Esta pesquisa foi suportada em parte pela American Heart Association conceder 0950118G.to MC-W.

1. Preparando-se anti-PECAM-1 anticorpo-conjugado esferas magnéticas (Dynabeads) <ol><li> Prepare BSA 0,1% em PBS misturando 50 mg BSA em 50 ml de PBS usando um vortex até BSA dissolve. Esterilizar por filtração através de filtro de seringa de 0,22 mM. Armazenar a 4 ° C. </li><li> Na capa TC, a transferência de 200 mL de bem ressuspenso ovelhas Dynabeads anti-IgG de rato em um tubo Eppendorf 1,5 ml e lavar as contas: em tubo de Monte MPC e deixe descansar por cerca de 1 min para permitir que as contas de sedimentos. Aspirar o sobrenadante, remova o tubo do MPC e ressuspender as contas em 1 ml de estéril 0,1% BSA / PBS. Pipeta cima e para baixo para voltar a suspender contas. </li><li> Repita lavar mais três vezes, para um total de quatro lavagens. </li><li> Retire o tubo do MPC e ressuspender as contas em 500 mL de 0,1% BSA / PBS. </li><li> Adicionar 10 ml de rato anti-rato PECAM-1 anticorpos (CD-31) para o tubo. </li><li> Tombo durante a noite a 4 ° C na sala de frio ou 2 horas em temperatura ambiente. </li><li> Lave as contas com estéril 0,1% BSA / PBS, conforme descrito no passo 1,2 quatro vezes. </li><li> Retire o tubo do MPC e ressuspender as contas em 200 mL de 0,1% BSA / PBS. Loja anti-PECAM-1 anticorpo-conjugado Dynabeads a 4 ° C por até 1 mês. </li></ol> 2. Isolar do mouse em células endoteliais pulmonares de camundongos neonatal Antes de prosseguir com o protocolo de purificação de tecido, IACUC aprovação do Comitê do procedimento é necessário. <ol><li> Prepare o seguinte: <ul><li> 15 ml de 1 mg / ml solução C / D em DMEM. Filtro com 0,22 mM filtro de seringa e quente a 37 ° C. </li><li> 50 ml com tubo de 15 ml DMEM, armazenar no gelo </li><li> Isolamento Media (IM), contendo 20% FBS e 1x penicilina / estreptomicina, em DMEM </li><li> 2% de gelatina. Mix 2 g de pele bovina de gelatina com 100 ml de água dd, 15 autoclave minutos e armazenar a 4 ° C. Quente a 37 ° C para liquefazer antes de frascos de revestimento TC. </li></ul></li><li> Anestesiar três filhotes de 6-8 dia de idade usando uma injeção IP de ketamina (100 mg / kg) e xilazina (10 mg / kg). Verificar a eficácia da anestesia e dissecar animais, um por um, como segue: filhotes Pin para embarcar, embeber a pele com álcool 70% e remover a pele da área do peito com uma tesoura de dissecção estéril. Gaiola do consumo de costela para expor os pulmões. </li><li> Assepticamente consumo lobos pulmonares individual, não tendo o cuidado de dissecar os brônquios e qualquer tecido conjuntivo visível em torno dos pulmões. Combine todos os pulmões em gelada DMEM. Euthanize os filhotes. </li><li> Trabalhando na capa TC, remova lobos pulmonares de DMEM, coloque em um prato de 100 milímetros TC e aspire DMEM excesso. </li><li> Com uma tesoura estéril, o tecido finamente picada cortando ~ 100 vezes. Transferência para 15 ml de solução C / D quentes para a digestão enzimática. Incubar 45 minutos a 37 ° C em um rotador. </li><li> Na capa TC, aspirar a suspensão tecido digerido em uma seringa de 20 ml com 14 g clumps cânula anexado e triturar em uma suspensão de células individuais, pelo menos 12 vezes. </li><li> Passe a suspensão do tecido através de um filtro de células 70 mm e lavar o filtro com 15 ml IM para parar a digestão. </li><li> Pellet suspensão de células por centrifugação a 400x g por 5 minutos. </li><li> Aspirar o sobrenadante e ressuspender sedimento em 3 ml de 0,1% BSA / PBS. </li><li> Transferência de suspensão de células a 5 tubos de poliestireno ml de fundo redondo e adicionar 22,5 mL anti-PECAM-1 anticorpo-conjugado Dynabeads. Tombo em temperatura ambiente por 12 minutos. </li><li> Casaco um frasco T75 com 2 mL de 2% de gelatina e aspirado de excesso de gelatina. Permitir que a gelatina para secar dentro do capô TC por cerca de 30 minutos antes de as células de revestimento. </li><li> Após a incubação talão é completo (passo 2.10), divididos igualmente suspensão de células em três tubos de Eppendorf e montar no MPC. </li><li> Quando as contas tiverem sedimentado usando o MPC (~ min 1), recolher o sobrenadante, remova o tubo do MPC, lave contas com ~ 1 ml de 0,1% BSA / PBS e remontar no MPC. </li><li> Repita quatro vezes lavagem (total 5 lavagens) </li><li> Contas ressuspender em 