Gli autori desiderano ringraziare il Sig. Josh Basford e il dottor Xiao Li-min per l&#39;assistenza tecnica. Questo lavoro è stato sostenuto in parte da sovvenzioni NIH (DK70992 e DK92779 alla ML).

1. Preparazione Tutti i buffer sono preparati al momento utilizzando una tecnica sterile e sterilizzato per filtrazione con un filtro di 0,22 micron Corning. <ol><li> Preparare perfusione Buffer I aggiungendo la seguente soluzione salina bilanciata di Hank (HBSS, senza Ca 2 + e Mg 2 +, vedi Tabella 1): Mg 2 + (MgCl 2) a 0,9 mM, EDTA a 0,5 mM e HEPES a 25 mM. </li><li> Preparare perfusione tampone II aggiungendo il seguente HBSS (con Ca 2 + e Mg 2 +, vedi Tabella 1): HEPES a 25 mM. </li><li> Preparare perfusione tampone II plus collagenaseII: Sciogliere collagenasi II (1000 U), 300 ml di perfusione tampone II e conservare la soluzione in acqua calda-bagno prima di perfusione. Questa soluzione deve essere utilizzata entro 30 minuti perché l&#39;attività della collagenasi II è diminuito con il tempo. </li><li> Preparare terreno completo William: Aggiungere il seguenteE a media Williams &#39;: L-glutammina 2 mM, siero bovino fetale (FBS) al 5%, a 100 nM insulina, desametasone e 100 nm, penicillina 100 IU / ml e streptomicina a 100 mg / ml. </li><li> Questi buffer deve essere riscaldata per 30 minuti in bagno d&#39;acqua a 42 ° C, una temperatura ottimale corrispondente ad una temperatura di uscita alla cannula di 37 ° C. </li></ol> 2. Perfusione Rat Liver per l&#39;isolamento <ol><li> Il sistema di perfusione costituito dalla pompa, autoclavabile tubazione di silastic e un bagno di acqua (vedi Figura 1). Pre-impostare la portata della pompa peristaltica perfusione a 10 ml / min. </li><li> Anestetizzare un ratto adulto (300 g di peso corporeo) con intraperitoneale (ip) ketamina (87 mg / kg di peso corporeo) più xilazina (13 mg / kg). Profondità dell&#39;anestesia deve essere monitorata mediante pizzico punta. Quando il topo non risponde più agli stimoli nocivi, radersi i capelli addominale e preparazione l&#39;addome con Betadine ed etanolo. Inserisci attraverso una incisione mediana. </li><li> Esporrela vena portale epatica attentamente spostando il visceri all&#39;esterno destro della cavità addominale, ed inserire un 18-gauge angiocath nella vena porta (vedi Figura 1). </li><li> Collegare il tubo perfusato all&#39;ago e avviare l&#39;infusione in situ a una portata bassa (10 ml / min) con pre-riscaldato (37 ° C) Buffer I perfusione. </li><li> Se eseguita correttamente, il fegato deve immediatamente iniziare a sbollentare. Una volta cannulazione successo è confermata, effettuare un taglio a vena cava inferiore (IVC) per consentire efflusso (Figura 1). Un ulteriore test per la cannulazione di successo può essere eseguita applicando una leggera pressione con un tampone sterile sulla IVC, tutti i lobi del fegato dovrebbe iniziare rapidamente a gonfiarsi. </li><li> Aumentare la velocità del flusso di 25 ml / min. Il fegato dovrebbe diventare di colore pallido. </li><li> Passare il liquido di perfusione di perfusione tampone II plus collagenasi II senza interruzione del flusso per altri 6 minuti. </li> &lt;li&gt; Periodicamente (5-10 volte durante la digestione) applicare una pressione con un tampone alla IVC per intervalli di 5 secondi. Il fegato si gonfiano, che porta alla dissociazione maggiore epatica cella, a sua volta riduzione del tempo totale di digestione, e aumentando resa finale. </li><li> Dopo la perfusione collagenasi, fegato dovrebbe iniziare a guardare molle. Sezionare il libero fegato, posto in un pre-refrigerato beaker sterile con 20 ml di terreno completo William, e poi portarla al cofano coltura cellulare dei tessuti. </li></ol> 3. Cella di isolamento degli epatociti <ol><li> All&#39;interno del cofano coltura cellulare, usare un raschietto cella per disperdere delicatamente le cellule in terreno completo di William all&#39;interno di una piastra di Petri sterile. </li><li> Filtrare la dispersione cellulare attraverso un filtro 100 dimensione dei pori cella um in una provetta conica da 50 ml per rimuovere tessuto connettivo e frammenti di tessuto non digerito. </li><li> Sospendere cellule in 40 ml di mezzo completo William e centrifugare a 50 xg per 3 min a 4 ° C. </li><li> Aspirare il supernatante, edelicatamente risospendere le cellule in mezzo completo 40 ml freddo di William per lavare le cellule. Ripetere centrifugazione. </li><li> Aspirare il surnatante e risospendere delicatamente le cellule con 25 ml di terreno completo di William. Aggiungere 25 ml di soluzione di Percoll 90% in PBS nel tubo e mescolare delicatamente. </li><li> Centrifugare a 200 xg per 10 min a 4 ° C. Aspirare le cellule morte dalla parte superiore del gradiente perché le cellule vitali rimanere sul fondo del gradiente Percoll. </li><li> Sospendere il pellet di cellule in 30 ml caldo terreno completo di William, e poi ripetere la centrifugazione e risospendere il pellet cellulare in 20 ml caldo terreno completo di William. </li><li> Contare le cellule all&#39;interno della sospensione cellulare utilizzando un emocitometro e determinare la vitalità cellulare mediante colorazione con blu tripano. </li></ol> 4. Cultura degli epatociti <ol><li> Diluire le cellule con mezzo completo caldo William a concentrazione preferita, ad esempio 2,5 x 10 5 cellule / ml. Celle di dischi ad un volume desiderato su piastre di coltura cellulare, per esempio. 