preparation of a basic 3d reconstructed intestinal mucosa model for microscopic evaluation of fixed intestinal equivalents

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

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.

Watch this Scientific Journal Video about Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents at JoVE.com

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

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

首先，选择包含带有 0.4 微米过滤器的插件的 24 孔板。使用移液管，在过滤器插件下方和上方加入 500 微升 HBSS。然后关闭多孔板并将其置于培养箱中两个小时。孵育后，小心地从平板中吸出HBSS。在无菌的 50 毫升管中在冰上的 DMEM 中制备无细胞胶原蛋白溶液。在膜过滤器上方加入 250 微升胶原蛋白，并将盖子放在盘子上。让溶液在室温下聚合。接下来，从培养箱中取出 L-929 成纤维细胞培养物。向烧瓶中加入两毫升预热的胰蛋白酶EDTA溶液，并将其放入培养箱中三到五分钟。将细胞悬液转移到无菌的 15 毫升管中，并以 645 g 离心 5 分钟。使用真空泵吸出上清液并将沉淀重悬于一毫升DMEM中。将 20 微升细胞悬液加入含有 20 微升台盼蓝溶液的试管中。并使用细胞计数室来评估细胞密度。接下来，从培养箱中取出 U-937 单核细胞培养物。离心细胞悬浮液，并将沉淀重悬于一毫升RPMI中。确保细胞均匀悬浮后，使用细胞计数室对细胞进行计数。接下来，在50微升DMEM中制备分别含有50,000 L-929和15,000 U-937细胞的细胞悬液。将每个 50 微升细胞悬液加入 450 微升胶原蛋白中并充分混合。用 500 微升胶原细胞溶液覆盖预先编码的无细胞固有层。关闭板并将其放入培养箱中两个小时，使溶液凝固。接下来，从培养箱中取出 Caco-2 细胞培养物。使用真空泵吸出培养基并用 10 毫升 PBS 冲洗细胞。加入五毫升PBS胰蛋白酶EDTA溶液，并将其放入培养箱中5至8分钟。然后离心细胞悬浮液，并将沉淀重悬于一毫升DMEM中。计数后，在50微升DMEM中制备含有150,000个Caco-2细胞的细胞悬液。将板放入层流罩中 10 分钟，然后将其转移到培养箱中。30 分钟后，在重建模型上方加入 500 微升 DMEM，在过滤器下方加入 500 微升，并将板放回培养箱。第二天，小心地用 500 微升新鲜的 DMEM 分别在过滤器上方和下方更换培养基。

preparation-basic-3d-reconstructed-intestinal-mucosa-model-for

Research

JoVE Journal

Biology

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

154 次观看.  Alma Mater Studiorum-University of Bologna. 首先，选择包含带有 0.4 微米过滤器的插件的 24 孔板。使用移液管，在过滤器插件下方和上方加入 500 微升 HBSS。然后关闭多孔板并将其置于培养箱中两个小时。孵育后，小心地从平板中吸出HBSS。在无菌的 50 毫升管中在冰上的 DMEM 中制备无细胞胶原蛋白溶液。在膜过滤器上方加入 250 微升胶原蛋白，并将盖子放在盘子上。让溶液在室温下聚合。接下来，从培养箱中?...

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

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 ...

科研

生物学

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.

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.