Yazarlar, teknik yardım için Sayın Josh Basford ve Dr Xiao-min Li teşekkür etmek istiyorum. Bu çalışma NIH hibe (DK70992 ve ML DK92779) tarafından kısmen desteklenmiştir.

1. Hazırlık Tüm tamponları taze steril teknik kullanılarak hazırlanmıştır ve bir Corning 0.22 mikron filtre kullanılarak sterilize filtresi. <ol><li> Hank Dengeli Tuz Çözeltisi için aşağıdaki ekleyerek Ben tampon Perfüzyon hazırlayın (HBSS, Ca 2 olmadan + ve Mg 2 +, bkz. Tablo 1): 25 0.5 mM ve HEPES 0,9 mM EDTA için Mg 2 + (MgCl2) mM. </li><li> (HBSS için aşağıdaki ekleyerek Perfüzyon tampon II hazırlayın Ca 2 + ve Mg 2 +, 25 mM HEPES: Tablo 1) bakın. </li><li> Perfüzyon tampon II artı collagenaseII hazırlayın: kollajenaz II (1000 U) 300 ml Perfüzyon tampon II ile çözülür ve perfüzyon önce su banyosunda çözüm sıcak tutun. Kollajenaz II aktivite süresi olan azalma nedeniyle, bu çözelti 30 dakika içinde kullanılmalıdır. </li><li> William komple Orta hazırlayın: Aşağıdaki ekle; insülin ile 100 nM, dekzametazon, 100 nM, penisilin 100 IU / ml streptomisin ve ila 100 mg / ml &#39;ye L-glutamin 2 mM, fetal bovin serumu (FBS)% 5 ila için: Williams&#39; Orta E. </li><li> Bunlar, tamponlar 42 az su banyosunda 30 dakika boyunca ısıtılmış edilmelidir ° C, 37 kanülün bir çıkış sıcaklığı tekabül eden en uygun ° C. </li></ol> 2. Karaciğer İzolasyon için Rat Perfüzyon <ol><li> Perfüzyon sistemi pompa, otoklavlanabilir silastik tüp ve su banyosu (bkz. Şekil 1) oluşur. 10 ml / dak perfüzyon peristaltik pompa önceden ayarlanabilir debi. </li><li> Intraperitoneal (ip) ketamin (87 mg / kg vücut ağırlığı) artı Xylazine (13 mg / kg) ile bir yetişkin sıçan (300 g vücut ağırlığı) uyuşturmak. Anestezi derinliği ayak tutam tarafından takip edilmelidir. Rat artık zararlı uyaran tepki verdiğinde, karın saç ve hazırlık betadin ve etanol ile karın tıraş. Ensizyon yoluyla girin. </li><li> Ortaya çıkarmakdikkatli karın boşluğunun sağ dış organları hareket ve hepatik portal ven (bkz. Şekil 1) içine 18-gauge angiocath eklemek tarafından hepatik portal ven. </li><li> Iğne perfüzat boru bağlayın ve önceden ısıtılmış (37 ° C) Perfüzyon Tampon ben düşük bir akış hızı (10 ml / dak) in situ olarak infüzyon başlatabilir. </li><li> Düzgün bir şekilde gerçekleştirildiği takdirde, karaciğer anında mazur başlamalıdır. Başarılı kateterizasyonu onaylandıktan sonra, inferior vena kava (IVC) bir kesim efflux (Şekil 1) izin olun. Başarılı kateterizasyonu için bir başka test IVC üzerine steril bez ile hafif bir basınç uygulayarak yapılabilir; karaciğerin tüm loblar hızla şişmeye başlaması gerekir. </li><li> 25 ml / dak akış hızını artırmak. Karaciğer rengi soluk haline gelmelidir. </li><li> Ek bir 6 dakika için akış kesinti olmaksızın Perfüzyon tampon II artı kollajenaz II perfüzyon çözüm açın. </li> &lt;li&gt; Periyodik (sindirim sırasında 5-10 kez) 5 saniye aralıklarla için IVC için bez ile basınç uygulayın. Karaciğer da toplam sindirim süresini azaltmak, ve nihai verimi artırarak, gelişmiş karaciğer hücre ayrışma yol şişer. </li><li> Kollajenaz perfüzyon sonra, karaciğer Duygusal aramaya başlamalıdır. 20 ml William tam medyum ile bir pre-soğutulmuş steril bir behere karaciğer içermeyen, yerinde incelemek ve sonra da doku kültürü hücre ardına al. </li></ol> 3. Hepatosit Hücre İzolasyon <ol><li> Hücre kültürü kaput içinde, nazikçe steril bir petri içinde William komple Orta içine hücreleri dağıtmak için bir hücre kazıyıcı kullanın. </li><li> Bağ dokusu ve sindirilmemiş doku parçaları ortadan kaldırmak için bir 50 ml Konik tüpü içine, bir 100 mikron gözenek büyüklüğü hücre süzgecinden hücre dispersiyon filtre. </li><li> 4 ° C&#39;de 3 dakika süreyle 50 x g&#39;de 40 ml William tam medyum ve santrifüj hücreleri askıya </li><li> Süpernatant aspire, veyavaşça hücreleri yıkamak için 40 ml soğuk William&#39;ın tam Orta hücrelerin yeniden askıya. Santrifüj tekrarlayın. </li><li> Süpernatant aspire ve yavaşça 25 ml William&#39;ın tam Orta ile tekrar askıya hücreler. Tüpe PBS içinde 