Apoio: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

Declaração de Ética: Uso dos espécimes tumorais humanas neste protocolo foi aprovado pela Universidade de Iowa Institutional Review Board (IRB). 1. Setup no dia anterior Tumor Colheita <ol><li> Placas de cultura de células: casaco Num capuz de cultura estéril, adicionar suficiente poli-L-ornitina (solução a 0,01%) para cobrir o fundo de uma cultura de poliestireno / poço de plástico / prato, substituir as tampas, e deixar repousar à temperatura ambiente durante pelo menos 2 hr. Lâminas de vidro ou de lamelas pode ser substituído por poliestireno para experiências que requerem imagiologia. </li><li> Após 2 horas, remove-L-ornitina poli solução por sucção e permitir que as placas a secar completamente em um estéreis capuzes cultura. Adicionar solução de laminina suficiente (10 ug / ml, em Ca 2 + / Mg 2 + livre HBSS [HBSS-/ -]) para cobrir completamente as placas de cultura, substituir as tampas, em seguida, ou 1) em lugar frio a 4 ° C frigorífico durante a noite, ou 2) colocar em 37 ° C incubadora durante pelo menos 2 horas. Se as placasserão armazenadas durante a noite ou mais, envolver as placas em filme plástico para evitar a evaporação / dessecação. </li></ol> 2. Configuração do Dia da Colheita Tumor <ol><li> Prepare Meios de Cultura: Meios é feita fresca no dia da colheita e processamento do tumor, armazenado a 4 ° C, e aqueceu-se a 37 ° C na incubadora imediatamente antes da mídia adições / modificações. </li><li> Meios de cultura de Schwannoma é de alta glucose (4,5 mg / ml) DMEM (com vermelho de fenol) com 0,1 mg / ml de penicilina e 0,1 mg / ml de estreptomicina; Mídia N2 complementar diluição final de 1:100; A insulina bovina de 5 ug / ml; Soro fetal bovino a 10% volume total. Filtre todos os meios de comunicação através do filtro de 0,22 mM. </li><li> Prepare Specimen Transport Refrigerador: Enviar um cheios de gelo refrigerador com 1-2 (dependendo da quantidade de tumor a ser colhida) estéreis de 50 ml tubos cônicos com 25 ml de HBSS com Ca 2 + / Mg 2 + (HBSS + / +) para a sala de cirurgia para a transferência de amostra de tumor e de transporte. </li><li> Prepare indivtubos de dissociação tumor idual - preencher estéreis 2 ml tubos redondos de fundo com 1 ml de HBSS + / + e colocar no gelo. (Os tubos serão utilizados para dissecção cortante / dissociação de amostras tumorais). </li><li> Para dissociação, prepare aproximadamente um tubo de dissociação para cada 0,5 cm 3 de tecido colhido; por exemplo, uma amostra do tumor medindo 1,0 x 1,0 x 1,0 cm (1 cm3) vai exigir dois tubos HBSS + / + de dissociação. </li><li> Esterilização luz ultravioleta: tesoura Local de tecido, pequenas pinças, cabo de bisturi, 1x prato normal de Petri, P1000 pipeta e pontas de pipeta em uma capa de cultura estéril, e deixar sob a luz ultravioleta para ~ 15 min antes da chegada e processamento de tecido do tumor. </li></ol> 3. Espécime Isolamento e Transporte <ol><li> Isolar amostras de tumor por ressecção cirúrgica. </li><li> Colocar imediatamente as amostras de tumor para o HBSS + / + gelada, tão rapidamente quanto possível após a retirada do paciente. Uma vez que todos tissue as amostras são coletadas, as amostras de transporte (no gelo) do teatro operatório para o laboratório para processamento posterior. </li></ol> 4. Dissociação de tecidos e trituração <ol><li> Seguindo uma técnica estéril padrão numa câmara de fluxo laminar, trazer tubo cónico com a amostra de tumor no capuz de cultura, e lava-se as amostras por inversão do tubo e do tumor de 50x. Permitir que os fragmentos a cair para o fundo do tubo, e, em seguida, remover HBSS + / +. Recarga com 30 ml HBSS + / +, e agite / inverter 50x novamente. Repita remoção inversão / media para um total de 4x. </li><li> Re-suspender fragmentos do tumor em 30 ml HBSS + / +, pela última vez, em seguida, despeje a mistura em uma placa de Petri sterile100 mm, e