This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

NOTA: Este estudo foi aprovado pelos Comitês de Ética apropriados. 1. cultura primária de pulmão fibroblastos humanos <ol><li> Coleta de tecido pulmonar: <ol><li> Obter amostras de tecido pulmonar humanos da sala de operação cirúrgica diretamente. Recolha cerca de 1 cm a 3 quarteirões do tecido pulmonar cancerosos e não cancerosos, com a amostra não-cancerosas recolhido o mais longe possível do tumor. </li><li> Suspender as amostras em meio de Eagle modificado por Dulbecco (DMEM) suplementado com 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina e 0,25 ng / mL de anfotericina B (meio isento de soro). Manter as amostras em suspensão na estéreis tubos de 50 ml e transferir para o laboratório. NOTA: Os fibroblastos são altamente migratório e proliferativo em comparação com outros tipos de células, tais como epiteliais, células endoteliais e do músculo liso. O método excrescência aproveita esses recursos de fibroblastos, e outgrocélulas asa de cortes de tecido são abundantes em fibroblastos. Depois de várias passagens de células, outros tipos de células não conseguem sobreviver e propagar em DMEM com 10% de soro fetal bovino (FBS), resultando em culturas de células de fibroblastos purificadas. As células podem ser usadas 3-7 passagens seguintes cultura primária. </li></ol></li><li> Cultura explante e condicionado para crescimento celular: <ol><li> Execute cultura primária de fibroblastos, utilizando um protocolo previamente notificados com modificações 5. </li><li> No laboratório, colocar a amostra de tecido em 10 cm de prato de cultura de tecidos sem meio de cultura e cortado em pequenos troços ~ 2-3 mm de tamanho usando fórceps esterilizados e bisturi. Durante este processo, evitar que a secção seca, de outro modo a eficiência da excrescência da célula é prejudicado. </li></ol></li><li> A separação da camada de células epiteliais e do tecido conjuntivo: <ol><li> Embeber as secções de tecido cortado em meio de cultura contendo 2,000 PU / ml de dispase I e cultura durante 16 horas a 4 ° C. &lt;/ Li&gt; <li> Transferem-se as secções de tecido para um prato, e separar a camada de células epiteliais do tecido conjuntivo adjacente. Processe as seções em uma bancada limpa para evitar a contaminação de microorganismos. NOTA: Se for necessário a cultura primária de células epiteliais, executar o passo 1.3. Se apenas os fibroblastos estão a ser isolado, omitir o passo 1.3 porque a cultura explante e celulares passagens subsequentes são desfavoráveis ​​para as células epiteliais, que produzem populações de fibroblastos purificados. </li></ol></li><li> Penhora de cortes de tecido: <ol><li> Picar os tecidos em um milímetro pedaços usando dois bisturis. Se as peças não são suficientemente pequenas, elas retirar mais facilmente a partir da placa, e as células irão falhar a ultrapassar na superfície do prato. </li><li> Coloque pequenas secções de tecido pulmonar afastados um do outro para manter um espaço vazio em torno de cada peça. Adicione arranhões na superfície do prato de cultura de tecido usando uma lâmina de bisturi para facilitar a ligação de secções de tecido. NOTA: CoçarTambém parece facilitar a excrescência de células, proporcionando faixas ao longo do qual as células podem migrar. <ol><li> Em alternativa, colocar as peças de tecidos picados, individualmente, em cada poço de uma placa de 6 poços, e cobri-los com uma lamela de cobertura. Fixe a tampa de deslizamento para a superfície da placa usando graxa de silicone. </li><li> Coloque graxa de silicone em dois pontos 1.5 cm de distância em cada poço, utilizando um pino estéril. Cobertura de pedaços de tecido aumenta a eficácia do crescimento de células e propagação de células após diversas passagens. </li></ol></li></ol></li><li> Subsequente cultura explante: <ol><li> Adicionar cuidadosamente meio de cultura para o prato antes de as secções de tecido de secar, como crescimento celular eficaz é perdido para a maior parte, se as amostras secarem. Não despeje o meio muito rapidamente como as secções de tecido seria separar. </li><li> Células de cultura em DMEM com 10% de FBS. Use médio suficiente apenas para cobrir o tecido, mas não muito que ele permite flutuante, como flutuabilidade adicional promove a separação doprato. </li></ol></li><li> Passagem Cell: <ol><li> Lidar com o meio de cultura com cuidado em todos os momentos como movimentos repetidos da placa de cultura pode levar ao descolamento dos cortes de tecido. </li><li> Colocar as placas de cultura numa incubadora a 37 ° C durante 5-7 dias. Atualizar o meio todos os dias, e lidar com as placas de cultura minimamente durante as médias alterações. Os fibroblastos superar a partir da borda das secções de tecido ao longo das próximas semanas. NOTA: ~ Após 2-3 semanas, os fibroblastos superando quase atingem confluência, dependendo da quantidade de área de superfície. </li><li> Lavar as células com PBS 1x, e adicionar 1 ml de tripsina I do prato. </li><li> Após tripsinização, separar as células utilizando 9 ml de DMEM suplementado com FBS a 10%. Recolher as células em suspensão no meio de um tubo de 10 ml, juntamente com as secções de tecido. A seguir à centrifugação para formar sedimentos celulares, ressuspender as