Agradecemos a U. Ganapati para a criação e testes da linha celular mStrawberry OP9. Além disso, agradecemos H. Shafifor sua assistência com o co-cultura. HP é apoiado por uma bolsa do Instituto Nacional do Coração, Pulmão e Sangue (F31HL087714) (HP). O conteúdo deste trabalho é da exclusiva responsabilidade do autor e não representam, necessariamente, a posição oficial do Instituto Nacional do Coração, Pulmão e Sangue ou o National Institutes of Health (NIH). O Fluxo UCLA Facility Núcleo Citometria é suportado pelo NIH (CA-16042 e AI-28697), o Jonsson Cancer Center, da UCLA AIDS Institute, e da Escola de Medicina da UCLA.

1. Preparação de células-tronco embrionárias <ol><li> Cultura de células ES de acordo com protocolo padrão para sua linha de celular particular. Certifique-se de preparar células ES suficiente para chapa para do mStrawberry OP9 células para o dia 0 (células-tronco embrionárias será banhado em 1x10 ^ 3 células por cm 2 no Dia 0) </li></ol> 2. Preparação de células OP9 <ol><li> Antes de crescimento mStrawberry OP9 células, preparar o OP9 meios de crescimento OP-9 meio de cultura celular <table border="1"><tbody><tr><td></td><td> Estoque </td><td> Conc final. </td><td> Volume (mL) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS (OP-9 testado) </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamina </td><td> 100X (200mm) </td><td> 2mM </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table> Armazenar a 4 ° C e usar dentro de uma semana de preparação </li><li> A fim de manter mStrawberry OP9 células, meios de comunicação devem ser trocados a cada dois dias e células devem ser repicadas antes de atingir células confluência (subcultura em 70-90% confluência). mStrawberry OP9 culturas de células são cultivadas nunca além de 90% confluência como isso irá resultar em sua diferenciação em adipócitos. Veja a Figura 3. <ol class="lalpha"><li> Subcultura células por células de lavar 2X com PBS (1x ou com PBS 1x + 0,1% com Tripsina-EDTA) </li><li> Adicionar pré-aquecido 0,1% Tripsina-EDTA a 37 ° C por 5 minutos (ou até retirar células) </li><li> Quando as células têm destacado, adicione OP-9 meio de crescimento. Transferência de células para um tubo de 50 ml cônico. </li><li> Células pelota a 300 xg por 5 minutos em temperatura ambiente. Elimine o sobrenadante. </li><li> Ressuspender as células em fresco mStrawberry OP9 meios de crescimento </li><li> Células em placas a uma densidade mínima de 10 4 células / cm 2 </li><li> Crescer mStrawberry OP9 células a confluência de partida três dias antes do dia necessários para o cultivo de células co-ES (dia 0, dia 5, ou dia8 / 9 do experimento) </li></ol></li></ol> 3. ES / mStrawberry OP9 Co-cultura <ol><li> Prepare media Co-cultura de diferenciação <table border="1"><tbody><tr><td></td><td> Estoque </td><td> Final </td><td> Volume (mL) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamina </td><td> 200mM </td><td> 2mM </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table></li><li> mStrawberry OP9 células (dia -3) <ol class="lalpha"><li> SEMPRE Subculture OP9 células em -3 Dia, para que você tenha camadas confluentes de células mStrawberry OP9 para 0 Dia da co-cultura. Ver Figura 4b. Placa mStrawberry OP9 células em 1x10 4 cm / 2 para confluência no Dia 0. Certifique-se de subcultura células extra para o dia 5 e dia 09/08 para o prosseguimento da co-cultura. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 co-cultura-Set-Up (Dia 0) <ol class="lalpha"><li class="jove_content"> Lave as células ES 2x com PBS </li><li> Trypsinize células-tronco embrionárias com pré-aquecido 0,25% de tripsina-EDTA, incubar por 5 min @ 37 ° C </li><li> Adicionar mídia Co-cultura de diferenciação para as células </li><li> Pellet células ES em 400xg, 5-7min, RT. Elimine o sobrenadante </li><li> Ressuspender as células em meios de co-cultura de diferenciação </li><li> Remova a mídia da mStrawberry OP9 células </li><li> Adicionar mídia Co-cultura de diferenciação para frascos mStrawberry OP9 </li><li> Placa de 1x10 3 células ES / cm 2 no balão da mStrawberry OP9 células. Incubar as células a 37 ° C, 5% CO 2 com a umidade. </li></ol></li><li> mStrawberry OP9 células (dia 2) <ol class="lalpha"><li> MStrawberry subcultura OP9 para que você tenha camadas confluentes de células mStrawberry OP9 para o dia 5 de co-cultura. Placa mStrawberry OP9 células em 1x10 4 cm / 2 para confluência no dia 5 (como no passo 3.2). Certifique-se de subcultura células extra para o dia 09/08, se necessário (como no passo 2.2). </li></ol></li><li> ES / mStrawberry OP9 co-cultura (dia 3) <ol class="lalpha"><li> Cuidadosamente remova o meio de co-cultura e substituir por meio de diferenciação fresco co-cultura </li></ol></li><li> ES / mStrawberry OP9 co-cultura (dia 5) <ol class="lalpha"><li> Lavar cuidadosamente co-culturas 2x com PBS </li><li> Trypsinize células com pré-aquecido 0,25% Tripsina-EDTA </li><li> Lave as células off frasco com media Co-cultura de diferenciação </li><li> Células de pelotização em 400xg, 7min, RT. Elimine o sobrenadante </li><li>Adicionar mídia Co-cultura de diferenciação para as células. Use uma agulha calibre 21 bluntended para ressuspender as células e para assegurar a suspensão única célula </li><li> Contagem de células utilizando um hemocitômetro. Deve ter aumento de 100-150 vezes no número de células ES desde o dia 0. </li><li> Re-semente centímetros 6.48x10 02/04 - 7.76x10 4 / cm 2 co-cultura de células em uma nova camada de confluentes, mStrawberry OP9 células </li><li> Dia 5 ENSAIOS-Restante co-cultura de células pode ser utilizado para análise de citometria de fluxo, cytopreps (30K células), ensaios de chip, etc </li></ol></li><li> ES / mStrawberry OP9 co-cultura (dia 09/08 e dia 12) <ol class="lalpha"><li> Remova a mídia de diferenciação do frasco e salvar. Lavar com PBS 1x monocamada para remover as células hematopoiéticas em suspensão. Não jogue fora as células hematopoiéticas. </li><li> Trypsinize células aderentes com pré-aquecido 0,25% Tripsina-EDTA </li><li> Lave as células off frasco com media diferenciação ES e adicione a células hematopoiéticas, encontradas em suspensão = </li><li> Células de pelotização em 400xg, 7min, RT. Elimine o sobrenadante </li><li> Adicionar Co-cultura de mídia ou PBS diferenciação para as células. Use uma agulha calibre 21 bluntended para ressuspender as células e para garantir a única célula de células em suspensão Conde utilizando um hemocitômetro. </li><li> Co-cultura pode ser continuado por 14 dias para avaliar mais maduro progenitores hematopoiéticos por re-semeadura co-cultura de células em uma nova camada de confluentes, mStrawberry OP9 células no dia 8 ou 9 e depois novamente no dia 12. Re-semente número de células otimizado com base na recuperação de células necessárias para o dia de ensaio. </li></ol></li><li> Avaliação do ES derivadas progênie (dia 14/01). ES derivadas progênie pode ser avaliado em qualquer dia da co-cultura pelo fluxo de classificar para fora mStrawberry células negativas (ES derivadas progênie). <ol class="lalpha"><li> Remova a mídia de diferenciação do frasco e salvar. Lavar com PBS 1x monocamada para remover as células hematopoiéticas em suspensão. Não jogue fora as células hematopoiéticas. </li><li> Trypsinize células aderentes com pré-aquecido 0,25% Tripsina-EDTA </li><li> Lave as células off frasco com media Co-cultura de diferenciação e adicionar às células hematopoiéticas, encontradas em suspensão </li><li> Células de pelotização em 400xg, 7min, RT. Elimine o sobrenadante </li><li> Ressuspender as células com 21-gauge agulha bluntended e contar </li><li> Progênie ES-derivada (mStrawberry células negativas) podem ser classificados de fluxo positivo mStrawberry OP9 células. Progênie ES-derivados podem ser caracterizados por análise de citometria de fluxo, cytopreps, ensaios chip, etc </li></ol></li></ol> 4. Resultados representativos: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Figura 1" /> Figura 1. ES / mStrawberry OP9 Sistema de Co-cultura. Nosso laboratório utiliza um in vitro de células ES sistema de co-cultura para o desenvolvimento do modelo hematopoiéticas. Neste sistema de co-cultura, as células ES diferenciar, em hemangioblastos no 5 º dia, quando colocado em uma camada confluente de células mStrawberry OP9. Como o produto co-cultura, precursores hematopoiéticos estão presentes no dia 8, enquanto terminalmente diferenciadas linhagens hematopoiéticas aparecem por dia 14. Setas indicam dia de diferenciação quando passaging de ES derivadas progênie em novas camadas confluentes de células em mStrawberry OP9 necessário. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Figura 2" /> Figura 2. Diferenciação de células ES adequada. Células-tronco embrionárias são semeados em uma camada confluente de mStrawberry OP9 no Dia 0. ES derivadas progênie começa a tornar-se visível no dia 3 de diferenciação, com ES derivadas progênie parecendo um aglomerado de células com uma borda definida ou começando a se formar um padrão whorl. Hemangioblastos, que é o precursor comum mesodérmica para as linhagens endoteliais e hematopoiéticas, deve aparecer como amontoados espirais de células no dia 5. Para co-cultura períodos superiores a cinco dias, a ES-progênie derivada deve aparecer como aglomerados de células hematopoiéticas. Dois a quatro grupos unicelulares, no dia 07/06, deve aparecer e crescer em clusters maiores célula Dia 09/08 de co-cultura. Progênie ES outros derivados também são encontrados fortemente ligada à camada de células aderentes do estroma. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Figura 3" /> Figura 3. Diferenciação celular sem sucesso ES. Os resultados deste co-cultura refletem diferenciação celular imprópria ES. Observe como a progênie ES derivados não aparecem em um padrão whorl no dia 5. A ausência de células em um padrão whorl indica a formação hemangioblast impróprio. Se hemangioblast não se desenvolvem adequadamente, e depois continuou co-cultura vai resultar na diferenciação de linhagem hematopoiéticas atrofiado. Co-culturas com formação hemangioblast indevido deve ser descartada. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Figura 4" /> Figura 4. mStrawberry OP9 culturas de células. (A) mStrawberry células são subcultivadas em OP970-90% confluência. (B) Para a diferenciação adequada, as células ES e ES-progênie derivadas são colocadas em uma camada muito confluentes de mStrawberry OP9 células. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Figura 5" /> Figura 5. Representante ES / mStrawberry OP9 perfil de fluxo co-cultura. Todos os descendentes ES-derivados são mStrawberry células negativas e podem ser classificadas a partir do fluxo de mStrawberry OP9 células. porta celular mStrawberry negativa foi determinada usando um ES/OP9 tradicionais co-cultura.

