Name: Forfatter Spotlight: Undersøgelse af virkningerne af forbindelser på tarmvæv ved hjælp af 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

Tak til Umberto Veronesi Foundation for et stipendium, der støtter forskerarbejde.

1. Udarbejdelse af den grundlæggende 3D rekonstruerede tarmslimhindemodel
BEMÆRK: Hele proceduren skal udføres i en steril laminar strømningshætte. Alle trin i proceduren, der involverer anvendelse af celleinkubatoren, betyder, at kulturer inkuberes ved 37 °C i en befugtet atmosfære indeholdende 5% CO2 (medmindre andet er angivet i protokollen).
<ol><li>Forudgående forberedelse af cellelinjerne anvendt i tarmmodelsystemet<ol><li>Seed L929 musefibroblastceller i ...

Konstruktion af en 3D-tarmslimhindemodel med paraffinindlejring

<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. ;. 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+intestinal+barrier+and+current+techniques+for+the+assessment+of+gut+permeability.">The intestinal barrier and current techniques for the assessment of gut permeability.</a> Cells. 9 (8), 1909 (1909).</li><li>Bednarek, R. <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+methods+for+measuring+the+permeability+of+cell+monolayers.">In vitro methods for measuring the permeability of cell monolayers.</a> Methods and Protocols. 5 (1), 17 (2022).</li><li>Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+intestinal+barrier+by+nutrients:+The+role+of+tight+junctions.">Regulation of the intestinal barrier by nutrients: The role of tight junctions.</a> Animal Science Journal. 91 (1), e13357 (2020).</li><li>Guibourdenche, 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=Food+contaminants+effects+on+an+in+vitro+model+of+human+intestinal+epithelium.">Food contaminants effects on an in vitro model of human intestinal epithelium.</a> Toxics. 9 (6), 135 (2021).</li><li>Panwar, S., Sharma, S., Tripathi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+barrier+integrity+and+dysfunctions+in+maintaining+the+healthy+gut+and+their+health+outcomes.">Role of barrier integrity and dysfunctions in maintaining the healthy gut and their health outcomes.</a> Frontiers in Physiology. 12, 715611 (2021).</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=Pro-inflammatory+effect+of+gliadins+and+glutenins+extracted+from+different+wheat+cultivars+on+an+in+vitro+3D+intestinal+epithelium+model.">Pro-inflammatory effect of gliadins and glutenins extracted from different wheat cultivars on an in vitro 3D intestinal epithelium model.</a> International Journal of Molecular Sciences. 22 (1), 172 (2020).</li><li>Fedi, A., 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=In+vitro+models+replicating+the+human+intestinal+epithelium+for+absorption+and+metabolism+studies:+A+systematic+review.">In vitro models replicating the human intestinal epithelium for absorption and metabolism studies: A systematic review.</a> Journal of Controlled Release. 335, 247-268 (2021).</li><li>De Fazio, L., 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=Necrotizing+enterocolitis:+Overview+on+in+vitro+models.">Necrotizing enterocolitis: Overview on in vitro models.</a> International Journal of Molecular Sciences. 22 (13), 6761 (2021).</li><li>Jubelin, C., 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=Three-dimensional+in+vitro+culture+models+in+oncology+research.">Three-dimensional in vitro culture models in oncology research.</a> Cell &amp; Bioscience. 12 (1), 155 (2022).</li><li>Russell, W., Burch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+principles+of+humane+experimental+technique.">The principles of humane experimental technique.</a> Available online. , (2023).</li><li>Ingber, D. 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 grundlæggende rekonstruerede tarmslimhindemodelsystem, der præsenteres her (figur 6), kombinerer fysiologisk kompleksitet (mere fysiologisk relevante 3D-cellekulturer indeholdende et Caco-2-monolag med en ECM-rig lamina propria-støtte indeholdende fibroblaster og monocytter) med eksperimentel enkelhed (ved hjælp af kommercielle humane cellelinjer til fremstilling af standardiseret og let gentageligt system)13. Som sådan anses dette modelsystem for at være et...