25 ml% 90 Percoll çözüm ekleyin ve hafifçe karıştırın. </li><li> 4 ° C&#39;de 10 dakika süreyle 200 x g&#39;de santrifüje Canlı hücreler Percoll degrade altında kalacaktır, çünkü degrade üstünden ölü hücreleri aspire. </li><li> 30 ml ılık William&#39;ın tam Orta hücre pelet Askıya ve sonra santrifüj tekrarlayın ve 20 ml sıcak William&#39;ın tam Ortamda hücre pelletini yeniden askıya. </li><li> Bir hemasitometre kullanarak hücre süspansiyonu içindeki hücreleri saymak ve Tripan mavisi ile hücre canlılığı belirler. </li></ol> 4. Hepatosit Kültür <ol><li> Tercih edilen konsantrasyon sıcak William komple Orta hücreleri seyreltilir, örneğin 2.5 x 10 5 hücre / ml. Hücre kültürü plakalar üzerinde istenilen hacimde Tabak hücreler, örneğin. 5 x 10 5 hücre / 2 ml / kuyu, 6 kuyu / plaka; veya 2.5 iyi x 10 5 hücre / 1,5 ml /, 12 kuyu / plakası. Un kaplı plaka karaciğer hücre kültürleri için uygundur. </li><li> Hepatositler tam hücre boyutu büyüyecek ve son bir izdiham elde etmek için yeterli alan sağlarken&gt;% 85 canlı hücreler ile tipik bir hazırlık olarak, hücre yoğunluğu hücre-hücre teması sağlayan yaklaşık% 60-70 izdiham, ulaşmalıdır % 90-95. </li><li> İyi bir merkezi bölgeye toplamak için hücreler için eğilimi en aza indirmek için, başka bir deyişle, hepatositlerin eşit bir tek tabaka oluşturmak üzere (hücre inkübatöründe bir hava akımı içinde nedeniyle büyük olasılıkla), plakaları içinde kalmasına izin Bir kuluçka koymadan önce 30 dakika boyunca hücre kültürü kaput. </li><li> Kültür 37 hücreleri% 95 hava ve% 5 CO2 ile nemlendirilmiş bir atmosferinde ° C. 4-h kültürden sonra, hücreler ya aynı serumu içeren ortam içinde kalır olabilir ya da serum-f ile orta yerineree ortam, örneğin HepatoZYME-SFM (bkz. Tablo 1). Serum içermeyen aracı madde, serum içermeyen aracı madde kullanılır hormonlar hiçbir yan etki ile hücre morfolojisi temin edilmesine yardımcı olur. </li><li> Hücreleri deney önce kurtarmak ve en az bir gece büyümeye olanak sağlar. Bu kritik enzimler (örneğin, P450) fonksiyonu korumaya yardımcı olabilir, çünkü 24 saat içinde test için hücreleri kullanmanızı öneririz. </li><li> Gerekirse, 2 gün aralıklarla büyüme medyumu yerini alır. </li></ol> 5.. Temsilcisi Sonuçlar Koşulları düzenli olarak bir sıçan karaciğer hazırlık başına 1.0 x 10 8 hücrelerinin hücre hasat oluşturmak nitelendirdi. Tripan mavisiyle çıkarma ile ölçülen hepatositlerin yaşayabilirliği 88 ~% 96 aralığında olduğu, sürekli. Şekil 2, 4 saat tohumlama sonrası hepatositlerin agrega ve form küme tasvir. En çok izole hücreler tipik monolayer büyüme düzleşir ve yaymak. 24 saat olarak, hücre kenarları tanımlandığı gibidir, hücre yüzeyinde oldukça düz ve lipid damlacıkları görülebilir. Hücreler 1-3 hücrelerinin merkezinde bulunan yuvarlak nükleol, ve hücreler arasında çekirdekler görüntü tutarlıdır boyutu (Şekil 2) sahiptir. <img alt="Şekil 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> Şekil 1. Karaciğer perfüzyon şeması. Bir 18-gauge angiocath karaciğer portal damar içine yerleştirilir, daha sonra bir perfüzat boru iğne bağlanır. Başarılı kateterizasyonu onaylandıktan sonra, inferior vena kava (IVC) bir kesim efflux izin olun. <img alt="Şekil 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> Şekil 2. Zamanla kültür hepatosit (4 saat ile 24 saat), büyütme x 200 morfolojisi. 24 saat kültüre sonra, hücreler tipik monolayer büyüme yayılır ve hücreler arasındaki kavşaklar doğrusaldır. <table border = &quot;1&quot;&gt; <tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> HBSS (Ca 2 olmadan + ve Mg 2 +) </td><td> Invitrogen </td><td> 14174 </td></tr><tr><td> HBSS (Ca2 + ve Mg ile birlikte 2 +) </td><td> Invitrogen </td><td> 14025 </td></tr><tr><td> Williams&#39;ın Orta D </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td> Worthington </td><td> LS004176 </td></tr><tr><td> Hücre süzgeç (100 mikron) </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> Tablo 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>

