma madura com projeções longas, que eram ideais para estudos de imagem ou função. Embora a maioria das impurezas e detritos celulares pudessem ser removidos devido à mudança de mídia, certos resíduos ligados ao revestimento de poli-D-lys...

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

Este protocolo tenta estabelecer um protocolo repetível para neurônios primários e isolamento de glia da bexiga de rato para novos experimentos celulares.

Isolamento e Cultura dos Neurônios Primários e Glia da Bexiga Urinária de Ratos Adultos

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 visualizações.  Guangzhou University of Chinese Medicine. Este protocolo tenta estabelecer um protocolo repetível para neurônios primários e isolamento de glia da bexiga de rato para novos experimentos celulares.

Vídeo: 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.

Isolamento de Neural Stem / células progenitoras da Região Periventricular do rato adulto e Medula Espinhal Humano

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

Biologia do Desenvolvimento

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.

Isolamento Microglia primária a partir de cultura mista de células gliais de tecido de cérebro de rato neonatal

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

Imunologia e Infecção

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.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e 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 ...

Neurociência

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.

Isolamento e cultivo de células neurais embrionárias de ratos: um protocolo rápido

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.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

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.

Preparação de culturas mista glial primários de rato adulto Spinal Cord Tissue

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.

Isolamento de gânglios das raízes dorsais e cultura primária para estudar a liberação de neurotransmissores

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.

Obtenção de Microglia Humana de Tecido Cerebral Humano Adulto

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.

Isolamento e cultura de neurônios primários e glia de uma bexiga urinária de rato

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.

Isolamento de células-tronco progenitoras neurais da região periventricular da medula espinhal de um rato adulto

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