Notas técnicas: <ul><li> Gelificación se inicia por la adición de sales. Por lo tanto, es fundamental que no sales de entrar en contacto con PuraMatrix hasta la gelificación se desea, la exposición a las sales de la fuerza iónica muy baja (por ejemplo, &lt;0,05 M) causará gelificación irreversible. </li><li> Soluciones sobrantes de acciones que se prepara puede ser utilizado en una fecha posterior siempre que no hayan estado en contacto con las sales. Usted no necesita preocuparse por sonicación repetir. </li><li> Las burbujas de aire se puede quitar PuraMatrix alícuotas en tubos estériles, seguido por centrifugación. </li><li> Tenga mucho cuidado durante los cambios de los medios de comunicación como la aspiración puede alterar la cama PuraMatrix. </li></ul> Materiales: BD PuraMatrix péptido RAD16 hidrogel-1% (Cat # 354 250): La concentración de péptido total de la mezcla no diluida es de 10 mg / ml. <ul><li> Agua baño de ultrasonido o Vortex </li><li> 96 y placa de cultivo celular </li><li> Medios de cultivo celular </li><li> Estéril filtrado H 2 O </li><li> Filtrado estéril de sacarosa al 20% </li><li> Las células (todas menos 5.000 células / pocillo): OVCAR-5 </li></ul> Todos los pasos se deben realizar en condiciones asépticas. Procedimiento: <ol><li> Sonicar la PuraMatrix 1% (RAD16) en un baño de ultrasonido agua durante 30 minutos para reducir la viscosidad antes de su uso. </li><li> La solución PuraMatrix 1% ahora se puede alícuotas en tubos estériles para simplificar los experimentos futuros. <ol class="lalpha"><li> Evitar la formación de burbujas de aire, si se forman sólo hacen girar por los tubos estériles que contengan PuraMatrix a 1000 rpm (300 xg) durante 5 minutos. </li><li> La concentración de péptido total de la mezcla no diluida es de 10 mg / ml. </li></ol></li><li> Dos grupos de cuatro pozos se sembraron a 2,5 mg / ml y 1,25 mg / ml de concentración de péptido total, respectivamente. </li><li> Diluir la PuraMatrix en cada pocillo conjunto con 0,2 micras estéril sacarosa filtrado del 20% y el 13,3% de sacarosa con el total de concentraciones de péptido de 5 mg / ml y 2,5 mg / ml, respectivamente, para alcanzar una concentración de 2x BD PuraMatrix en 10% de sacarosa. <ol class="lalpha"><li> Hacer 20% de sacarosa, diluir 10 g de sacarosa, entonces el volumen de 50 ml de agua. </li><li> 13% y 10% de sacarosa se puede preparar por dilución de la solución de reserva del 20%. <ol class="lroman"><li> 13% de sacarosa (por cada 10 ml): 6,5 ml de sacarosa al 20% + 3,5 ml estéril H 2 O. </li><li> 10% de sacarosa (por cada 10 ml): 5,0 ml de sacarosa al 20% + 5.0 ml estéril H 2 O. </li></ol></li></ol></li><li> Prepare una mezcla maestra de péptido PuraMatrix basado en el número de pozos tiene la intención de la placa. (Véase el siguiente ejemplo.) Luego alícuotas en tubos individuales que contienen 25 ml de la mezcla maestra. <ol class="lalpha"><li> La mezcla maestra debe estar preparado el doble de la concentración final deseada de péptido total, ya que se diluye con un volumen igual de células durante el recubrimiento. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="la figura 1" /></li></ol></li><li> Preparar las suspensiones de células de cultivos celulares de células trypsinizing monocapa. <ol class="lalpha"><li> 2 ml de 0,05% de tripsina EDTA se puede utilizar para cada frasco T-75. </li></ol></li><li> Neutralizar la tripsina mediante la adición de medios de cultivo celular que contiene 10% inactivado por calor suero fetal bovino (uso de al menos 2 ml de los medios de comunicación por ml de tripsina utilizada en el paso anterior.) </li><li> Centrifugar las células a 1000 rpm (~ 300 xg) durante 5 minutos para crear un sedimento celular. <ol><li> Resuspender las células en estéril de sacarosa al 10% se filtra. </li><li> Contar las células y girar de nuevo a 1000 rpm (~ 300 xg) durante 5 minutos para eliminar pequeñas cantidades de sal en los medios de comunicación. </li></ol></li><li> Resuspender las células OVCAR-5 en el 10% de sacarosa en 2,0 x 10 5 células / ml por lo que el número final de células por pozo será de 5.000 células en cada pocillo. Los siguientes pasos deben llevarse a cabo rápidamente. </li><li> Añadir 25 l de la x 2.0 10 5 células / ml de suspensión en el primer tubo individuales con 25 l de la mezcla maestra de PuraMatrix como se muestra en el esquema de arriba. <ol class="lalpha"><li> Rápidamente la mezcla con la pipeta (~ 5 veces arriba y abajo) en el tubo sin introducir burbujas de aire. </li><li> A continuación, una pipeta de 50 l de suspensión celular / PuraMatrix mezcla en el pozo apropiado de un plato de 96 pozos. </li><li> Iniciar la gelificación de la PuraMatrix BD pipeteando suavemente 150 medios de comunicación l cultivo de células (RPMI + 10% FBS) al lado del pozo <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="la figura 2" /></li></ol></li><li> Repita los pasos descritos en el 8 hasta que todos los pozos se han plateado. </li><li> Después de 30 minutos muy suavemente quitar ~ 2 / 3 de los medios de comunicación con una pipeta 200 l para equilibrar el pH del hidrogel. <ol class="lalpha"><li> NO USE una aspiradora, ya que esto INTERRUMPIR LA MATRIZ </strong&gt; </li><li> Coloque la punta de la pipeta en el pozo para que no toque la cama PuraMatrix y retire con cuidado los medios de comunicación desde el pozo. </li><li> Se debe tener cuidado para no alterar la matriz durante el cultivo de células en PuraMatrix. </li></ol></li><li> Muy suavemente añadir 150 l de nuevo medio de cultivo celular al lado de las células y que contiene encapsulado en PuraMatrix. </li><li> Cambiar el medio de cultivo celular en cada pocillo cada 48 horas con medio de cultivo fresco con cuidado de repetir los pasos 10 y 11. </li></ol> Los resultados representativos: <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg"><img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="la figura 1" border="0" /></a> Figura 1. OVCAR-5 células fueron encapsulados como se describe en el protocolo anterior. Las imágenes fueron adquiridas con un invertido Olympus FV1000 microscopio equipado con Spectra-Physics DeepSee Ti: Sapphire láser sintonizado a 750 nm. Sesiones de formación de imágenes se llevaron a cabo los días 1, 3, 5 y 7 días después de galvanoplastia. A larga distancia de trabajo, el agua mojando objetivo de 40x (Olympus LUMPLFLN 0,8 NA) fue utilizado para la imagen a través de la PuraMatrix. Volúmenes tridimensionales fueron recogidos por la adquisición de las pilas de la imagen en 1,47 micras pasos axial. Dos no descanned detectores de recogida de la emisión de la autofluorescencia de violeta (440 hasta 490 nm) y verde (510-550 nm) filtros de paso de banda. Las imágenes finales y películas profundidad de la pila se procesaron con ImageJ mediante la combinación de los dos canales de detección.

