Name: 著者スポットライト:3Dヒト細胞株モデルを用いた腸組織に対する化合物の影響の調査
Uploaded: 2023-09-01T13:00:00
Description: Watch this Scientific Journal Video about Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models at JoVE.com

ウンベルト・ヴェロネージ財団の研究者活動を支援するフェローシップに感謝します。

1. 基本的な3D再建腸粘膜モデルの作成
注意: 手順全体は、滅菌層流フードで実行する必要があります。細胞インキュベーターの使用を含む手順のすべてのステップは、培養物が5%CO2 を含む加湿雰囲気中で37°Cでインキュベートされることを意味します(プロトコルに別段の記載がない限り)。
<ol><li>腸管モデル系で使用する細胞株の事前調製<ol><li>2 mM L-グル...

パラフィン包埋法による3次元腸管粘膜モデルの構築

<ol>
	<li>Chelakkot, C., Ghim, J., Ryu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+regulating+intestinal+barrier+integrity+and+its+pathological+implications.">Mechanisms regulating intestinal barrier integrity and its pathological implications.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-9 (2018).</li><li>Schoultz, I., Keita, &. #. 1. 9. 7. ;. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+time+for+reviewer+3+to+request+human+organ+chip+experiments+instead+of+animal+validation+studies.">Is it time for reviewer 3 to request human organ chip experiments instead of animal validation studies.</a> Advanced Science. 7 (22), 2002030 (2020).</li><li>Jung, S. M., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+small+intestine+for+studying+intestinal+diseases.">In vitro models of the small intestine for studying intestinal diseases.</a> Frontiers in Microbiology. 12, 767038 (2022).</li><li>Nitsche, K. S., M&#252;ller, I., Malcomber, S., Carmichael, P. L., Bouwmeester, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementing+organ-on-chip+in+a+next-generation+risk+assessment+of+chemicals:+a+review.">Implementing organ-on-chip in a next-generation risk assessment of chemicals: a review.</a> Archives of Toxicology. 96 (3), 711-741 (2022).</li><li>Kekilli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midkine+level+may+be+used+as+a+noninvasive+biomarker+in+Crohn's+disease.">Midkine level may be used as a noninvasive biomarker in Crohn's disease.</a> Turkish Journal of Medical Sciences. 50, 324-329 (2020).</li><li>Truzzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermidine-eugenol+supplement+preserved+inflammation-challenged+intestinal+cells+by+stimulating+autophagy.">Spermidine-eugenol supplement preserved inflammation-challenged intestinal cells by stimulating autophagy.</a> International Journal of Molecular Sciences. 24 (4), 4131 (2023).</li><li>Truzzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+supplements+safe?+Effects+of+gallic+and+ferulic+acids+on+in+vitro+cell+models.">Are supplements safe? Effects of gallic and ferulic acids on in vitro cell models.</a> Nutrients. 12 (6), 1591 (2020).</li><li>Buckley, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+multiple+tight+junctional+complexes+in+human+airway+epithelial+cells.">Visualisation of multiple tight junctional complexes in human airway epithelial cells.</a> Biological Procedures. Online. 20, 3 (2018).</li><li>Ghosh, R., Gilda, J. E., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+necessity+of+and+strategies+for+improving+confidence+in+the+accuracy+of+western+blots.">The necessity of and strategies for improving confidence in the accuracy of western blots.</a> Expert Review of Proteomics. 11 (5), 549-560 (2014).</li></ol>

ここで紹介する基本的な再建腸粘膜モデルシステム(図6)は、生理学的複雑さ(線維芽細胞と単球を含むECMに富む固有層支持体を持つCaco-2単層を含む、より生理学的に関連性のある3D細胞培養)と実験の単純さ(市販のヒト細胞株を使用して標準化され、再現性のあるシステムを作製する)を兼ね備えています13.そのため、このモデルシステムは、潜在的な医?...