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Diferenciação adequada de células ES na camada confluente de mStrawberry OP9 células resulta na produção de Flk1 hemangioblastos + no dia 5 de co-cultura. Hemangioblastos deve aparecer como amontoados espirais de células, como observado na Figura 1. Para o restante da co-cultura, da progênie ES derivadas visível deve aparecer como aglomerados de células hematopoiéticas (Figura 1). No Day6 / 7 03:58 grupamentos celulares deve aparecer e crescer em clusters célula maior (tanto aderentes e em suspensão) no dia 09/08 de co-cultura. Progênie ES outros derivados também são encontrados fortemente ligada dentro da camada de células aderentes do estroma. Embora não incluída no protocolo, é essencial para pré-testar o FBS utilizado no meio de crescimento ES, o mStrawberry OP9 meios de crescimento e da ES / mStrawberry OP9 media diferenciação. Devidamente testados soro deve garantir um crescimento adequado de mStrawberry OP9 células, bem como, a diferenciação adequada de células ES em co-cultura até o dia 5 de diferenciação. Como uma alternativa ou para além mStrawberry OP9 células, OP9 células podem ser irradiados, em 8000 rads, antes da semeadura na confluência (7.8x10 4 células / cm 2). Irradiação de células OP9 permite a eliminação dos repetidos passos repique, bem como, permite a eliminação quase completa do velho, contaminando OP9 células de ensaios a jusante. OP9 células foram projetadas para expressar a proteína mStrawberry transduzindo OP9 células com 3ug/mL de um vector lentiviral mStrawberry expressar sob o controle de um promotor CMV (CMV pRRLsin-vector, vector UCLA core). Padrão ES/OP9 co-cultura protocolos implica a colocação de células-tronco embrionárias em uma camada confluente OP9 células, bem como, um dia passo replating 5, a fim de reduzir a idade, contaminando OP9 células de passagem de célula. Além disso, somente as células hematopoiéticas, encontradas em suspensão são removidos para a avaliação da ES derivadas progênie. No entanto, com tanto passo um repique ea colheita de células em suspensão potencialmente apenas um perde um número substancial de desenvolvimento ES-derivado células hematopoiéticas. Esta questão torna-se importante abordar quando se tenta caracterizar defeitos hematopoiéticas associadas com linhas ES nocaute. A fim de contornar este problema nosso laboratório usado uma versão modificada co-cultura, através do uso de mStrawberry expressando OP9 células. Isso garante que todos os descendentes ES são transferidos para o co-cultura continuou no dia 5, e para a colheita de todas as progênies derivadas ES para ensaios de downstream. Esta alteração prevê uma ferramenta útil na avaliação e caracterização de progênie hematopoiéticas derivadas de humanos e linhas ES mouse. Este ES-mStrawberry OP9 co-cultura recapitula o modelo de vários estágios primitivos e hematopoiese definitiva. Ao estudar as fases iniciais do desenvolvimento hematopoiético, muitas pessoas usam a BL-CFC (Blast-como células formadoras de colônia) de ensaio, que avaliam o desenvolvimento do hemangioblast da célula ES. Enquanto o ensaio BL-CFC é uma ferramenta útil na investigação de desenvolvimento hematopoiético cedo, o mStrawberry-ES sistemas de co-cultura apresenta utilitário aumentar à medida que permite a avaliação da hemangioblast, bem como, mais adulto linhagens hematopoiéticas. Trabalho anterior usando OP9/ES tradicionais e protocolos de diferenciação EB mostrou que fatores adicionais, talvez necessárias, como a superexpressão de HoxB4, a fim de obter a longo prazo repovoar células-tronco hematopoiéticas in vitro. Trabalho adicional é necessário avaliar se mStrawberry negativo, classificado ES derivadas populações contêm verdadeiras células-tronco hematopoéticas.

