Los autores desean agradecer al Sr. Josh Basford y el Dr. Li Xiao-min para la asistencia técnica. Este trabajo fue apoyado en parte por subvenciones del NIH (DK70992 y DK92779 a ML).

1. Preparación Todos los tampones se preparan utilizando una técnica estéril y esterilizarse por filtración utilizando un filtro de 0,22 micras Corning. <ol><li> Preparar perfusión búfer mediante la adición de las siguientes acciones para la solución salina equilibrada de Hank (HBSS, sin Ca 2 + y Mg 2 +, ver Tabla 1): Mg 2 + (MgCl 2) a 0,9 mM, EDTA a 0,5 mM, y HEPES a 25 mM. </li><li> Preparar perfusión tampón II mediante la adición de las siguientes acciones para HBSS (con Ca 2 + y Mg 2 +, ver Tabla 1): HEPES a 25 mM. </li><li> Preparar buffer de perfusión II más collagenaseII: Disolver la colagenasa II (1000 U) con 300 ml de perfusión II de amortiguación y mantener la solución caliente en baño de agua antes de la perfusión. Esta solución debe ser utilizado dentro de 30 minutos debido a la actividad de la colagenasa II disminuyó con el tiempo. </li><li> Preparar medio completo de William: Agregue el siguienteMedio a E de Williams: L-glutamina a 2 mM, suero bovino fetal (FBS) al 5%; insulina a 100 nM, dexametasona a 100 nM, penicilina a 100 UI / ml y estreptomicina a 100 mg / ml. </li><li> Estos tampones debe ser calentado durante 30 minutos en el baño de agua a 42 ° C, una temperatura óptima que corresponde a una temperatura de salida en la cánula de 37 ° C. </li></ol> 2. Rata perfusión para el aislamiento del hígado <ol><li> El sistema de perfusión se compone de la bomba, el tubo de silastic autoclave y un baño de agua (ver Figura 1). Pre-establecer el caudal de la bomba de perfusión peristáltica a 10 ml / min. </li><li> Se anestesia una rata adulta (300 g de peso corporal) con intraperitoneal (ip) cetamina (87 mg / kg de peso corporal), además de xilazina (13 mg / kg). Profundidad de la anestesia debe ser supervisada por pizca dedo del pie. Cuando la rata ya no responde a los estímulos nocivos, afeitarse el vello abdominal y preparación del abdomen con betadine y etanol. Entrar a través de una incisión en la línea media. </li><li> Exponerla vena portal hepática moviendo cuidadosamente las vísceras al exterior derecho de la cavidad abdominal, e insertar un angiocath 18-calibre en la vena portal hepática (ver Figura 1). </li><li> Conectar el tubo perfundido a la aguja e iniciar la infusión in situ a un caudal bajo (10 ml / min) con pre-calentado (37 ° C) tampón de perfusión yo. </li><li> Si se realiza correctamente, el hígado debe inmediatamente comenzar a blanquear. Una vez que la canulación exitosa se ​​confirma, hacer un corte en la vena cava inferior (VCI) para permitir el flujo de salida (Figura 1). Una prueba más para la canulación exitosa se puede realizar aplicando una ligera presión con el hisopo estéril de la vena cava inferior, todos los lóbulos del hígado debe rápidamente comienzan a hincharse. </li><li> Aumentar la velocidad de flujo de 25 ml / min. El hígado debe ser de color pálido. </li><li> Cambie la solución de perfusión a la perfusión tampón II, más colagenasa II sin interrupción del flujo durante 6 minutos. </li> &lt;li&gt; Periódicamente (5-10 veces durante la digestión) de ejercer presión con el hisopo para la vena cava inferior para intervalos de 5 segundos. El hígado se hincha, dando lugar a una mayor disociación celular hepática, a su vez reduce el tiempo total de digestión y aumentando el rendimiento final. </li><li> Después de la perfusión de colagenasa, el hígado debe comenzar a verse pesado. Diseccionar el hígado sin lugar, en un vaso de precipitados estéril previamente enfriado con 20 ml de medio completo de Guillermo, y luego llevarlo a la campana de cultivo de tejidos de células. </li></ol> 3. El aislamiento celular de los hepatocitos <ol><li> Dentro de la campana de cultivo celular, utilizar un rascador de células para dispersar suavemente las células en medio completo de William dentro de una placa de Petri estéril. </li><li> Se filtra la dispersión de células a través de un poro 100 micras filtro de células de tamaño en un tubo cónico de 50 mL con el fin de eliminar los tejidos conectivos y fragmentos de tejido no digeridas. </li><li> Suspender las células en 40 ml de medio completo de William y centrifugar a 50 xg durante 3 min a 4 ° C. </li><li> Aspirar el sobrenadante, ysuavemente volver a suspender las células en medio completo 40 ml de William frío para lavar las células. Repetir la centrifugación. </li><li> Aspirar el sobrenadante, y suavemente volver a suspender las células con 25 ml de medio completo de William. Añadir 25 ml de solución de Percoll al 90% en PBS en el