We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

Nota: Para este estudo, as células estromais mesenquimatosas do pulmão (LR-MSC) a partir de tecido de pulmão distai foram isoladas, quer com base na sua expressão de marcadores de superfície celular (CD45 negativo, CD31 negativo, Sca-1 elevada, EpCAM neg) 14,15, usando explante out-crescimento de 16, ou usando a digestão enzimática 17. Aderente LR-MSC foram cultivadas em DMEM com glucose elevada e sem glutamina, 15% de FBS, glutamina mM de antibiótico / antimicótico 1x e 2 (meio completo daqui em diante) e incubou-se a 37 ° C, 5% de CO 2. LR-MSC foram passadas 3-4 vezes para assegurar uma população de células com características de crescimento relativamente homogéneas para o ensaio de migração. 1. Preparar uma monocamada confluente <ol><li> Para separar as MSCs a partir de placas de cultura padrão de 100 mm, lavar as células com 10 ml de 37 ° C pré-aquecido PBS, aspirar o PBS e adicionar 4 ml de tripsina (0,25%). Incubar em 3-5 min a 37 ° C incubadora. Aspirar o cells para um tubo cónico, adicionar um volume igual de meio completo e centrifugação a 300 g durante 5 min para sedimentar as células para baixo. Nota: de incubação mais longo em tripsina pode alterar os receptores da superfície celular e, potencialmente, afectar a migração. </li><li> Aspirar media e células ressuspender em meio completo fresco. Contagem de células, excluindo as células mortas usando exclusão com azul de tripano e as células do estroma placa 500.000 mesenquimais em 4 ml de meio completo numa célula de 60 mm de cultura prato. Nota: Dependendo da fonte das células, os testes podem ser necessários para determinar o número óptimo de células necessárias. </li><li> Incubar as células até placas são 90% confluentes para fixação celular ideal e proliferação, normalmente 48-72 horas para MSC. Nota: 100% de confluência não é recomendado, uma vez que muitos inibição células experiência de contato dependentes de ancoragem, o que pode alterar a sua capacidade migratória. Placas com menos de 80% de confluência irá resultar em fotos inutilizáveis ​​porque o software de análise de imagem irá interpretar lacunas entre as células como parte do perímetro do zero (veja a Figura 1D). </li><li> Após as células terem atingido confluência de 90%, danificar as células conforme for apropriado para a aplicação. Para avaliar o dano celular após a exposição à fumaça de cigarro extrato solúvel (CSE), geram 100% CSE, chamando a fumaça de um cigarro Universidade de Kentucky pesquisa (3R4F) através de 25 ml de meio completo em um Kitasato mais de 2 min 18. </li><li> Incubam-se as células durante 24 horas em 4 ml de 1-4% de CSE diluído em meio completo. Após incubação, lavam as células duas vezes com 4 ml de PBS estéril quente, antes de substituir com 4 ml de meio completo fresco. Nota: danificar as células com alta concentração de extrato de fumaça de cigarro (&gt; 5%) pode reduzir substancialmente confluência. </li></ol> 2. Criação da Raspadinha <ol><li> Em um ambiente estéril, retire a tampa da placa de 60 milímetros de MSCs danificados, e estabelecer uma borda em linha reta (por exemplo, uma régua de plástico esterilizado) acRoss da borda superior da placa. Sem remover a mídia, use um estéril ponta da pipeta 200 mL guiado pelo straight-edge estéril para fazer um a quatro arranhões paralelos em toda a placa aproximadamente 5 milímetros e 50 mm de comprimento. Nota: Uma borda reta minimiza a quantidade de ajustes de rotação necessários para a placa quando está no palco microscópio. É imperativo que os arranhões aparecer completamente vertical (ou horizontal) na tela. Gerando múltiplas arranhões em uma placa 60 mm não altera o microambiente de uma forma que afecta a migração de células (dados não mostrados), mas podem ser uma consideração para outros tipos de células. O uso de pequenos poços não é recomendado uma vez que a variabilidade de células crescendo perto da borda do poço se torna mais pronunciada e pode afectar a geração uniforme de arranhões. </li><li> Aspirar os meios e lava-se suavemente as placas com 2 ml de meio completo pré-aquecido para impedir que as células adiram ao centro do zero, e para remover célulass que foram soltos pelo coçar. Substitua com 4 ml de meio completo fresco. </li><li> Usando um marcador permanente estéril, etiquetar cada arranhão de 1-4 no lado inferior da placa no local que não interfira com a imagiologia do arranhões. Nota: É importante que as imagens do mesmo zero são comparadas antes e após a migração, já que pode haver variação na largura zero. </li></ol> 3. Tendo Imagens iniciais da Raspadinha Nota: Se o ensaio de zero é efectuada em várias placas, recomenda-se que as imagens iniciais de todos os arranhões em uma única placa são tomadas antes arranhões são feitas na placa seguinte. <ol><li> Usar um microscópio de contraste de fase invertida com uma câmara para gerar as imagens necessárias para análise. </li><li> Posicione a placa de cultura no microscópio, e use o objectivo de baixa potência (10X) para localizar zero # 1. Se a condensação sobre a tampa da placa de cultura está a impedir uma clara image das células, utilizar um conjunto de fase aquecida a 37 ° C. </li><li> Mantendo a zero completamente horizontal e no centro do campo de vista, obter o número de imagens necessárias para abranger 90% do comprimento do zero. Obter imagens de partes distintas do zero com nenhuma sobreposição entre as imagens. Nota: A refracção da luz nas bordas da placa de cultura superexpõe as imagens, o que os torna impossível de analisar. Portanto, não tirar fotos a partir do primeiro e duram 5% do comprimento do zero. </li><li> Salve imagens de cada arranhão em uma pasta separada. </li><li> Repita os passos de 3,2-3,4 para arranhões 2, 3 e 4. </li><li> Retorno placas para a incubadora imediatamente após a imagem inicial. Deixe as células migram para 4-7 horas. Nota: 7 horas é o ideal quando se avalia MSCs danificadas com CSE. A incubação de células durante mais de 7 horas não é recomendada porque a proliferação de células pode actuar como uma variável de confusão durante a migração celular (ver Nota Opcional 8.2 abaixo). </li></ol><pclass = &quot;jove_title&quot;&gt; 4. Analisando iniciais de raspadinhas Imagens <ol><li> Baixe e instale o ImageJ Ferida plugin de instrumento de cura (consulte a Tabela de Materiais / Equipamentos). </li><li> Abra o arquivo de imagem em ImageJ, clique em &quot;Processo&quot; e &quot;Find Edges&quot; para destacar as células ao redor do zero para a Ferramenta de cicatrização de feridas para calcular a área de rascunho (Figura 2B). Em seguida, clique em &quot;Imagem&quot;, &quot;Ajuste&quot;, &quot;Limiar de cores&quot; e no menu dropdown rotulado &quot;Limiar de cores&quot; altere o valor para &quot;B &amp; W&quot;. </li><li> Dentro do menu &quot;Limiar de cores&quot;, ajuste ambos os controles deslizantes de nível de luminosidade até um contraste perfeito entre zero e celular frente a é alcançado. Para permitir o maior alcance ao tentar criar contraste na imagem, mova o controle deslizante inferior completamente para a direita (imagem totalmente preta), e em seguida, ajuste o controle deslizante superior para gerar o máximo de contraste. </li><li> Ajuste lentamente o topo brideslizante lho da esquerda para a direita até que a imagem limpa mostra o contorno do zero (Figura 2C). Uma vez que foram identificadas as bordas do zero, clique em &quot;Mais Ferramentas&quot; na barra de ferramentas ImageJ, selecione &quot;MRI_Wound_Healing_Tool&quot; no menu suspenso, e pressione o botão &quot;m&quot; (que aparecerá na barra de ferramentas ImageJ) para destacar a área de rascunho e saída da área computada em uma janela pop-resultados out (Figura 2D). </li><li> Copiar e colar todos os números da janela de resultados (Figura 2E) em uma planilha excel. Certifique-se de separar os dados de arranhões individuais. </li></ol> 5. Tendo Imagens Finais da Raspadinha <ol><li> Depois que as células migraram (4-7 hr), repita os passos de 3,2-3,4. Imagem das placas na mesma ordem em que foram riscados de modo que as células em cada placa tiveram o mesmo tempo para migrar. Nota: As células podem ser trabalhada em vários pontos de tempo, dependendo da sua sensividade de ambas as mudanças de temperatura e de pH. Alternativamente, imagens de células vivas pode ser usado para seguir as células em tempo real, à medida que migram. </li></ol> 6. Analisando finais de raspadinhas Imagens <ol><li> Repita os passos de 4,2-4,5 para imagens finais zero. </li></ol> 7. Cálculo da área de rascunho para quantificar as Migrações <ol><li> Calcula-se a área média pré-migração calculando a média dos valores dos respectivos riscos iniciais da área. </li><li> Calcule a quantidade total de migração subtraindo a área de migração final para cada imagem, calculado na etapa 6, a partir da área de pré-migração média (passo 7.1). Gerar uma lista de valores para cada um zero que representa a alteração na migração. </li><li> Reúnem-se os dados a partir de múltiplos arranhões dentro do mesmo grupo de teste (ou seja, todas as placas e todos os arranhões em células expostas a 0% de CSE). Use um teste de análise de variância para analisar as diferenças entre os grupos experimentais. </li><li> Se as células são lentos para migrare necessitam de um tempo de incubação mais longo após arranhão (superior a 10-12 h), executar um ensaio de incorporação de BrDU ou EdU 19 para determinar se as células são submetidos a qualquer proliferação durante o período em que o ensaio de migração está a ocorrer. Escolha um tempo de ensaio de migração para evitar a proliferação celular significativa. </li></ol> 8. Opcional <ol><li> Para avaliar se a senescência celular é significativo que ocorre como resultado do procedimento de coçar, realizar um ensaio de beta-galactosidase associada a senescência em uma placa de ensaio em tempos predeterminados após arranhão 19. </li></ol>

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The authors declare that they have no competing financial interests.

