著者は、技術支援のために氏ジョシュバスフォード博士シャオ分のLiに感謝します。この作品は、NIHの助成金（MLへDK70992およびDK92779）によって部分的にサポートされていました。

1。準備すべてのバッファは、新たに滅菌技術を用いて調製されており、コーニング0.22μmのフィルターを用いて濾過滅菌。 <ol><li> 0.5〜25 mMとHEPES、0.9 mMのEDTAにはMg 2 +（MgCl 2の ）：ハンクス平衡塩溶液（HBSSは、Ca 2 +およびMg 2なしで+、 表1を参照）に以下を加えることで、 私をバッファ灌流を準備します。 mMの。 </li><li> （HBSSに以下を追加することにより、 灌流緩衝液IIを準備するのCa 2 +およびMg 2 +、 25 mMのHEPESに： 表1）を参照してください。 </li><li> 灌流緩衝液IIプラスcollagenaseIIを準備します 。コラゲナーゼII（1000 U）300ミリリットル灌流バッファーIIで溶解し、灌流前に水浴中で解決策を暖かく保つ。コラゲナーゼIIの活性は時間とともに減少するので、この溶液を30分以内に使用する必要があります。 </li><li>ウィリアムの完全培地を準備します。次の行を追加します。100nmにインスリン、デキサメタゾンは100nM、ペニシリン100 IU / mlおよびストレプトマイシン〜100 mg / mlに、L-グルタミン2mMのに、5％ウシ胎児血清（FBS）：ウィリアムズのミディアムEへ。 </li><li>これらのバッファは、42℃水浴中で30分間加温する必要があります°C、37のカニューレの出口温度に対応する最適な温度は℃、 </li></ol> 2。肝臓の分離のためのラット灌流<ol><li>灌流システムは、ポンプ、オートクレーブシラスティックチューブや水浴（ 図1を参照）で構成されています。 10ミリリットル/分に蠕動灌流ポンプの流量を事前に設定します。 </li><li>腹腔内（ip）ケタミン（87 mg / kg体重）とキシラジン（13 mg / kg体重）で成体ラット（300 g体重）を麻酔。麻酔の深さはつま先のピンチによって監視されるべきである。ラットは、もはや有害刺激に応答しない場合は、腹部の毛と準備betadineとエタノールで腹部を剃る。正中切開を介して入力してください。 </li><li>さらす慎重に腹腔の右外側に内臓を移動することで、肝門脈、と（ 図1を参照）肝門静脈に18ゲージangiocathを挿入します。 </li><li>針に灌流チューブを接続し、予め温めておいた（37℃） 潅流緩衝液Iで低流量（10ml /分）で、in situで注入を開始します。 </li><li>適切に実行した場合、肝臓は即座に湯通しを開始する必要があります。一度成功したカニュレーションが確認され、流出（ 図1）を可能にするために下大静脈（IVC）で切断してください。成功したカニ​​ュレーションのためにさらなるテストがIVCに滅菌綿棒で軽い圧力を適用することによって行うことができます。肝臓のすべての葉はすぐに膨潤を開始する必要があります。 </li><li> 25ミリリットル/分の流量を増加させます。肝臓は色が蒼白になる必要があります。 </li><li>追加の6分間の流れが中断されない、 灌流緩衝液IIプラスコラゲナーゼIIへの灌流液を切り替えます。 </li> &lt;LI&gt;定期的に（消化中の5-10倍）は5秒間隔でIVCに綿棒で圧力を適用します。肝臓は、順番に合計消化時間を短縮し、最終的な収率を増加させる、強化された肝細胞解離につながる、膨張します。 </li><li>コラゲナーゼ灌流した後、肝臓はどろどろ見て開始する必要があります。 20ミリリットルウィリアムの完全培地であらかじめ冷却し、滅菌ビーカーに肝臓フリー、場所を分析し、組織細胞培養フードにそれを取る。 </li></ol> 3。肝細胞の分離<ol><li>細胞培養フード内に、穏やかに滅菌シャーレ内にウィリアムの完全培地に細胞を分散させるために、セルスクレーパーを使用しています。 </li><li>結合組織および未消化の組織片を除去するために、50 mlコニカルチューブに100μmの細孔径のセルストレーナーを介して細胞分散をフィルタリングします。 </li><li> 4℃で3分間、50×gで40ミリリットルウィリアムの完全培地と遠心で細胞をサスペンド</li><li>上清を吸引し、穏やかに細胞を洗浄するには、40 mlの冷ウィリアムの完全培地で細胞を再懸濁する。遠心操作を繰り返します。 </li><li>上清を吸引し、ゆっくりと25ミリリットルウィリアムの完全培地で再懸濁細胞。チューブにPBSで25ミリリットルの90％パーコール溶液を加え、穏やかに混ぜる。 </li><li> 4℃で10分間、200 xgで遠心分離生細胞は、Percoll勾配の最下部に残っているため、グラデーションの上から死んだ細胞を吸引します。 </li><li> 30ミリリットル暖かいウィリアムの完全培地中で細胞ペレットを一時停止し、遠心分離を繰り返し、20ミリリットル暖かいウィリアムの完全培地中で細胞ペレットを再懸濁する。 </li><li>血球計算板を用いた細胞懸濁液内の細胞をカウントし、トリパンブルー染色により細胞の生存を決定します。 </li></ol> 4。肝細胞培養<ol><li>好ましい濃度に暖かいウィリアムの完全な媒体、例えば2.5×10 5細胞/ mlで細胞を希釈します。細胞培養プレート上の任意のボリュームで板細胞、例えば。 5×10 5細胞/ 2ミリリットル/ウェル、6ウェル/プレート、または、2.5×10 5細胞/ 1.5ミリリットル/ウェル、12ウェル/プレート。非コーティングしたプレートは、肝細胞の培養には問題ありません。 </li><li> &gt; 85％、生細胞の典型的な予備校では、細胞密度は、肝細胞は、完全な細胞の大きさに成長するための十分なスペースを維持しながら、細胞間接触を可能にする約60〜70％コンフルエントに達するとの最終的な合流が得られるはず90から95パーセント。 </li><li>井戸の中心部に集約するセルの傾向を最小限に抑えるために、換言すれば、肝細胞のさえ単分子膜を形成するために（細胞インキュベーターの空気の流れの内部に起因する可能性が最も高い）、プレートの内部に残るようにインキュベーターに置く前に30分間細胞培養フード。 </li><li> 37℃で細胞を培養温度95％空気および5％CO 2の加湿大気中のC。 4時間培養後、細胞は、同じ血清含有培地に残ったか、血清fで培地を交換する希土類元素の媒体、例えばHepatoZYME-SFM（ 表1を参照）。無血清培地は無血清培地が使用されているホルモンからも副作用の影響で細胞の形態を維持するのに役立ちます。 </li><li>細胞は実験前にを回復し、少なくとも一晩成長することができます。これは重要な酵素（例えば、P450の）の機能を維持するのを助けるかもしれないので私達は24時間以内にテスト用の細胞を使用することをお勧めします。 </li><li>必要に応じて、2日間隔で培地を交換してください。 </li></ol> 5。代表的な結果条件は、定期的に1匹のラットの肝臓から調製当たり1.0×10 8細胞の細胞収穫を生成する説明します。トリパンブルー排除法によって測定した肝細胞の生存率は88〜96％の範囲内で一貫していた。 図2に、4時間の播種後肝細胞集合体とフォームのクラスタに示されている。ほとんどの単離された細胞は、典型的な単層成長に平らに広がる。 24時間では、セルの端が定義されて、細胞の表面はかなり滑らかで、脂肪滴が表示されています。細胞は1〜3セルの中央に位置する円形で核小体、、、、細胞間の核ディスプレイ一貫性のある大きさ（ 図2）を持っています 。 <img alt="図1" src="/files/ftp_upload/3917/3917fig1.jpg" /> 図1肝灌流の図。 18ゲージangiocathは、肝臓の門脈に挿入され、その後灌流チューブを針に接続されています。一度成功したカニ​​ュレーションが確認され、下大静脈（IVC）のカットが流出できるようになります。 <img alt="図2" src="/files/ftp_upload/3917/3917fig2.jpg" /> 2時間以上培養肝細胞の形態（4時間〜24時間）、倍率×200 図 。 24時間培養後、細胞は、典型的な単層の成長に広がり、細胞間の接合部は直線状です。 <table bordeR = &quot;1&quot;&gt; <tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> HBSS（Ca 2 +を 、およびMg 2 +なし） </td><td>インビトロジェン</td><td> 14174 </td></tr><tr><td> HBSS（Ca 2 +を 、およびMg 2 +） </td><td>インビトロジェン</td><td> 14025 </td></tr><tr><td>ウィリアムズのミディアムE </td><td>インビトロジェン</td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td>ワージントン</td><td> LS004176 </td></tr><tr><td>セルストレーナー（100μm以下） </td><td> BD </td><td> 352360 </td></tr><tr><td> HepatoZYME-SFM </td><td>インビトロジェン</td><td> 17705 </td></tr><tr><td>パーコール</td><td>シグマ</td><td> P4937 </td></tr><tr><td> Angiocath（18ゲージ） </td><td> BD </td><td> 381705 </td></tr></tbody></table> 表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>

