Les auteurs tiennent à remercier M. Josh Basford et le Dr Xiao-Min Li pour l&#39;assistance technique. Ce travail a été soutenu en partie par des subventions du NIH (DK70992 DK92779 et à ML).

1. Préparation Tous les tampons sont fraîchement préparés en utilisant une technique stérile et stérilisé par filtration à l&#39;aide d&#39;un filtre Corning um 0,22. <ol><li> Préparer perfusion je tampon en ajoutant ce qui suit à la solution saline équilibrée de Hank (HBSS, sans Ca 2 + et Mg 2 +, voir le tableau 1): Mg 2 + (MgCl 2) à 0,9 mM, EDTA à 0,5 mM, et HEPES à 25 mM. </li><li> Préparer un tampon de perfusion II en y ajoutant ce qui suit à HBSS (avec Ca 2 + et Mg 2 +, voir le tableau 1): HEPES à 25 mM. </li><li> Préparer un tampon de perfusion II plus collagenaseII: Dissoudre la collagénase II (1000 U) avec 300 ml de tampon de perfusion II et de garder la solution chaude dans l&#39;eau dans la baignoire avant la perfusion. Cette solution doit être utilisée dans les 30 minutes parce que l&#39;activité de la collagénase II, a diminué avec le temps. </li><li> Préparer le milieu complet de Guillaume: Ajoutez la ligne suivanteà E moyen Williams: L-glutamine 2 mM, sérum de veau fœtal (FBS) à 5%, l&#39;insuline à 100 nM, la dexaméthasone à 100 nm, la pénicilline à 100 UI / ml et de streptomycine à 100 mg / ml. </li><li> Ces tampons doivent être chauffés pendant 30 minutes dans le bain-marie à 42 ° C, une température optimale correspondant à une température de sortie à la canule de 37 ° C. </li></ol> 2. Perfusion Rat pour l&#39;isolement du foie <ol><li> Le système de perfusion se compose de la pompe, autoclavable tube silastic et un bain d&#39;eau (voir Figure 1). Pré-régler le débit de la pompe de perfusion péristaltique à 10 ml / min. </li><li> Anesthésier un rat adulte (300 g de poids corporel) avec intrapéritonéale (ip) kétamine (87 mg de poids corporel / kg) ainsi que la xylazine (13 mg / kg). Profondeur de l&#39;anesthésie doit être surveillée par pincement de l&#39;orteil. Lorsque le rat ne répond plus aux stimulus nociceptif, raser les poils du ventre et de préparation de l&#39;abdomen avec de la Bétadine et l&#39;éthanol. Entrez par une incision médiane. </li><li> Exposerla veine porte hépatique en prenant soin de déplacer le viscères à l&#39;extérieur, à droite de la cavité abdominale, et insérer une angiocath de calibre 18 dans la veine porte hépatique (voir Figure 1). </li><li> Raccordez le tuyau perfusat à l&#39;aiguille et de lancer la perfusion in situ à un débit faible (10 ml / min) avec pré-chauffée (37 ° C) tampon de perfusion je. </li><li> S&#39;il est effectué correctement, le foie doit instantanément commencer à blanchir. Une fois canulation réussie est confirmée, faire une coupe à la veine cave inférieure (VCI) pour permettre à efflux (Figure 1). Un autre test pour la canulation réussie peut être réalisée en appliquant une légère pression avec un tampon stérile sur la veine cave inférieure; tous les lobes du foie devraient rapidement commencer à gonfler. </li><li> Augmenter le débit à 25 ml / min. Le foie doit être de couleur pâle. </li><li> Mettez la solution de perfusion à la perfusion de tampon II, plus de la collagénase II sans interruption de la circulation pendant 6 minutes supplémentaires. </li> &lt;li&gt; périodiquement (5-10 fois lors de la digestion) appliquer une pression avec un tampon à l&#39;IVC pour 5 secondes d&#39;intervalle. Le foie se gonflent, conduisant à la dissociation accrue des cellules hépatiques, à son tour réduire le temps de digestion totale, et en augmentant le rendement final. </li><li> Après perfusion de collagénase, le foie doit commencer à regarder en bouillie. Disséquer le foie libre, dans un bécher de pré-réfrigérés stérile avec 20 ml de milieu complet de Guillaume, puis le prendre à la hotte de la culture des tissus cellulaires. </li></ol> 3. Isolement des hépatocytes <ol><li> Dans la hotte de culture cellulaire, utiliser un grattoir à cellules en douceur disperser les cellules dans un milieu complet de Guillaume dans un boîte de Pétri stérile. </li><li> Filtrer la dispersion des cellules à travers une 100 um taille de cellule crépine pores dans un tube conique de 50 ml pour retirer les tissus conjonctifs et des fragments de tissus non digérées. </li><li> Suspendre les cellules dans 40 ml de milieu complet de William et centrifuger à 50 xg pendant 3 min à 4 ° C. </li><li> Aspirer le surnageant etdoucement remis en suspension les cellules dans un milieu complet 40 ml froid William à laver les cellules. Répétez centrifugation. </li><li> Aspirer le surnageant, et doucement remis en suspension les cellules avec 25 ml de milieu complet de William. Ajouter 25 ml de solution de Percoll 90% dans du PBS dans le tube et