<ol>
	<li>Gelain, F., Bottai, D., Vescovi, A., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+self-assembling+Peptide+nanofiber+scaffolds+for+adult+mouse+neural+stem+cell+3-dimensional+cultures.">Designer self-assembling Peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.</a> PLoS One. 1, e119-e119 (2006).</li></ol>

puramatrix encapsulation of cancer cells

Evidencia creciente sugiere que las células del cáncer de cultivo en tres dimensiones con mayor precisión recapitula la complejidad de la biología del tumor. Muchos de estos modelos utilizan la membrana basal reconstituida procede de animales que contienen una cantidad variable de factores de crecimiento y citoquinas que pueden influir en el crecimiento de estos modelos de cultivo celular. A continuación, describimos en detalle la preparación y el uso de PuraMatrix, un auto disponible en el mercado montaje de gel de péptido que carece de origen animal y material de los agentes patógenos para encapsular y propagar el cáncer de ovario línea celular, OVCAR-5. Comenzaremos por describir la forma de preparar el PuraMatrix antes de su uso. A continuación, se demuestra cómo mezclar la PuraMatrix y la suspensión celular para encapsular las células en el hidrogel. Tras la adición de medios de cultivo celular o inyección en un entorno fisiológico, el componente de péptidos de PuraMatrix rápidamente sí se constituye en un hidrogel en 3D que muestra a escala nanométrica estructura fibrosa con un tamaño de poro promedio de 500 a 200 nm<sup&gt; 1</sup&gt;. Además, se demuestra cómo propagar cultivos crecidos en PuraMatrix encapsulado. Cuando se encapsula en PuraMatrix, OVCAR-5 células se ensamblan en tres dimensiones las estructuras acinares que se asemejan más a la morfología de los nódulos micrometástasis observado en la clínica de monocapa en modelos in vitro. Mediante microscopía confocal que ilustran la aparición del representante OVCAR-5 células encapsuladas en PuraMatrix el día 1, 3, 5, y 7 placas mensaje. El uso de PuraMatrix a las células cancerosas cultura a mejorar nuestra comprensión de la enfermedad y nos permitirá evaluar la respuesta al tratamiento en los sistemas modelo más predictivo clínicamente.

Watch this Scientific Journal Video about PuraMatrix Encapsulation of Cancer Cells at JoVE.com

PuraMatrix Encapsulation of Cancer Cells

Este video muestra la forma de encapsular las células de cáncer en la cultura PuraMatrix, un auto disponible en el mercado montaje de gel de péptido.

PuraMatrix encapsulación de las células cancerosas

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Research

JoVE Journal

Biology

16.6K vistas.  Harvard Medical School. Este video muestra la forma de encapsular las células de cáncer en la cultura PuraMatrix, un auto disponible en el mercado montaje de gel de péptido.

Video: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

Título encapsulación celular a través de gotitas

Investigación

Biología

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Aislamiento y análisis de las células madre hematopoyéticas de la placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

La encapsulación celular en hidrogeles 3D para ingeniería de tejidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

El aislamiento de células madre de cáncer de páncreas xenoinjertos

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Citometría de flujo de purificación de las células de ratón meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

La visualización de las células intersticiales de Cajal (ICC) de red en ratones

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...