Os autores gostariam de agradecer ao Sr. Josh Basford e Dr. Xiao Li-min para assistência técnica. Este trabalho foi financiado em parte pelo NIH subvenções (DK70992 e DK92779 ao ML).

1. Preparação Todos os tampões são preparadas de fresco utilizando uma técnica estéril e esterilizado por filtração utilizando um filtro de Corning iM 0,22. <ol><li> Preparar Perfusion tamponar I, adicionando o seguinte para a Solução Salina Equilibrada de Hank (HBSS, sem Ca 2 + e Mg 2 +, ver Tabela 1): Mg 2 + (MgCl2) a 0,9 mM, EDTA a 0,5 mM, e tampão HEPES a 25 mM. </li><li> Preparar tampão de perfusão II pela adição do que se segue para HBSS (com Ca 2 + e Mg 2 +, ver Tabela 1): HEPES a 25 mM. </li><li> Preparar tampão de perfusão II mais collagenaseII: Dissolver colagenase II (1000 U) com 300 ml de Perfusão tampão II e manter a solução quente em banho-maria antes de perfusão. Esta solução deve ser utilizada dentro de 30 min, porque a actividade de colagenase II diminuiu com o tempo. </li><li> Prepare Médio completo William: Adicione o seguintea E Médio Williams &#39;: L-glutamina a 2 mM, soro fetal bovino (FBS) a 5%; de insulina e 100 nm, a dexametasona 100 nM, penicilina a 100 UI / ml e estreptomicina a 100 mg / ml. </li><li> Estes tampões deve ser aquecido durante 30 minutos em banho-maria a 42 ° C, uma temperatura óptima correspondente a uma temperatura de saída na cânula de 37 ° C. </li></ol> 2. Perfusão Rato para Isolamento de Fígado <ol><li> O sistema de perfusão consiste na bomba, tubagem silástica autoclavável e um banho de água (ver Figura 1). Pré-ajustado a taxa de fluxo da bomba peristáltica para perfusão 10 ml / min. </li><li> Anestesiar um rato adulto (300 g de peso corporal) com intraperitoneal (ip) de cetamina (87 mg de peso corporal / kg) mais xilazina (13 mg / kg). Profundidade da anestesia deve ser monitorado por pitada pés. Quando o rato já não responde ao estímulo nocivo, raspar o cabelo abdominal e preparar o abdômen com betadine e etanol. Digite através de uma incisão na linha média. </li><li> Expora veia porta hepática por cuidadosamente movendo as vísceras para o exterior direito da cavidade abdominal, e inserir uma angiocath de 18 gauge para a veia porta hepática (ver Figura 1). </li><li> Ligue o tubo de perfusato à agulha e iniciar a infusão in situ em uma baixa taxa de fluxo (10 ml / min), com pré-aquecido (37 ° C) Tampão I de perfusão. </li><li> Se realizada corretamente, o fígado deve imediatamente começar a branquear. Uma vez que a canulação bem sucedida é confirmada, fazer um corte em veia cava inferior (IVC) para permitir que o efluxo (Figura 1). Um outro teste para a canulação bem sucedida pode ser realizada pela aplicação de pressão de luz com zaragatoa estéril sobre a VCI; todos os lóbulos do fígado deve rapidamente começar a inchar. </li><li> Aumentar a taxa de fluxo de 25 ml / min. O fígado deve tornar-se pálido. </li><li> Alternar a solução de perfusão para perfusão tampão II mais colagenase II sem interrupção do fluxo para um adicional de 6 minutos. </li> &lt;li&gt; periodicamente (5-10 vezes durante a digestão) aplicar pressão com uma compressa para o IVC para intervalos de 5 segundos. O fígado inchará, conduzindo a maior hepática dissociação celular, por sua vez reduz o tempo de digestão total, e aumentando o rendimento final. </li><li> Após a perfusão colagenase, fígado devem começar a olhar piegas. Dissecar o fígado lugar, livre em um copo pré-refrigerados estéril com 20 ml de meio de William completa, e depois levá-la ao tecido capa de cultura de células. </li></ol> 3. Isolamento das células de hepatócitos <ol><li> Dentro do capuz de cultura de células, usar um raspador de células suavemente para dispersar as células em meio completo de William dentro de uma placa de Petri estéril. </li><li> Filtra-se a dispersão célula através de um coador de 100 uM de poro de tamanho de célula dentro de um tubo cónico de 50 ml a fim de remover os tecidos conjuntivos e fragmentos de tecido não digerido. </li><li> Suspender as células em 40 ml de meio completo de William e centrifugar a 50 xg durante 3 min a 4 ° C. </li><li> Aspirar o sobrenadante, edelicadamente ressuspender as células em meio completo 40 ml frio William para lavar células. Repita centrifugação. </li><li> Aspirar o sobrenadante e gentilmente re-suspender as células com 25 ml de meio de William completa. Adicionar 25 ml de solução de Percoll 90% em PBS para