tumor separar em 0,5 centímetros 3 agrupamentos. Se fragmentos de tumores maiores são encontradas, usar a tesoura de tecido ou bisturi (# 11 ou # 15 lâminas) para cortar em pedaços menores. </li><li> Use a pinça para colocar os 0,5 centímetros 3 grupos de tecido para o indivíduo HBSS + / + cheia, 2 ml de fundo redondo cônicatubos ai, tudo no gelo. Use as tesouras de tecidos para triturar os fragmentos tumorais em sub-fragmentos de um milímetro-a suspensão deve ter uma aparência &#39;snowglobe&#39; (Figura 1). Picar o tumor apenas até que a maioria dos fragmentos são sub-1 mm; não &#39;sobre-mince&#39; as amostras tumorais. Coloque o tubo de tecido completamente picada de volta no gelo, e repita com todos os tubos de amostras adicionais. </li><li> Transfira todas as amostras tumorais fragmentadas em um único tubo cônico de 15 ml. A pipeta P1000 com um corte / modificados ponta estéril funciona bem para essa etapa. Use adicional HBSS + / + para lavar / enxaguar os 2 tubos ml de dissociação para garantir que todos amostra de tumor foi coletado em tubo de 15 ml. Girar mistura em centrífuga a 23 ° C, a 73 g durante 5 min. </li><li> Sucção fora HBSS + / +, deixando o sedimento celular. Re-suspender fragmentos em 1:1 mistura de 0,25% de tripsina: 0,2% de colagenase (dissolvido em HBSS-/ -) - adicionar apenas o suficiente para manter fragmentos do tumor em apertada suspension. Substitua o parafuso-top, e coloque em 37 ° C incubadora por 30 min. Agitar a mistura de células de pelo menos uma vez durante a incubação de manter fragmentos em suspensão. Coloque o meio de cultura Schwannoma na incubadora para aquecê-la neste momento também. </li><li> Após a incubação, adicionar 100 mL de FBS a cada tubo para inactivar a tripsina, e depois centrifugar a amostra a 23 ° C, 73 g durante 5 min. Com uma pipeta, cuidadosamente sucção fora tanto quanto possível sobrenadante sem perturbar pellet celular. Adicionar o meio de cultura aquecido (N2/insulin/5% de FBS) aos fragmentos de tumor a uma razão final de 01:02 tumor: meio de cultura (por exemplo, se houver um ml de fragmentos tumorais, adicionar 2 ml de aqueceu mídia). </li><li> Preparar para a trituração do tumor, cortando a ponta fora da ponta de uma pipeta P1000 com um diâmetro de cerca de 1-2 mm. Lentamente triturar a suspensão de células usando dicas progressivamente menores e menores de pipeta de diâmetro, até que você possa facilmente pipetar a solução através de um unaltponteira ered P1000. Mover-se imediatamente a uma ponta menor, uma vez que pode facilmente pipetar a solução tumor através da ponta - não mais de triturar a amostra. Se um único pedaço maior de blocos de tecido de aspiração durante a trituração, tocando a ponta da pipeta na parte inferior do tubo cónico permite frequentemente a trituração contínua. </li></ol> 5. Celular chapeamento <ol><li> Ona 0,5 mm 3 fragmento de tecido é suficiente para semear um prato de 100 mm x, ou 4 x lâminas 8-well/4-well. Pouco antes de adicionar a mistura media / celular, remova a solução laminina das placas de cultura, mas não deixe as placas seco. </li><li> Para cada placa de 100 mm, dissolve-se o fragmento de tumor em 10 ml de meio de cultura aquecido. </li><li> Para cada poço de uma 4-bem corrediça, utilizar 750 ul do meio de cultura aquecido (3 ml para toda a lâmina). </li><li> Para cada poço de um de 8 poços corrediça, utilizar 400 ul do meio de cultura aquecido (3,2 ml de toda a lâmina). </li><li> Incubar as culturas a 37 &amp;# 160; ° C, 100% de humidade, 6% de CO 2. Deixe as culturas sozinho por pelo menos 36 horas, e, em seguida, alterar a mídia, se a solução é amarelado (ácido). Caso contrário, alterar a mídia em 72 horas, em seguida, a cada 2-3 dias ou quando os meios de comunicação tornam-se ácida. Se a mídia se torna ácido diariamente durante vários dias seguidos, isso é geralmente uma indicação do potencial de contaminação por bactérias ou fungos. </li></ol>