1 ml de VascuLife com engs Mv-Vida e penicilina Fatores Kit 1x / estreptomicina (VL) por tubo, misturando bem. </li><li> Placa em um pré-revestido T75 frasco e trazer volume total em cada frasco de 10 ml com VL. </li><li> Mudança de mídia no dia seguinte, permita que um dia cheio de crescimento, e depois mudar metade dos media em dias alternados. Quando as células são&gt; 70-80% confluentes ou mais (geralmente após 3-4 dias de cultura), eles podem ser classificados com anti-ICAM-2 Dynabeads. </li></ol> 3. Preparando-se anti-ICAM-2-anticorpo conjugado Dynabeads <ol><li> Dynabeads anti-ICAM-2-anticorpo conjugado são usados ​​para purificar as células que foram selecionados com anti-PECAM-1 anticorpo-conjugado Dynabeads e cultivadas até confluentes. Este procedimento é realizado conforme descrito para Dynabeads anti-PECAM-1 anticorpo-conjugado (1, acima), exceto que anti-mouse ICAM-2 (CD-102) anticorpo é usado em vez de anti-PECAM-1 anticorpos. </li><li> Dynabeads anti-ICAM-2-anticorpo conjugado pode ser armazenado a 4 ° C por atéa 1 mês. </li></ol> 4. Classificação células de camundongo Lung endotelial com anti-ICAM-2 Dynabeads <ol><li> Brasão T75 frasco de cultura de tecido com 2% de gelatina. </li><li> Aspirar a mídia do frasco T75 confluente com células e lave com 8 ml PBS. </li><li> Aspirar PBS, adicionar 2 ml de tripsina 0,05% / EDTA e deixe células detach (cerca de 5 minutos). Toque levemente frascos para ajudar destacando. </li><li> Quando as células têm destacado, adicionar 2 ml IM para inibir células de tripsina e raspar para separar as células restantes aderentes. Transferência de suspensão de células para um tubo de 15 ml e spin down por 5 min a 400x g. </li><li> Aspirar a mídia e celulares ressuspender sedimento em 2 ml de 0,1% BSA / PBS. </li><li> Transferir para um tubo de poliestireno 5 ml de fundo redondo e adicionar 10 ml anti-ICAM-2 Dynabeads. Tumble por 12 minutos em temperatura ambiente. </li><li> Dividido em duas células em suspensão de 1,5 ml tubos Eppendorf e montar no MPC. </li><li> Aspirar o sobrenadante após a contas tenham aderido por um minuto. Remova os tubos de MPC e ressuspender cada tubo 1 ml em 0,1% BSA / PBS. Remontar na MPC. </li><li> Repita quatro vezes lavagem (total de 5 lavagens). </li><li> Ressuspender cada tubo com 1 ml VL e realizar uma contagem de células. </li><li> Células em placas a 250-350K células por frasco T25 e / ou 750K-1 milhão de células por frasco T75. </li><li> Traga o volume para 5 ml em T25 VL frascos, e 10 ml em frascos T75. </li><li> Mudança de mídia todos os dias. Células podem ser usadas para experimentos em que a cultura atinge cerca de 80% confluência, geralmente em 2-3 dias. </li></ol> 5. Resultados representante Normalmente, após 6-7 dias desde o preparo inicial das células, somos capazes de obter cerca de 1,2-1,5 x 10 6 células endoteliais pulmonares de três filhotes de 6-8 dias de idade. Células normais de exposição &quot;paralelepípedos&quot; morfologia em microscopia de luz e mostrar VE-caderina (CD144) coloração em junções célula-célula, que é característica de células endoteliais (Figura 1). Maioria dos MLECs expressar VE-caderina e VEGFR2 como demonstrado por FACS análise (Figura 2). Geralmente, nós usá-los para experimentos dentro de duas semanas após o isolamento MLEC inicial. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="Figura 1" /> Figura 1. Análise microscópica de monocamada MLEC confluentes. (A) imagem de microscopia de luz mostra a morfologia das células em cultura de paralelepípedos típico de células endoteliais. Uniforme estruturas redondas localizada na área perinuclear das células são muitas as esferas magnéticas utilizado para o isolamento MLEC. (B) endotelial específico VE caderina-se localizada nas junções célula-célula, como mostrado por microscopia confocal. Bar é representante da 20Hm. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="Figura 2" /> Figura 2 FACS análise de MLECs cultivadas marcado com anticorpos específicos para marcadores endoteliais específicos:. VE-caderina (CD144) ou VEGFR2, como indiciado, seguido por ficoeritrina conjugada anticorpo secundário, confirma a identidade de MLECs endoteliais isoladas. Linha vermelha indica isotipo específico de controle, linha verde indica anti-VE caderina-ou anti-VEGFR2 --IgG específicos.

