Thanks to the Umberto Veronesi Foundation for a fellowship supporting researcher work.

1. Preparation of the basic 3D reconstructed intestinal mucosa model
NOTE: The entire procedure must be carried out in a sterile laminar flow hood. All steps in the procedure involving the use of the cell incubator signify that cultures are incubated at 37 &#176;C in a humidified atmosphere containing 5% CO2 (unless stated otherwise in the protocol).
<ol>
	<li>Prior preparation of the cell lines used in the intestinal model system
	<ol>
		<li>Seed L929 mouse fibroblast cells at a concentration of 5 x 105 in 5 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 2 mM L-glutamine, 10% Fetal Bovine Serum (FBS), and 1% Penicillin-Streptomycin (Pen-Strep) in a&#160;F25 flask and culture it in an incubator 4 days before the construction of the intestinal models. After 48 h, remove the medium using a pipette. Then add fresh medium (5 mL), and incubate the cells further for 48 h. 
		NOTE: The cells must be 80% confluent before being used.</li>
		<li>Seed Caco-2 cells (concentration of 2 x 106) in 10 mL of DMEM medium (with 10% FBS and 1% Pen-Strep) in a&#160;F75 cell flask and culture it in an incubator 4 days before the construction of the intestinal mucosa model. After 48 h, change the medium as described in step 1.1.1 and further incubate for 48 h to a confluency of 80%. 
		NOTE: The cells must be in an active proliferating phase: not too sparse nor too confluent. A 50-60% confluence is recommended. The cells must not be sown the day before making the model because this could slow down the proliferative capacity of the cells, which would then not take root perfectly in the reconstructed 3D model.</li>
		<li>Add U937 cells that grow in suspension (concentration of 1 x 106) to 10 mL of Roswell Park Memorial Institute (RPMI) medium (containing 2 mM L-glutamine, 1% sodium pyruvate, 10% FBS, and 1% Pen-Strep) in a F75 cell flask 2 days prior to the model set-up, and place in an incubator for 48 h.</li>
	</ol>
	</li>
	<li>Preparation of the Transwell co-culture plate inserts
	<ol>
		<li>Select a 24-well plate containing inserts with 0.4 &#181;m filters. 
		NOTE: The 0.4 &#181;m filter is a standard choice in drug transport studies. The 3 &#181;m and 8 &#181;m filter sizes are not recommended to prevent any possible losses of the collagen-embedded cells.</li>
		<li>Using a pipette, hydrate the Transwell filters (henceforth referred to as membrane inserts) with 500 &#181;L of Hanks&#39; Balanced Salt Solution (HBSS) below and above the filter insert.</li>
		<li>Close the multi-well plate and place it in an incubator for a minimum of 2 h. 
		NOTE: The membrane inserts can remain in the HBSS for up to 24 h. This operation can be performed the day before constructing the model. It is important that the membrane inserts are completely hydrated and that the filter does not dry out, as this could make it harder for the sample to adhere properly.</li>
		<li>Remove the plates from the incubator after 2 h (or 24 h). Carefully aspirate the HBSS from above and below the membrane inserts using a pipette and leave it to dry for 10 min.</li>
	</ol>
	</li>
	<li>Preparation of the cell-free collagen lamina propria of the basic 3D intestinal model system (DAY 1)
	<ol>
		<li>Prepare a cell-free collagen solution in a 50 mL sterile tube on ice containing the following components in DMEM: 10% Fetal Bovine Serum (FBS), 200 mM L-glutamine, 1% sodium bicarbonate and 1.35 mg/mL Type 1 rat tail collagen. 
		NOTE: All these components must be kept on ice and added to the DMEM with cooled pipette tips. The Type 1 rat tail collagen must be added last as this polymerizes with increasing temperature and pH. The amount of cell-free collagen solution prepared will depend on the number of intestinal equivalents required.</li>
		<li>Add 250 &#181;L of the solution to each plate insert (above the membrane filter) for the number of intestinal equivalents selected and place the lid over the multi-well plate. Allow the collagen solution to polymerize at room temperature (RT) under the laminar flow hood. 
