Esta pesquisa foi financiada pelo Estado de Bankhead Coley-Science Team Grant da Flórida (2BT03), os Institutos Nacionais de Saúde / Instituto Nacional do Câncer (1R21CA164322-01) e Equipe de Ciência Grant do Moffitt Cancer Center. Este trabalho foi apoiado em parte pela facilidade de Pesquisa Translacional do núcleo no H. Lee Moffitt Cancer Center &amp; Research Institute, uma Comprehensive Cancer Center NCI designado (P30-CA076292). O acesso a células primárias só foi possível através do Protocolo de Total Care Cancer no Moffitt Cancer Center.

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	<li>Salmon, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+differential+sensitivity+of+human-tumor+stem+cells+to+anticancer+drugs.">Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.</a> N Engl J Med. 298, 1321-1327 (1978).</li><li>Suggitt, M., Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50+years+of+preclinical+anticancer+drug+screening:+empirical+to+target-driven+approaches.">50 years of preclinical anticancer drug screening: empirical to target-driven approaches.</a> Clin Cancer Res. 11, 971-981 (2005).</li><li>Kirshner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112, 2935-2945 (2008).</li><li>Durie, B. G., Jacobson, J., Barlogie, B., Crowley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnitude+of+response+with+myeloma+frontline+therapy+does+not+predict+outcome:+importance+of+time+to+progression+in+southwest+oncology+group+chemotherapy+trials.">Magnitude of response with myeloma frontline therapy does not predict outcome: importance of time to progression in southwest oncology group chemotherapy trials.</a> J Clin Oncol. 22, 1857-1863 (2004).</li><li>Harousseau, J. L., Attal, M., Avet-Loiseau, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complete+response+in+multiple+myeloma.">The role of complete response in multiple myeloma.</a> Blood. 114, 3139-3146 (2009).</li><li>Khin, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+assay+for+chemosensitivity+in+multiple+myeloma.">A preclinical assay for chemosensitivity in multiple myeloma.</a> Cancer Res. 74, 56-67 (2014).</li><li>Misund, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measurement+of+drug+sensitivity+of+myeloma+cells+co-cultured+with+bone+marrow+stromal+cells.">A method for measurement of drug sensitivity of myeloma cells co-cultured with bone marrow stromal cells.</a> J Biomol Screen. 18, 637-646 (2013).</li><li>Ramasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+experimental+model+to+evaluate+the+concomitant+effect+of+drugs+on+the+tumour+microenvironment+and+cancer+cells.">Fluorescence-based experimental model to evaluate the concomitant effect of drugs on the tumour microenvironment and cancer cells.</a> Br J Haematol. 157, 564-579 (2012).</li><li>McMillin, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-specific+bioluminescence+platform+to+identify+stroma-induced+changes+to+anticancer+drug+activity.">Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.</a> Nat Med. 16, 483-489 (2010).</li><li>Koh, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cells+for+microscopy+using+cytospin.">Preparation of cells for microscopy using cytospin.</a> Methods Enzymol. 533, 235-240 (2013).</li><li>Al-Quran, S. Z., Yang, L., Magill, J. M., Braylan, R. C., Douglas-Nikitin, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bone+marrow+plasma+cell+infiltrates+in+multiple+myeloma:+the+added+value+of+CD138+immunohistochemistry.">Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.</a> Hum Pathol. 38, 1779-1787 (2007).</li><li>Najar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+promote+or+suppress+the+proliferation+of+T+lymphocytes+from+cord+blood+and+peripheral+blood:+the+importance+of+low+cell+ratio+and+role+of+interleukin-6.">Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.</a> Cytotherapy. 11, 570-583 (2009).</li><li>Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+i+trialist.">Phase i trialist.</a> Lancet Oncol. 13, 236 (2012).</li></ol>

The authors have no conflict of interest to disclose.

Em resumo, este é um método poderoso e alta taxa de transferência para quantificar a quimiossensibilidade de culas do MM primárias e ex vivo farmacodinâmica da droga através de uma reconstrução da medula óssea. Os passos críticos dentro deste protocolo são a semeadura adequada dos poços e concentrando adequada (ver Figura 2 para os resultados esperados): assegurar que as células estromais MM e são distribuídos uniformemente, que as células estromais são aderentes ao fundo do po...

