Die Autoren danken Herrn Dr. Josh Basford und Xiao-Min Li, um technische Unterstützung zu danken. Diese Arbeit wurde zum Teil durch Zuschüsse NIH (DK70992 DK92779 und zu ML) unterstützt.

1. Vorbereitung Alle Puffer werden frisch zubereitet und mit steriler Technik und sterilisiert mit einem Corning 0,22 um Filter zu filtern. <ol><li> Bereiten Perfusionspuffer ich, indem Sie folgende an Hanks Balanced Salt Solution (HBSS ohne Ca 2 + und Mg 2 +, siehe Tabelle 1): Mg 2 + (MgCl 2) bis 0,9 mm, EDTA bis 0,5 mm, und HEPES bis 25 mM. </li><li> Bereiten Perfusionspuffer II, indem Sie folgende an HBSS (mit Ca 2 + und Mg 2 +, siehe Tabelle 1): 25 mM HEPES zu. </li><li> Bereiten Perfusionspuffer II plus collagenaseII: Lösen Sie Kollagenase II (1000 U) mit 300 ml Perfusionspuffer II und hält die Lösung in warmen Wasserbad vor Perfusion. Diese Lösung sollte innerhalb von 30 Minuten verwendet werden, da die Aktivität der Kollagenase II nahm mit der Zeit. </li><li> Bereiten Williams komplette Medium: Fügen Sie die folgendeWilliams &#39;Medium E: L-Glutamin zu 2 mM, fötales Rinderserum (FBS) bis 5%, Insulin bis 100 nM bis 100 nM Dexamethason, Penicillin und 100 IU / ml und Streptomycin bis 100 mg / ml. </li><li> Diese Puffer sind für 30 Minuten im Wasserbad bei 42 ° C erwärmt werden, eine optimale Temperatur entsprechend einer Austrittstemperatur an der Kanüle von 37 ° C </li></ol> 2. Ratten-Leber-Perfusion für Isolation <ol><li> Die Perfusion System besteht aus der Pumpe, autoklavierbar Silasticschlauch und einem Wasserbad (siehe Abbildung 1). Voreinstellung der Durchflussmenge der peristaltischen Perfusionspumpe bis 10 ml / min. </li><li> Anesthetize einer erwachsenen Ratte (300 g Körpergewicht) mit intraperitoneale (ip) Ketamin (87 mg / kg Körpergewicht) plus Xylazin (13 mg / kg). Narkosetiefe sollte von Zeh Prise überwacht werden. Wenn die Ratte reagiert nicht mehr auf schädliche Reize, rasieren, Haare und Bauch-prep der Bauch mit Betadine und Ethanol. Geben Sie durch einen Einschnitt an der Mittellinie. </li><li> Aussetzendie Pfortader durch vorsichtiges Bewegen der Eingeweide nach rechts außerhalb der Bauchhöhle, und fügen Sie eine 18-Gauge-Angiocath in die Pfortader (siehe Abbildung 1). </li><li> Schließen Sie das Perfusat Schlauch an der Nadel und starten Sie die Infusion in situ bei einer niedrigen Flussrate (10 ml / min) mit vorgewärmten (37 ° C) Perfusionspuffer ich. </li><li> Wenn richtig ausgeführt, sollte die Leber sofort beginnen zu blanchieren. Nach erfolgreicher Kanülierung bestätigt wird, machen einen Schnitt am unteren Hohlvene (IVC) zu Efflux (Abbildung 1) zu ermöglichen. Ein weiterer Test für die erfolgreiche Kanülierung kann durch leichten Druck mit einem sterilen Tupfer auf die IVC durchgeführt werden, alle Leberlappen sollten schnell anfangen zu schwellen. </li><li> Erhöhen der Strömungsgeschwindigkeit auf 25 ml / min. Die Leber sollte blass in der Farbe. </li><li> Schalten Sie die Perfusionslösung zu Perfusionspuffer II plus Kollagenase II ohne Unterbrechung der Strom für weitere 6 Minuten. </li> &lt;li&gt; In regelmäßigen Abständen (5-10 mal während der Verdauung) Druck ausüben mit Tupfer auf die IVC für die 5-Sekunden-Intervallen. Die Leber wird anschwellen, was zu erhöhten Leber-Zell-Dissoziation, die wiederum reduziert gesamte Dauer der Verdauung und die Erhöhung Endausbeute. </li><li> Nach Kollagenase Perfusion, sollte Leber beginnen, sich matschig. Sezieren die Leber frei, in eine vorgekühlte sterile Becher mit 20 ml Vollmedium Williams, und dann bringen Sie es zum Gewebezellkultur Kapuze. </li></ol> 3. Hepatocyte Zellisolation <ol><li> Innerhalb der Zellkultur Haube, mit einem Zellschaber vorsichtig dispergieren die Zellen in vollständigem Medium Williams in einer sterilen Petrischale. </li><li> Filtern Zelldispersion durch ein 100 um Porengröße Zelle Sieb in eine 50 ml konisches Rohr, um das Bindegewebe und unverdauten Gewebe-Fragmente zu entfernen. </li><li> Suspend-Zellen in 40 ml Vollmedium Williams und bei 50 Zentrifuge × g für 3 min bei 4 ° C </li><li> Den Überstand aspirieren undvorsichtig resuspendieren Zellen in Vollmedium 40 ml kaltem Williams, um Zellen zu waschen. Wiederholen Sie die Zentrifugation. </li><li> Saugen Sie den Überstand und resuspendieren sanft Zellen mit 25 ml Vollmedium Williams. 