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

Costruzione di un modello 3D della mucosa intestinale con inclusione di paraffina

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.

Autore in primo piano: Indagine sugli effetti dei composti sul tessuto intestinale utilizzando modelli 3D di linee cellulari umane 

Sistema modello intestinale tridimensionale (3D) di base con una componente immunitaria

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  

Preparazione di un modello di base della mucosa intestinale ricostruita in 3D per la valutazione microscopica di equivalenti intestinali fissi

Per iniziare, selezionare una piastra a 24 pozzetti contenente inserti con filtri da 0,4 micron. Utilizzando una pipetta, aggiungere 500 microlitri di HBSS sotto e sopra gli inserti del filtro. Quindi chiudere la piastra a più pozzetti e metterla in un'incubatrice per due ore.Dopo l'incubazione, aspirare con cautela l'HBSS dalla piastra. Preparare una soluzione di collagene priva di cellule in una provetta sterile da 50 millilitri in DMEM su ghiaccio. Aggiungere 250 microlitri di collagene sopra il filtro a membrana e posizionare il coperchio sul piatto.Lasciare polimerizzare la soluzione a temperatura ambiente. Successivamente, rimuovere la coltura cellulare di fibroblasti L-929 dall'incubatrice. Aggiungere due millilitri di una soluzione di tripsina EDTA preriscaldata al matraccio e metterlo in un'incubatrice per tre-cinque minuti.Trasferire la sospensione cellulare in una provetta sterile da 15 millilitri e centrifugare a 645 G per cinque minuti. Utilizzando una pompa a vuoto, aspirare il surnatante e risospendere il pellet in un millilitro di DMEM. Aggiungere 20 microlitri di sospensione cellulare in una provetta contenente 20 microlitri di soluzione Trypan Blue.E usa una camera di conteggio delle cellule per valutare la densità cellulare. Successivamente, rimuovere la coltura cellulare monocitaria U-937 dall'incubatore. Centrifugare la sospensione cellulare e risospendere il pellet in un millilitro di RPMI.Dopo essersi assicurati che le cellule siano sospese in modo omogeneo, contarle utilizzando una camera di conteggio delle cellule. Successivamente, preparare sospensioni cellulari contenenti rispettivamente 50.000 cellule L-929 e 15.000 cellule U-937 in 50 microlitri di DMEM. Aggiungere ogni sospensione cellulare da 50 microlitri a 450 microlitri di collagene e mescolare accuratamente.Sovrapporre la lamina propria priva di cellule precodificata con 500 microlitri di soluzione cellulare di collagene. Chiudere la piastra e metterla in un'incubatrice per due ore per consentire alla soluzione di solidificarsi. Successivamente, rimuovere la coltura cellulare Caco-2 dall'incubatrice.Utilizzando una pompa a vuoto, aspirare il mezzo e risciacquare le celle con 10 millilitri di PBS. Aggiungere cinque millilitri di soluzione PBS tripsina EDTA e metterla in un'incubatrice per cinque-otto minuti. Quindi centrifugare la sospensione cellulare e risospendere il pellet in un millilitro di DMEM.Dopo il conteggio, preparare una sospensione cellulare contenente 150.000 cellule Caco-2 in 50 microlitri di DMEM. Posizionare la piastra in una cappa a flusso laminare per 10 minuti prima di trasferirla nell'incubatrice. Dopo 30 minuti, aggiungere 500 microlitri di DMEM sopra il modello ricostruito e 500 microlitri sotto il filtro e rimettere la piastra nell'incubatrice.Il giorno successivo, sostituire con cura il terreno con 500 microlitri di DMEM fresco rispettivamente sopra e sotto il filtro.

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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 ...