<ol>
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História das culturas in vitro Schwannoma Esforços para estabelecer em culturas in vitro schwannoma começou apenas algumas décadas após o neuroma acústico inicial ressecção cirúrgica aberta ocorreu em 1894 12. As primeiras culturas foram gravadas por Kredel na década de 1920, que tentou em vão crescer tumor picada pelo &quot;método da gota pendurado&quot; 12 . Mais tarde, os drs. Murray e Stout publicou sua experi?...

Schwannomas vestibulares (VSS) representam Schwann célula (SC) tumores do nervo vestibular, comprometendo 10% de todas as neoplasias intracranianas 1-3. VSes ocorrer na neurofibromatose tipo 2 (NF2), formas ou esporádica ou familiar, tanto associados inativando defeitos no supressor de tumor NF2 gene. Tratamento para VSS é geralmente a ressecção cirúrgica ou radiocirurgia, no entanto, a morbidade de tais procedimentos incluem surdez, neuropatias faciais, vazamento de fluido espinhal, desequilíbrio, e rebrota tumor 1. Além disso nem todos os pacientes são aceitáveis ​​candidatos à cirurgia ou radioterapia. Tal morbidade significativa, bem como a falta de terapias alternativas tem impulsionado a investigação sobre a biologia molecular única de VSes na esperança de desenvolver novos tratamentos 3,4. Cultura celular permite a análise rápida, fácil, e em profundidade do comportamento molecular e celular e de triagem do potencial terapêutico comlibras. Historicamente, a falta de acesso a amostras de tecido fresco e ao fato de que as células schwannoma não são imortalizadas têm dificultado significativamente o uso de culturas primárias para a investigação de schwannoma tumorigênese. Para superar o fornecimento restrito de culturas primárias, a linha celular imortalizada HEI193 VS foi gerado através de transdução com o HPV E6 e E7 oncogenes 5. Este transdução oncogénica introduzido alterações moleculares e fenotípicas significativas para as células, o que limita a sua utilização como um modelo para tumores schwannoma humanos. Culturas SC derivados de linhas de camundongos transgênicos que não possuem um gene NF2 funcional representam uma outra alternativa para investigar tumorigenesis SC NF2-dependente in vitro. Estas culturas no entanto deixar de recapitular a heterogeneidade dos VSes humano ou conta para o comportamento específico humano. A maioria das técnicas anteriores de cultura VS necessário tempos de processamento relativamente longas e complicadas técnicas de cultura seletivos 6-8.Aqui apresentamos um protocolo simples e reprodutível para a primária VS cultura de células com processamento completo em menos de 3 horas, com 95% de pureza das células tumorais, como determinado por imunocoloração.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-Ornithine, 0.01% Solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4957</td>
			<td style="width:167px;">Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laminin Mouse Protein&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">23017-015 / L2020</td>
			<td>Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.2% Collagenase (dissolved in HBSS -/-)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C2674</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPS 100mm Round Culture Dish</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 4 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256521</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 8 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256522</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Suction Filter Flask</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP99500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">50ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2mL Round Bottom Tube</td>
			<td style="width:71px;">USA Scientific</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Scissor</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Handle</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">10004-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#11 Scalpel Blade</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-9801-11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-Tissue Culture 100mm Round Petri Dish</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-820-904</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P1000 Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">P3963-1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Serological Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">R3073-2P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile, Non-Filtered P1000 Pipette Tips</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TD1250R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulated Ice Cooler</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture Hood&#160;</td>
			<td style="width:71px;">Baker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge&#160;</td>
			<td style="width:71px;">Eppendorf -5810R</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) +/+ (w/ Ca2+, Mg2+)&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">24020-117</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) -/- (w/out Ca2+, Mg2+)</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14170-112</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM), High Glucose, w/ Phenol Red</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Supplement</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">17502-048</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin - Streptomycin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15140-163</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Insulin (1mg/ml 200x)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I6634</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
	</tbody>
</table>