25 ml 90% Percoll-Lösung in PBS in die Röhre und vorsichtig mischen. </li><li> Zentrifugieren bei 200 xg für 10 min bei 4 ° C Absaugen toten Zellen aus dem oberen Ende des Gradienten, da die lebensfähigen Zellen am Boden der Percoll-Gradienten bleiben. </li><li> Hängen Sie das Zellpellet in 30 ml warmem Vollmedium Williams, und wiederholen Sie dann Zentrifugation und Resuspendieren des Zellpellets in 20 ml warmem Vollmedium Williams. </li><li> Zählen der Zellen in der Zellsuspension unter Verwendung eines Hämozytometers und bestimmen die Lebensfähigkeit der Zellen durch Trypan-Blau-Färbung. </li></ol> 4. Hepatozytenkultur <ol><li> Verdünnte Zellen mit Vollmedium warmen Williams auf bevorzugte Konzentration, z. B. 2,5 × 10 5 Zellen / ml. Tafel Zellen bei einer gewünschten Volumen auf Zellkulturplatten, zB. 5 x 10 5 Zellen / 2 ml / Well, 6 Vertiefungen / Platte, oder 2,5 x 10 5 Zellen / 1,5 ml / gut, 12 Vertiefungen / Platte. Un-beschichtete Platte ist für Leber-Zellkulturen in Ordnung. </li><li> In einem typischen Prep mit&gt; 85% lebensfähige Zellen, sollte die Zelldichte erreichen etwa 60-70% Konfluenz, die für die Zell-Zell-Kontakt ermöglicht und gleichzeitig genügend Raum für die Hepatozyten bis zu ihrer vollen Größe der Zellen wachsen, und eine endgültige Zusammenfluss von 90-95%. </li><li> Um eine gleichmäßige Monoschicht von Hepatozyten zu bilden, mit anderen Worten, die Tendenz zu aggregieren Zellen zu minimieren im zentralen Bereich der Wanne (wahrscheinlich aufgrund der Luft-Strömung innerhalb des Zellinkubator), dass die Platten im Inneren bleiben die Zellkultur Haube für 30 Minuten, bevor Sie sie in einem Brutkasten. </li><li> Kultur wurden die Zellen bei 37 ° C in einer befeuchteten Atmosphäre von 95% Luft und 5% CO 2. Nach 4-stündiger Kultur konnte die Zellen entweder in dem gleichen Serum enthaltenden Medium bleiben oder zu ersetzen mit Serum-free Medium, zB HepatoZYME-SFM (siehe Tabelle 1). Das serumfreie Medium können die Zellmorphologie ohne negative Auswirkungen von Hormonen, wenn ein Serum-freies Medium verwendet wird, aufrechtzuerhalten. </li><li> Erlauben Zellen zu erholen und wachsen mindestens über Nacht vor dem Experiment. Wir empfehlen, um die Zellen für den Test innerhalb von 24 Stunden zu verwenden, da dies zur Erhaltung der Funktion des kritischen Enzyme (z. B. P450). </li><li> Ersetzen des Wachstumsmediums Zeitabständen von 2 Tagen, wenn nötig. </li></ol> 5. Repräsentative Ergebnisse Die beschriebenen Bedingungen regelmäßig zu generieren Zelle Ernten von 1,0 x 10 8 Zellen pro Präparat aus einer Rattenleber. Die Lebensfähigkeit der Hepatozyten durch Trypanblau-Ausschluss gemessen war durchweg im Bereich von 88 ~ 96%. Wie in 2, die Hepatozyten zusammen und bilden Cluster nach 4 Stunden Seeder dargestellt. Die meisten isolierten Zellen abflachen und in typischen Monolage Wachstum verteilt. Von 24 Stunden, die Kanten der Zellen definiert werden, die Oberfläche von Zellen ziemlich glatt, und Lipidtröpfchen sind. Die Zellen haben ein bis drei Nucleoli, die rund sind, in der Mitte der Zellen befindet, und die Kerne Anzeige einheitliche Größe zwischen den Zellen (2). <img alt="1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Abbildung 1. Ein Diagramm der Leberperfusion. Eine 18-Gauge-Angiocath in die Pfortader der Leber eingeführt wird, so wird ein Perfusat Schlauch mit der Nadel verbunden. Nach erfolgreicher Kanülierung bestätigt wird, machen einen Schnitt am unteren Hohlvene (IVC), damit Efflux. <img alt="2" src="/files/ftp_upload/3917/3917fig2.jpg" /> 2. Morphologie der kultivierten Hepatocyten im Laufe der Zeit (4 Stunde bis 24 Stunden), Vergrößerung 200. Nach 24 Stunden lang kultiviert, die Zellen in typischen Monoschicht Wachstum verbreitet, und die Verbindungen zwischen den Zellen linear sind. <table border = &quot;1&quot;&gt; <tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> HBSS (ohne Ca 2 + und Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (mit Ca 2 + und Mg 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> Williams &#39;Medium E </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Zellsieb (100 um) </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> Tabelle 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>