5 x 10 5 cellule / 2 ml / pozzetto, 6 pozzetti / piastra, o 2,5 x 10 5 cellule / 1,5 ml / pozzetto, 12 pozzetti / piastra. Un rivestita con lastra è bene per colture di cellule epatiche. </li><li> In una preparazione tipica con&gt; 85% cellule vitali, la densità cellulare dovrebbe raggiungere circa 60-70% di confluenza, che consente di contatto cellula-cellula mantenendo uno spazio sufficiente per gli epatociti crescere delle dimensioni delle cellule piena e dare un finale di confluenza 90-95%. </li><li> Al fine di formare un monostrato anche di epatociti, in altre parole, per minimizzare la tendenza per le cellule di aggregazione nella zona centrale del pozzo (più probabilmente dovuta al flusso d&#39;aria all&#39;interno della cella incubatore), le piastre consentono di rimanere all&#39;interno la cultura cappa cellulare per 30 minuti prima di metterle in una incubatrice. </li><li> Coltivare le cellule a 37 ° C in atmosfera umidificata al 95% aria e 5% CO 2. Dopo 4 h-coltura, le cellule potrebbero o rimanere nello stesso medium contenente siero o sostituire il mezzo con siero-free media, ad esempio HepatoZYME-SFM (vedi Tabella 1). Il mezzo privo di siero aiuta a mantenere la morfologia delle cellule senza effetti negativi da ormoni quando un terreno privo di siero viene utilizzato. </li><li> Consentono alle cellule di recuperare e crescere almeno per una notte prima della sperimentazione. Si consiglia di utilizzare le cellule per il test entro 24 ore perché questo può aiutare a preservare la funzione degli enzimi critici (ad esempio, P450). </li><li> Sostituire il mezzo di crescita a intervalli di 2 giorni, se necessario. </li></ol> 5. Risultati rappresentativi Le condizioni descritte generare periodicamente raccolti cellule di 1.0 x 10 8 cellule per la preparazione di un fegato di ratto. La vitalità degli epatociti misurata mediante esclusione con Trypan blu era costantemente all&#39;interno dell&#39;intervallo di 88 ~ 96%. Come illustrato in figura 2, l&#39;aggregato epatociti e cluster di forma dopo 4 ore la semina. Cellule più isolate appiattire e diffuso in crescita monostrato tipica. Entro 24 ore, i bordi delle cellule sono definite, superficie delle cellule è abbastanza regolare, e goccioline lipidiche sono visibili. Le cellule hanno da uno a tre nucleoli, di forma rotonda, situati al centro delle cellule, e la dimensione di visualizzazione nuclei coerente tra cellule (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Figura 1. Un diagramma di perfusione del fegato. Una 18-gauge angiocath viene inserito nella vena porta di fegato, poi un tubo perfusato è collegato all&#39;ago. Una volta cannulazione successo è confermato, fare un taglio alla vena cava inferiore (IVC) per consentire efflusso. <img alt="Figura 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> Figura 2. Morfologia di epatociti coltivati ​​nel tempo (4 ore a 24 ore), ingrandimento x 200. Dopo la coltura per 24 ore, le cellule diffuse in crescita monostrato tipico, e le giunzioni tra le cellule sono lineari. <table border = &quot;1&quot;&gt; <tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> HBSS (senza Ca 2 +, e Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (con Ca 2 +, e Mg 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> E Media Williams </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Cella filtro (100 micron) </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> Tabella 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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Coltura primaria di epatociti è un modello in vitro ampiamente utilizzato per studiare vari aspetti della fisiologia del fegato e della patologia. Ad esempio, la cultura primaria viene utilizzata per valutare l&#39;espressione e la funzione di enzimi che metabolizzano i farmaci inclusi i citocromi P450, metabolismo dei farmaci, interazioni farmaco-farmaco e dei meccanismi di citotossicità e genotossicità 3-7. Il protocollo descrive l&#39;isolamento e la coltura di cellule epatiche di ratto è adat...