mélanger délicatement. </li><li> Centrifuger à 200 xg pendant 10 min à 4 ° C. Aspirer les cellules mortes de la partie supérieure du gradient parce que les cellules viables restent au bas de l&#39;gradient de Percoll. </li><li> Suspendre le culot cellulaire dans 30 ml de milieu chaud William complète, puis répétez la centrifugation et remettre en suspension le culot cellulaire dans 20 ml de milieu chaud William complète. </li><li> Compter les cellules dans la suspension cellulaire à l&#39;aide d&#39;un hémocytomètre et de déterminer la viabilité cellulaire par coloration au bleu trypan. </li></ol> 4. Culture des hépatocytes <ol><li> Diluer les cellules avec du milieu complet chaude de William à la concentration préférée, par exemple 2,5 x 10 5 cellules / ml. Cellules de la plaque à un volume souhaité sur plaques de culture cellulaire, par exemple. 5 x 10 5 cellules / 2 ml / puits, 6 puits / plaque; ou 2,5 x 10 5 cellules / 1,5 ml / puits, 12 puits / plaque. Un plaque revêtue est très bien pour des cultures de cellules hépatiques. </li><li> Dans une préparation typique avec&gt; 85% de cellules viables, la densité cellulaire devrait atteindre environ 60-70% de confluence, ce qui permet de contact cellule-cellule tout en conservant suffisamment d&#39;espace pour les hépatocytes de croître à leur taille de la cellule complète et donner un confluent de la finale 90-95%. </li><li> Afin de former une monocouche même des hépatocytes, en d&#39;autres termes, pour réduire au minimum la tendance pour les cellules à se regrouper dans la zone centrale du bien (probablement dû à l&#39;intérieur de l&#39;air de la cellule incubateur), permettre aux plaques de rester à l&#39;intérieur le capot de culture cellulaire pendant 30 minutes avant de les placer dans un incubateur. </li><li> Culture des cellules à 37 ° C dans une atmosphère humidifiée d&#39;air de 95% et 5% de CO 2. Après 4-h culture, les cellules peuvent soit rester dans le même milieu contenant du sérum ou de remplacer le milieu avec sérum-free moyenne, par exemple HepatoZYME-GDF (voir tableau 1). Le milieu sans sérum aide à maintenir la morphologie des cellules, sans effets néfastes d&#39;hormones quand un milieu sans sérum est utilisé. </li><li> Laisser les cellules de récupérer et de se développer au moins une nuit avant l&#39;expérimentation. Nous vous recommandons d&#39;utiliser les cellules pour le test dans les 24 heures parce que cela peut aider à préserver la fonction des enzymes essentiels (par exemple, P450). </li><li> Remplacez le milieu de croissance à 2 jours d&#39;intervalle, si nécessaire. </li></ol> 5. Les résultats représentatifs Les conditions décrites générer régulièrement les récoltes de cellules de 1,0 x 10 8 cellules par la préparation de l&#39;un foie de rat. La viabilité des hépatocytes mesurées par exclusion du bleu trypan a été constamment au sein de la gamme de 88 ~ 96%. Comme le montre la figure 2, l&#39;ensemble des hépatocytes et de cluster sous forme après 4 h d&#39;ensemencement. La plupart des cellules isolées aplatir et la propagation de la croissance monocouche typique. Par 24 h, les bords de cellules sont définis, la surface de cellules est relativement lisse, et les gouttelettes lipidiques sont visibles. Les cellules ont un à trois nucléoles, qui sont ronds, situés au centre des cellules, et la taille d&#39;affichage des noyaux cohérente entre les cellules (Figure 2). <img alt="Figure 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Figure 1. Un diagramme de la perfusion du foie. Un angiocath de calibre 18 est inséré dans la veine porte du foie, un tube perfusat est relié à l&#39;aiguille. Une fois canulation réussie est confirmée, faire une coupe à la veine cave inférieure (VCI) pour permettre à efflux. <img alt="Figure 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> Figure 2. Morphologie des hépatocytes cultivés au fil du temps (4 h à 24 h), grossissement x 200. Après culture pendant 24 heures, les cellules réparties de la croissance monocouche typique, et les jonctions entre les cellules sont linéaires. <table border = &quot;1&quot;&gt; <tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td></tr><tr><td> HBSS (sans Ca 2 + et Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (avec Ca 2 + et Mg 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> Milieu E Williams </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Cellule de filtrage (100 um) </td><td> BD </td><td> 352360 </td></tr><tr><td> HepatoZYME-GDF </td><td> Invitrogen </td><td> 17705 </td></tr><tr><td> Percoll </td><td> Sigma </td><td> P4937 </td></tr><tr><td> Angiocath (calibre 18) </td><td> BD </td><td> 381705 </td></tr></tbody></table> Tableau 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>

