Name: Autor destacado: Investigación de los efectos de los compuestos en el tejido intestinal utilizando modelos de líneas celulares humanas en 3D
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

Gracias a la Fundación Umberto Veronesi por una beca de apoyo al trabajo de los investigadores.

1. Preparación del modelo básico de mucosa intestinal reconstruida en 3D
NOTA: Todo el procedimiento debe realizarse en una campana de flujo laminar estéril. Todas las etapas del procedimiento que impliquen el uso de la incubadora celular significan que los cultivos se incuban a 37 °C en una atmósfera humidificada que contiene un 5% deCO2 (a menos que se indique lo contrario en el protocolo).
<ol><li>Preparación previa de las líneas celulares utilizadas en el sis...

Construcción de un modelo 3D de mucosa intestinal con inclusión en parafina

<ol>
	<li>Chelakkot, C., Ghim, J., Ryu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+regulating+intestinal+barrier+integrity+and+its+pathological+implications.">Mechanisms regulating intestinal barrier integrity and its pathological implications.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-9 (2018).</li><li>Schoultz, I., Keita, &. #. 1. 9. 7. ;. 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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>

El sistema modelo básico de mucosa intestinal reconstruida que se presenta aquí (Figura 6) combina la complejidad fisiológica (cultivos celulares 3D más relevantes desde el punto de vista fisiológico que contienen una monocapa de Caco-2 con un soporte de lámina propia rica en MEC que contiene fibroblastos y monocitos) con la simplicidad experimental (utilizando líneas celulares humanas comerciales para producir un sistema estandarizado y fácilmente repetible)13</sup...

La barrera epitelial intestinal, un revestimiento interno de una célula de espesor que contiene diferentes tipos de células epiteliales, constituye la primera barrera o interfaz defensiva física entre el medio externo e interno del cuerpo 1,2. Los enterocitos de tipo cilíndrico constituyen el tipo más abundante de células epiteliales. Estos son responsables de mantener la integridad de la barrera epitelial a través de las interacciones entre varios componentes de la barrera, incluidas las uniones estrechas (TJ), desempeñando un papel importante en el endurecimiento de la barrera 1,3. La estructura de la TJ consiste en proteínas de placa intracelular, como la zonula occludens (ZO) y la cingulina, que cooperan con las proteínas transmembrana, incluidas las ocludinas, las claudinas y las moléculas de adhesión de unión (JAM) que forman una estructura similar a una cremallera que une estrechamente a las células vecinas 3,4. Las proteínas transmembrana regulan la difusión paracelular pasiva de compuestos pequeños y excluyen las moléculas grandes tóxicas.
Los compuestos alimentarios potencialmente tóxicos y los contaminantes alimentarios estimulan la producción de citoquinas inflamatorias que alteran la permeabilidad epitelial, activando las células inmunitarias y causando inflamación crónica del tejido intestinal 5,6,7. Por el contrario, se ha informado que varios fitoquímicos antioxidantes y antiinflamatorios reducen la expresión de citocinas inflamatorias y mejoran la integridad de la barrera intestinal de TJ a través de la restauración de la expresión y el ensamblaje de la proteína TJ 4,6,8. Por lo tanto, la regulación de la integridad de la barrera epitelial por compuestos beneficiosos y dañinos ha visto un aumento en el uso de modelos in vivo e in vitro destinados a imitar la barrera intestinal para el cribado farmacéutico y los estudios de toxicidad. Esto es particularmente relevante dado el creciente interés en comprender la fisiopatología de las enfermedades intestinales intestinales (EII), la enterocolitis necrotizante y el cáncer, que pueden ser simuladas en modelos experimentales 8,9,10.
Ha habido una demanda para el desarrollo de modelos in vitro basados en células con el fin de lograr el objetivo de las "3R" en la experimentación con animales. Estos incluyen alternativas de reemplazo al uso de animales, reducción en el número de animales utilizados y refinamiento en la adopción de métodos que alivian la angustia 11,12,13. Además, los mecanismos moleculares, celulares y fisiológicos subyacentes entre los modelos humanos y murinos (siendo los roedores las especies más utilizadas) son distintivos, lo que lleva a la controversia sobre la eficacia de los modelos murinos como predictores en las respuestas humanas12,13. Entre las numerosas ventajas de los modelos in vitro de líneas celulares humanas se incluyen la experimentación restringida a objetivos, la observación directa y el análisis continuo13.
Las monocapas de tipo unicelular en cultivos bidimensionales (2D) han servido como modelos poderosos. Sin embargo, estos no pueden reproducir con precisión la complejidad fisiológica de los tejidos humanos 8,13,14. Como resultado, los sistemas de cultivo 3D se están desarrollando con mejoras cada vez mayores para recapitular la complejidad fisiológica de los tejidos intestinales sanos y enfermos como herramientas de evaluación de riesgos de próxima generación13,14. Estos modelos incluyen andamios 3D Transwell con diversas líneas celulares, cultivos de organoides y dispositivos microfluídicos (intestino en chip) que utilizan tanto líneas celulares como organoides (derivados de tejidos sanos y enfermos)8,13,14.
El protocolo 3D de "tejido sano" equivalente intestinal presentado en el presente estudio se basó en lograr un equilibrio entre la complejidad fisiológica y la simplicidad experimental13. El modelo es representativo de un andamio 3D de Transwell, compuesto por tres líneas celulares (enterocitos [la línea Caco-2 del adenocarcinoma de colon de referencia] con un componente inmune de soporte [monocitos U937 y fibroblastos L929]), constituyendo un sistema estandarizado y fácilmente repetible aplicable para el cribado preliminar de moléculas dietéticas de interés sobre la integridad de la barrera epitelial intestinal y la respuesta inmunitaria. El protocolo incluye la inclusión en parafina para la evaluación microscópica de la luz de la integridad de la barrera epitelial utilizando equivalentes intestinales fijos. La ventaja del enfoque actual es que se pueden teñir numerosas secciones de los tejidos incrustados para múltiples parámetros a partir de un solo experimento.

