Este trabajo fue financiado por los Institutos Nacionales de Salud Grant 5KO8DK085136-02 a JY.

1. Obtener el tejido del colon Ratón Un protocolo para llevar a cabo experimentos con animales, que detalla los fundamentos y objetivos de la investigación, fue presentado y aprobado por nuestra oficina institucional de Investigación Animal de Supervisión. <ol><li> La eutanasia del ratón con isoflurano inhalado seguido por dislocación cervical. </li><li> Haga una incisión en la línea media ventral, retraer el intestino delgado de distancia e identificar el colon. Repliegue la vejiga y el útero (si existe) lateralmente e identificar el hueso púbico y cortar con tijeras finas para exponer el recto. </li><li> Diseccionar el mesenterio dorsal del recto fuera del colon y movilizar el colon desde el ano hasta el ciego, y seccionar para desenchufar el colon desde el intestino delgado. </li><li> Coloque la muestra en solución salina tamponada con fosfato enfriada con hielo (PBS). </li><li> Llene una jeringa de 10 ml con PBS enfriado con hielo y lavar el colon luz varias veces para borrar su contenido, a continuación, cortar la opa de colonn en sentido longitudinal con tijeras. </li><li> Coloque el colon en un tubo cónico de 50 ml que contiene 20 ml de PBS enfriado en hielo. El colon no tiene que ser colocado en cualquier orientación particular. Lave el colon mediante la sustitución de la PBS hasta que el líquido es transparente, sin partículas. </li></ol> Humano Un protocolo para obtener tejido humano de pacientes quirúrgicos, detallando la importancia de la obtención de este tejido, así como una evaluación de los riesgos y beneficios potenciales, fue presentado y aprobado por nuestra oficina institucional del Programa de Protección de la Investigación Humana. <ol><li> Colaboración de investigación con un cirujano colorrectal es necesario ser capaz de obtener tejidos humanos para su investigación. Un protocolo para obtener tejido del colon humano debe ser presentado y aprobado por la junta de revisión institucional. </li><li> Escrito el consentimiento informado se obtuvo a partir de la paciente antes del procedimiento quirúrgico. </li><li> El cirujano realizará una resección de colon en el standmoda ard. Una vez que el colon se ha retirado del paciente, el espécimen es llevado inmediatamente a la patología. Un 0,5 en la sección de espesor total de la pared del colon que no sea necesario para la evaluación patológica se proporciona y se coloca inmediatamente en helado de PBS. Este espécimen no debe incluir el mesenterio del colon y apéndices epiploicos debe retirar también, ya que la presencia de grasa va a afectar el proceso de la digestión. </li></ol> 2. Dénudé células epiteliales <ol><li> Incubar toda la colon de ratón en 25 ml de EDTA 5 mM en HBSS (temperatura ambiente) en un tubo cónico de 50 ml. Colocar el tubo cónico en un C agitando baño de aire 37 ° (250 rpm) durante 15 min. Después de 15 minutos, se vierte con cuidado el líquido y vuelva a llenar el tubo con 25 ml de EDTA 5 mM, repita para un total de 5 lavados. Para colon humano, después de obtener una tira de ½ pulgada de la pared del colon del patólogo cortar el colon con tijeras en cuatro trozos de igual tamaño. Coloque dos de las piezas en un tubo cónico de 50 ml, y secolocar las dos piezas restantes de tejido de colon en un tubo cónico de 50 ml separado. A