O objectivo deste método é para caracterizar a sensibilidade à droga de mieloma múltiplo (MM), as células primárias a um painel de agentes ex vivo, tão próximo quanto possível das condições fisiológicas, e com suficiente precisão que estes resultados podem ser usados ​​para identificar, resistente à quimioterapia sub populações dentro a carga tumoral e, em última instância, para parametrizar modelos computacionais desenvolvidos para estimar a resposta clínica. Modelos computacionais são ferramentas poderosas para analisar sistemas complexos, tais como interações cancer-host-terapia. No entanto, os modelos são apenas tão bom quanto os dados utilizados para parametrizar-los. Infelizmente, a maioria dos dados disponíveis na literatura não podem ser utilizados na sua forma actual para parametrizar tais modelos, uma vez que muitas vezes são obtidos em condições experimentais que se excluem mutuamente. Assim, novas experiências são muitas vezes necessários com o objetivo de obter o conjunto de parâmetros experimentais necessários. A maioria dos ensaios de viabilidade, no entanto, são destrutivos e thnos limitada a um pequeno número de pontos de tempo, muitas vezes, apenas um. Isso prejudica enormemente o uso de ensaios quimiossensibilidade para parametrizar modelos computacionais, uma vez que tais experiências não fornecem informações sobre a dinâmica temporal do sistema. Isto é especialmente verdadeiro com as células cancerosas primárias, que são muitas vezes limitadas a alguns milhões por amostra, e têm uma vida útil curta após a biópsia, limitando assim o número de condições experimentais disponíveis. Além disso, a maioria dos ensaios de viabilidade exigir a separação do cancro das não-cancro (estroma), as células no momento da quantificação, o que aumenta o trabalho extra para o protocolo, perturba ainda mais as células, e limita o número de experiências que pode ser realizado . Há 40 anos, Salmon e colaboradores uma proposta de seu famoso ensaio de formação de colónias vitro para avaliação de chemosensitivity de células tumorais. Infelizmente este ensaio encontrou o sucesso misto, devido ao pequeno número de mye múltiplaloma (MM) amostras de doentes que foram capazes de formar colónias em condições de controlo: Estimou-se que apenas um pequeno subgrupo de 0,001% a 0,1% de culas de MM foram capazes de replicação, sendo denominado como células estaminais mieloma múltiplo 2. Isto limita significativamente a taxa de sucesso do ensaio e do número de fármacos que pode ser testado de uma só vez, até mesmo nos modelos mais recentes 3. Esta questão é muito mais importante agora, quando agentes MM (padrão ou experimentais) são mais numerosos do que há quatro décadas. Uma das principais limitações destes primeiros ensaios é a sua saída dicotomizada: ou um paciente é &quot;sensível&quot; ou &quot;resistentes&quot; a um fármaco particular. Não são fornecidas informações sobre o grau de sensibilidade e heterogeneidade da população do tumor. Assim, os pacientes com pequenos sub-populações de células de crescimento rápido resistentes seriam classificados como &quot;sensíveis&quot; à terapia, mas recaída em breve, e, portanto, não benAFE do tratamento. Dada a importância da duração da resposta na sobrevida global (OS) de pacientes com câncer de 4,5, é claro como estes ensaios não foram capazes de estimar corretamente OS de forma consistente. A fim de contornar estas limitações, e para ser capaz de gerar modelos computacionais específicas do paciente que se estimar a resposta clínica personalizado para um painel de drogas, nós desenvolvemos um método para teste de sensibilidade ao fármaco de forma não destrutiva de mieloma múltiplo (MM) linhas celulares e culas do MM primários em uma reconstrução ex vivo do microambiente da medula óssea, incluindo a matriz extracelular e estroma 6. Este ensaio, no entanto, tinha a limitação de depender de um slide microfluídico comercial particular, cujas dimensões e custo restringiu o número de experimentos ou drogas que poderiam ser realizados de uma só vez em uma determinada peça de equipamento. O sistema aqui descrito estende este ensaio original em um alto-throughput plataforma de dose-resposta organotípica, in vitro para rastreio de drogas, com base num algoritmo de análise de imagem digital para quantificar de forma não destrutiva a viabilidade celular. Cada poço em 384 ou 1536 poços placas é uma reconstrução em 3D do microambiente da medula óssea, incluindo células primárias mM, matriz extracelular, e do estroma e factores de crescimento derivados do paciente. Microscopia vivo e análise de imagem digital são utilizados para detectar acontecimentos de morte celular em diferentes concentrações de fármaco, os quais são utilizados para gerar as superfícies de dose-resposta. A partir dos dados in vitro, um modelo matemático identifica o tamanho e a quimiossensibilidade de sub-populações dentro a carga tumoral do paciente, e pode ser utilizado para simular a forma como o tumor responderá à droga (s) em condições fisiológicas, num regime clínico 6 ( Figura 1). As principais inovações desta plataforma são: (a) pequeno número de células cancerosas necessários (1.000-10.000 por concent drogaração); (B) avaliação da eficácia do fármaco em condições fisiológicas (matriz extracelular, estroma, fatores de crescimento derivado do paciente); (C) Nenhuma toxicidade de viabilidade marcadores já que apenas 7 de imagens de