Name: 作者聚焦：使用 3D 人类细胞系模型研究化合物对肠道组织的影响
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

感谢翁贝托·韦罗内西基金会（Umberto Veronesi Foundation）为研究人员工作提供奖学金。

1. 基本3D重建肠黏膜模型的制备
注意：整个过程必须在无菌层流罩中进行。涉及使用细胞培养箱的程序中的所有步骤都意味着将培养物在含有5%CO2 的湿润气氛中在37°C下孵育（除非方案中另有说明）。
<ol><li>肠道模型系统中使用的细胞系的预先制备<ol><li>在 F25 烧瓶中以 5 x 105 的浓度接种 L929 小鼠成纤维细胞，在 5 mL 含有 2 mM L-谷氨酰胺、10% 胎牛血清 ...

石蜡包埋的 3D 肠粘膜模型的构建

<ol>
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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>

这里介绍的基本重建肠粘膜模型系统（图6）结合了生理复杂性（含有Caco-2单层的生理上更相关的3D细胞培养物，以及含有成纤维细胞和单核细胞的富含ECM的固有层支持物）和实验简单性（使用商业人类细胞系产生标准化且易于重复的系统）13.因此，该模型系统被认为是小鼠模型的合适替代品，旨在评估潜在药物/食品成分对肠道屏障完整性的影响。在这方面...

肠上皮屏障是包含不同类型上皮细胞的单细胞厚内膜，构成了身体外部和内部环境之间的第一个物理防御屏障或界面 1,2。柱状肠细胞是最丰富的上皮细胞类型。它们负责通过包括紧密连接 （TJ） 在内的多个屏障组件之间的相互作用来维持上皮屏障的完整性，在屏障收紧中起着重要作用 1,3。TJ 结构由细胞内斑块蛋白组成，例如闭塞带 （ZO） 和扣带蛋白，与跨膜蛋白（包括闭塞蛋白、claudins 和连接粘附分子 （JAM））合作，形成紧密连接相邻细胞的拉链状结构 3,4。跨膜蛋白调节小化合物的被动细胞旁扩散，排除有毒的大分子。
潜在有毒的食物化合物和食物污染物会刺激炎性细胞因子的产生，从而破坏上皮通透性，激活免疫细胞并引起慢性肠道组织炎症 5,6,7。相比之下，据报道，各种抗氧化和抗炎植物化学物质可通过恢复 TJ 蛋白表达和组装来降低炎性细胞因子表达并增强肠道 TJ 屏障完整性 4,6,8。因此，有益和有害化合物对上皮屏障完整性的调节已经看到体内和体外模型的使用增加，这些模型旨在模拟肠道屏障进行药物筛选和毒性研究。鉴于人们对了解肠病 （IBD）、坏死性小肠结肠炎和癌症的病理生理学的兴趣日益浓厚，这一点尤其重要，这些都可以在实验模型中模拟 8、9、10。
为了实现动物试验中“3R”的目标，人们一直需要开发基于细胞的体外模型。这些措施包括替代使用动物的替代品，减少使用的动物数量，以及改进采用减轻痛苦的方法11,12,13。此外，人类和小鼠模型（啮齿动物是使用最广泛的物种）之间的潜在分子、细胞和生理机制是独特的，导致关于小鼠模型作为人类反应预测因子的功效的争议12,13。体外人类细胞系模型的众多优点包括靶标限制实验、直接观察和连续分析13。
二维 （2D） 培养物中的单细胞型单层已成为强大的模型。然而，这些不能精确地再现人体组织的生理复杂性8,13,14。因此，3D培养系统正在开发中，并不断改进，以概括健康和患病肠道组织的生理复杂性，作为下一代风险评估工具箱13,14。这些模型包括具有不同细胞系的 3D Transwell 支架、类器官培养物和使用细胞系和类器官（来自健康和患病组织）的微流体装置（肠芯片）8,13,14。
本研究中提出的 3D“健康组织”肠道等效方案基于在生理复杂性和实验简单性之间取得平衡13。该模型代表了 3D Transwell 支架，由三种细胞系（肠细胞 [金标准结肠腺癌 Caco-2 系]和支持免疫成分 [U937 单核细胞和 L929 成纤维细胞]）组成，构成了一个标准化且易于重复的系统，适用于初步筛选对肠上皮屏障完整性和免疫反应感兴趣的膳食分子。该协议包括石蜡包埋，用于使用固定肠道等效物对上皮屏障完整性进行光学显微镜评估。本方法的优点是可以使包埋组织的许多切片从单个实验中对多个参数进行染色。