腸管上皮関門は、異なる種類の上皮細胞を含む1細胞厚の内層であり、身体の外部環境と内部環境との間の最初の物理的防御障壁または界面を構成する1,2。円柱型腸細胞は、上皮細胞の中で最も豊富なタイプを構成します。これらは、タイトジャンクション(TJ)を含むいくつかのバリアコンポーネント間の相互作用を通じて上皮バリアの完全性を維持する役割を担っており、バリアの引き締めに重要な役割を果たしています1,3。TJ構造は、閉塞帯帯(ZO)やシングリンなどの細胞内プラークタンパク質からなり、オクルージン、クローディン、接合接着分子(JAM)などの膜貫通タンパク質と協働して、隣接する細胞を緊密につなぐジッパー状の構造を形成します3,4。膜貫通型タンパク質は、低分子化合物の受動的な傍細胞拡散を調節し、有毒な高分子を排除します。
潜在的に有毒な食品化合物や食品汚染物質は、炎症性サイトカイン産生を刺激し、上皮透過性を破壊し、免疫細胞を活性化し、慢性的な腸組織の炎症を引き起こします5,6,7。対照的に、さまざまな抗酸化および抗炎症性植物化学物質は、TJタンパク質の発現と組み立ての回復を通じて、炎症性サイトカインの発現を減少させ、腸のTJバリアの完全性を高めることが報告されています4,6,8。したがって、有益な化合物と有害な化合物の両方による上皮バリアの完全性の調節により、医薬品スクリーニングおよび毒性試験のための腸管バリアを模倣することを目的としたin vivoおよびin vitroモデルの両方の使用が増加しています。これは、腸管疾患(IBD)、壊死性腸炎、および癌の病態生理学を理解することへの関心が高まっていることを考えると、特に関連性があります8,9,10。
動物実験における「3R」の目的を達成するために、細胞ベースのin vitroモデルの開発が求められています。これらには、動物の使用に代わる代替手段、使用される動物の数の削減、および苦痛を軽減する方法の採用の改良が含まれます11,12,13。さらに、ヒトモデルとマウスモデル(げっ歯類が最も広く使用されている種)の根底にある分子、細胞、および生理学的メカニズムは独特であり、ヒトの応答における予測因子としてのマウスモデルの有効性に関する論争につながっています12,13。in vitroヒト細胞株モデルの多くの利点には、標的制限実験、直接観察、および連続分析が含まれます13。
2次元(2D)培養における単細胞型単分子膜は、強力なモデルとして役立っています。しかし、これらはヒト組織の生理学的複雑さを正確に再現することはできない8,13,14。その結果、次世代のリスク評価ツールボックスとして、健康な腸組織と病気の腸組織の両方の生理学的複雑さを再現するために、ますます改善された3D培養システムが開発されています13,14。これらのモデルには、多様な細胞株を備えた3Dトランズウェルスキャフォールド、オルガノイド培養物、細胞株とオルガノイド(健康な組織と病気の組織の両方に由来する)の両方を使用したマイクロ流体デバイス(腸管オンチップ)が含まれます8,13,14。
本研究で提示された3D「健康な組織」腸と同等のプロトコルは、生理学的複雑さと実験の単純さのバランスをとることに基づいていました13。このモデルは、3つの細胞株(腸細胞[ゴールドスタンダードの結腸腺癌Caco-2株]と支持免疫成分[U937単球およびL929線維芽細胞])で構成され、腸上皮バリアの完全性と免疫応答に関心のある食物分子の予備スクリーニングに適用可能な標準化された再現性のあるシステムを構成する3Dトランズウェルスキャフォールドの代表例です。プロトコルは固定腸の等量を使用して上皮障壁の完全性の光学顕微鏡評価のためのパラフィンの埋め込みを含んでいる。本アプローチの利点は、1回の実験で複数のパラメータについて、包埋組織の多数の切片を染色することができることである。