Çıkar çatışması ilan etti.

Karaciğer primer kültürü yaygın karaciğer fizyolojisi ve patolojisi çeşitli yönlerini incelemek için kullanılan in vitro model olduğunu. Örneğin, primer kültür P450 sitokromlar, ilaç metabolizması, ilaç-ilaç etkileşimleri ve sitotoksisite ve genotoksisite 3-7 mekanizmaları da dahil olmak üzere ilaç metabolize eden enzimlerin ifade ve fonksiyonunu değerlendirmek için kullanılır. Sıçan karaciğer hücrelerinin izolasyonu ve kültür tarif protokolü 8. Aiken <em...

isolation and primary culture of rat hepatic cells

İlköğretim hepatosit kültürü yaygın karaciğer fonksiyonu, hastalık, patofizyolojisi, farmakoloji ve diğer ilgili konularda temel araştırmalarda kullanılan bir araçtır. Sağlam hepatosit izolasyonu için iki adım kollajenaz perfüzyon dayanan yöntemi ilk kez 1969 1 Berry ve Friend tarafından tanıtıldı ve o zamandan beri birçok değişiklikler geçirmiştir. En sık kullanılan teknik Seglenin 1976 2 tarafından tanımlanmıştır. Esasen, hepatositlerde portal ven yoluyla olmayan bir recirculating kollajenaz perfüzyon ile anestezi yetişkin sıçan ayrışmış bulunmaktadır. İzole hücreleri daha sonra, bir 100 mikron gözenek boyutu örgü naylon filtre aracılığıyla süzülmüş ve plakalar üzerinde kültürlenmiştir. 4-saatlik kültür sonra, orta kültürüne ek süre için serum içeren ya da serum içermeyen aracı madde, örneğin HepatoZYME-SFM ile değiştirilir. Bu prosedürler iyi metin ile daha çok video gösterdiği edilebilir cerrahi ve steril kültür adımları gerektirir. Here, biz çok sayıda canlı hepatositlerin nesil sürekli izin video ve yazılı bir protokol, her iki tarafından bu işlemler için ayrıntılı adımlar belgelemek.

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

İlköğretim hepatositler bir fizyolojik ilgili deneysel sistem biyokimyasal, moleküler ve metabolik fonksiyonlarını değerlendirmek için değerli bir araç sağlar. Biz sürekli to1.0 × 10 kadar yaşayabilir hepatositler üretir yerinde karaciğer perfüzyon, sıçan için güvenilir bir protokol tarif<sup&gt; 8</sup88 ~% 96 arasında hücre canlılığı ile hazırlık başına&gt; hücreler.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

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 Görüntüleme.  University of Cincinnati College of Medicine. İlköğretim hepatositler bir fizyolojik ilgili deneysel sistem biyokimyasal, moleküler ve metabolik fonksiyonlarını değerlendirmek için değerli bir araç sağlar. Biz sürekli to1.0 × 10 kadar yaşayabilir hepatositler üretir yerinde karaciğer perfüzyon, sıçan için güvenilir bir protokol tarif 8 hücreler.

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.

Yetişkin Sıçan Kalp miyositler İlköğretim Kültür

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

Biyoloji

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.

Primer Tümörler gelen Bağışıklık Hücre İzolasyonu

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.

Fare Primer Pankreas Asiner Hücreleri İzolasyon ve Kültür

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

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.

Sıçan Adrenokortikal hücreleri ve deneyleri Steroidogenic fonksiyonların birincil kültür

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.

Minyatür Domuzlardan Birincil Aort Endotel Hücrelerinin İzolasyon ve Kültürü

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.

İzolasyon, Transfeksiyon ve Yetişkin Fare ve Sıçan Kardiyomiyositlerinin Uzun Süreli Kültürü

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.

Viral Olmayan Gen Tedavisi için Memeli Primer Pigment Epitel Hücrelerinin İzolasyonu, Kültürü ve Genetik Mühendisliği

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.

Primer Sıçan Hepatositlerinin Çok Parametreli Perfüzyon Kontrolü ile İzolasyonu

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