O protocolo descrito aqui fornece um método quantitativo robusto, padronizado para executar e analisar testes de zero. Ensaios simples arranhão são rotineiramente utilizados em muitas áreas diferentes de estudo para analisar a migração celular. No entanto, tradicionalmente o ensaio zero faltou um set-up e quantificação protocolo padronizado, o que levou a problemas com a reprodutibilidade 7,10. Muitas das modificações e otimizações para melhorar o ensaio zero reduziram essas questões, incluindo i...

A migração celular é altamente coordenada e vital para muitos processos fisiológicos, tais como desenvolvimento do tecido, reparação e regeneração, bem como processos patológicos tais como a metástase do cancro e arteriosclerose 1. Uma compreensão da migração celular é particularmente relevante para as terapias emergentes utilizadas para reparar tecidos danificados e tratar condições patológicas, incluindo as tecnologias de transplante de células e tecidos artificial enxerto 2. Dado o interesse atual no papel de células estromais mesenquimais (MSCs) na mediação de reparação de tecidos 3, quantificando a capacidade migratória destas células usando um ensaio que é rigorosamente quantificável, adaptável e relação custo-benefício é de interesse. É importante ressaltar que tal ensaio deve ser suficientemente sensível para detectar mudanças relativamente sutis na capacidade migratória celular após dano. Os métodos actuais para quantificar a migração de células, incluindo ensaios de risco, os ensaios de migração trans-poços (CH Boydenâmbares), matrizes micropillar e ensaios zona de exclusão de células possuem uma série de limitações na reprodutibilidade, personalização, quantificação e custo-efetividade 1,4,5. O ensaio zero otimizado aqui descrito demonstra resultados robustos, capacidade de análise quantificáveis ​​e baseados em imagem, relação custo-eficácia e capacidade de adaptação a outras aplicações. Os ensaios de raspadinhas têm sido utilizados em várias capacidades para avaliar a migração celular e proliferação em diferentes condições experimentais 5. O ensaio implica semeando as células designadas, cultivá-las para completar ou próximo da confluência, e coçar a monocamada resultante com uma agulha estéril ou ponta de pipeta 6. Análise é mais vulgarmente efectuada por comparação da largura do zero ao longo de múltiplos pontos de tempo em locais seleccionados aleatoriamente 7-9. Apesar de seu uso proeminente, o ensaio do zero é confundida por problemas com a reprodutibilidade e quantificação. A variabilidade nageração de zero a não apenas altera o microambiente das células, mas também pode impedir a migração de células ao danificar a superfície da placa e da matriz extracelular subjacente 5. Os ensaios são frequentemente realizados ao longo de 7 a 12 h, no entanto, para linhas de células exibindo migração mais lenta e os tempos de ensaio mais longos, a proliferação torna-se uma variável de confusão 7,10. Por último, as células senescentes gerados pelo processo de coçar pode libertar factores que interferem com a sinalização extracelular necessária para fechar a fenda na monocamada 1. Optimizar a zero ensaio requer a criação de uma diferença consistente, que não interfere com as propriedades de superfície, minimizando o comprimento de tempo de ensaio, e a prevenção da morte de células indesejáveis ​​durante a manipulação. O ensaio à base de batente consiste numa optimização do ensaio de uma zona de exclusão de células. Este ensaio utiliza uma rolha colocado no meio do poço que exclui o crescimento celular, mas permite que as células a ser revestida em volta da zona de exclusão central.Para avaliar a migração, a tampa é removida, e a zona de exclusão resultante proporciona uma superfície para a migração ocorra. No entanto, este ensaio é difícil para personalizar ou adaptar a 10 e para algumas aplicações, esta técnica também pode ser um custo proibitivo. Em contraste com os ensaios de zero e os seus derivados, ensaios de trans-migração bem (ou ensaios de câmara de Boyden) avaliar a migração através da quantificação do número de células que se movem de uma câmara, através de um filtro de membrana microporosa, para uma câmara contendo agentes quimiotácticos 8,11, 12. Esta técnica tem utilidade limitada para as células aderentes como as MSCs porque após a migração através da membrana porosa, as células aderem ao lado da membrana exposta aos agentes quimiotácticos, e pode ser difícil de quantificar com precisão. Enquanto o ensaio é capaz de analisar alguns padrões de migração tridimensionais, os tipos de células restritas para o qual é capaz de quantificar com precisão limite de migração celular sua utilidade 10. Outra alternativa para ensaios de zero usa uma rede de micro-pilar, que mede a motilidade celular através de um espaço tridimensional utilizando a capacidade das células para se deformar e migram para a matriz como um substituto. Polydimethlysiloxane (PDMS) elastómeros curados sobre um molde preciso e tratada com ozono e fibronectina produz um microambiente homogénea e não degradável. Espaçamento Micro-pilar também pode ser variado conforme necessário para avaliar a capacidade das células para introduzir a matriz 4. O molde é criado por meio de gravura de ião reactivo profundo de bolachas de silício para criar uma versão da matriz negativa de alta relação de aspecto 13. Enquanto o ensaio é reforçada pela sua capacidade de personalização, capacidade de modelar a migração tridimensional, e análise através da visualização direta de células que migram, dificuldade em criar micro-pilar matrizes impede economicamente seu uso generalizado. O ensaio zero otimizado descrito neste protocolo oferece uma eficiente, cosmétodo t-eficaz para produzir arranhões consistentes que podem ser analisados ​​usando o software disponível gratuitamente. Em vez de medidas de largura simples, feitas em toda a zero antes e depois da migração celular, o software permite ao usuário determinar áreas totais scratch antes e depois da migração. Este avanço limita a questão de tentar determinar onde a zero as medidas de largura devem ser tomadas, e se a largura do zero é uniforme ao longo dela de comprimento. Além disso, a optimização cuidadosa dos números de células, confluência celular e o tipo e grau do dano infligido nas células é discutida, a fim de optimizar ainda mais o ensaio.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">DMEM (high glucose, no added glutamine)</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">31053-036</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fetal Bovine Serum, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3091002</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Antibiotic/antimycotic, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SV3007901</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:527px;">Glutamine, 200mM, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3003401</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">TypeLE cell dissociation reagent</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">12563-01</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Dulbecco&#39;s Phosphate Buffered Saline, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3002802</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Falcon standard 60mm tissue culture dishes</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">08-772B</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fisherbrand Sterile 200ul pipet tips</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td>02-707-502</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Inverted light microscope with camera attachment</td>
			<td style="width:221px;">Variable</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:527px;">ImageJ software&#160;</td>
			<td style="width:221px;">http://imagej.nih.gov/ij/</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:527px;">MRI_Wound_Healting plugin</td>
			<td style="width:221px;"><a href="http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool">http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool</a></td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Statistical analysis software</td>
			<td style="width:221px;">Microsoft Excel</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
	</tbody>
</table>

optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

Os ensaios scratch aqui apresentadas foram realizadas utilizando residentes pulmão células estromais mesenquimais murino (LR-MSCs) e isolado como referência nas notas de protocolo. O LR-MSC foram semeadas a uma densidade de 500000 células em 60 mm de cultura de tecidos prato, e cultivadas a 90% de confluência em 48 horas. Para gerar danos, 4% de CSE (descrito acima) foi incubado com as células durante 24 horas após o período de sementeira inicial, mas antes do ensaio zero. Para cada ensaio, o zero foi gerado usa...

Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Otimização do Ensaio Wound zero para detectar mudanças na Murino estromais mesenquimais migração celular após a lesão por Extract fumo do cigarro Solúvel

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

optimization-wound-scratch-assay-to-detect-changes-murine-mesenchymal

Research

JoVE Journal

Developmental Biology

16.8K visualizações.  College of the Holy Cross. The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Vídeo: Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Zebrafish queratinócitos explantes para estudar migração celular Collective e reepitelização na cicatrização de feridas cutâneas

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Biologia do Desenvolvimento

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Avaliação da migração de células endoteliais após a exposição a produtos químicos tóxicos

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ensaio para avaliar contrátil Função de células mesenquimais que a transição epitelial-mesenquimal Sofreu

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolamento e expansão de CTM / células estromais derivadas de placenta humana Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Produção e Administração de Therapeutic-tronco mesenquimais / celular estromal (MSC) Spheroids condicionadas em culturas 3-D Sob Condições Xeno-livres

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolamento e Caracterização de células mesenquimais do estroma a partir de cordão umbilical humano e Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Um dispositivo para executar a migração celular / cicatrização de feridas em uma placa de 96 poços

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Um Ensaio de Blot de Ponto de 5 mC Quantificando o Nï¿½el de Metilaï¿½o de ADN da Dediferenciaï¿½o de Condrocitos<em&gt; In Vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

O ensaio de co-cultura anel aórtico: Uma ferramenta conveniente para avaliar o potencial angiogênico das células estromais mesenquimais em Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Visualização da migração celular tangencial no desenvolvimento Tectum óptica de Chick

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...