1. Protocolo a ser usado Multi-Frascos para cultura de células <ol><li> Preparação dos vasos e adição de suspensão de células <ol><li> Prepare meio de crescimento celular, conforme necessário. </li><li> Line up multi-Flask verticalmente em seu lado com a tampa virada para cima na superfície de trabalho (capela de fluxo laminar estéril). <ol><li> Estes estão disponíveis em frascos de 3 e 5 camadas formatos e usar um padrão de fluxo de trabalho semelhante a um balão de T-175. </li></ol></li><li> Solte e remova as tampas. Adicionar a quantidade necessária de pré-aquecido médio em frasco utilizando uma pipeta de 50 ou 100 mL ou por vazamento. <ol><li> Pipetas que são ≤ 10 ml pode chegar ao fundo do vaso quando Multi-Frascos são colocados verticalmente, com tampa virada para cima. Pipetas ≥ 10ml possa chegar até o navio imediatamente após o logotipo no Flask Multi-, proporcionando assim um portal conveniente para adicionar maiores volumes de mídia para navio. </li></ol></li><li> Para evitar bolhas de meio, permitir fluxo de líquido para flow ao longo da parede interna da tampa multi-Flask (logo-side). </li><li> Adicionar suspensão celular a partir de um estoque de células concentrados em meio de crescimento através da camada superior usando uma pipeta de 10 mL e frascos cap. Dicas: - Transporte Multi-Frascos em um carrinho para o site incubadora e executar as etapas restantes. - A densidade de semeadura de células irá variar dependendo do tipo de célula, médio e precisam duração cultura. Comece com a densidade de semeadura e volume de mídia ao usado no padrão T-175 frascos e multiplicar por 3 ou 5, dependendo do Flask Multi-formato utilizado. </li></ol></li><li> Mistura de células <ol><li> Mix posição: Segure o Flask Multi-ereto com o logotipo voltado para você e vire anti-horário para um ângulo de 45 ° com a porta mix voltado para você. </li><li> Exploração no mesmo ângulo, incline ligeiramente Multi-Flask da frente para trás (pescoço longe de você) até que o líquido na camada superior drena totalmente downwards através da porta mix. Pivot no mix porta lateral. </li><li> Da mesma forma, agite levemente Flask Multi-de trás para frente (pescoço para você) até meio ralos plenamente da camada inferior para a parte superior através da porta mix. Repita os passos 1.2.2 e 1.2.3 mais uma vez para assegurar a mistura adequada. </li><li> Traga Multi-Flask de volta à posição mix (Passo 1.2.1) e prossiga para a Etapa 1,3 </li><li> Alternativamente, suspensão de células podem ser preparados externamente a partir do Flask Multi-e suspensão de células pode ser adicionado ao recipiente utilizando uma pipeta ou por gentil vazamento. <ol><li> Porta permite misturar em recipiente de mistura de células com a mídia e elimina a necessidade de grandes volumes de células em suspensão externamente. </li><li> Ele também permite equalização de mídia em todas as camadas da Flask Multi- </li></ol></li></ol></li><li> Equilíbrio de fluidos <ol><li> Depois de misturar / adição de suspensão de células, coloque Flask Multi-verticalmente sobre uma superfície de trabalho plana para equalizar o volume de líquido equally em todas as camadas. </li></ol></li><li> Líquido partição em cada camada <ol><li> Segure o frasco Multi-com o logotipo voltado para você e gire no sentido horário para um ângulo de 45 ° para particionar o líquido para cada uma das camadas. Dica: - Para melhores resultados, é importante o uso de uma superfície de trabalho apartamento em Passos 1,3-1,4 </li></ol></li><li> Transporte <ol><li> Uma vez que a mídia contendo suspensão de células é particionado, transporte Flask Multi-na mesma 45 ° de ângulo (sentido horário) como no Passo 1.4.1 com a mídia longe do porto mix em incubadora. Esta posição também é adequado ao remover frascos de incubadora para a visualização de um microscópio. </li></ol></li><li> Colocando Multi-Flask em incubadora prateleira <ol><li> Segurando Multi-Flask no ângulo de 45 ° (sentido horário, longe da porta-mix), gentilmente gire-o para baixo horizontalmente na superfície incubadora (usando o canto longe do porto como um mixpivot). Lay Multi-Frasco com logotipo voltado para cima. <ol><li> Falcon design Multi-Flask permite que navios a serem empilhados e os mantém no lugar por uma costela resistente empilhamento. </li></ol></li></ol></li><li> Distribuição das células e reagentes <ol><li> Depois de colocar Multi-Flask sobre uma superfície plana de trabalho, gentilmente rock e para trás e para os lados para distribuir