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

体内和体外肠道模型在研究炎症性肠道疾病的病理生理学、潜在有益物质的药理学筛选以及潜在有害食品成分的毒性研究方面的使用有所增加。与此相关的是，目前需要开发基于细胞的体外模型来替代动物模型。在这里，提出了一个使用细胞系的基本“健康组织”三维（3D）肠道等效模型的方案，具有提供实验简单性（标准化和易于重复的系统）和生理复杂性（Caco-2肠细胞与U937单核细胞和L929成纤维细胞的支持免疫成分）的双重好处。该协议还包括用于固定肠道当量的光学显微镜评估的石蜡包埋，从而提供了从单个实验中分析多个视觉参数的优势。苏木精和伊红 （H&E） 染色切片显示 Caco-2 柱状细胞在对照处理中形成紧密且规则的单层，用于验证该模型作为实验系统的功效。使用麸质作为促炎食品成分，从切片中分析的参数包括单层厚度减少，以及与底层基质 （H&E） 的破坏和分离、闭合蛋白染色所示的紧密连接蛋白表达降低（可统计学量化），以及迁移的 U937 细胞的免疫激活，如分化簇 14 （CD14） 染色和 CD11b 相关分化为巨噬细胞所证明的那样。如使用脂多糖模拟肠道炎症所示，可以测量的其他参数是粘液染色和细胞因子表达增加（如中间因子），这些参数可以在固定前从培养基中提取。基本的三维 （3D） 肠粘膜模型和固定切片可推荐用于炎症状态和屏障完整性研究，并可以分析多个视觉可量化参数。

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.

作者聚焦：使用 3D 人类细胞系模型研究化合物对肠道组织的影响 

具有免疫成分的基本三维 （3D） 肠道模型系统

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.

第一个重要方面是确定用于实验目的的基本 3D 肠道等效粘膜的可接受性。这是使用组织学和组织病理学实验室中最广泛使用的染色剂进行的，即苏木精（染色核材料深蓝紫色）和伊红（染色细胞质材料深浅不一的粉红色）。首先对未经处理的对照进行H&E染色，该对照在与实验处理相同的条件和时间范围内培养。通过可视化细胞的模式、形状和结构，该肠道细胞模型系统的成功取决于 Caco-2 细胞在 ...

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

在这里，我们描述了构建一个基本的三维（3D）肠道细胞系模型系统和一个石蜡包埋方案，用于固定肠道当量的光学显微评估。对选定的蛋白质进行染色可以分析来自单个实验的多个视觉参数，以潜在地用于临床前药物筛选研究。

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 ...

工作范围是利用这些体外三维人类细胞系模型来研究肠道组织上有益和有益化合物的事实。这些模型可用于进一步研究理解，也可以用作基本药物模型。使用植物衍生的化合物亚精胺和丁香酚，无论是单独还是以补充剂形式一起使用，我们使用这些模型测量自噬的有益增加和炎症的减少。然后，我们还将能够展示从某些小麦品种中提取的麸质对肠道组织的潜在有害影响。我们目前正在使用价格经济、标准化且易于重复的体外系统，该系统可以更好地反映人类肠道的生理机能，作为动物模型的替代品。由于我们也使用商业人类细胞系而不是植物来源的细胞，因此该研究的反应更适用于普通人群。该协议完全基于不同线路的使用。这些使模型最标准化和可重现。此外，同时培养不同的细胞系、肠细胞、成纤维细胞和免疫系统细胞，允许更接近患者体内发生的细胞反应。

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.

科研

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.

制备基本3D重建肠粘膜模型，用于固定肠道当量的显微评估

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.