paraffin embedding of a basic 3d reconstructed intestinal mucosa model for immune-histochemical staining

石蜡包埋的 3D 肠粘膜模型的构建

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

Watch this Scientific Journal Video about Paraffin Embedding of a Basic 3D Reconstructed Intestinal Mucosa Model for Immune-Histochemical Staining at JoVE.com

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

用于免疫组织化学染色的基本 3D 重建肠粘膜模型的石蜡包埋

对于细胞的石蜡包埋，将石蜡机设置为 58 摄氏度。使用钳子，将膜插入物转移到无菌 24 孔板的清洁孔中。在过滤器上方加入 500 微升 37% 缓冲福尔马林和 PBS，在过滤器下方加入 1 毫升。盖上盖子，将盘子放在通风橱中两个小时。一旦固有层从膜插入物中分离出来，将肠粘膜转移到含有 25 毫升 35% 乙醇的烧杯中，并孵育 10 分钟。在增加乙醇浓度下脱水后，将样品置于 50 毫升二甲苯或组织学清除剂中 10 至 20 分钟。样品透明后，将它们放入金属组织盒支架中，然后将它们浸入加热机器内的液体石蜡中。45 分钟后，使用切片机切下四微米切片。将切割部分放在载玻片上，并在 37 摄氏度的烤箱中干燥 24 小时。可接受的 3-D 肠道等效粘膜模型由 Caco-2 细胞组成，这些细胞在富含细胞外基质的固有层上方形成紧密且规则的单层。不可接受的模型包括那些显示过度生长、上皮层紊乱、畸形或缺乏上皮层形成的模型。与对照组相比，高麸质含量小麦蛋白的促炎作用破坏了细胞单层并减小了上皮层的厚度。对照组的闭塞蛋白含量显著高于暴露于麸质蛋白的组。U937单核细胞的CD14和CD11b染色表明，高面筋含量的小麦蛋白激活了单核细胞，并诱导其迁移和分化为巨噬细胞。在脂多糖攻击下，Caco-2细胞增加了粘液的产生和炎症标志物中间因子的释放。

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JoVE Journal

Biology

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

155 次观看. 对于细胞的石蜡包埋，将石蜡机设置为 58 摄氏度。使用钳子，将膜插入物转移到无菌 24 孔板的清洁孔中。在过滤器上方加入 500 微升 37% 缓冲福尔马林和 PBS，在过滤器下方加入 1 毫升。盖上盖子，将盘子放在通风橱中两个小时。一旦固有层从膜插入物中分离出来，将肠粘膜转移到含有 25 毫升 35% 乙醇的烧杯中，并孵育 10 分钟。在增加乙醇浓度下脱水后，将样品置于 50 ?...

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

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.

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 ...