Pas de conflits d&#39;intérêt déclarés.

Culture primaire d&#39;hépatocytes est un modèle in vitro largement utilisée pour étudier divers aspects de la physiologie et la pathologie du foie. Par exemple, la culture principale est utilisée pour évaluer l&#39;expression et la fonction de enzymes métabolisant les médicaments, y compris cytochromes P450, métabolisme des médicaments, des interactions médicamenteuses, et les mécanismes de cytotoxicité et la génotoxicité 3-7. Le protocole décrivant l&#39;isolement et la cul...

isolation and primary culture of rat hepatic cells

La culture des hépatocytes primaires est un outil précieux qui a été largement utilisé dans la recherche fondamentale de la fonction hépatique, la maladie, la physiopathologie, la pharmacologie et autres sujets connexes. La méthode basée sur la perfusion de collagénase en deux étapes pour l&#39;isolement des hépatocytes intacts a été introduite par Berry et ami en 1969 1 et, depuis lors, a subi de nombreuses modifications. La technique la plus couramment utilisée a été décrite par Seglenin 1976 2. Essentiellement, les hépatocytes sont dissociées de rats adultes anesthésiés par une perfusion de collagénase non-recirculation par la veine porte. Les cellules isolées sont ensuite filtrée à travers un 100 um de taille des pores du filtre en nylon de maille, et mis en culture sur des plaques. Après 4 heures de culture, le milieu est remplacé par du milieu contenant du sérum ou sans sérum, par exemple HepatoZYME-GDF, un délai supplémentaire pour la culture. Ces procédures nécessitent des étapes de la culture chirurgicales et stérile qui peut être mieux démontrée par la vidéo que par le texte. Havant, nous documentons les étapes détaillées pour ces procédures à la fois par la vidéo et protocole écrit, ce qui permet de manière cohérente dans la génération des hépatocytes viables dans de grands nombres.

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

Hépatocytes primaires constituent un outil précieux pour évaluer biochimiques, moléculaires fonctions, et métabolique dans un système physiologiquement pertinente expérimentale. Nous décrivons un protocole fiable pour le rat en perfusion du foie in situ, ce qui génère constamment hépatocytes viables jusqu&#39;à à 1,0 × 10<sup&gt; 8</sup&gt; Cellules par la préparation avec la viabilité des cellules entre 88 ~ 96%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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

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55.4K vues.  University of Cincinnati College of Medicine. Hépatocytes primaires constituent un outil précieux pour évaluer biochimiques, moléculaires fonctions, et métabolique dans un système physiologiquement pertinente expérimentale. Nous décrivons un protocole fiable pour le rat en perfusion du foie in situ, ce qui génère constamment hépatocytes viables jusqu'à à 1,0 × 10 8 Cellules par la préparation avec la viabilité des cellules entre 88 ~ 96%.

Vidéo: 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.

Culture primaire des adultes myocytes cœur de rat

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

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Biologie

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.

Isolement des cellules immunitaires des tumeurs primitives

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.

Isolement et culture de cellules primaires de souris acineuses du pancréas

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.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

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.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

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.

Culture primaire de cellules Corticosurrénaliennes Rat et tests des fonctions Stéroïdogéniques

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.

Isolement et culture des cellules endothéliales aortiques primaires des porcs miniatures

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.

Isolation, transfection et culture à long terme des cardiomyocytes adultes de souris et de rats

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.

Isolement, culture et génie génétique de cellules épithéliales pigmentaires primaires de mammifères pour la thérapie génique non virale

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.

Isolement des hépatocytes primaires de rat avec contrôle de perfusion multiparamétrique

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