basic three-dimensional (3d) intestinal model system with an immune component

炎症性腸疾患の病態生理学の研究、潜在的に有益な物質の薬理学的スクリーニング、および潜在的に有害な食品成分の毒性研究のために、 in vivo および in vitro 腸モデルの使用が増加しています。関連して、動物モデルに代わる細胞ベースの in vitro モデルの開発が現在求められています。ここでは、細胞株を使用した基本的な「健康な組織」の三次元(3D)腸と同等モデルのプロトコルが、実験のシンプルさ(標準化され、容易に再現可能なシステム)と生理学的複雑さ(U937単球とL929線維芽細胞の支持免疫成分を持つCaco-2腸細胞)の両方を提供するという二重の利点を備えています。このプロトコルには、固定腸当量物の光学顕微鏡評価のためのパラフィン包埋も含まれており、それによって1回の実験から複数の視覚パラメータを分析するという利点を提供します。ヘマトキシリンとエオシン(H&E)で染色した切片は、対照処理でCaco-2柱状細胞がタイトで規則的な単層を形成していることを示すもので、実験システムとしてのモデルの有効性を検証するために使用されます。グルテンを炎症誘発性食品成分として使用し、切片から分析したパラメータには、単層の厚さの減少、基礎となるマトリックス(H&E)からの破壊と剥離、オクルージン染色(統計的に定量化可能)から示されるようなタイトジャンクションタンパク質発現の減少、分化クラスター14(CD14)染色とCD11b関連のマクロファージへの分化から証明される遊走U937細胞の免疫活性化が含まれます。リポ多糖類を用いて腸の炎症をシミュレートすることによって示されるように、測定可能な追加のパラメータは、粘液染色の増加および固定前に培地から抽出することができるサイトカイン発現(ミッドカインなど)である。基本的な3次元(3D)腸粘膜モデルと固定切片は、複数の視覚的に定量化可能なパラメータを分析する可能性を備えた炎症状態とバリアの完全性の研究に推奨できます。

The intestinal epithelial barrier, a one-cell-thick internal lining containing different types of epithelial cells, constitutes the first physical defensive barrier or interface between the outside and the internal milieu of the body1,2. Columnar-type enterocytes constitute the most abundant type of epithelial cells. These are&#160;responsible for maintaining epithelial barrier integrity through interactions between several barrier components, including tight junctions (TJs), playing a significant role in barrier tightening1,3. The TJ structure consists of intracellular plaque proteins, such as zonula occludens (ZO) and cingulin, cooperating with transmembrane proteins, including occludins, claudins, and junctional adhesion molecules (JAMs) that form zipper-like structure tightly linking the neighboring cells3,4. The transmembrane proteins regulate the passive paracellular diffusion of small compounds and exclude toxic large molecules.
Potentially toxic food compounds and food contaminants stimulate inflammatory cytokine production that disrupts the epithelial permeability, activating immune cells and causing chronic intestinal tissue inflammation5,6,7. In contrast, various anti-oxidant and anti-inflammatory phytochemicals have been reported to reduce inflammatory cytokine expression and enhance intestinal TJ barrier integrity through the restoration of TJ protein expression and assembly4,6,8. Hence, the regulation of epithelial barrier integrity by both beneficial and harmful compounds has seen an increase in the use of both in vivo and in vitro models aimed at mimicking the intestinal barrier for pharmaceutical screening and toxicity studies. This is particularly relevant given the increasing interest in understanding the pathophysiology of intestinal bowel diseases (IBD), necrotizing enterocolitis, and cancer, which can be simulated in experimental models8,9,10.
There has been a demand for the development of cell-based in vitro models in order to achieve the objective of the &#8220;3Rs&#8221; in animal testing. These include replacement alternatives to the use of animals, reduction in the number of animals used, and refinement in adopting methods that alleviate distress11,12,13. Moreover, the underlying molecular, cellular, and physiological mechanisms between human and murine models (rodents being the most widely used species) are distinctive, leading to controversy regarding the efficacy of the murine models as predictors in human responses12,13. Numerous advantages of in vitro human cell-line models include target-restricted experimentation, direct observation, and continuous analysis13.
Single-cell-type monolayers in two-dimensional (2D) cultures have served as powerful models. However, these cannot precisely reproduce the physiological complexity of human tissues8,13,14. As a result, 3D&#160;culture systems are being developed with ever-increasing improvements to recapitulate the physiological complexity of both healthy and diseased intestinal tissues as next-generation risk assessment toolboxes13,14. These models include 3D Transwell scaffolds with diverse cell lines, organoid cultures, and microfluidic devices (intestine-on-chip) using both cell lines and organoids (derived from both healthy and diseased tissues)8,13,14.&#160;
The 3D &#8220;healthy tissue&#8221; intestinal equivalent protocol presented in the present study was based on striking a balance between physiological complexity and experimental simplicity13. The model is representative of a 3D Transwell scaffold, comprised of three cell lines (enterocytes [the gold-standard colon adenocarcinoma Caco-2 line] with a supporting immune component [U937 monocytes and L929 fibroblasts]), constituting a standardized and easily repeatable system applicable for the preliminary screening of dietary molecules of interest on intestinal epithelial barrier integrity and immune response. The protocol includes paraffin embedding for light microscopic evaluation of epithelial barrier integrity using fixed intestinal equivalents. The advantage of the present approach is that numerous sections of the embedded tissues can be made to stain for multiple parameters from a single experiment.