continuación, se incuba con 25 ml de EDTA 5 mM en HBSS y realizar lavados, como se describe anteriormente. </li><li> Enjuague el colon dos veces en 20 ml de PBS enfriado en hielo. </li><li> Coloque el tejido de colon de ratón en un nuevo tubo cónico de 50 ml que contiene 20 ml de medio RPMI-5, 10 U de dispasa, y 2000 U de colagenasa D (temperatura ambiente). Para el tejido del colon humano, utilice el doble de la cantidad de dispasa y colagenasa (20 U dispasa, 4.000 U de colagenasa D). </li><li> Coloque el tubo en un baño de aire 37 ° C agitando orientada verticalmente (Si se coloca horizontalmente, hay demasiada aireación y daño celular). Agitar a 250 rpm durante un máximo de 60 min. Después de la exposición a la dispasa y colagenasa D, el tejido del colon se iniciará de digerir y el tejido del colon se comenzará a verse fibrosa. Esto por lo general toma alrededor de 30 minutos. Cuando el colon tiene esta apariencia, extirpar el colon de las enzimas. </li><li> Agitar cada tubo hacia arriba y abajo 2-4 veces para romper el tejido. PEllet el tejido a 200 xg en una centrífuga de mesa (4 ° C) durante 5 min. Vierta cuidadosamente el sobrenadante y deséchelo. </li></ol> 3. Aislamiento miofibroblastos y Cultura <ol><li> Resuspender cada sedimento mediante la adición de 10 ml de tampón de lisis ACK (47 ° C). Centrifugar de nuevo a 200 xg durante 5 minutos (4 º C), verter y desechar el sobrenadante. </li><li> Resuspender cada sedimento mediante la adición de 10 ml de RPMI-5 con una pipeta. </li><li> Pase medio de la suspensión de células (5 ml) a través de un colador de malla 70 micras utilizando una pipeta de 10 ml en una placa de TC tratados con 100 mm. A continuación, añadir otros 15 ml de RPMI-5. Repita el procedimiento con los 5 ml restantes de la suspensión celular en una placa de 100 mm TC-tratada diferente. Por lo tanto, uno de colon de ratón producirá dos platos TC tratados 100 mm. Una tira de ½ pulgada de tejido de colon humano producirá un total de cuatro platos TC-tratada de 100 mm. </li><li> Coloque las placas de cultivo de tejidos en una incubadora de CO2 un 10% a 37 ° C. Las condiciones de cultivo son los mismos fo el ratón y células humanas primarias. </li><li> Después de 3 horas, lavar suavemente las células no adherentes con dos cambios de HBSS. La flotación de escombros se debe lavar suavemente. Las células adherentes se componen de células epiteliales, macrófagos y miofibroblastos. Una semana después de la siembra, sólo miofibroblastos brecha, macrófagos y células epiteliales entran en senescencia después de la primera paso. Añadir fresco RPMI-5 y crecer las células en cultivo celular. </li><li> Las células se pasaron por tratamiento con tripsina durante 5 min y se dividen 2:01. </li></ol> Abreviaturas HBSS: solución salina equilibrada de Hank; RPMI-5: Medios de Roswell Park Memorial Institute; ACK: amonio-cloruro y potasio; FCS: suero fetal de ternero; TC-tratada: el cultivo de tejidos tratados Soluciones Tampón de lisis ACK - (4,15 g NH 4 Cl, 0,5 g de KHCO3, 18,6 mg de Na 2 EDTA, 400 ml de H2O, ajustar el pH a 7.2 a 7.4 con HCl 1 N). Esterilizable Filtroelectrónico a través de un filtro de 0,2 micras y se almacenan a 4 ° C. RPMI-5 - (para hacer 500 ml: 454,5 ml RPMI, 25 ml de FCS, 5 ml de 200 mM L-glutamina, 5 ml 1 M HEPES, pH 7,4, 5 ml piruvato de sodio 100 mM, 5 ml 100x Pen-Strep, 500 ml 50 mM B-ME en PBS).