uniformemente as células em superfícies de cultura de tomar cuidado para não derramar líquido de cada camada. Pilha de frascos. <ol><li> Este balão é feito de material translúcido e células sobre a última camada pode ser facilmente visualizado em um microscópio. </li></ol></li></ol></li><li> Media Exchange <ol><li> Aspirar enquanto inclinando Flask Multi-o para a esquerda (mistura de porta-side) com o logotipo voltado para você. </li><li> Em seguida, incline o frasco Multi-à direita, continuando a aspirar todos os meios de comunicação residual. </li><li> Adicionar a quantidade apropriada de mídia e siga as etapas 1,3-1,7 </li></ol></li></ol> 2.Colheita de células multi-Frascos <ol><li> Dissociar células, line up Multi-Frascos verticalmente e levar cada um a posição Mix (Passo 1.2.1). Inverta suavemente frascos com pescoço de frente para você, de modo a permitir que a media de drenagem para a camada superior da Multi-Flask. </li><li> Inserir um 5 ou 10 mL ponta de aspiração através do pescoço até chegar a nível de líquido perto do porto mix. Aspirar meio esgotado. </li><li> Adicionar dissociação tampão reagente / lavar e trazer para Mix posição como no Passo 1.2.1, então prossiga e siga os passos de 1,3-1,7. Neutralize com meio de crescimento / agente neutralizante e despeje suspensão de células em um tubo de receber ou inverter frasco de modo que as células de drenagem para a camada superior do navio e recolher as células usando uma pipeta de 10 mL. Dica: Para aumentar a recuperação de células ou reagentes, trazer Multi-Flask para misturar posição (Passo 1.2.1), inverta com o pescoço para operador para permitir a drenagem completa de mídia da Al<html> l camadas para a camada superior. Então, Flask Multi-inclinação no sentido horário para ângulo de 45 ° (longe da porta mix) enquanto o Flask Multi-permanece invertida. Use uma pipeta (10-10 mL) para coletar os reagentes restantes. </li></ol> 3. Recomenda volume de trabalho na Falcon Multi-Frascos Meio de crescimento 3-Layer: 75-150 mL por Multi-Flask 5-Layer: 125-250 mL por Multi-Flask Dissociação agente Layer-3: ≥ 15 mL por Multi-Flask 5-Layer: ≥ 25 mL por Multi-Flask Dica: Comece com um volume de meio utilizado no padrão T-175 frascos e multiplicar por 3 ou 5 vezes, dependendo do Flask multi-formato avaliados para que mL por unidade de superfície continua a mesma. 4. Resultados representativos: 1. Projeto de Falcon Flask Multi- files/ftp_upload/3418/3418fig1.jpg &quot;/&gt; . Figura 1: Flask Multi-Cultura de Células embarcações estão disponíveis em uma 3 - e 5-camada formato empilhável para scale-up de células fornecendo 525 e 875 cm 2 de superfície de crescimento, respectivamente. Pipeta de acesso facilita a adição e remoção de células e reagentes em e fora do navio. Presença de port-mix permite rápida em recipiente de mistura e equalização de media em todas as camadas da Multi-Flask. 2. Rendimento das células usando multi-Flask: Estas embarcações estão disponíveis em 3 camadas e 5 camadas formatos que correspondem a 3 e 5 vezes a área da superfície da T-175 frascos. Assim, ≥ 3 e 5 vezes (130 ± 6,8 x 10 6 e 218 ± 23,6 x 10 6 células, respectivamente) o número de BHK-21 (baby hamster kidney), as células foram cultivadas e se recuperou de Multi-Flask comparado a T-175 frascos (43,2 ± 3,5 x 10 6 células; Fig.2A). Rendimento celular por unit área de superfície era equivalente em 3 - e 5-Multi-camada de frascos e T-175 frascos para BHK-21, LNCaP (humanas da próstata linhagem celular de adenocarcinoma), Hep-G2 (linha celular humana hepatocarcinoma), Ecopack 2-293 (humanos linhagem de células renais) cultivadas por um período de 48-96h (Fig.2B) em meio de crescimento (35 ml por camada), como recomendado pelo fornecedor de células (ATCC, Sigma e / ou Clontech). As células foram enumeradas em um contador de Vi-CELL automatizado (1). <img alt="Figura 2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> Figura 2A: três e cinco vezes o número de células BHK-21 foram cultivadas e se recuperou de 3 - e 5-layer Multi-Frascos em relação ao T-175 frascos. Rendimento esperado foi determinado utilizando rendimento celular média do controle T-175 frascos multiplicado por três e cinco vezes para a 3 - e 5-layer Multi-Frascos, respectivamente (n = 4 frascos / formato). <img alt="Figura 2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /><br/&gt; Figura 2B: rendimento das células por cm 2 foi equivalente em 3 - e 5-Multi-camada de frascos e T-175 frascos para BHK-21, LNCaP, HepG2 e EcoPack2-293 células. Cada barra representa média de 4-6 frascos. Células BHK-21 (11.000 cells/cm2) foram cultivadas por 72 horas, as células LNCaP (20.000 cells/cm2) e EcoPack2-293 células (~ 35.000 cells/cm2) foram cultivadas por 96 horas e HepG2 células (25.000 células / cm 2 ) foram cultivadas por 48 horas antes da colheita. 