著者スポットライト:3Dヒト細胞株モデルを用いた腸組織に対する化合物の影響の調査 

免疫成分を用いた基本的な3次元(3D)腸管モデルシステム

The scope of the work is to use these in vitro three-dimensional human cell line models to investigate the fact of beneficial as well as helpful compounds on intestinal tissue. The models can be used to further research understanding and also as a basic drug model. Using the plant-derived compounds, spermidine and eugenol, both separately and together in supplement form, we measure beneficial increases in autophagy and decreases in inflammation using these models.Then we will also able to show the potentially harmful effect of gluten extracted from certain wheat varieties on intestinal tissues. We are currently using economically priced, standardized, and easily repeatable in vitro system that better reflect the physiology of the human intestine as an alternative to animal models. Since we also use commercial human cell line rather than plant-derived cells, the responses from the study are more applicable to the general population.The protocol is based entirely on the use of different lines. These renders the model most standardized and reproducible. Furthermore, the simultaneous cultivation of different cell lines, enterocytes, fibroblasts, and immune system cells, allows for cellular responses that are closer to what happens in the patient.

最初の重要な側面は、実験目的で基本的な3D腸当量粘膜の受容性を決定することです。これは、組織学および組織病理学の研究室で最も広く使用されている染色、すなわちヘマトキシリン(核物質を濃い青紫色に染色)およびエオシン(ピンクのさまざまな色合いの細胞質物質を染色)で行います。H&E染色は、まず未処理のコントロールで行い、実験的処理と同じ条件と時間枠で培養します。細胞?...

Watch this Scientific Journal Video about Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models at JoVE.com

ここでは、基本的な3次元(3D)腸細胞株モデルシステムの構築と、固定された腸当等物の光学顕微鏡的評価のためのパラフィン包埋プロトコルについて説明します。選択したタンパク質を染色することで、1回の実験で複数の視覚的パラメータを解析し、前臨床薬物スクリーニング研究に活用することができます。

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

この研究の範囲は、これらのin vitroの3次元ヒト細胞株モデルを使用して、腸組織上の有益で有用な化合物の事実を調査することです。このモデルは、研究の理解を深めるために、また基本的な薬物モデルとして使用することができます。植物由来の化合物であるスペルミジンとオイゲノールを別々に、またはサプリメントの形で一緒に使用し、これらのモデルを使用してオートファジーの有益な増加と炎症の減少を測定します。次に、特定の小麦品種から抽出されたグルテンが腸組織に及ぼす潜在的に有害な影響を示すこともできます。現在、動物モデルの代替として、ヒト腸の生理機能をよりよく反映した、経済的に価格が高く、標準化され、再現性が容易なin vitroシステムを使用しています。また、植物由来の細胞ではなく、市販のヒト細胞株を使用しているため、この研究からの回答は、一般の人々により適用可能です。このプロトコルは、完全に異なる回線の使用に基づいています。これにより、モデルが最も標準化され、再現性が高まります。さらに、異なる細胞株、腸細胞、線維芽細胞、免疫系細胞を同時に培養することで、患者に起こることにより近い細胞応答が可能になります。