isolation and primary culture of rat hepatic cells

La cultura degli epatociti primario è uno strumento prezioso che è stato ampiamente utilizzato nella ricerca di base della funzione epatica, patologia, la fisiopatologia, farmacologia e altri argomenti correlati. Il metodo basato su due fasi perfusione collagenasi per l&#39;isolamento di epatociti intatti è stato introdotto da Berry e Friend nel 1969 1 e, da allora, ha subito molte modifiche. La tecnica più comunemente usata è stata descritta da Seglenin 1976 2. Essenzialmente, epatociti sono dissociate da ratti adulti anestetizzati da un non-ricircolo perfusione con collagenasi attraverso la vena porta. Le cellule isolate vengono quindi filtrata attraverso un filtro a pori 100 um nylon dimensioni delle maglie, e coltivate su piastre. Dopo 4 ore di coltura, il terreno viene sostituito con terreno contenente siero o privo di siero, ad esempio HepatoZYME-SFM, per il tempo aggiuntivo di cultura. Queste procedure richiedono operazioni chirurgiche cultura e sterili che possono essere meglio dimostrato dal video che da testo. Here, ci documentare i passaggi dettagliati di queste procedure sia da video e protocollo scritto, che consentono costantemente la generazione di epatociti vitali in gran numero.

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

Epatociti primari costituirà un valido strumento per valutare le funzioni biochimiche, molecolari e metaboliche in un sistema fisiologicamente rilevante sperimentale. Noi descriviamo un protocollo affidabile per la perfusione di fegato di ratto in situ, che genera costantemente epatociti vitali fino to1.0 × 10<sup&gt; 8</sup&gt; Cellule per la preparazione con la vitalità delle cellule tra 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

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 Views.  University of Cincinnati College of Medicine. Epatociti primari costituirà un valido strumento per valutare le funzioni biochimiche, molecolari e metaboliche in un sistema fisiologicamente rilevante sperimentale. Noi descriviamo un protocollo affidabile per la perfusione di fegato di ratto in situ, che genera costantemente epatociti vitali fino to1.0 × 10 8 Cellule per la preparazione con la vitalità delle cellule tra 88 ~ 96%.

Video: Isolation and Primary Culture of Rat Hepatic Cells

Primary Culture of Adult Rat Heart Myocytes

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

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

Cultura primario di miociti adulti Cuore Rat

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

Ricerca

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 di cellule immunitarie di tumori primari

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 del mouse principale acinose pancreatiche Cellule

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 colture primarie di cellule di topo aortica endoteliali

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 dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

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.

Colture primarie di cellule corticosurrenali ratto e saggi di Steroidogenic funzioni

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 delle cellule endoteliali aortiche primarie dai suini in 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, trasfezione e cultura a lungo termine di cardiomiociti per topi e ratti adulti

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, coltura e ingegneria genetica delle cellule epiteliali pigmentate primarie dei mammiferi per la terapia genica 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.

Isolamento degli epatociti primari di ratto con controllo della perfusione multiparametrica

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