3. Distribuição de mídia entre camadas de Multi-Flask Meio de cultura de células (DMEM; Invitrogen) foi adicionado em 5 camadas Multi-Frascos (250 mL/5-layer navio) e dividida em camadas de acordo com protocolo descrito acima. Distribuição de mídia foi medido por furos em cada camada e mídia bombeado para fora de camadas individuais. Peso de líquido recuperado de cada camada foi encontrado para ser relativamente uniforme de camada a camada, como mostrado na Figura 3. Eles são os seguintes: 510,8 ± 0,73, 50,3 ± 0,58, 50,21 ± 0,13, 49,88 ± 0,35, 49,45 ± 0,37 (gm). <img alt="Figura 3" src="/files/ftp_upload/3418/3418fig3.jpg" /> Figura 3. Uniform distribuição de mídia em cada uma das cinco camadas de uma camada de 5-Multi-Flask. Meio de cultura celular foi adicionado em Multi-Frascos (250 navio ml/5-layer), equilibrada e dividida em camadas individuais. A mídia foi bombeado para fora através de buracos perfurados em camadas individuais e peso líquido foi gravado a partir de cada camada (n = 6 frascos). 4. Distribuição celular entre as camadas de Multi-Flask As células podem ser adicionados e misturados dentro da Multi-Flask. Nós distribuição simulada de células entre as camadas de Multi-Flask usando pérolas (10μm; PolySciences Inc.), semelhante em tamanho às células. Suspensão de esferas foi adicionado em Multi-Flask navio utilizando uma pipeta de 10 mL através da camada superior e misturado com a mídia no vaso como described no protocolo acima. Distribuição de talão foi medido por furos em cada camada e fluido contendo suspensão de esferas foi bombeado para fora de camadas individuais. Concentração talão recuperado de cada camada foi lido em um contador Coulter e registrados. Mostrado a seguir são equivalentes inter-layer distribuições talão em 3 camadas Multi-Frascos (Fig.4A). A mistura de porta-permite a distribuição homogênea de células e reagentes entre Flask Multi-camadas. <img alt="Figura 4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> Figura 4A: distribuição do grânulo em cada uma das três camadas de uma camada de 3-Multi-Flask. Uma suspensão de grânulos (3,6 x10 6 / ml) foi adicionado ao meio dispensada em Multi-Frascos (suspensão de esferas: volume de mídia é 1:10, vol: vol) e mistos, seguido pelo equilíbrio e as etapas de partição utilizando o protocolo descrito. Concentração talão recuperado de cada camada (meio bombeado através de furos em cada camada) foi lido em uma contagem Coulterer e registrados. Mostrado a seguir são equivalentes inter-layer distribuições talão em 3 - Multi-layer Frascos (n = 5 frascos) Mostrados abaixo são imagens representativas de Ecopack 2-293 padrões de coloração de células cultivadas em&gt; confluência de 80% em 3 camadas de Multi-Frascos em meios de crescimento suplementado. Monocamadas de células foram fixadas e coradas com cristal violeta e Multi-camadas Flask foram então cortadas e imagens digitalizadas (2). Nota padronização celular, foi homogênea em todas as camadas da Flask-Multi (Fig.4B). Resultados semelhantes foram obtidos com vários tipos de células avaliados (dados não mostrados). <img alt="Figura 4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> Figura 4B: Esta figura ilustra o crescimento de células homogêneas entre as camadas de Multi-Frascos. Ecopack-2-293 células cultivadas a&gt; confluência de 80% em 3-Multi-camada de frascos e T-175 foram fixados e corados com violeta de cristal. Multi-Flask vasos foram cortados e cada camada manchada foi digitalizada. 5 Air oferta em Flask-Multi.: Análise de media gasto com BioProfile FLEX analisador (3,4) (Nova Biomedical) de EcoPack2-293 células cultivadas por 96 horas não revelou nenhuma diferença na saturação do ar de células cultivadas em 5 camadas Multi-Frascos vs T-175 frascos (81,03 ± 1,9 vs 83,4 ± 5,8% ambiente O 2). <img alt="Figura 5" src="/files/ftp_upload/3418/3418fig5.jpg" /> Figura 5: Air saturação (% ambiente O 2) dos meios de comunicação passaram foram semelhantes em pré-mixed media de 5 camadas Multi-Frascos vs T-175 frascos. EcoPack2-293 células foram semeadas a uma densidade de 35.000 células / cm 2 e cultivadas por 96 horas antes da análise de media (n = 3 frascos).