basic-three-dimensional-3d-intestinal-model-system-with-an-immune

Research

JoVE Journal

Biology

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

生物学

Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents

To begin, select a 24-well plate containing inserts with 0.4 micron filters. Using a pipette, add 500 microliters of HBSS below and above the filter inserts. Then close the multi-well plate and place it in an incubator for two hours.After incubation, carefully aspirate the HBSS from the plate. Prepare a cell-free collagen solution in a sterile 50 milliliter tube in DMEM on ice. Add 250 microliters of collagen above the membrane filter and place the lid on the plate.Allow the solution to polymerize at room temperature. Next, remove the L-929 fibroblast cell culture from the incubator. Add two milliliters of a pre-warmed trypsin EDTA solution to the flask and place it in an incubator for three to five minutes.Transfer the cell suspension to a sterile 15 milliliter tube and centrifuge at 645 G for five minutes. Using a vacuum pump, aspirate the supernatant and resuspend the pellet in one milliliter of DMEM. Add 20 microliters of cell suspension into a tube containing 20 microliters of Trypan Blue solution.And use a cell counting chamber to evaluate the cell density. Next, remove the U-937 monocyte cell culture from the incubator. Centrifuge the cell suspension and resuspend the pellet in one milliliter of RPMI.After ensuring cells are homogeneously suspended, count them using a cell counting chamber. Next, prepare cell suspensions containing 50, 000 L-929 and 15, 000 U-937 cells respectively in 50 microliters of DMEM. Add each 50 microliter cell suspension to 450 microliters of collagen and mix thoroughly.Overlay the pre-coded cell-free lamina propria with 500 microliters of the collagen cell solution. Close the plate and place it in an incubator for two hours to allow the solution to set. Next, remove the Caco-2 cell culture from the incubator.Using a vacuum pump, aspirate the medium and rinse the cells with 10 milliliters of PBS. Add five milliliters of PBS trypsin EDTA solution and place it in an incubator for five to eight minutes. Then centrifuge the cell suspension and resuspend the pellet in one milliliter of DMEM.After counting, prepare a cell suspension containing 150, 000 Caco-2 cells in 50 microliters of DMEM. Place the plate in a laminar flow hood for 10 minutes before transferring it to the incubator. After 30 minutes, add 500 microliters of the DMEM above the reconstructed model and 500 microliters below the filter and return the plate to the incubator.The next day, carefully replace the medium with 500 microliters of fresh DMEM above and below the filter respectively.

固定腸当物の顕微鏡的評価のための基本的な3D再構成腸粘膜モデルの作成

Paraffin Embedding of a Basic 3D Reconstructed Intestinal Mucosa Model for Immune-Histochemical Staining

For paraffin embedding of cells, set the paraffin machine to 58 degrees Celsius. Using pliers, transfer the membrane inserts to clean wells of a sterile 24-well plate. Add 500 microliters of 37%buffered formalin and PBS above the filter, and one milliliter below the filter.Close the lid and leave the plate in a fume hood for two hours. Once the lamina propria is detached from the membrane insert, transfer the intestinal mucosa into a beaker containing 25 milliliters of 35%ethanol, and incubate for 10 minutes. After dehydration in increasing ethanol concentrations, place the samples in 50 milliliters of xylene or a histological clearing agent for 10 to 20 minutes.Once the samples are transparent, place them in a metal tissue cassette holder and submerge them in liquid paraffin inside a heated machine. After 45 minutes, use a microtome to cut four micron sections. Place the cut section on the slides and dry them in an oven at 37 degrees Celsius for 24 hours.An acceptable 3-D intestinal equivalent mucosa model consists of Caco-2 cells formed in a tight and regular monolayer above the extracellular-matrix-rich lamina propria. Unacceptable models include those showing excessive growth, disorganized epithelial layers, malformation or lack of epithelial layer formation. The proinflammatory effect of wheat protein with a high gluten content disrupted the cellular monolayer and reduced the thickness of the epithelial layer compared to the control group.The occludin protein content was significantly higher in the control group than in the group exposed to gluten protein. CD14 and CD11b staining of U937 monocytes showed that wheat protein with a high gluten content activated the monocytes, and induced their migration and differentiation into macrophages. Under the lipopolysaccharide challenge, Caco-2 cells increased mucus production and the release of the inflammatory marker midkine.