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

Ha aumentado el uso de modelos intestinales in vivo e in vitro para estudiar la fisiopatología de las enfermedades inflamatorias intestinales, para el cribado farmacológico de sustancias potencialmente beneficiosas y para los estudios de toxicidad de componentes alimentarios potencialmente nocivos. Cabe destacar que existe una demanda actual para el desarrollo de modelos in vitro basados en células que sustituyan a los modelos animales. Aquí, se presenta un protocolo para un modelo básico de equivalente intestinal tridimensional (3D) de "tejido sano" utilizando líneas celulares con el doble beneficio de proporcionar simplicidad experimental (sistema estandarizado y fácilmente repetible) y complejidad fisiológica (enterocitos Caco-2 con un componente inmune de soporte de monocitos U937 y fibroblastos L929). El protocolo también incluye la inclusión en parafina para la evaluación microscópica de luz de equivalentes intestinales fijos, lo que proporciona la ventaja de analizar múltiples parámetros visuales a partir de un solo experimento. Para verificar la eficacia del modelo como sistema experimental se utilizan cortes teñidos con hematoxilina y eosina (H&E) que muestran las células cilíndricas de Caco-2 formando una monocapa apretada y regular en tratamientos de control. Utilizando el gluten como componente alimentario proinflamatorio, los parámetros analizados en las secciones incluyen la reducción del grosor de la monocapa, así como la disrupción y el desprendimiento de la matriz subyacente (H&E), la disminución de la expresión de la proteína de unión estrecha como se muestra en la tinción de ocludina (cuantificable estadísticamente) y la activación inmune de las células U937 migratorias como se evidencia en la tinción del grupo de diferenciación 14 (CD14) y la diferenciación relacionada con CD11b en macrófagos. Como se muestra mediante el uso de lipopolisacáridos para simular la inflamación intestinal, los parámetros adicionales que se pueden medir son el aumento de la tinción de moco y la expresión de citocinas (como la midquina) que se pueden extraer del medio antes de la fijación. El modelo básico tridimensional (3D) de la mucosa intestinal y las secciones fijas pueden recomendarse para estudios de estado inflamatorio e integridad de la barrera con la posibilidad de analizar múltiples parámetros visuales cuantificables.

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 destacado: Investigación de los efectos de los compuestos en el tejido intestinal utilizando modelos de líneas celulares humanas en 3D 

Sistema Modelo Intestinal Tridimensional Básico (3D) con un Componente Inmune

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.

El primer aspecto importante es determinar la aceptabilidad de la mucosa intestinal equivalente básica en 3D con fines experimentales. Esto se realiza con la tinción más utilizada en los laboratorios de histología e histopatología, a saber, la hematoxilina (tiñe el material nuclear de color azul-púrpura intenso) y la eosina (tiñe el material citoplasmático de diferentes tonos de rosa). La tinción H&E se realiza primero en un control no tratado, que se cultiva en las mismas condiciones y plazos que los tratamien...

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

Aquí describimos la construcción de un sistema modelo básico tridimensional (3D) de líneas celulares intestinales y un protocolo de inclusión de parafina para la evaluación microscópica óptica de equivalentes intestinales fijos. La tinción de proteínas seleccionadas permite el análisis de múltiples parámetros visuales de un solo experimento para su uso potencial en estudios preclínicos de detección de fármacos.

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 ...

El objetivo del trabajo es utilizar estos modelos de líneas celulares humanas tridimensionales in vitro para investigar el hecho de compuestos beneficiosos y útiles en el tejido intestinal. Los modelos se pueden utilizar para ampliar la comprensión de la investigación y también como modelo básico de fármacos. Utilizando los compuestos derivados de plantas, espermidina y eugenol, tanto por separado como juntos en forma de suplemento, medimos los aumentos beneficiosos de la autofagia y la disminución de la inflamación utilizando estos modelos.Entonces también podremos mostrar el efecto potencialmente dañino del gluten extraído de ciertas variedades de trigo sobre los tejidos intestinales. Actualmente estamos utilizando sistemas in vitro de precio económico, estandarizados y fácilmente repetibles que reflejan mejor la fisiología del intestino humano como alternativa a los modelos animales. Dado que también utilizamos líneas celulares humanas comerciales en lugar de células derivadas de plantas, las respuestas del estudio son más aplicables a la población general.El protocolo se basa enteramente en el uso de diferentes líneas. Esto hace que el modelo sea más estandarizado y reproducible. Además, el cultivo simultáneo de diferentes líneas celulares, enterocitos, fibroblastos y células del sistema inmune, permite respuestas celulares más cercanas a lo que sucede en el paciente.

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.

Investigación

Biología

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.

Preparación de un modelo básico de mucosa intestinal reconstruida en 3D para la evaluación microscópica de equivalentes intestinales fijos

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.