Name: Forfatter Spotlight: Undersøke effekten av forbindelser på tarmvev ved hjelp av 3D-humane cellelinjemodeller
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

Takk til Umberto Veronesi Foundation for et stipend som støtter forskerarbeid.

1. Utarbeidelse av den grunnleggende 3D rekonstruerte tarmslimhinnemodellen
MERK: Hele prosedyren må utføres i en steril laminær strømningshette. Alle trinnene i prosedyren som involverer bruk av celleinkubatoren betyr at kulturer inkuberes ved 37 ° C i en fuktet atmosfære som inneholder 5% CO2 (med mindre annet er angitt i protokollen).
<ol><li>Tidligere forberedelse av cellelinjene som brukes i tarmmodellsystemet<ol><li>Seed L929 mus fibroblastceller i en konsen...

Konstruksjon av en 3D-tarmslimhinnemodell med parafininnstøping

<ol>
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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>

Det grunnleggende rekonstruerte modellsystemet for tarmslimhinner som presenteres her (figur 6) kombinerer fysiologisk kompleksitet (mer fysiologisk relevante 3D-cellekulturer som inneholder et Caco-2-monolag med en ECM-rik lamina propria-støtte som inneholder fibroblaster og monocytter) med eksperimentell enkelhet (ved bruk av kommersielle humane cellelinjer for å produsere standardiserte og lett repeterbare systemer)13. Dette modellsystemet anses derfor som et egn...

Tarmepitelbarrieren, en encelletykk indre foring som inneholder forskjellige typer epitelceller, utgjør den første fysiske defensive barrieren eller grensesnittet mellom utsiden og kroppens indre miljø 1,2. Columnar-type enterocytter utgjør den mest tallrike typen epitelceller. Disse er ansvarlige for å opprettholde epitelbarrierens integritet gjennom interaksjoner mellom flere barrierekomponenter, inkludert tette kryss (TJ), som spiller en betydelig rolle i barrierestramming 1,3. TJ-strukturen består av intracellulære plakkproteiner, slik som zonula occludens (ZO) og cingulin, som samarbeider med transmembranproteiner, inkludert okkludiner, klaudiner og junctional adhesjonsmolekyler (JAMs) som danner glidelåslignende struktur som tett forbinder nabocellene 3,4. De transmembrane proteiner regulerer passiv paracellulær diffusjon av små forbindelser og utelukker giftige store molekyler.
Potensielt giftige matforbindelser og matforurensninger stimulerer inflammatorisk cytokinproduksjon som forstyrrer epitelpermeabiliteten, aktiverer immunceller og forårsaker kronisk tarmvevbetennelse 5,6,7. I motsetning til dette har forskjellige antioksidanter og antiinflammatoriske fytokjemikalier blitt rapportert å redusere inflammatorisk cytokinekspresjon og forbedre intestinal TJ-barriereintegritet gjennom restaurering av TJ-proteinuttrykk og montering 4,6,8. Derfor har reguleringen av epitelbarriereintegritet av både gunstige og skadelige forbindelser sett en økning i bruken av både in vivo og in vitro-modeller rettet mot å etterligne tarmbarrieren for farmasøytiske screening- og toksisitetsstudier. Dette er spesielt relevant gitt den økende interessen for å forstå patofysiologien ved tarmtarmsykdommer (IBD), nekrotiserende enterokolitt og kreft, som kan simuleres i eksperimentelle modeller 8,9,10.
Det har vært etterspørsel etter utvikling av cellebaserte in vitro-modeller for å oppnå målet om "3R" i dyreforsøk. Disse inkluderer erstatningsalternativer til bruk av dyr, reduksjon i antall dyr som brukes, og forbedring i å ta i bruk metoder som lindrer nød 11,12,13. Videre er de underliggende molekylære, cellulære og fysiologiske mekanismene mellom humane og murine modeller (gnagere er den mest brukte arten) særegne, noe som fører til kontrovers angående effekten av murine modellene som prediktorer i menneskelige responser12,13. Mange fordeler med in vitro humane cellelinjemodeller inkluderer målbegrenset eksperimentering, direkte observasjon og kontinuerlig analyse13.
Encelle-type monolayers i todimensjonale (2D) kulturer har tjent som kraftige modeller. Disse kan imidlertid ikke nøyaktig reprodusere den fysiologiske kompleksiteten til menneskelig vev 8,13,14. Som et resultat utvikles 3D-kultursystemer med stadig økende forbedringer for å rekapitulere den fysiologiske kompleksiteten til både sunt og sykt tarmvev som neste generasjons risikovurderingsverktøykasser13,14. Disse modellene inkluderer 3D Transwell stillas med forskjellige cellelinjer, organoidkulturer og mikrofluidiske enheter (tarm-på-chip) som bruker både cellelinjer og organoider (avledet fra både sunt og sykt vev)8,13,14.
3D "sunt vev" intestinal ekvivalent protokoll presentert i denne studien var basert på å finne en balanse mellom fysiologisk kompleksitet og eksperimentell enkelhet13. Modellen er representativ for et 3D Transwell-stillas, bestående av tre cellelinjer (enterocytter [gullstandardadenokarsinomet Caco-2-linjen] med en støttende immunkomponent [U937-monocytter og L929-fibroblaster]), som utgjør et standardisert og lett repeterbart system som gjelder for foreløpig screening av diettmolekyler av interesse for tarmepitelbarriereintegritet og immunrespons. Protokollen inkluderer parafininnebygging for lysmikroskopisk evaluering av epitelbarriereintegritet ved bruk av faste tarmekvivalenter. Fordelen med den nåværende tilnærmingen er at mange deler av det innebygde vevet kan gjøres for å flekke for flere parametere fra et enkelt eksperiment.