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Los autores tienen nada que revelar.

La técnica general es similar cuando se utiliza el ratón o de colon humano. Esta técnica también se puede utilizar para aislar miofibroblastos en el intestino delgado. Los detalles específicos para el aislamiento de miofibroblastos de 1. ratón y 2. colon humano se discuten a continuación. 1. Ratón Colon Irrigación del colon de ratón se facilita uniendo un 4 en, 16 G Popper aguja de extremo romo a un extremo del colon. Esto también ayuda al cortar el colon...

La regulación de la función del tracto gastrointestinal (GI), implica complejas interacciones dinámicas entre la capa de la mucosa y su mesénquima subyacente. Estas interacciones normalmente sirven para mantener la homeostasis, pero también puede conducir al desarrollo de enfermedades en condiciones patológicas 1. Los miofibroblastos son una subpoblación de células del estroma situado subyacente a la capa epitelial que se comunican de una manera paracrina y la zona subpoblaciones de células para regular una serie de procesos del tracto GI críticos que incluyen la regeneración de la mucosa, la reparación, y la fibrosis 2. La línea celular 18Co es un ejemplo de una línea celular humana de colon miofibroblastos que ha sido ampliamente utilizado para estudiar la función de miofibroblastos ya que comparte muchas de las características de primaria en miofibroblastos in situ. Estos incluyen la expresión de la proteína de un liso-actina de músculo (α-SMA) y vimentina, así como la expresión de un númerode receptores de la superficie celular tales como el receptor del factor de crecimiento epidérmico, o receptores para el ácido 3,4 lisofosfatídico. Debido a estas características ultraestructurales comunes, así como similitudes en la función biológica 5, la línea celular de 18Co se ha utilizado ampliamente para estudiar la función de miofibroblastos en el contexto de muchos estados de enfermedad tales como la enfermedad inflamatoria intestinal o 3,6 cáncer colorrectal. Sin embargo, hay limitaciones inherentes y preocupaciones al utilizar una línea celular. Estos incluyen la inestabilidad genotípica con el tiempo que diferencia líneas celulares a partir de células primarias, alterando el fenotipo y la función biológica de la célula, incluyendo las tasas de crecimiento y las interacciones con otras poblaciones celulares. Las líneas celulares también carecen de los componentes normales de la microambiente GI (epitelial, estromal, y componentes vasculares), que es una limitación importante para su uso. Por lo tanto, las conclusiones extraídas de la investigación experimental línea celular requiere una validación adicional de rsk de una mala interpretación. Miofibroblastos primarias pueden ser obtenidas a partir de tejido de colon humano y el ratón para confirmar los hallazgos experimentales identificados en una línea celular 7. El método fue descrito originalmente por Mahida, et al. 8, y el protocolo es una de las muchas técnicas bien descritos que se pueden utilizar para aislar miofibroblastos primarios de ratón y de colon humano 9. Para cualquier modelo de ratón de la enfermedad colónica, esta técnica se puede utilizar para aislar miofibroblastos primarias para estudiar cómo interactúan con las células vecinas o contribuir a tanto normal o patológica GI procesa 10,11. Esta técnica se puede utilizar para estudiar cómo miofibroblastos contribuyen a la curación de la mucosa, la fibrosis, y el desarrollo de los adenomas y carcinomas colorrectales. Miofibroblastos primarios también se pueden obtener a partir de tejido de colon humano que ha sido resecado en el momento de la operación de (enfermedad inflamatoria del intestino, estenosis, diverticulitis) benigno o maligno Condiciones. Células primarias aisladas de tejido de colon humano y de ratón se pueden cultivar en cultivo celular y se utilizan en un número limitado de pasajes.

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

El miofibroblastos es una célula estromal del tracto gastrointestinal (GI) que ha ido ganando considerable atención por su papel crítico en muchas funciones gastrointestinales. Mientras que varias líneas celulares myofibroblast están disponibles comercialmente para estudiar estas células in vitro, los resultados de investigación desde una línea de células expuestas a condiciones experimentales de cultivo de células tienen limitaciones inherentes debido a la naturaleza excesivamente reduccionista de la obra. El uso de miofibroblastos primaria ofrece una gran ventaja en términos de confirmación de los hallazgos experimentales identificados en una línea celular. Aislamiento de miofibroblastos primarios a partir de un modelo animal permite el estudio de los miofibroblastos en condiciones que imitan más estrechamente se estudió el estado de enfermedad. Aislamiento de miofibroblastos primarios de tejido del colon humano ofrece sin duda los datos experimentales más relevantes, ya que las células vienen directamente de los pacientes con la enfermedad de base. Se describe una técnica bien establecida que puede ser utilized aislar miofibroblastos primarias de ratón y el tejido del colon humano. Estas células aisladas se han caracterizado a ser actina alfa del músculo liso y vimentina-positivo, y negativo-desmina, consistente con los miofibroblastos subepiteliales intestinales. Células miofibroblastos primarias se pueden cultivar en cultivo celular y se utilizan para fines experimentales más de un número limitado de pasajes.

Una vez aislado, miofibroblastos humanas primarias se pueden cultivar en cultivo celular y se utilizan en un número limitado de pasajes (hasta el paso 4). Estas células se caracterizan por ser un suave-actina y vimentina positivo muscular, y desmina-negativo (Figura 1A), en consonancia con los miofibroblastos subepiteliales intestinales 5,7. También tienen una morfología estrellada característica (Figura 1B). Miofibroblastos primarias se pueden utilizar para confirmar lo...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

El miofibroblastos es una célula estromal influyente del tracto gastrointestinal que regula importantes procesos fisiológicos en los estados tanto normales y de enfermedad. Se describe una técnica que permite el aislamiento de miofibroblastos primarios de ratón y tejido de colon humano, que puede ser utilizado para la experimentación in vitro.

Aislamiento de Miofibroblastos primarias de ratón y el tejido del colon humano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Research

JoVE Journal

Biology

19.4K vistas.  UCLA.  El miofibroblastos es una célula estromal influyente del tracto gastrointestinal que regula importantes procesos fisiológicos en los estados tanto normales y de enfermedad. Se describe una técnica que permite el aislamiento de miofibroblastos primarios de ratón y tejido de colon humano, que puede ser utilizado para la experimentación in vitro. 

Video: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Aislamiento de los núcleos neuronales a partir de tejido cerebral humano post-mortem

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Investigación

Biología

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Aislamiento de Miofibroblastos de Mouse y Esófago Humano

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Aislamiento y caracterización de fibroblastos cardíacos adultos y miofibroblastos

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

Generación De Monocapas Epiteliales Murinas Primarias De Colon De Criptas Intestinales

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...