Name: using caco-2 cells to study lipid transport by the intestine
Uploaded: 2015-08-20T14:00:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

のCaco-2細胞の1メンテナンス<ol><li>正規の組織培養皿を用いて、 <ol><li> 37℃の水浴中にバイアルを配置することによって、凍結バイアルからのCaco-2細胞を解凍し、直ちに予め温めた増殖培地（15％ウシ胎児血清（FBS、10 mlを含む10cmの組織培養皿に追加））ダルベッコ改変イーグル培地（DMEM）で。 <ol><li> （15分間）、それらが分離されるまで、37℃で0.05％3mlのトリプシン/ 0.53 mMのエチレンジアミン四酢酸（EDTA）で細胞をインキュベートすることにより、6：たCaco-2細胞は、50〜70％コンフルエンスに達したときに、それらを1分割。細胞凝集を回避するために、穏やかなピペッティングにより細胞を数回混ぜます。予熱した増殖培地10 mlを入れた10cmの組織培養皿にトリプシン処理した細胞を0.5mlを加えます。 </li></ol></li><li>静かに左右方向に続く前後方向に皿を数回振ります。料理を旋回しないでくださいように、不等細胞分散をもたらすことができます。 </li><li> 5％CO 2を供給し、37℃のインキュベーターで平らな面に皿を置きます。傾斜面は、また、不等細胞分散をもたらすことができます。 </li><li>インキュベーションを開始した後、増殖培地1日を変更します。 </li><li>細胞を監視し、それらがコンフルエンスに達する日を記録。細胞は、それらが分割された日から合流点に到達するのは約1週間かかります。 </li><li>細胞がコンフルエンスに到達する前に週に二度、増殖培地を変更します。細胞がコンフルエンスに達した後は、一日おきに成長培地を変更します。細胞は、7日後にコンフルエントになったら、毎日の成長培地を変更します。 注：彼らは13日後コンフルエント（ 図1）である場合、細胞が実験のための準備ができています。細胞は、最終的にリポタンパク質の生産にあまり有効になりますので、8コンフルエント後の13から17日間の間にあるセルを使用。実験を行う方法については、手順3に進みます。</li></ol></li><li>透過膜システムを使用して<ol><li>ステップ1.1.1.1で説明したように、透過膜インサートでのCaco-2細胞を播種します。上記。その細胞増殖が（回復期）通常遅いため凍結バイアルから細胞を使用しないでください。簡単に説明すると、予め温めた増殖培地の10ミリリットルが含まれている頂端チャンバー（上部コンパートメント）にトリプシン処理した細胞の0.5ミリリットルを追加します。また、基底外側室（下部コンパートメント）に予熱した増殖培地の10ミリリットルを追加します。 注：ベストプラクティスについては、側底チャンバーに最初にして頂端チャンバーに増殖培地を追加します。古いメディアを取り外すときは、頂端メディアの前に側底メディアを削除します。それが膜を破裂としてポリカーボネート膜を突っついは避けてください。 </li><li>によるポリカーボネート膜を介して、悪い細胞の可視性のために、同様の密度との定期的な組織培養皿中のCaco-2細胞を播種すると、細胞コンフルエンスを判断するためにこの料理を使用しています。 </li><li>のフォローTEPS上記1.1.2-1.1.6。 </li></ol></li></ol> 2.遺伝子の過剰発現<ol><li>定期的なトランスフェクション法を用いて、 <ol><li> 1.1.1.1.-1.1.4上記で説明したように組織培養皿上でのCaco-2細胞をシード。 注：細胞が約40〜50％コンフルエントである場合、それらは、トランスフェクトされる準備ができています。 </li><li>マイクロチューブ中の還元血清培地の472μlにトランスフェクション試薬の67μlを添加します。管壁と希釈されていないトランスフェクション試薬との接触を避けてください。 </li><li> pLL3.7の23μgのトランスフェクション試薬/低血清培地混合物に緑色蛍光タンパク質（EGFP）9を強化加えます。 </li><li> RTで20分間混合物をインキュベートします。 </li><li> DMEM、10％FBSの8ミリリットルでのCaco-2細胞の増殖培地を交換してください。 </li><li>滴下皿にDNA /トランスフェクション試薬/縮小血清培地の混合物を追加します。 </li><li>軽く前方およびBaで料理を数回振ります左右方向に続くckward方向。 </li><li> 37℃のインキュベーターで皿を置きます。 </li><li>成長培地10mlのインキュベーションを開始した後の日と皿の中でメディアを交換してください。 </li><li>遺伝子発現を監視します。 <ol><li> 4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）染色することなく、遺伝子発現をモニターします。蛍光顕微鏡を使用して、緑である細胞の割合を決定します。パーセンテージは、トランスフェクション効率を表します。 </li><li> DAPI染色と遺伝子発現を監視します。 <ol><li>リン酸緩衝生理食塩水（PBS）で3回細胞を洗浄します。 </li><li> PBS中の4％ホルムアルデヒド（室温で10分間のインキュベーション）を10mlを加えることにより細胞を固定します。 </li><li> PBSで細胞を3回洗浄します。 </li><li> 500 DAPI、1％BSA、0.01％ジギトニン：1を含む8mlのPBSで室温で15分間、暗所で細胞をインキュベートします。 </li><li> PBSで細胞を3回洗浄します。 </li><li> U蛍光顕微鏡を歌う、青色であるものに緑/青の相対的な（ 図2上部パネル）である細胞の数を決定します。計算された割合は、トランスフェクション効率を表します。 </li></ol></li></ol></li></ol></li><li>レンチウイルス過剰発現システム10を使用して<ol><li> 15cmの組織培養皿（それらの増殖培地としてDMEM中10％FBS）中に種子ヒト胚腎臓（HEK）293T細胞。 注：細胞が60〜70％コンフルエントである場合、それらは、トランスフェクトされる準備ができています。 </li><li>マイクロチューブ11に減少血清培地の1,133μlにトランスフェクション試薬の162μlを添加します。管壁と希釈されていないトランスフェクション試薬との接触を避けてください。 </li><li>トランスフェクション試薬/ RにpLL3.7のeGFP（または他の構造物）、のRev応答エレメント（RRE）の15.6μgの、REVの6.0μgの、および水疱性口内炎ウイルス糖タンパク質（VSVG）の8.4μgの24μgのを追加します。析出血清培地混合物。 </li><li> RTで20分間混合物をインキュベートします。 </li><li> DMEM、10％FBSを16 mlの増殖培地を交換してください。 </li><li>滴下皿にDNA /トランスフェクション試薬/縮小血清培地の混合物を追加します。 </li><li>静かに左右方向に続く前後方向に皿を数回振ります。それは、この時点で剥離しHEK293T細胞のいくつかを見るために、通常です。 </li><li>インキュベーターで皿を置きます。 </li><li>インキュベーションの24時間後、増殖培地を25 mlの皿の中でメディアを交換してください。 </li><li>翌日、レンチウイルス（最初のコレクション）を含む増殖培地を収集します。 </li><li>より多くのレンチウイルス（第2集）を収集するための手順を繰り返し2.2.10。 </li><li> （5分間、2,500×gで）第一および第二の収集をプールし、遠心分離により細胞破片を除去。 </li><li>使い捨てボトルトップフィルター（0.45μmの孔）を持つコレクションをフィルタリングし、RT（JA-20ローター）で2時間31,400×gでコレクションを遠心分離することにより、ウイルスを濃縮します。ペレットが配置されている遠心分離管の底部をマーク。上清を除去し、約15分間室温で反転チューブを残します。 </li><li> 1×PBS100μlのウイルス含有ペレットを再懸濁します。泡を避けます。 </li><li> -80℃で濃縮したウイルスを保管してください。凍結融解の繰り返しは避けてください。 </li><li>滴定によってのCaco-2細胞を形質導入するために必要なウイルスの最適量を決定します。コストを節約するために、代わりに滴定のための10cmの組織培養皿の（増殖培地/ウェルの0.3ミリリットル）を24ウェルプレートを使用しています。 <ol><li>濃縮したウイルスの増加量を加える（0、1、2、5、10、25、50μL/ウェル）を40％〜70％コンフルエントのCaco-2細胞へのポリブレン（最終濃度=5μg/ mlの）を補充しました。 1.1.2で説明したように静かに24ウェルプレートを振ります。 </li><li>それらの遺伝子発現（ステップ2.1.10。）（ 図2下のを監視4パネル）。 </li></ol></li><li>導入遺伝子の発現は、一般的に維持されるので、将来の実験のために形質導入​​された細胞を維持し、継続的な遺伝子発現（ステップ2.1.10。）を監視します。 </li></ol></li></ol> 3.リポタンパク質分泌を刺激<ol><li>定期的な組織培養ディッシュを使用して<ol><li>オレイン酸、レシチン40 mg及びタウロコール酸ナトリウム48mgの50mgを混合することにより、100Xの脂質混合物を準備します。 （8皿のための十分な）PBSで900μlにボリュームをもたらします。勢いよく脂質混合物をボルテックスし。 </li><li>増殖培地の89.1ミリリットルに100Xの脂質混合物の900μlを添加します。混合し、脂質含有培地をフィルタリングします。オレイン酸、レシチン、およびタウロコール酸ナトリウムの最終濃度（1×）は、それぞれ2.0ミリメートル、1.36ミリメートル、および1.0 mMです。 </li><li>細胞への脂質含有培地10mlのを追加します。 </li><li> 37で4時間脂質含有培地で細胞を培養しますCのインキュベーターを°。 </li><li> PBSで細胞を3回洗浄します。 </li><li>リポタンパク質の分泌を収集するために、細胞を増殖培地を10mlを加えます。 </li><li> 37℃のインキュベーターで2時間インキュベートします。 </li><li>リポ蛋白質含有培地を収集します。 </li></ol></li><li>透過膜システムを使用して<ol><li>手順に従ってください3.1.1-3.1.2脂質混合物を調製しました。 </li><li>頂端チャンバへの脂質含有培地10ml及び側底チャンバへの成長培地10mlのを追加します。 </li><li> 37℃のインキュベーター内で4時間インキュベートします。基底外側チャンバ内にリポ蛋白質含有培地を収集します。 </li></ol></li></ol> 4.リポタンパク質の分離（図3） <ol><li>遠心分離（5分間2,000×gで）によってリポタンパク質含有培地から細胞破片を除去。 </li><li> 50mlのチューブにメディアをデカントします。 </li><li>メディアへの塩化ナトリウムの5.95グラムを追加します。 </li><li> SAの場合mpleはリポタンパク質の完全性を維持するために1プロテアーゼインヒビターカクテル錠を追加、TEMによって分析されます。 </li><li>水で23ミリリットル（1.2グラム/ mlの濃度のNaCl溶液）にボリュームをもたらします。 </li><li>完全に溶質を溶解させます。 </li><li>ポリカーボネート超遠心管にソリューションのすべてをデカント。 </li><li>ゆっくり水0.5ml（1.0グラム/ mlの濃度）と1.2グラム/ mlの濃度の溶液を重ねます。 </li><li>体積重量ではなく、チューブのバランスをとります。 </li><li>ゆっくりT865ローターにチューブをロードします。 </li><li> 4℃で24時間、429460×gで超遠心機でサンプルをスピン。 </li><li>すぐに穏やかにピペッティングすることによって、トップ0.5ミリリットルの溶液を分離します。できるだけまだチューブを保管してください。 </li></ol> 5. TEM分析<ol><li> 2％リンタングステン酸の5ミリリットル（V / W）を準備し、pHを6.0に調整します。 </li><li>シリンジフィルター（0.2μmの孔サイズ）で2％リンタングステン酸をフィルタリングします。 </li><li>無菌皿にリポタンパク質サンプルの20μlのを削除します。 </li><li>同じ皿に次のサンプルを濾過し、2％リンタングステン酸20μlのを削除します。 </li><li>ゆっくり鈍い側が試料上に載っているとEMグリッドをドロップします。 </li><li> 1分間室温でインキュベートします。 </li><li>ゆっくりグリッドからサンプルを除去するために、濾紙上のEMグリッドの側をタップします。 </li><li>ゆっくり鈍い側は2％リンタングステン酸で休んでEMグリッドをドロップします。 </li><li> 1分間室温でインキュベートします。 </li><li>ゆっくりグリッドからリン酸を除去し、濾紙上のEMグリッドの側をタップします。 </li><li> TEMを使用して、リポタンパク質（ 図4）の画像を取り込みます。 </li></ol>