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, 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=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+vascular+endothelial+cadherin+(7B4/cadherin-5),+an+endothelium-specific+cadherin.">Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.</a> Arterioscler Thromb Vasc Biol. 15, 1229-1239 (1995).</li><li>Zachary, I., Gliki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+transduction+mechanisms+mediating+biological+actions+of+the+vascular+endothelial+growth+factor+family.">Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family.</a> Cardiovasc Res. 49, 568-581 (2001).</li><li>Diglio, C. A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. E., Gale, D., White, G. C., Vansluys, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+angiogenesis,+endothelial+migration,+proliferation,+and+MAPK+signaling+in+Rap1b-deficient+mice.">Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice.</a> Blood. 111, 2647-2656 (2008).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo Medical College of Wisconsin IACUC Comissão sob o protocolo AUA # 00001206.

Microvascular ECs têm provado ser um modelo útil em muitas áreas da biologia vascular e acredita-se ser mais fisiologicamente relevante para o estudo (angiogênese, por exemplo) do que HUVECs amplamente estudada 3. Anteriormente, foi relatado que microvascular ECs podem ser obtidas no rim, coração de tecido adiposo, pele, retina, cérebro, gliomas, e também de pulmão 2, 4-7, 10, 11. No entanto, um método consistente e confiável para isolamento de microvascular CEPs ainda é necessária. O procedimento apresentado aqui é uma modificação de um protocolo previamente publicado 2, que difere da maioria dos procedimentos publicados na medida em que usa filhotes ao invés de animais adultos. Isso é crucial para o sucesso de isolamento, como células de animais jovens têm um maior potencial de proliferação e culturas derivadas de seus tecidos tendem a produzir maior número de células. Uma semana de idade os animais parecem ser o ideal, mas os ratos mais jovens (4 dias de idade) também pode ser usado. Além disso, os números mais altos ou mais baixos de filhotes pode ser usado se for necessário, como temos sido bem sucedidos em isolar MLECs de um filhote de cachorro. No entanto, enquanto scaling cima ou para baixo o processo de isolamento, a densidade de células de revestimento deve permanecer inalterada, já que esta é também crítico para a proliferação celular. O processo de purificação passo 2 de MLECs usando esferas magnéticas conjugadas primeiro a anti-PECAM-1 e do anti-ICAM-2 anticorpos é muito mais eficiente para a obtenção de culturas puras CE que o descrito anteriormente procedimentos única etapa de purificação. Este protocolo elimina a necessidade de usar técnicas manuais muito trabalhoso, a centrifugação de gradiente e FACS menos eficiente de classificação para as células descartando a contaminação, como fibroblastos, células do sangue e células musculares lisas. A população resultando MLEC pode ser usado para análise in vitro de respostas angiogênico, permeabilidade vascular e transmigração de leucócitos, cicatrização de feridas, bem como análise bioquímica de vias de sinalização. Se necessário, pode ser MLECs semeadas em diferentes superfícies, como lamínulas fibronectina ou gelatina revestido de imunofluorescência ou eletrodo matrizes de trans-endotelial resistência (TER) medições. Resultados obtidos podem ser diretamente comparado com os fenótipos observados in vivo, que é especialmente importante no contexto do crescente número de knockout e linhas de ratinhos transgénicos. Implementação bem sucedida deste método para análise in vitro de mecanismos celulares subjacentes defeitos observados in vivo, já foi apresentada 12.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of pulmonary endothelial cells from neonatal mice