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Primärkultur von Hepatozyten ist ein in vitro Modell in großem Maße verwendet, um verschiedene Aspekte der Leber Physiologie und Pathologie zu studieren. Zum Beispiel wird Primärkultur verwendet, um die Expression und Funktion der Wirkstoff-Enzyme einschließlich Cytochrom P450, Arzneimittel-Metabolismus, Wechselwirkungen zwischen Medikamenten, und die Mechanismen der Zytotoxizität und Genotoxizität 3-7 zu bewerten. Das Protokoll beschreibt die Isolierung und Kultur von Ratten-Leberzellen ausge...

isolation and primary culture of rat hepatic cells

Primäre Hepatozyten-Kultur ist ein wertvolles Werkzeug, die ausgiebig in der Grundlagenforschung der Leberfunktion, Krankheiten, Pathophysiologie, Pharmakologie und anderer verwandter Fächer eingesetzt. Das Verfahren auf Zwei-Schritt-Kollagenase-Perfusion für die Isolation von intakten Hepatozyten Basis wurde erstmals von Berry und Freund in 1969 1 eingeführt und seit damals hat viele Veränderungen durchgemacht. Das am häufigsten verwendete Technik wurde von Seglenin 1976 2 beschrieben. Im Wesentlichen sind Hepatozyten getrennt von erwachsenen Ratten betäubt durch eine nicht rezirkulierenden Kollagenase Perfusion durch die Pfortader. Die isolierten Zellen werden dann durch ein 100 um Porengröße Mesh Nylonfilter filtriert und auf Platten kultiviert. Nach 4-Stunden-Kultur wird das Medium mit serumhaltigem oder serumfreiem Medium, z. B. HepatoZYME-SFM, für eine längere Kultur ersetzt. Diese Verfahren erfordern chirurgische und sterile Kultur Schritte, die besser von Video werden als durch Text kann unter Beweis gestellt. Here, dokumentieren wir die detaillierten Schritte für diese Verfahren sowohl von Video-und schriftlichen Protokoll, das konsequent erlauben bei der Erzeugung von lebensfähigen Hepatozyten in großen Stückzahlen.

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

Primäre Hepatozyten ein wertvolles Instrument, um biochemische, molekulare und metabolische Funktionen in einem physiologisch relevanten experimentellen Systems zu evaluieren. Wir beschreiben ein zuverlässiges Protokoll für Ratte in situ Leberperfusion, die konsequent erzeugt lebensfähigen Hepatozyten bis bis 1,0 × 10<sup&gt; 8</sup&gt; Zellen pro Präparat mit der Lebensfähigkeit der Zellen zwischen 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

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 Aufrufe.  University of Cincinnati College of Medicine. Primäre Hepatozyten ein wertvolles Instrument, um biochemische, molekulare und metabolische Funktionen in einem physiologisch relevanten experimentellen Systems zu evaluieren. Wir beschreiben ein zuverlässiges Protokoll für Ratte in situ Leberperfusion, die konsequent erzeugt lebensfähigen Hepatozyten bis bis 1,0 × 10 8 Zellen pro Präparat mit der Lebensfähigkeit der Zellen zwischen 88 ~ 96%.

Serie: 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.

Primäre Kultur der erwachsenen Ratte Herz Myozyten

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

Forschung

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.

Isolierung von Immunzellen aus Primärtumoren

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.

Isolierung und Kultivierung von primären Maus Azinuszellen des Pankreas

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.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

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.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

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.

Primärkultur von Ratte Nebennierenrindeninsuffizienz Zellen und Assays Steroidogenic Funktionen

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.

Isolation und Kultur der primären Aorten-Endothelzellen von Miniaturschweinen

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, Transfektion und Langzeitkultur von Erwachsenenmaus und Rattenkardiomyozyten

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.

Isolierung, Kultur und Gentechnik von primären Pigmentepithelzellen von Säugetieren für die nicht-virale Gentherapie

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.

Isolierung primärer Rattenhepatozyten mit Multiparameter-Perfusionskontrolle

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