tubo y mezclar suavemente. </li><li> Centrifugar a 200 xg durante 10 min a 4 ° C. Aspirar las células muertas de la parte superior del gradiente porque las células viables se mantienen en el fondo del gradiente de Percoll. </li><li> Suspender el sedimento celular en 30 ml de medio completo cálida de Guillermo, y luego repetir la centrifugación y vuelva a suspender el pellet de células en 20 ml de medio completo cálida de Guillermo. </li><li> Cuenta las células dentro de la suspensión celular utilizando un hemocitómetro y determinar la viabilidad celular por tinción con azul tripán. </li></ol> 4. Cultivo de hepatocitos <ol><li> Diluir las células con medio completo caliente de William a la concentración preferida, por ejemplo, 2,5 x 10 5 células / ml. Células de placas en un volumen deseado sobre placas de cultivo celular, por ejemplo. 5 x 10 5 células / 2 ml / pocillo, 6 pocillos / placa, o 2,5 x 10 5 células / ml 1,5 / Bueno, 12 pozos / placa. Sin revestir la placa está muy bien para cultivos de células hepáticas. </li><li> En una preparación típica con&gt; 85% células viables, la densidad celular debe alcanzar aproximadamente 60-70% de confluencia, que permite el contacto célula-célula mientras se mantiene un espacio suficiente para los hepatocitos para crecer a su tamaño de la célula completa y producir una confluencia final de 90-95%. </li><li> Con el fin de formar una monocapa incluso de los hepatocitos, en otras palabras, para minimizar la tendencia de las células a agregarse en la zona central del pozo (probablemente debido a la parte interior del flujo de aire de incubadora de células), permiten que las placas permanecen en el interior la campana de cultivo celular durante 30 minutos antes de colocarlos en una incubadora. </li><li> Cultivar las células a 37 ° C en una atmósfera humidificada de 95% de aire y 5% de CO 2. Después de 4-H cultivo, las células o bien podría permanecer en el mismo medio que contenía suero o sustituir el medio con suero-fmedio de REE, por ejemplo, HepatoZYME-SFM (ver Tabla 1). El medio libre de suero ayuda a mantener la morfología de las células sin efectos adversos de las hormonas cuando un medio libre de suero se utiliza. </li><li> Permiten a las células para recuperarse y crecer al menos una noche antes de la experimentación. Recomendamos el uso de las células de prueba dentro de 24 horas ya que esto puede ayudar a preservar la función de enzimas críticas (por ejemplo, P450). </li><li> Sustituir el medio de crecimiento a intervalos de 2 días, si es necesario. </li></ol> 5. Los resultados representativos Las condiciones descritas regularmente generar cosechas de células de 1,0 x 10 8 células por preparación de un hígado de rata. La viabilidad de los hepatocitos medidos por exclusión con azul tripano fue consistentemente dentro del rango de 88 ~ 96%. Como se muestra en la Figura 2, el agregado hepatocitos y clúster forma después de la siembra de 4 horas. Mayoría de las células aisladas se aplanan y se extendió en el crecimiento de monocapa típica. Por 24 horas, los bordes de las células se definen, superficie de las células es bastante suave, y gotas de lípidos son visibles. Las células tienen de uno a tres nucleolos, de forma redonda, situada en el centro de las células, y el tamaño de visualización núcleos coherente entre las células (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Figura 1. Un diagrama de la perfusión del hígado. Un angiocath 18-calibre se inserta en la vena porta del hígado, a continuación, un tubo perfundido está conectado a la aguja. Una vez que la canulación exitosa se confirma, hacer un corte en la vena cava inferior (VCI) para permitir el flujo de salida. <img alt="Figura 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> Figura 2. Morfología de los hepatocitos en cultivo a través del tiempo (4 horas a 24 horas), aumento x 200. Después se cultivaron durante 24 h, las células se extienden en el crecimiento de monocapa típica, y las uniones entre las células son lineales. <table border = &quot;1&quot;&gt; <tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> HBSS (sin Ca 2 + y Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (con Ca 2 + y Mg 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> E medio de Williams </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Colagenasa II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Cell colador (100 m) </td><td> BD </td><td> 352360 </td></tr><tr><td> HepatoZYME-SFM </td><td> Invitrogen </td><td> 17705 </td></tr><tr><td> Percoll </td><td> Sigma </td><td> P4937 </td></tr><tr><td> Angiocath (18-calibre) </td><td> BD </td><td> 381705 </td></tr></tbody></table> Tabla 1.