利害の衝突が宣言されません。

肝細胞の初代培養は、広く肝臓生理学と病理学のさまざまな側面を勉強するために使用されるin vitroモデルである。例えば、初代培養は、チトクロームP450、薬物代謝、薬物相互作用し、細胞毒性および遺伝毒性3-7のメカニズムを含む薬物代謝酵素の発現と機能を評価するために使用されます。修正を加えたラット肝細胞の単離および培養は、エイケンらの以前のレポー?...

isolation and primary culture of rat hepatic cells

初代肝細胞培養は、広範囲に肝機能、疾患、病態生理学、薬理学および他の関連科目の基礎研究に使用されている貴重なツールです。無傷の肝細胞の単離のための二段階コラゲナーゼ灌流に基づく方法は、1969年1ベリーと友人によって導入されたと、それ以来、多くの変更を受けています。最も一般的に使用される技術はSeglenin 1976年2によって記述されていた。本質的に、肝細胞は門脈を介して非循環コラゲナーゼ灌流により麻酔成体ラットから分離しています。単離された細胞は、100μmの細孔サイズのメッシュナイロンフィルターを通して濾過し、プレート上に培養される。 4時間培養後、培地は、培養への追加時の血清含有又は無血清培地、例えば、HepatoZYME-SFM、置き換えられます。これらの手順は、優れたテキストよりもビデオで実証することができる外科手術、無菌培養工程を必要とします。 HERE、我々は大量に実行可能な肝細胞の生成に一貫してできるように、ビデオと書かれたプロトコルの両方でこれらの手順の詳細な手順を文書化します。

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

初代肝細胞は、生理学的に関連する実験系の生化学的、分子生物学、代謝機能を評価するための貴重なツールを提供しています。我々は一貫してto1.0×10アップ可能な肝細胞を生成する現場肝灌流、ラットの信頼性の高いプロトコルを記述する<sup&gt; 8</sup88〜96％の間に細胞の生存と準備当たり&gt;細胞。

ラット肝細胞の単離及び初代培養

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

55.3K ビュー.  University of Cincinnati College of Medicine. 初代肝細胞は、生理学的に関連する実験系の生化学的、分子生物学、代謝機能を評価するための貴重なツールを提供しています。我々は一貫してto1.0×10アップ可能な肝細胞を生成する現場肝灌流、ラットの信頼性の高いプロトコルを記述する 8細胞。

ビデオ: 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.

成体ラットの心筋細胞の初代培養

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

生物学

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.

原発腫瘍から免疫細胞の分離

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.

マウスプライマリ膵臓腺房細胞の単離と培養

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.

マウス大動脈内皮細胞の単離および初代培養

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.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

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.

初代培養ラット副腎皮質細胞とステロイド ホルモン産生機能の試金

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.

ミニチュアブタからの原発性大動脈内皮細胞の単離と培養

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, 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.

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

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.

多パラメータ灌流制御による原発性ラット肝細胞の単離

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

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

ブタ網膜色素上皮細胞の初代培養

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...