Células endoteliais fornecer um modelo de pesquisa útil em muitas áreas da biologia vascular. Desde o seu primeiro isolamento 1, as células endoteliais da veia umbilical humana (HUVECs) têm se mostrado conveniente, fácil de obter e cultura, e, portanto, são as células mais amplamente estudada endoteliais. No entanto, para a pesquisa focada em processos como os outros permeabilidade a angiogênese, ou muitos, células endoteliais microvasculares (ECs) são um modelo muito mais fisiologicamente relevante para o estudo 2. Além disso, a ECS isolados de camundongos knockout fornecer uma ferramenta útil para a análise de proteínas ex vivo função. Várias abordagens para isolar e microvascular cultura CEPs de origem diferentes foram relatados até agora 3-7, mas o isolamento consistente e cultura de pura CEPs ainda é um grande problema técnico em muitos laboratórios. Aqui, nós fornecemos um protocolo passo a passo sobre um método confiável e relativamente simples de isolar e cultivar células endoteliais pulmonares do rato (MLECs). Nesta abordagem, o tecido pulmonar obtido a partir de 6 - a 8 dias de idade os filhotes é o primeiro corte em pedaços, digeridos com colagenase / dispase (C / D) de soluções e dispersas mecanicamente em uma única célula de suspensão. MLECS são purificados a partir de suspensão celular usando a seleção positiva com anti-PECAM-1 anticorpo conjugado a Dynabeads usando um concentrador de Partícula Magnética (MPC). Tais células purificadas são cultivadas em gelatina revestidos de cultura de tecidos (CT) pratos até que eles se tornam confluentes. Nesse ponto, as células são mais purificado utilizando Dynabeads acoplado a anti-ICAM-2 de anticorpos. MLECs obtidas com este protocolo exibem um fenótipo de paralelepípedos, como visualizado por contraste de fase microscopia de luz, e seu fenótipo endotelial foi confirmada através da análise FACS com anti-VE-caderina 8 e anti-VEGFR2 9 anticorpos e coloração de imunofluorescência de VE caderina. Em nossas mãos, este procedimento de isolamento de duas etapas de forma consistente e confiável rende uma população pura de MLECs, que pode ser mais culta. Este método vai permitir aos investigadores para aproveitar o crescente número de knockout e camundongos transgênicos para correlacionar diretamente os estudos in vivo com os resultados de experimentos in vitro realizados em MLECs isolados e, assim, ajudar a revelar os mecanismos moleculares dos fenótipos vasculares observados in vivo.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Aqui, nós descrevemos um protocolo para isolamento e cultura de células endoteliais pulmonares de camundongos. Este método compreende mecânico e dissociação enzimática do tecido pulmonar, bem como um processo de purificação passo 2, utilizando anti-PECAM-1 e anti-ICAM-2 anticorpos conjugados com esferas magnéticas, que produz uma população de células endoteliais pura de origem principalmente microvascular.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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JoVE Journal

Biology

46.9K visualizações.  BloodCenter of Wisconsin. Aqui, nós descrevemos um protocolo para isolamento e cultura de células endoteliais pulmonares de camundongos. Este método compreende mecânico e dissociação enzimática do tecido pulmonar, bem como um processo de purificação passo 2, utilizando anti-PECAM-1 e anti-ICAM-2 anticorpos conjugados com esferas magnéticas, que produz uma população de células endoteliais pura de origem principalmente microvascular.

Vídeo: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolamento de Valvular Células Endoteliais

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Biologia

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolamento e Cultura de Neonatais rato Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento de Células Embrionárias ventriculares endoteliais

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolamento de artéria pulmonar células musculares lisas de camundongos recém-nascidos

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Geração e cultura de sangue Conseqüência células endoteliais do sangue periférico humano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento de células de camundongo Coronária endoteliais

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...