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Todos os autores são funcionários da Becton Dickinson, Segmento de Biociências, Discovery Unidade Labware.

O presente estudo demonstra o aumento da produtividade que a concepção Flask Multi-oferece aos pesquisadores. Embora seja importante seguir as etapas descritas anteriormente para desempenho otimizado ao usar Multi-Frascos, há poucos passos críticos neste protocolo que são considerados mais essenciais. Estes incluem: (i) mistura de células e reagentes usando a porta mix dentro do vaso (ii) o transporte de Multi-Flask em um ângulo de 45 ° no sentido horário após particionamento líquido para cada uma das camadas (iii) que Flask-Multi pós plano de particionamento para o incubadora. Uso adequado de Multi-Flask leva à produção de uma população de células homogêneas dentro de cada navio que é cultivada em um ambiente que é congruente a T-175 frascos (5). Estas embarcações fornecer células 3 e 5 vezes mais em um espaço semelhante ao do frasco T-175. O navio 3 e 5 camadas fornece 525 e 875 centímetros 2 de área de crescimento, respectivamente, e oferecem espaço e trabalho savings para os usuários. Tratamento de superfície de tecido Cultura é comparável ao padrão frascos permitindo scale-up, sem a necessidade de re-otimização das condições de cultura existente ou comprometer a homogeneidade, qualidade ou desempenho de células (6, 7). Isto também prevê a comparabilidade com os dados coletados anteriormente. Esses vasos podem ser também revestido com reagentes tais como colágeno, fibronectina, poli-D-lisina para fornecer um substrato para o crescimento especializados apego e diferenciação de certos tipos de células, tais como hepatócitos 8, 9 kertainocytes, células-tronco cultivadas em soro de mídia livre formulações. Soluções de revestimento pode ser removido com a retenção de líquido residual mínima, de modo a reduzir o desperdício de reagentes valiosos, bem como a remoção eficaz de solução de revestimento antes de a cultura de células. O recomendado volume de mídia ideal para células de cultura em frascos multi-faixa ,142-0,287 ml / cm 2, que se traduzem em 25-50 ml por camada. Ao contrário de um outro frasco de múltiplas camadas, tseu produto oferece uma combinação porta-in que permite que a embarcação rápida no vaso de mistura, bem como distribuição uniforme de células e reagentes dentro e entre as camadas de cada navio. Linhagens de células diversas, culturas primárias e células-tronco foram ampliados de forma eficiente usando Multi-Frascos. Estes navios são particularmente vantajoso em aplicações que exigem um grande número de células, como no alto throughput screening, a produção de vacinas, transfections vetor viral e terapia celular. Economia de tempo, espaço, trabalho e geração de resíduos são reduzidos ganhos chave do Flask multi-versus culturas convencionais em vasos de camada única. Podemos cultura cerca de três vezes o número de células colhidas de 5, T-175 frascos no mesmo espaço, com 3, 5 camadas Multi-Frascos. Esta vantagem na economia de espaço não se limita apenas a T-175, mas também pode ser estendido para outros navios: um aparelho de garrafa padrão rolo que se encaixa em incubadoras de casas comuns de laboratório rol ~ 4garrafas de Ler (2200 ml) cada um dos quais fornece 850 centímetros 2 de superfície. Na mesma área, ~ 20, 5 camadas Multi-Frascos podem ser alojados proporcionando assim cinco vezes a superfície de crescimento para células de cultura. Além disso, com um aumento do &quot;Go-Green&quot; consciência, opções para reduzir a geração de resíduos é altamente desejável. A esse respeito, há um 38% (5, T-175 frascos pesam ~ 640g enquanto que uma, Flask Multi-5 camadas pesa ~ 400g) diminuição da geração de resíduos utilizando Multi-Frascos em relação ao T-175 frascos e levar essas vantagens a diminuir em armazenamento dos resíduos e custos de eliminação e resultar em economia de custos para o usuário.