campo claro é usado, portanto, não há necessidade de transferir células com fluorescência 8 ou 9 bioluminescência; (D) proporciona um efeito de imagem contínua de droga como uma função da concentração e do tempo de exposição (farmacodinâmica); e (e) entre a integração in vitro e modelos computacionais evolutivos, para estimar o resultado clínico 6 (Silva et al., em preparação). O factor essencial que permite que o algoritmo de análise de imagem digital para discernir cancro a partir de células do estroma é a sua diferente afinidade para o fundo do poço: culas do MM não aderem, e permanecem em suspensão na matriz, enquanto que as células estromais aderir ao fundo do bem e em seguida, alongamento. Esta separação ocorre mesmo que MM e estroma umre inoculadas ao mesmo tempo, e ocorre durante o processo de S / N de incubação. O girar para baixo no centrifugador seguinte semeadura da placa acelera ainda mais o processo. Como consequência, as células MM aparecem na imagem como discos brilhantes redonda rodeada por um anel de escuro, enquanto que o estroma mantém um perfil baixo e um tom mais escuro (Figura 2). Assim, o ensaio na sua forma actual é o mais adequado para as células de cancro não-aderentes, embora ajustes no algoritmo poderia ser feito para o cancro e estroma separados com base em outras características morfológicas, tais como o tamanho e forma. Neste protocolo, descrevem dois tipos de matriz extracelular (colagénio e da matriz da membrana basal), bem como dois tipos de co-culturas, com (derivadas de medula óssea as células estaminais mesenquimais) BMSC e HUVEC, representando os nichos intersticial e perivascular da medula óssea , respectivamente. Usando uma placa de 384 poços, 5 concentrações por droga e duas repetições, fomos capazes de test até 31 drogas diferentes com a amostra de um único paciente. Em placas de 1536 poços este número é 127. A Figura 3 ilustra a disposição actualmente usada para a propagação de células Manual, enquanto que a Figura 1 representa uma carta a distribuição óptima utilizando um pipetador robotizado. No desenho da Figura 3, cada placa de 384 poços pode transportar três amostras de doentes com sete drogas diferentes, ou uma amostra de pacientes testados com 21 drogas. Cada droga é representado por 5 concentrações, e cada condição é semeada em duplicado. 4 poços de controlo (sem fármaco adicionado) são semeadas para cada conjunto de sete medicamentos (por exemplo, poços 36-39, 126-129 e 200-203). Para garantir a eficácia dos fármacos utilizados, uma linha de células de mieloma humano (por exemplo, H929 ou MM1.S) também é semeado, e drogados em duplicado na concentração mais elevada de cada um dos fármacos utilizados poços (76-89). O controlo positivo linha celular assegura que as drogas utilizadas têm uma potência adequada, uma vez que as suas curvas de resposta de dose são ac comparávelexperimentos de Ross. Dois poços foram semeadas com uma linha de células de mieloma como controlo para as condições ambientais, em outras palavras, para detectar possíveis problemas na incubadora de bancada durante imagiologia poços (75 e 90). A enumeração dos poços segue um padrão em zigue-zague, a fim de reduzir a distância percorrida pela fase motorizada do microscópio, o que reduz o tempo de aquisição e reduz o desgaste do equipamento.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>AMG</td>
			<td>AMAFD1000 + AMC1000</td>
			<td>Digital microscope equipped with motorized stage and bench top incubator.</td>
		</tr>
		<tr>
			<td>384-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3683 SIGMA</td>
			<td>Corning CellBIND 384 well plates 
			384 well plate, polystyrene, CellBIND surface, sterile, clear flat bottom, black, w/lid</td>
		</tr>
		<tr>
			<td>1,536-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3832 ALDRICH</td>
			<td>Corning 1536 well plates, low base, surface treatment CellBIND, sterile</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-1440-02 SIGMA</td>
			<td>For in vitro isolation of lymphocytes from human peripheral blood.</td>
		</tr>
		<tr>
			<td>CD138 magnetic beads</td>
			<td>Miltenyi&#160;</td>
			<td>130-051-301</td>
			<td>Antibody conjugated magnetic beads for selection of CD138+ cells.</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi&#160;</td>
			<td>&#160;130-042-401</td>
			<td>Column for separation of cells.</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi&#160;</td>
			<td>130-042-302</td>
			<td>Magnet for separation column.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E6909 SIGMA</td>
			<td>ECM Gel from Engelbreth-Holm-Swarm murine sarcoma</td>
		</tr>
		<tr>
			<td>Getrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td>LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Life Technologies</td>
			<td>C-003-25P-B</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC )</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>GIBCO</td>
			<td>11875-093</td>
			<td>RPMI 1640 Medium + L-Glutamine + Phenol Red</td>
		</tr>
		<tr>
			<td>FBS Heat inactivated</td>
			<td>GIBCO</td>
			<td>10082-147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>3.1mg/mL Bovine collagen type I</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td>Bovine Collagen Solution, Type I, 3 mg/ml, 100 ml</td>
		</tr>
		<tr>
			<td>Precision XS</td>
			<td>BIOTEK</td>
			<td>PRC3841</td>
			<td>Robotic pipettor system</td>
		</tr>
	</tbody>
</table>