dentro do tubo e misturar suavemente. </li><li> Centrifugar a 200 xg durante 10 min a 4 ° C. Aspirar as células mortas da parte superior do gradiente, porque as células viáveis ​​permanecem na parte inferior do gradiente de Percoll. </li><li> Suspender o pellet celular em 30 ml de Meio de William quente completo, e repita centrifugação e volte a suspender o pellet celular em 20 ml de Meio de William quente completo. </li><li> Contar as células dentro da suspensão de células utilizando um hemocitómetro e determinar a viabilidade celular por coloração com azul de tripano. </li></ol> 4. Cultura Hepatócito <ol><li> Diluir as células com meio completo William aquecer a concentração preferida, por exemplo, 2,5 x 10 5 células / ml. As células em placas a um volume desejado, em placas de cultura de células, por exemplo. 5 x 10 5 células / 2 ml / poço, 6 poços / placa, ou 2,5 x 10 5 células / 1,5 mL / poço, 12 poços / placa. Un revestido placa é boa para culturas de células hepáticas. </li><li> Numa preparação típica, com&gt; 85% de células viáveis, a densidade de células deve atingir cerca de 60-70% de confluência, o que permite contacto célula-célula, mantendo espaço suficiente para os hepatócitos a crescer ao seu tamanho de célula completa e produzir um final de confluência 90-95%. </li><li> De modo a formar uma monocamada até mesmo de hepatócitos, por outras palavras, para minimizar a tendência para que as células agregado na área central do poço (mais provavelmente devido ao interior do fluxo de ar de célula incubadora), permitem que as placas de permanecer no interior o capuz de cultura de células durante 30 minutos antes de colocá-los numa incubadora. </li><li> Cultura das células a 37 ° C numa atmosfera humidificada de 95% de ar e 5% de CO 2. Depois de 4-h de cultura, as células podem permanecer em meio contendo soro iguais ou substituir o meio com soro-free médio, por exemplo, HepatoZYME-SFM (ver Tabela 1). O meio isento de soro ajuda a manter a morfologia das células, sem efeitos adversos de hormonas quando um meio isento de soro é utilizado. </li><li> Permitir que as células para recuperar e crescer, pelo menos, durante a noite antes da experimentação. Nós recomendamos usar as células para o teste dentro de 24 horas, pois isso pode ajudar a preservar a função das enzimas essenciais (por exemplo, P450s). </li><li> Substituir o meio de crescimento em intervalos de 2 dias, se necessário. </li></ol> 5. Os resultados representativos As condições descritas regularmente gerar colheitas de células de 1,0 x 10 células por 8 preparação de um fígado de rato. A viabilidade dos hepatócitos medidos por exclusão de azul de tripano foi consistentemente dentro da gama de 88 ~ 96%. Como representado na Figura 2, o agregado hepatócitos e aglomerado forma após a semeadura 4 hr. A maioria das células isoladas achatar e espalhar no crescimento típico monocamada. Por 24 h, as bordas de células são definidas, de superfície de células é bastante suave, e gotículas lipídicas são visíveis. As células têm de um a três nucléolos, de forma redonda, localizadas no centro das células, eo tamanho de exibição núcleos consistente entre as células (Figura 2). <img alt="A Figura 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Figura 1. Um diagrama da perfusão do fígado. Um angiocath de 18 gauge é inserido na veia porta do fígado, em seguida, uma tubagem de perfusato está ligado à agulha. Uma vez canulação sucesso é confirmado, faça um corte na veia cava inferior (VCI) para permitir que o efluxo. <img alt="A Figura 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> Figura 2. Morfologia de hepatócitos em cultura ao longo do tempo (4 hr a 24 hr), a ampliação x 200. Após cultura durante 24 h, as células se espalhar em crescimento em monocamada típico, e as junções entre as células são lineares. <table border = &quot;1&quot;&gt; <tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> HBSS (sem Ca 2 + e Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (com Ca 2 + e Mg 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> E Médio Williams </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Colegenase II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Celular filtro (100 mm) </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 gauge) </td><td> BD </td><td> 381705 </td></tr></tbody></table> Tabela 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>