<table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Catálogo # </td><td> Comentários </td></tr><tr><td> 3 camadas de cultura de tecidos tratados com 525 centímetros 2 </td><td> BD Biosciences </td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences Cultura de Células - BD Falcon Multi-Frascos</a> </td></tr><tr><td> 5 camadas da cultura de tecidos tratados 875 centímetros 2 </td><td> BD Biosciences </td><td> 353144 </td></tr><tr><td> 100ml pipeta </td><td> BD Biosciences </td><td> 357600 </td></tr><tr><td> 10 ml de pipeta </td><td> BD Biosciences </td><td> 357551 </td></tr><tr><td> 5 ml de aspiração pipeta </td><td> BD Biosciences </td><td&gt; 357501 </td></tr><tr><td> 50 tubo de polipropileno ml cônico </td><td> BD Biosciences </td><td> 352070 </td></tr><tr><td> T-175 Tecido frasco Cultura tratados com </td><td> BD Biosciences </td><td> 353028 </td></tr><tr><td> Gram Cristal Violeta </td><td> BD </td><td> 212525 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td></tr><tr><td> GMEM </td><td> Sigma </td><td> G5154 </td></tr><tr><td> RPMI-1640 </td><td> ATCC </td><td> 30-2001 </td></tr><tr><td> MEM </td><td> ATCC </td><td> 30-2003 </td></tr><tr><td> Tripsina-EDTA </td><td> Lonza </td><td> CC-5012 </td></tr><tr><td> Soro fetal bovino </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Triptose fosfato Caldo </td><td> Sigma </td><td> T8159 </td&gt; </tr><tr><td> Células BHK-21 </td><td> Sigma </td><td> 85011433 </td></tr><tr><td> Hep-G2, LNCaP </td><td> ATCC </td><td></td></tr><tr><td> Ecopack 2-293 </td><td> Clontech </td><td></td></tr><tr><td> Paraformaldeído </td><td> Microscopia Eletrônica de Ciências </td><td> 15710 </td></tr><tr><td> Bead poliestireno </td><td> PolySciences Inc. </td><td> 24628-20 </td></tr><tr><td> Bio-Flex Profile Analyzer </td><td> Nova BioMedical </td><td></td></tr></tbody></table>