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J. Cell Biol. 43, 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Saito, K., Kobayashi, K., Mizuno, Y., Fukuchi, Y., Furihata, T., Chiba, K., Schmidt, M., Schmitz, H. J., Baumgart, A., Guedon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+alpha+(PPARalpha)+agonists+induce+constitutive+androstane+receptor+(CAR)+and+cytochrome+P450+2B+in+rat+primary+hepatocytes.">Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists induce constitutive androstane receptor (CAR) and cytochrome P450 2B in rat primary hepatocytes.</a> Drug Metab. Pharmacokinet. 25, 108-111 (2010).</li><li>Gomez-Lechon, M. J., Donato, M. T., Castell, J. V., Jover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatocytes+in+primary+culture:+the+choice+to+investigate+drug+metabolism+in+man.">Human hepatocytes in primary culture: the choice to investigate drug metabolism in man.</a> Curr. Drug Metab. 5, 443-462 (2004).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malar. J. 6, 169 (2007).</li><li>Bu, S. Y., Mashek, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+long-chain+acyl-CoA+synthetase+5+mediates+fatty+acid+channeling+between+anabolic+and+catabolic+pathways.">Hepatic long-chain acyl-CoA synthetase 5 mediates fatty acid channeling between anabolic and catabolic pathways.</a> J. Lipid Res. 51, 3270-3280 (2010).</li><li>Aiken, J., Cima, L., Schloo, B., Mooney, D., Johnson, L., Langer, R., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+rat+liver+perfusion+for+optimal+harvest+of+hepatocytes.">Studies in rat liver perfusion for optimal harvest of hepatocytes.</a> J. Pediatr. Surg. 25, 140-144 (1990).</li><li>Jauregui, H. O., McMillan, P. N., Hevey, K., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+lectin+binding+to+adult+rat+hepatocyte+cell+surfaces.">A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces.</a> In Vitro Cell. 24, 401-412 (1988).</li><li>Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Trump, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+liver+cell+culture.+II.+Primary+culture.">Mouse liver cell culture. II. Primary culture.</a> In Vitro. 17, 926-934 (1981).</li><li>Hay, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operator-induced+contamination+in+cell+culture+systems.">Operator-induced contamination in cell culture systems.</a> Dev. Biol. Stand. 75, 193-204 (1991).</li></ol>

No hay conflictos de interés declarado.