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

Det har vært en økning i bruken av in vivo og in vitro intestinale modeller for å studere patofysiologien ved inflammatoriske tarmsykdommer, for farmakologisk screening av potensielt fordelaktige stoffer og for toksisitetsstudier på potensielt skadelige matkomponenter. Av relevans er det en nåværende etterspørsel etter utvikling av cellebaserte in vitro-modeller for å erstatte dyremodeller. Her presenteres en protokoll for en grunnleggende, "sunt vev" tredimensjonal (3D) tarmekvivalent modell ved bruk av cellelinjer med den doble fordelen av å gi både eksperimentell enkelhet (standardisert og lett repeterbart system) og fysiologisk kompleksitet (Caco-2 enterocytter med en støttende immunkomponent av U937-monocytter og L929-fibroblaster). Protokollen inkluderer også parafininnebygging for lysmikroskopisk evaluering av faste tarmekvivalenter, og gir dermed fordelen av å analysere flere visuelle parametere fra et enkelt eksperiment. Hematoksylin- og eosinfargede seksjoner som viser Caco-2-søylecellene som danner et tett og regelmessig monolag i kontrollbehandlinger, brukes til å verifisere effekten av modellen som et eksperimentelt system. Ved bruk av gluten som en pro-inflammatorisk matkomponent, inkluderer parametere analysert fra seksjoner redusert monolagtykkelse, samt forstyrrelse og løsrivelse fra den underliggende matriksen (H &E), redusert tett kryssproteinuttrykk som vist fra okkludinfarging (kvantifiserbar statistisk) og immunaktivering av migrerende U937-celler som vist fra klyngen av differensiering 14 (CD14) farging og CD11b-relatert differensiering i makrofager. Som vist ved bruk av lipopolysakkarid for å simulere tarmbetennelse, er ytterligere parametere som kan måles økt slimfarging og cytokinekspresjon (som midkine) som kan ekstraheres fra mediet før fiksering. Den grunnleggende tredimensjonale (3D) tarmslimhinnemodellen og faste seksjoner kan anbefales for inflammatoriske status- og barriereintegritetsstudier med mulighet for å analysere flere visuelle kvantifiserbare parametere.

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.

Forfatter Spotlight: Undersøke effekten av forbindelser på tarmvev ved hjelp av 3D-humane cellelinjemodeller 

Grunnleggende tredimensjonalt (3D) tarmmodellsystem med en immunkomponent

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.

Det første viktige aspektet er å bestemme akseptabiliteten til den grunnleggende 3D-tarmekvivalente slimhinnen for eksperimentelle formål. Dette utføres med den mest brukte flekken i histologi- og histopatologilaboratorier, nemlig hematoksylin (flekker kjernefysisk materiale dyp blålilla farge) og eosin (flekker cytoplasmatisk materiale varierende nyanser av rosa). H&E-fargingen utføres først på en ubehandlet kontroll, som dyrkes under de samme forholdene og tidsrammen som de eksperimentelle behandlingene. Fra å...

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

Her beskriver vi konstruksjon av et basalt tredimensjonalt (3D) tarmcellelinjemodellsystem og en parafininnebyggingsprotokoll for lysmikroskopisk evaluering av faste tarmekvivalenter. Farging av utvalgte proteiner tillater analyse av flere visuelle parametere fra et enkelt eksperiment for potensiell bruk i prekliniske legemiddelscreeningsstudier.

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 ...

Omfanget av arbeidet er å bruke disse in vitro tredimensjonale humane cellelinjemodellene for å undersøke faktumet av fordelaktige så vel som nyttige forbindelser på tarmvev. Modellene kan brukes til videre forskningsforståelse og også som en grunnleggende legemiddelmodell. Ved å bruke de planteavledede forbindelsene, spermidin og eugenol, både hver for seg og sammen i tilskuddsform, måler vi gunstige økninger i autofagi og reduksjoner i betennelse ved hjelp av disse modellene.Da vil vi også kunne vise den potensielt skadelige effekten av gluten ekstrahert fra visse hvetesorter på tarmvev. Vi bruker for tiden økonomisk priset, standardisert og lett repeterbart in vitro-system som bedre reflekterer tarmens fysiologi som et alternativ til dyremodeller. Siden vi også bruker kommersiell human cellelinje i stedet for planteavledede celler, er svarene fra studien mer gjeldende for den generelle befolkningen.Protokollen er helt basert på bruk av forskjellige linjer. Disse gjør modellen mest standardisert og reproduserbar. Videre tillater samtidig dyrking av forskjellige cellelinjer, enterocytter, fibroblaster og immunsystemceller cellulære responser som er nærmere det som skjer i pasienten.

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.

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.

Utarbeidelse av en grunnleggende 3D rekonstruert tarmslimhinnemodell for mikroskopisk evaluering av faste tarmekvivalenter

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.