scale-up of mammalian cell culture using a new multilayered flask

Um número crescente de aplicações baseadas em células requerem um grande número de células. Utilização de camada única T-frascos, que são adequados durante a pequena escala de expansão, pode tornar-se complicado, trabalhoso e demorado quando um grande número de células são obrigatórios. Para atender a essa necessidade, o desempenho de um novo navio cultura multi-camadas de células para facilitar escala fácil de células da camada única T-frascos serão discutidos. Os frascos testados estão disponíveis em 3 - e 5-camada formato e permitir que a cultura ea recuperação completa de três e cinco vezes o número de células, respectivamente, em comparação com T-175 frascos. Uma característica chave do BD Flask Multi-é uma porta de mix / equilíbrio que permite rápida em recipiente de mistura, bem como distribuição uniforme de células e reagentes dentro e entre as camadas de cada navio e consistentemente produzem células que podem ser cultivadas em um ambiente que é congruente a T-175 frascos. O projeto destes frascos Multi-também permite cacesso pipeta onvenient para adicionar reagentes e células diretamente no frasco, bem como a recuperação eficiente de células valiosas e reagentes e reduz o risco de contaminação devido a vazamento. Para aplicações onde o vazamento é preferível a pipetagem, o projeto permite a retenção de líquido residual mínima, de modo a reduzir o desperdício de valiosas células e reagentes.

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Células desempenham um papel fundamental e crescente na pesquisa e na descoberta e desenvolvimento de novas terapêuticas. Com esta crescente necessidade de maior número de células que precisam de maneiras mais eficientes e eficazes para o cultivo e colheita de células apego dependente. Um frasco de múltiplas camadas com as características certas pode servir a esse propósito.

Scale Up de Cultura de células de mamíferos utilizando um Flask New Multicamadas

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Nanyang Technological University

Research

JoVE Journal

Biology

37.5K visualizações.  BD Biosciences. Células desempenham um papel fundamental e crescente na pesquisa e na descoberta e desenvolvimento de novas terapêuticas. Com esta crescente necessidade de maior número de células que precisam de maneiras mais eficientes e eficazes para o cultivo e colheita de células apego dependente. Um frasco de múltiplas camadas com as características certas pode servir a esse propósito.

Vídeo: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

Ole Isacson: desenvolvimento de novas terapias para a doença de Parkinson

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser Microdissecção Captura de tecidos de mamíferos

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Development of New Therapeutic Applications Using Microfluidics

Desenvolvimento de novas aplicações terapêuticas Usando microfluídica

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Determinação do Número celular durante Cultura Celular usando o contador de células Scepter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Determinação da contagem de células de mamíferos, tamanho de célula e Saúde célula usando o Moxi Z Mini contador de células automatizado

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparação de Rat Serum Indicado para cultura de embriões de mamíferos Whole

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Entrega Protein direto para células de mamíferos utilizando células-permeável Cys<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt; Domínios zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...