an organotypic high throughput system for characterization of drug sensitivity of primary multiple myeloma cells

Neste trabalho, descrevemos uma nova abordagem que combina ensaios ex vivo de sensibilidade a drogas e análise de imagem digital para estimar chemosensitivity e heterogeneidade de mieloma múltiplo células derivadas de pacientes (MM). Esta abordagem consiste na sementeira de células primárias MM recentemente extraídos a partir de aspirados de medula óssea para as câmaras de microfluidos aplicadas em placas de poços múltiplos, cada um consistindo de uma reconstrução do microambiente da medula óssea, incluindo a matriz extracelular (colagénio ou matriz de membrana basal) e estroma (paciente- as células estaminais mesenquimais derivadas) ou células endoteliais de origem humana (HUVECs). As câmaras são drogados com diferentes agentes e concentrações, e são gravadas sequencialmente durante 96 horas por meio de microscopia de campo claro, em um microscópio motorizado equipado com uma câmera digital. Software de análise de imagem digital detecta as células vivas e mortas da presença ou ausência de movimento da membrana, e gera curvas de alteração da viabilidade como uma função óf concentração de fármaco e o tempo de exposição. Usamos um modelo computacional para determinar os parâmetros de quimio-sensibilidade da população do tumor para cada droga, bem como o número de sub-populações apresentam-se como uma medida da heterogeneidade de tumores. Estes modelos adaptados para o paciente pode, então, ser usado para simular regimes terapêuticos e estimar a resposta clínica.

O fluxo da experiência é brevemente descrito na Figura 1 Se todas as etapas são completadas com sucesso, as imagens obtidas por microscópio deve ser equivalente à Figura 2:. Vivo culas do MM deve ser claramente visto como discos brilhantes e BMSC ou HUVECs deve ser pouco visível e bem distribuídas no fundo. Como pode ser visto na Figura 2A, culas do MM variam em tamanho de um paciente para outro, mas, em geral, eles são menores do que as linhas celulares. Uma ve...

Watch this Scientific Journal Video about An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells at JoVE.com

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Um Sistema Organotípicas alta taxa de transferência para Caracterização de Drogas de sensibilidade de células de mieloma múltiplo primárias

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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Medicine

8.5K visualizações.  H. Lee Moffitt Cancer Center and Research Institute. We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Vídeo: An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema de co-cultura com uma fatia organotípicas Cérebro e esferóide 3D de carcinomas de células

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicina

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

High Throughput caracterização de células-tronco adultas modificadas para liberação de fatores terapêuticos como estratégia de neuroproteção

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Criação de um Modelo de Xenoenxerto Humano mieloma múltiplo na galinha para estudar o crescimento tumoral, invasão e angiogênese

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método otimizado de LC-MS/MS para a análise do elevado-throughput de amostras clínicas de Ivacaftor, seus principais metabolitos e Lumacaftor em fluidos biológicos de pacientes de fibrose cística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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 ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Análise de alto throughput de dados de mapeamento óptico usando o ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Um ensaio de eletroquimioluminescência de alto rendimento 7-plex rastreando simultaneamente diabetes tipo 1 e múltiplas doenças autoimunes

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

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 ...