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The author has nothing to disclose.

この論文では、使用できる2つのシステムは、Caco-2細胞は、すな​​わち、正規の組織培養皿や透過膜に記載されている維持します。透過膜システムを使用する利点は、頂端の分離および基底外側コンパートメント、および脂質混合物をインキュベートし、同時にリポタンパク質分泌を収集する能力を含みます。しかし、透過膜インサートは高価であり、それらのポリカーボネート膜は、良好...

4 -食事脂肪の腸管吸収に関する研究、および脂溶性薬物とビタミンは、リンパ瘻モデル1を用いて、in vivoで行うことができます。しかし、関連する外科技術は、困難でなく、高価なだけでなく、です。糞便分析に基づいて、in vivoでのアプローチを利用することができるが、それらは、胃腸管2,5パーセントの取り込みを決定するために主に使用されています。この論文に記載のin vitroモデルは、よりコスト効果的であり、関係する技術は、おそらくあまり挑 ​​戦しています。遺伝的修飾研究は、時間がかかり、それらは、このin vitroモデルを使用して行われる場合に、より経済的かつ少ないです。 腸細胞によって取り込まれる脂溶性物質は、リポタンパク質6,7にパッケージ化されているので、リポタンパク質を生成するために、このin vitroモデルの有効性が重要です。二つの主要なintestinaLのリポタンパク質は、カイロミクロン及び超低密度リポタンパク質（VLDL）です。脂質は、胃腸内腔に豊富に存在する場合、直径が80nm以上とリポタンパク質として定義されるキロミクロンは、小腸により厳密に製造されています。彼らは最大のリポタンパク質であるため、カイロミクロンは考えられる最も効率的な脂質トランスポーターです。カイロミクロン8を生成することができる。このインビトロモデルは、食事の脂肪吸収、消化管による脂溶性ビタミンの吸収、および親油性薬物の経口バイオアベイラビリティを研究するために使用することができます。リポタンパク質画分中の脂溶性分子、ビタミン、または薬物の存在は、小腸によるその吸収の指標です。前述のように、このモデルは、経口バイオアベイラビリティ、親油性薬剤6を改善するために使用することができます。 本稿では、Caco-2細胞はどのように脂質マイル、透過膜または通常の組織培養皿で維持されるべきである方法を説明しますリポタンパク質の産生を刺激するための固定装置は、レンチウイルス発現系は、効果的な過剰発現を達成するために使用する方法、調製方法、および単離されたリポタンパク質を分析するべきであるべきです。

<table><tbody><tr><td>DMEM</td><td>VWR</td><td>16750-112</td><td>Pre-warm the growth media in individual tissue culture dish before adding cells</td></tr><tr><td>FBS</td><td>Fisher</td><td>3600511</td><td>We do not heat inactivate our serum</td></tr><tr><td>Trypsin</td><td>VWR</td><td>45000-660</td><td>Cells can be washed with PBS prior to trypsin treatment&amp;nbsp;</td></tr><tr><td>Permeable membrane system</td><td>Fisher</td><td>7200173</td><td>10-cm dish, 3-mm pore size, polycarbonate membrane</td></tr><tr><td>10 cm dish</td><td>Fisher</td><td>08-772-E</td><td>Tissue culture dish</td></tr><tr><td>15 cm dish</td><td>Fisher</td><td>08-772-24</td><td>Tissue culture dish</td></tr><tr><td>24 well plate</td><td>Fisher</td><td>12565163</td><td>Tissue culture plate</td></tr><tr><td>Lipid-based transfection reagent</td><td>Fisher</td><td>PRE2691</td><td>Can be substituted with other transfection reagent</td></tr><tr><td>Reduced serum media</td><td>Invitrogen</td><td>11058021</td><td>For transfection</td></tr><tr><td>pLL3.7 eGFP</td><td>Addgene</td><td>11795</td><td>&lt;a href=&quot;https://www.addgene.org/11795/&quot;&gt;https://www.addgene.org/11795/&lt;/a&gt;</td></tr><tr><td>Bottle-top filter</td><td>Fisher</td><td>9761120</td><td>0.45 mm pore</td></tr><tr><td>Polybrene</td><td>Fisher</td><td>NC9840454</td><td>10 mg/ml</td></tr><tr><td>Oleic acid</td><td>Sigma</td><td>01383-5G</td><td>Prevent freeze-thaw cycle</td></tr><tr><td>Lecithin&amp;nbsp;</td><td>Fisher</td><td>IC10214625</td><td>Egg lecithin&amp;nbsp;</td></tr><tr><td>Sodium taurocholate</td><td>Fisher</td><td>NC9620276</td><td>Product discontinued; alternative catalog number: 50-121-7956</td></tr><tr><td>Protease inhibitor cocktail tablet (EDTA-free)</td><td>Fisher</td><td>5892791001</td><td>Used mainly for samples that need TEM analysis</td></tr><tr><td>Polycarbonate ultracentrifuge tube</td><td>Fisher</td><td>NC9696153</td><td>Reusing it multiple times will collapse the tube</td></tr><tr><td>Lid for ultracentrifuge tube</td><td>Fisher</td><td>NC9796914</td><td>A tool is required to remove the tube/lid from rotor</td></tr><tr><td>Syringe</td><td>Fisher</td><td>50-949-261</td><td>Disposable</td></tr><tr><td>Syringe filter</td><td>Fisher</td><td>09-719C</td><td>Pore size = 0.2 mm; nylon</td></tr><tr><td>Phosphotungstic acid</td><td>Fisher</td><td>AC208310250</td><td>For preparing 2% phosphotungstic acid, pH 6.0</td></tr><tr><td>Tweezer</td><td>Fisher</td><td>50-238-62</td><td>Extra fine and strong tips</td></tr><tr><td>Formvar/carbon grid</td><td>Fisher</td><td>50-260-34</td><td>Formvar/carbon film square grid 400 Copper</td></tr></tbody></table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

