Name: Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents
Uploaded: 2023-09-01T13:00:00
Description: 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

Konstruktion eines 3D-Darmschleimhautmodells mit Paraffineinbettung

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.

Autor im Rampenlicht: Untersuchung der Auswirkungen von Verbindungen auf das Darmgewebe mit Hilfe von 3D-Modellen menschlicher Zelllinien 

Grundlegendes dreidimensionales (3D) Darmmodellsystem mit einer Immunkomponente

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  

Erstellung eines einfachen 3D-rekonstruierten Darmschleimhautmodells zur mikroskopischen Beurteilung von fixierten Darmäquivalenten

Wählen Sie zunächst eine 24-Well-Platte mit Einsätzen mit 0,4-Mikron-Filtern. Geben Sie mit einer Pipette 500 Mikroliter HBSS unter und über die Filtereinsätze. Verschließen Sie dann die Multiwell-Platte und legen Sie sie für zwei Stunden in einen Inkubator.Nach der Inkubation wird das HBSS vorsichtig von der Platte abgesaugt. Bereiten Sie eine zellfreie Kollagenlösung in einem sterilen 50-Milliliter-Röhrchen in DMEM auf Eis zu. Geben Sie 250 Mikroliter Kollagen über den Membranfilter und legen Sie den Deckel auf die Platte.Lassen Sie die Lösung bei Raumtemperatur polymerisieren. Als nächstes entfernen Sie die L-929-Fibroblasten-Zellkultur aus dem Inkubator. Zwei Milliliter einer vorgewärmten Trypsin-EDTA-Lösung in den Kolben geben und drei bis fünf Minuten lang in einen Inkubator geben.Die Zellsuspension wird in ein steriles 15-Milliliter-Röhrchen überführt und fünf Minuten lang bei 645 g zentrifugiert. Mit einer Vakuumpumpe wird der Überstand abgesaugt und das Pellet in einem Milliliter DMEM resuspendiert. Geben Sie 20 Mikroliter Zellsuspension in ein Röhrchen mit 20 Mikrolitern Trypanblaulösung.Und verwenden Sie eine Zellzählkammer, um die Zelldichte zu bewerten. Als nächstes wird die U-937-Monozyten-Zellkultur aus dem Inkubator entnommen. Zentrifugieren Sie die Zellsuspension und resuspendieren Sie das Pellet in einem Milliliter RPMI.Nachdem Sie sichergestellt haben, dass die Zellen homogen suspendiert sind, zählen Sie sie mit einer Zellzählkammer. Als nächstes werden Zellsuspensionen mit 50.000 L-929- bzw. 15.000 U-937-Zellen in 50 Mikrolitern DMEM hergestellt. Geben Sie jeweils 50 Mikroliter Zellsuspension zu 450 Mikrolitern Kollagen und mischen Sie es gründlich.Überlagern Sie die vorkodierte zellfreie Lamina propria mit 500 Mikrolitern der Kollagenzelllösung. Verschließen Sie die Platte und legen Sie sie für zwei Stunden in einen Inkubator, damit die Lösung aushärten kann. Als nächstes entnehmen Sie die Caco-2-Zellkultur aus dem Inkubator.Saugen Sie das Medium mit einer Vakuumpumpe an und spülen Sie die Zellen mit 10 Millilitern PBS. Fügen Sie fünf Milliliter PBS-Trypsin-EDTA-Lösung hinzu und legen Sie sie für fünf bis acht Minuten in einen Inkubator. Zentrifugieren Sie dann die Zellsuspension und resuspendieren Sie das Pellet in einem Milliliter DMEM.Nach der Zählung wird eine Zellsuspension mit 150.000 Caco-2-Zellen in 50 Mikrolitern DMEM hergestellt. Legen Sie die Platte 10 Minuten lang in eine Laminar-Flow-Haube, bevor Sie sie in den Inkubator überführen. Nach 30 Minuten werden 500 Mikroliter DMEM über das rekonstruierte Modell und 500 Mikroliter unter den Filter gegeben und die Platte wieder in den Inkubator zurückgelegt.Ersetzen Sie das Medium am nächsten Tag vorsichtig durch 500 Mikroliter frisches DMEM über bzw. unter dem Filter.

Research

JoVE Journal

Biology

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 ...