peletes resultantes em meio novo. </li><li> Semente as células e os cortes de tecido para um novo culture prato, altura em que os tecidos não conseguem anexar ao prato, enquanto as células aderir e crescer. No dia seguinte, retire os pedaços de tecido flutuantes por aspiração, e mudar o meio. Fibroblastos anexados propagar com culturas subsequentes. </li></ol></li></ol> 2. tridimensional Co-cultura de fibroblastos humanos pulmão e células cancerosas <ol><li> Preparação de células e reagentes: <ol><li> Aproximadamente usar 2 x 10 5 células epiteliais e 2,5 x 10 5 fibroblastos por poço. Prepare o colagénio tipo IA (3 mg / ml, pH 3), FBS (100%), tampão de reconstituição (NaOH 50 mM, 260 mM de NaHCO3, 200 mM de HEPES), 5x de DMEM, meio DMEM suplementado com FBS a 10% para a cultura de fibroblastos, e um meio de co-cultura em 3D (uma mistura 1: 1 de meio de cultura de fibroblastos e meio de cultura para as células cancerosas). </li><li> Células de adenocarcinoma do pulmão A549, a cultura, os fibroblastos e a co-cultura 3D A549 em DMEM suplementado com FBS a 10%. </li></ol></li><li> O colagénio em gel de formação: <ol><li> Lavar fibroblastos em cultura em placa de 10 cm com 1x PBS, e adicionar 1 ml de tripsina I do prato. Após tripsinização durante ~ 5 min a 37 ° C, separar as células utilizando 9 mL de DMEM suplementado com FBS a 10%. Recolher as células em suspensão no meio num tubo de 10 ml e centrifugar para a 200-300 xg para formar sedimentos celulares. </li><li> Ressuspender as peletes resultantes em 100% de FBS a uma densidade de 5 x 10 5 / ml. </li><li> Prepare o gel de colagénio em gelo. Arrefecer os reagentes, pipetas e tubos no frigorífico antes do procedimento seguinte. Mesmo em gelo, a mistura solidifica, em certa medida, e o volume total é menor do que a soma de todos os constituintes. </li><li> Em cada poço, preparar o gel de colagénio por mistura de 0,5 ml de suspensão de fibroblastos (2,5 x 10 5 células) em FBS, 2,3 ml de colagénio do tipo IA, 670 ul de DMEM 5x, e 330 ul de tampão de reconstituição. Permitir que os geles para definir facilmente à temperatura ambiente. <ol><li> Vigorosamente pipeta de modo que a mi homogéneaxture. Evite a criação de bolhas durante o processo de pipetagem. </li></ol></li><li> Adicionar a mistura de (3 ml) a cada poço de uma placa de 6 poços e deixa-se gelatinizar na incubadora a 37 ° C, sem perturbação durante 30-60 min. </li></ol></li><li> Cultura de células do cancro no gel de colagénio: <ol><li> Execute co-cultura tridimensional usando um protocolo previamente notificados com modificações 6. </li><li> Ressuspender as células cancerosas em meio de co-cultura em 3D (ou em DMEM com FBS a 10%, no caso de células A549) a uma densidade de 1 x 10 5 / ml. </li><li> Pour 2 ml da solução de células ressuspenso (2 x 10 5 células) sobre a superfície de cada gel. Modifique o número de células de células cancerosas ou fibroblastos em co-cultura, dependendo das condições experimentais. </li></ol></li><li> Contração gel: <ol><li> Incubar géis noite a 37 ° C de modo que as células cancerosas podem aderir ao gel de colagénio. Separe cada gel, e gerar uma &quot;cultura flutuante &#39;em cada poço of a placa de 6 poços. NOTA: Ao utilizar uma cultura flutuante, várias modificações no tamanho de gel são induzidas por tensão mecânica e degradação do colagénio, que são mediados por interacção celular entre as células cancerosas e fibroblastos co-cultivadas. </li><li> Utilize uma agulha G 21 angular ou pequena espátula para separar cada um gel a partir do bordo do poço. A cada 2-3 dias, atualizar os poços com 2 ml de meio 3D co-cultura. Medir o tamanho de cada gel todos os dias durante 5 dias. </li></ol></li><li> Análise de géis de colagénio: <ol><li> Meça o conteúdo de colágeno de cada gel, utilizando métodos de quantificação de colágeno, tais como o ensaio Sircol. Realizar análise do transcriptoma ou RT-PCR quantitativo após a recolha de RNA a partir dos géis 5. </li></ol></li></ol> Cultura da Interface e Invasion Ensaio 3. Air-líquido <ol><li> Após cultura das géis de colagénio a 37 ° C durante 5 dias, expor os géis contraídos para o ar, colocando-os sobre uma malha (70 de tamanho de poro) no novo 6-placas de poços. Faça a malha a partir de filtros celulares disponíveis comercialmente usados ​​para citometria de fluxo. </li><li> Suavemente encher cada poço com 11 ml de 3D meio de co-cultura. </li><li> Posicionar os géis para a malha usando uma colher esterilizada, e remover qualquer meio remanescente dos géis. Nesta condição, submergir em geles a forma enquanto expor a superfície superior do gel de ar. Definir essa condição cultura como interface de ar líquido. NOTA: Depois de 5-7 dias de manutenção da cultura de interface ar-líquido, a 37 ° C na incubadora, as células cancerosas invasivas migrar para dentro do gel. Dependendo do tipo de célula e, como resultado de interacções celulares, graus variáveis ​​de alterações morfológicas em células, as células cancerosas são induzidas pela cultura de interface ar-líquido. </li><li> Para a avaliação histológica, fixar o gel em formol durante a noite a temperatura ambiente e incorporar em parafina. Mancha seções verticais (4 mm) com HE (H &amp; E). Realizar análise imuno-histoquímica on as seções 4. </li></ol>