Den intestinale epitelbarriere, en encelle tyk indre foring, der indeholder forskellige typer epitelceller, udgør den første fysiske defensive barriere eller grænseflade mellem ydersiden og det indre miljø i kroppen 1,2. Enterocytter af kolonnetype udgør den mest rigelige type epitelceller. Disse er ansvarlige for at opretholde epitelbarriereintegritet gennem interaktioner mellem flere barrierekomponenter, herunder stramme kryds (TJ'er), der spiller en væsentlig rolle i barrierestramning 1,3. TJ-strukturen består af intracellulære plakproteiner, såsom zonula occludens (ZO) og cingulin, der samarbejder med transmembranproteiner, herunder occludiner, claudiner og junctional adhæsion molekyler (JAM'er), der danner lynlåslignende struktur, der tæt forbinder nabocellerne 3,4. De transmembranproteiner regulerer den passive paracellulære diffusion af små forbindelser og udelukker giftige store molekyler.
Potentielt giftige fødevareforbindelser og fødevareforurenende stoffer stimulerer inflammatorisk cytokinproduktion, der forstyrrer epitelpermeabiliteten, aktiverer immunceller og forårsager kronisk tarmvævsbetændelse 5,6,7. I modsætning hertil er forskellige antioxidant- og antiinflammatoriske fytokemikalier blevet rapporteret at reducere inflammatorisk cytokinekspression og forbedre tarm-TJ-barriereintegriteten gennem genoprettelse af TJ-proteinekspression og samling 4,6,8. Derfor har reguleringen af epitelbarrierens integritet af både gavnlige og skadelige forbindelser set en stigning i brugen af både in vivo- og in vitro-modeller, der sigter mod at efterligne tarmbarrieren til farmaceutisk screening og toksicitetsundersøgelser. Dette er især relevant i betragtning af den stigende interesse for at forstå patofysiologien af tarmsygdomme (IBD), nekrotiserende enterocolitis og kræft, som kan simuleres i eksperimentelle modeller 8,9,10.
Der har været efterspørgsel efter udvikling af cellebaserede in vitro-modeller for at nå målet om "3R'erne" i dyreforsøg. Disse omfatter erstatningsalternativer til brugen af dyr, reduktion i antallet af anvendte dyr og forfining i vedtagelsen af metoder, der lindrer nød 11,12,13. Desuden er de underliggende molekylære, cellulære og fysiologiske mekanismer mellem humane og murinmodeller (gnavere er den mest anvendte art) karakteristiske, hvilket fører til kontroverser om effektiviteten af murine modeller som prædiktorer i humane reaktioner12,13. Talrige fordele ved in vitro humane cellelinjemodeller omfatter målbegrænsede eksperimenter, direkte observation og kontinuerlig analyse13.
Enkeltcelle-type monolag i todimensionelle (2D) kulturer har fungeret som kraftfulde modeller. Disse kan imidlertid ikke præcist reproducere den fysiologiske kompleksitet af humant væv 8,13,14. Som følge heraf udvikles 3D-kultursystemer med stadigt stigende forbedringer for at rekapitulere den fysiologiske kompleksitet af både sundt og sygt tarmvæv som næste generations risikovurderingsværktøjskasser13,14. Disse modeller inkluderer 3D Transwell-stilladser med forskellige cellelinjer, organoidkulturer og mikrofluidiske enheder (tarm-på-chip) ved hjælp af både cellelinjer og organoider (afledt af både sunde og syge væv)8,13,14.
3D "sundt væv" intestinal ækvivalent protokol præsenteret i denne undersøgelse var baseret på at finde en balance mellem fysiologisk kompleksitet og eksperimentel enkelhed13. Modellen er repræsentativ for et 3D Transwell-stillads, der består af tre cellelinjer (enterocytter [guldstandarden colon adenocarcinoma Caco-2-linje] med en understøttende immunkomponent [U937-monocytter og L929-fibroblaster]), der udgør et standardiseret og let gentageligt system, der kan anvendes til foreløbig screening af diætmolekyler af interesse for intestinal epitelbarriereintegritet og immunrespons. Protokollen inkluderer paraffinindlejring til lysmikroskopisk evaluering af epitelbarriereintegritet ved anvendelse af faste tarmækvivalenter. Fordelen ved den nuværende fremgangsmåde er, at adskillige sektioner af de indlejrede væv kan gøres til farvning for flere parametre fra et enkelt eksperiment.