Não há conflitos de interesse declarados.

Cultura primária de hepatócitos é um modelo in vitro amplamente utilizado para estudar vários aspectos da fisiologia do fígado e da patologia. Por exemplo, a cultura principal é usado para avaliar a expressão e função de enzimas metabolizadoras de drogas de citocromos P450, incluindo metabolismo de drogas, droga-droga e interações, os mecanismos de citotoxicidade e genotoxicidade 3-7. O protocolo descrevendo isolamento e cultura de células hepáticas de ratos é adaptado a partir...

isolation and primary culture of rat hepatic cells

Cultura primária de hepatócitos é uma ferramenta valiosa que tem sido amplamente utilizada na pesquisa básica da função hepática, doença de fisiopatologia, farmacologia e outros assuntos relacionados. O método baseado em duas etapas perfusão colagenase para o isolamento de hepatócitos intactas foi introduzido pela primeira vez por Berry e amigo em 1969 1 e, desde então, passou por muitas modificações. A técnica mais vulgarmente utilizado foi descrito por Seglenin 1976 2. Essencialmente, os hepatócitos são dissociados ratos adultos anestesiados por uma perfusão de colagenase não-recirculação através da veia portal. As células isoladas são então filtrada através de um filtro de tamanho de poro 100 uM de nylon de malha, e cultivadas em placas de. Após 4 horas de cultura, o meio é substituído com meio contendo soro ou isento de soro, por exemplo, HepatoZYME-SFM, por um tempo adicional para a cultura. Esses procedimentos exigem passos cirúrgicos e de cultura estéril, que pode ser melhor demonstrada por vídeo do que pelo texto. Here, nós documentamos os passos detalhados para estes procedimentos por vídeo e protocolo escrito, que permitem de forma consistente na geração de hepatócitos viáveis ​​em grandes números.

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

Hepatócitos primários proporcionar uma ferramenta valiosa para avaliar as funções bioquímicas, moleculares, e metabólicas em um sistema fisiologicamente relevante experimental. Nós descrevemos um protocolo confiável para rato em perfusão de fígado in situ, que sempre gera hepatócitos viáveis ​​até to1.0 × 10<sup&gt; 8</sup&gt; Células por preparação com viabilidade celular entre 88 ~ 96%.

Isolamento e cultura primária de células hepáticas de ratos

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 visualizações.  University of Cincinnati College of Medicine. Hepatócitos primários proporcionar uma ferramenta valiosa para avaliar as funções bioquímicas, moleculares, e metabólicas em um sistema fisiologicamente relevante experimental. Nós descrevemos um protocolo confiável para rato em perfusão de fígado in situ, que sempre gera hepatócitos viáveis ​​até to1.0 × 10 8 Células por preparação com viabilidade celular entre 88 ~ 96%.

Vídeo: 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.

Cultura primária de Miócitos Coração Adulto Rat

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

Biologia

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.

Isolamento de células imunes de tumores primários

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.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

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.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e 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.

Cultura primária de células Adrenocortical rato e ensaios das funções reguladora

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.

Isolamento e cultura de células endoteliais aórticas primárias de suínos em 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.

Isolamento, Transfecção e Cultura de Longo Prazo de Cardiomiócitos de Ratos Adultos e Ratos

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.

Isolamento, Cultura e Engenharia Genética de Células Epiteliais de Pigmento Primário mamífero para terapia genética não 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.

Isolamento de Hepatócitos Primários de Ratos com Controle de Perfusão Multiparâmetro

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