Cultivo primario de hepatocitos es un modelo in vitro utilizado para estudiar diversos aspectos de la fisiología y la patología del hígado. Por ejemplo, la cultura primaria se utiliza para evaluar la expresión y función de las enzimas que metabolizan las drogas incluyendo los citocromos P450, el metabolismo de drogas, las interacciones fármaco-fármaco, y los mecanismos de citotoxicidad y genotoxicidad 3-7. El protocolo que describe el aislamiento y cultivo de las células hepáticas d...

isolation and primary culture of rat hepatic cells

Cultivo de hepatocitos primaria es una herramienta valiosa que ha sido ampliamente utilizado en la investigación básica de la función hepática, la enfermedad, fisiopatología farmacología, y otros temas relacionados. El método basado en la perfusión de colagenasa de dos pasos para el aislamiento de hepatocitos intactos se introdujo por primera vez por Berry y Friend en 1969 1 y, desde entonces, ha sufrido muchas modificaciones. La técnica más utilizada fue descrito por Seglenin 1976 2. Esencialmente, los hepatocitos se disocian de ratas adultas anestesiadas por una perfusión de colagenasa no recirculante a través de la vena porta. Las células aisladas se filtra a través de un filtro de 100 micras de tamaño de poro de malla de nylon, y se cultivaron en placas. Después del cultivo 4-horas, el medio se reemplazó con medio que contenía suero o suero libre, por ejemplo HepatoZYME-MSB, por el tiempo adicional a la cultura. Estos procedimientos requieren pasos quirúrgicos cultura y estéril que puede ser mejor demostrado por el vídeo que por texto. HERE, que documenta los pasos detallados para estos procesos, tanto por video y protocolo escrito, que permiten consistente en la generación de hepatocitos viables en grandes cantidades.

Watch this Scientific Journal Video about Isolation and Primary Culture of Rat Hepatic Cells at JoVE.com

Isolation and Primary Culture of Rat Hepatic Cells

Hepatocitos primarios proporcionará una herramienta valiosa para evaluar las funciones bioquímicas, moleculares y metabólicos en un sistema experimental fisiológicamente relevantes. Se describe un protocolo fiable para la rata en la perfusión del hígado in situ, que constantemente genera hepatocitos viables hasta to1.0 × 10<sup&gt; 8</sup&gt; Células por la preparación con la viabilidad celular entre 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

isolation-and-primary-culture-of-rat-hepatic-cells

Research

JoVE Journal

Biology

55.4K vistas.  University of Cincinnati College of Medicine. Hepatocitos primarios proporcionará una herramienta valiosa para evaluar las funciones bioquímicas, moleculares y metabólicos en un sistema experimental fisiológicamente relevantes. Se describe un protocolo fiable para la rata en la perfusión del hígado in situ, que constantemente genera hepatocitos viables hasta to1.0 × 10 8 Células por la preparación con la viabilidad celular entre 88 ~ 96%.

Video: Isolation and Primary Culture of Rat Hepatic Cells

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

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 ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Aislamiento y cultivo de ratón primarias células acinares pancreáticas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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 ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Cultivo primario de células adrenocorticales rata y análisis de funciones Steroidogenic

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Aislamiento y cultivo de células endoteliales aórticas primarias de cerdos en miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Aislamiento, Transfección y Cultura a Largo Plazo de Cardiomiocitos adultos de ratón y rata

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Aislamiento, cultivo e ingeniería genética de células epiteliales de pigmento primario de mamíferos para terapia génica no viral

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Aislamiento de hepatocitos primarios de rata con control de perfusión multiparamétrico

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...