primary culture of human vestibular schwannomas

Schwannomas vestibulares (VSS) representam Schwann célula (SC) tumores do nervo vestibular, comprometendo 10% de todas as neoplasias intracranianas. VSes ocorrer na neurofibromatose tipo 2 (NF2), formas ou esporádica ou familiar, tanto associados inativando defeitos no supressor de tumor NF2 gene. Tratamento para VSS é geralmente a ressecção cirúrgica ou radiocirurgia, porém a morbidade desses procedimentos tem conduzido investigações sobre tratamentos menos invasivos. Historicamente, a falta de acesso a amostras de tecido fresco e ao fato de que as células schwannoma não são imortalizadas têm dificultado significativamente o uso de culturas primárias para a investigação de schwannoma tumorigênese. Para superar o fornecimento restrito de culturas primárias, a linha celular imortalizada HEI193 VS foi gerado através de transdução com o HPV E6 e E7 de oncogenes. Este transdução oncogénica introduzido alterações moleculares e fenotípicas significativas para as células, o que limita a sua utilização como um modelo para es humanostumores chwannoma. Nós, portanto, ilustrar um protocolo simplificado, reprodutível para a cultura do ser humano primário VS células. Esta técnica permite facilmente dominado investigações moleculares e celulares que recapitulam com mais precisão a complexidade da doença VS.

Fragmentação tumor correcta é essencial para a propagação óptima e excrescência em pratos de cultura de tecidos (Figura 1). Identificação de células de schwannoma, durante os primeiros dias após o revestimento é muitas vezes difícil, devido ao obscurecimento quantidades de detritos celulares e as células vermelhas do sangue. Até o 4 º ou 5 º dia, extensões celulares perto de fragmentos tumorais aderentes criará reconhecível &#39;laçar&#39; padrões entre os esc...

Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Primary Culture of Human Vestibular Schwannomas

Schwannomas vestibulares (VSS) são tumores não-malignos de células de Schwann (SC), origem associados com mutações no gene supressor de tumores de NF2. Apresentamos um protocolo reprodutível e eficiente para humano primário VS cultura celular que permite a manipulação experimental celular e molecular e análise e recapitula a heterogeneidade da doença humana.

Cultura primária de Vestibular Humano Schwanomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

primary-culture-of-human-vestibular-schwannomas

Research

JoVE Journal

Medicine

12.7K visualizações.  University of Iowa Hospitals and Clinics.  Schwannomas vestibulares (VSS) são tumores não-malignos de células de Schwann (SC), origem associados com mutações no gene supressor de tumores de NF2. Apresentamos um protocolo reprodutível e eficiente para humano primário VS cultura celular que permite a manipulação experimental celular e molecular e análise e recapitula a heterogeneidade da doença humana. 

Vídeo: Primary Culture of Human Vestibular Schwannomas

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pele da biópsia Explant Cultura para Derivação de primárias fibroblastos humanos

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Medicina

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Em organoide Cultura in vitro de primário do mouse tumores do cólon

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, cultura, e imagem de Fetal Humano pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional Cultura alginato-talão de células humanas adenoma pituitário

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e cultura de células endoteliais primárias de Artérias e Veias canina

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento de células humanas primárias do Decidual de membranas fetais de termo placentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Desequilíbrios musculares: Teste e treinamento de força funcional da limitação excêntrico em populações atléticas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

Vestibulotóxicos cirúrgico do rato para estudar o sistema Vestibular

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Caracterização de uma nova técnica de cultura de retina organotipípica humana

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...