図 13日間コンフルエント後のCaco-2細胞1が表示され、正常な。ドーム状構造体の外観および細胞内脂質小滴は、分化したCaco-2細胞の特徴です。のCaco-2細胞を播種時に均等に分散されていないときは、皿の特定の領域に凝集し、過増殖う。任意の細胞を含まない皿の中のいくつかの領域が存在することになります。旋回および傾斜面に皿を置くことは避けるべきです。?...

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.7K ビュー.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

ビデオ: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

コレステロール流出アッセイ

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

医学

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

携帯の障壁を通過する薬物送達システムの輸送を評価するためのモデルと方法

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

生物工学

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

脂肪食法による脂質飛沫異化作用の制御の探索

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

がん研究

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

免疫・感染学

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

生細胞における脂質交換アッセイ

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

生化学

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

ヒト腸オルガノイドにおける脂質液滴形成の特徴付けと定量化のための蛍光ベースアッセイ

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

プロテオリポソー​​ムベースの流出アッセイのClの単一分子特性を決定する<sup&gt; -</sup&gt;チャネルとトランスポーター

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

生物学

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内血網膜バリア透過性を評価する ためのin vitro モデルとしての内皮細胞トランスサイトーシスアッセイ

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

3Dで栽培ネイティブ腸とのCaco-2細胞において上皮輸送タンパク質の機能および発現を研究するための方法

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

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コレステロール流出アッセイ

携帯の障壁を通過する薬物送達システムの輸送を評価するためのモデルと方法

脂肪食法による脂質飛沫異化作用の制御の探索

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

生細胞における脂質交換アッセイ

ヒト腸オルガノイドにおける脂質液滴形成の特徴付けと定量化のための蛍光ベースアッセイ

プロテオリポソームベースの流出アッセイのClの単一分子特性を決定する

内血網膜バリア透過性を評価するためのin vitro モデルとしての内皮細胞トランスサイトーシスアッセイ

3Dで栽培ネイティブ腸とのCaco-2細胞において上皮輸送タンパク質の機能および発現を研究するための方法

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

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コレステロール流出アッセイ

携帯の障壁を通過する薬物送達システムの輸送を評価するためのモデルと方法

脂肪食法による脂質飛沫異化作用の制御の探索

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

生細胞における脂質交換アッセイ

ヒト腸オルガノイドにおける脂質液滴形成の特徴付けと定量化のための蛍光ベースアッセイ

プロテオリポソー​​ムベースの流出アッセイのClの単一分子特性を決定する

内血網膜バリア透過性を評価する ためのin vitro モデルとしての内皮細胞トランスサイトーシスアッセイ

3Dで栽培ネイティブ腸とのCaco-2細胞において上皮輸送タンパク質の機能および発現を研究するための方法

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

プロテオリポソームベースの流出アッセイのClの単一分子特性を決定する

内血網膜バリア透過性を評価するためのin vitro モデルとしての内皮細胞トランスサイトーシスアッセイ