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

Der har været en stigning i brugen af in vivo - og in vitro-tarmmodeller til undersøgelse af patofysiologien af inflammatoriske tarmsygdomme, til farmakologisk screening af potentielt gavnlige stoffer og til toksicitetsundersøgelser af potentielt skadelige levnedsmiddelkomponenter. Af relevans er der en aktuel efterspørgsel efter udvikling af cellebaserede in vitro-modeller til erstatning af dyremodeller. Her præsenteres en protokol for en grundlæggende, "sundt væv" tredimensionel (3D) tarmækvivalent model ved hjælp af cellelinjer med den dobbelte fordel at give både eksperimentel enkelhed (standardiseret og let gentageligt system) og fysiologisk kompleksitet (Caco-2 enterocytter med en understøttende immunkomponent af U937 monocytter og L929 fibroblaster). Protokollen inkluderer også paraffinindlejring til lysmikroskopisk evaluering af faste tarmækvivalenter, hvilket giver fordelen ved at analysere flere visuelle parametre fra et enkelt eksperiment. Hæmatoxylin og eosin (H&E) farvede sektioner, der viser Caco-2 søjleformede celler, der danner et tæt og regelmæssigt monolag i kontrolbehandlinger, bruges til at verificere effektiviteten af modellen som et eksperimentelt system. Ved anvendelse af gluten som en proinflammatorisk fødevarekomponent inkluderer parametre analyseret fra sektioner reduceret monolagtykkelse samt forstyrrelse og løsrivelse fra den underliggende matrix (H&E), nedsat tæt forbindelsesproteinekspression som vist ved okklusionfarvning (kvantificerbar statistisk) og immunaktivering af migrerende U937-celler som det fremgår af klyngen af differentiering 14 (CD14) farvning og CD11b-relateret differentiering i makrofager. Som vist ved at bruge lipopolysaccharid til at simulere tarmbetændelse, er yderligere parametre, der kan måles, øget slimfarvning og cytokinekspression (såsom midkin), der kan ekstraheres fra mediet før fiksering. Den grundlæggende tredimensionelle (3D) tarmslimhindemodel og faste sektioner kan anbefales til inflammatoriske status- og barriereintegritetsundersøgelser med mulighed for at analysere flere visuelle kvantificerbare parametre.

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: Undersøgelse af virkningerne af forbindelser på tarmvæv ved hjælp af 3D-humane cellelinjemodeller 

Grundlæggende tredimensionelt (3D) tarmmodelsystem 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 vigtige aspekt er at bestemme acceptabiliteten af den grundlæggende 3D intestinale ækvivalente slimhinde til eksperimentelle formål. Dette udføres med den mest anvendte plet i histologi- og histopatologilaboratorier, nemlig hæmatoxylin (pletter nukleart materiale dyb blålilla farve) og eosin (pletter cytoplasmatisk materiale varierende nuancer af pink). H&E-farvningen udføres først på en ubehandlet kontrol, som dyrkes under samme forhold og tidsramme som de eksperimentelle behandlinger. Fra visualise...

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 konstruktionen af et grundlæggende tredimensionelt (3D) tarmcellelinjemodelsystem og en paraffinindlejringsprotokol til lysmikroskopisk evaluering af faste tarmækvivalenter. Farvning af udvalgte proteiner tillader analyse af flere visuelle parametre fra et enkelt eksperiment til potentiel anvendelse i prækliniske lægemiddelscreeningsundersøgelser.

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 ...

Formålet med arbejdet er at anvende disse in vitro tredimensionelle humane cellelinjemodeller til at undersøge det faktum, at gavnlige såvel som nyttige forbindelser på tarmvæv. Modellerne kan bruges til yderligere forskningsforståelse og også som en grundlæggende lægemiddelmodel. Ved hjælp af de planteafledte forbindelser, spermidin og eugenol, både separat og sammen i tilskudsform, måler vi gavnlige stigninger i autofagi og fald i inflammation ved hjælp af disse modeller.Så vil vi også kunne vise den potentielt skadelige virkning af gluten ekstraheret fra visse hvedesorter på tarmvæv. Vi bruger i øjeblikket økonomisk prissat, standardiseret og let gentageligt in vitro-system, der bedre afspejler tarmens fysiologi som et alternativ til dyremodeller. Da vi også bruger kommercielle humane cellelinjer snarere end planteafledte celler, er svarene fra undersøgelsen mere anvendelige for den generelle befolkning.Protokollen er udelukkende baseret på brugen af forskellige linjer. Disse gør modellen mest standardiseret og reproducerbar. Desuden giver samtidig dyrkning af forskellige cellelinjer, enterocytter, fibroblaster og immunsystemceller mulighed for cellulære reaktioner, der er tættere på, hvad der sker i patienten.

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.

Udarbejdelse af en grundlæggende 3D rekonstrueret tarmslimhindemodel til mikroskopisk evaluering af faste tarmækvivalenter

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.