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The authors declare that they have no competing financial interests.

FAC formam um dos principais componentes da MEC células cancerosas e não apenas fornecer um andaime para o tumor, mas também participar activamente no desenvolvimento do tumor 7. As evidências acumuladas desvenda o impacto da FAC ou suas moléculas relacionadas à eventual prognóstico, com destaque para os papéis críticos de progressão do tumor CAF mediada 8. No estudo anterior, foram empregados método consequência de isolar FAC pulmonares 4. Nesta ...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:267px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:471px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DMEM</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:471px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FBS</td>
			<td style="width:297px;">GIBCO</td>
			<td style="width:471px;">10437</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Collagen type IA</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;">CELL-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Reconstitution buffer</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cover slip</td>
			<td style="width:297px;">NUNC</td>
			<td style="width:471px;">174934</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Silicone grease</td>
			<td style="width:297px;">Dow Corning Toray</td>
			<td style="width:471px;">High vacuum grease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dispase I</td>
			<td style="width:297px;">WAKO</td>
			<td style="width:471px;">386-02271</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">6-well plate</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">353046</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell strainer (70 &#956;m)</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">352350</td>
		</tr>
	</tbody>
</table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Este método de co-cultura, imitando o microambiente tumoral, é uma ferramenta útil para pesquisar as interacções entre as células cancerosas e fibroblastos embebidos no gel de colagénio. No estudo anterior, os três parâmetros foram avaliados neste modelo experimental: contração gel de colágeno, a invasão das células do câncer e alteração morfológica. Proliferação de células cancerosas também foi estimado usando Ki67 immunostaining 4. Amostras de tecido pulmonar foram colhidos a partir de...

Watch this Scientific Journal Video about Three-dimensional Co-culture Model for Tumor-stromal Interaction at JoVE.com

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensional modelo de co-cultura para Interaction Tumor estromal-

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

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17.2K visualizações.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Vídeo: Three-dimensional Co-culture Model for Tumor-stromal Interaction

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Geração Experimental de Carcinoma Associado fibroblastos (FAC) de Fibroblastos Humanos mamária

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Medicina

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensional (3D) Tumor Esferóide Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Impressão tridimensional de uma anomalia complexa da aorta

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Modelo de guia de impressão tridimensional assistida Vertebroplastia Percutânea (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Imagem tridimensional de alta resolução da Vasculatura do Footpad em um Modelo de Gangrena Hindlimb Murine

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...