OP-9 celular Manutenção de Stock Componentes Médio <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega Scientific </td><td> FB01 </td><td> Pré-testados </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25-005-CI </td><td> 200nM </td></tr><tr><td> Tripsina-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 Diferenciação Celular stock Componentes Médio <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> Pré-testados </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25-005-CI </td><td> 200mM </td></tr><tr><td> PBS (sem Ca 2 + e Mg 2 +) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

A diferenciação in vitro de células-tronco embrionárias para um destino de células hematopoiéticas é útil quando se estuda populações de células que são de difícil acesso e in vivo para caracterizar os primeiros genes envolvidos na hematopoiese, sem ter que lidar com letalidades embrionárias. O ES/OP9 sistema de co-cultura foi originalmente projetada para produzir progênie hematopoiéticas, sem o excesso de produção de macrófagos, como a linha de células do estroma OP9 é derivada da calvária de ratos mutantes osteopetrose que a falta funcional M-CSF. A in vitro ES/OP9 sistema de co-cultura pode ser usado, a fim de recapitular desenvolvimento hematopoiético cedo. Quando cultivadas em OP9 células do estroma, células-tronco embrionárias se diferenciam em Flk-1 + hemangioblastos, progenitores hematopoiéticos e, finalmente, maduro, linhagens terminalmente diferenciadas. O padrão ES/OP9 protocolo de co-cultura implica a colocação de células-tronco embrionárias em uma camada confluente de células OP9, bem como, periódicos passos repique, a fim de remover o velho, contaminando OP9 células. Além disso, os protocolos atuais envolvem avaliar apenas as células hematopoiéticas, encontradas na suspensão e não são otimizados para a avaliação da ES-derivados de progênie em cada dia de diferenciação. No entanto, com passos repique ea colheita de células em suspensão apenas um potencialmente perde uma grande parte ES derivadas progênie e desenvolvimento de células hematopoiéticas. Esta questão torna-se importante abordar quando se tenta caracterizar defeitos hematopoiéticas associadas com linhas ES nocaute. Aqui nós descrevemos uma versão modificada ES / mStrawberry OP9 co-cultura, que permite a eliminação de contaminantes OP9 células de ensaios a jusante. Este método permite a avaliação completa de todas as progênies derivadas ES em todos os dias de co-cultura, resultando em um padrão de diferenciação hematopoiética, que mais directamente corresponde ao padrão de diferenciação hematopoiética observado dentro do embrião.

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Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 células permitir uma avaliação completa de todos os descendentes ES-derivados de co-cultura.

Modificado ES / OP9 Co-Protocol Cultura Fornece Caracterização melhorada de Progênie Hematopoiéticas

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

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Vídeo: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Congelamento células ES

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Biologia

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células ES: Cultura A partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

Cell-ES derivadas Neuroepiteliais culturas de células

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagação de-tronco embrionárias humanas (ES) Células

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing das células hematopoiéticas da medula óssea

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subclonagem Além disso Inserção (SPI) - Um Método recombineering Novel para a construção rápida de Gene Segmentação Vetores

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...