		NOTE: Apart from the transition to a more solid phase, polymerization is also evident from a change in color from yellow to pink. Polymerization is usually complete within 10-15 min.</li>
	</ol>
	</li>
	<li>Preparation, cell counting, and addition of fibroblast (L929) and monocyte (U937) cells to the model system (DAY 1)
	<ol>
		<li>Remove the L929 cell culture (step 1.1.1) from the incubator. Using a vacuum pump, aspirate the medium, replace it with 5 mL of sterile phosphate buffer saline (PBS; without Ca and Mg), and rinse the cells.</li>
		<li>Aspirate the PBS using a vacuum pump. Add 2 mL of a pre-prepared trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA in PBS) and place in an incubator for 3-5 min.</li>
		<li>Use an inverted microscope (for example, an Eclipse Ts2, Nikon) to establish whether the cells are detaching from the adhesion surface. If this is occurring, immediately add 2 mL of DMEM (containing 10% FBS) to block the trypsin reaction and rinse the cells.</li>
		<li>Transfer the cell solution to a sterile 15 mL tube and centrifuge at 645 x g for 5 min. Using a vacuum pump, aspirate the supernatant. 
		NOTE: Care is needed not to disturb the pellet. The effect of DMEM was tested on the L929 and U937 cells and was not shown to have adverse effects on growth.</li>
		<li>Add 1 mL of DMEM to the pellet and suspend the cells. 
		NOTE: The cells must be homogeneously suspended in the solution.</li>
		<li>Remove 20 &#181;L of the cells in DMEM and add 20 &#181;L of Trypan blue solution. Remove 20 &#181;L of the mix and evaluate cell density microscopically using a cell counting chamber.</li>
		<li>Remove the U937 cell culture (step 1.1.3) from the incubator. Centrifuge the cell solution at 645 x g for 5 min. Using a vacuum pump, aspirate the supernatant to avoid disturbing the pellet.</li>
		<li>Suspend the cells in 1 mL of RPMI. As with the L929 cells, make sure the cells are homogeneously suspended in the solution.</li>
		<li>Similarly, establish the cell density as reported in step 1.4.6.</li>
		<li>Prepare a collagen solution as described in step 1.3.1. 
		NOTE: The amount of solution to be made must take into consideration 450 &#181;L for each insert (or model).</li>
		<li>Prepare a solution of DMEM to contain a count of 50,000 L929 cells and 15,000 U937 cells, respectively, in a volume of 50 &#181;L for each intestinal equivalent model to be constructed. 
		NOTE: The number of cells is a critical factor. Too many of both cell types would result in a lamina propria excessively full of cells that would not be adequately organized. An inferior number of fibroblasts (which generate collagen) would result in a less compact lamina propria, whereas too few monocytes would impede the immune response to stimuli. Provided the correct count of cells is present within each 50 &#181;L aliquot - a total volume of 600 &#181;L can be prepared for the 12 filter inserts present in each 24 well-plate.</li>
		<li>Add each 50 &#181;L aliquot containing the cells to 450 &#181;L of collagen solution in step 1.4.10. Mix thoroughly.</li>
		<li>Overlay the pre-coated cell-free collagen lamina propria with 500 &#181;L of the collagen-containing cell solution for each model. 
		NOTE: It is important to rapidly add the volumes to each insert, and as such, it is advisable to limit the number of reconstructed intestinal mucosa models to 12 or fewer at a time.</li>
		<li>Close the plate and place it in an incubator for 2 h to allow the solution to set.</li>
	</ol>
	</li>
	<li>Preparation, cell counting, and addition of epithelial Caco-2 cells to the intestinal model (DAY 1)
	<ol>
		<li>Remove the Caco-2 cell culture (step 1.1.2) from the incubator. Repeat the procedures from step 1.4.1 using 10 mL of PBS for preliminary rinsing. Then add 5 mL of a pre-prepared trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA in Ca- and Mg-free PBS) and place in an incubator for 5-8 min.</li>
		<li>Repeat steps 1.4.3 (but using 5 mL of DMEM to block the trypsin reaction) through to 1.4.6 to count the cells using the cell counting chamber.</li>
		<li>Prepare a solution of DMEM to contain a count of 150,000 Caco-2 cells in 50 &#181;L. 
		NOTE: The number of cells used is a critical point. Exceeding 150,000 cells could result in a compact disorganized epithelial layer, whereas with too little (less than 100,000), the cells struggle to grow and do not adequately cover the basement membrane creating a discontinuous intestinal epithelial layer. Make sure the cells are effectively suspended. The tip of a 200 &#181;L pipette can be used to ensure homogenous distribution. Given that 12 models can be constructed at any given time, a 600 &#181;L cell solution can be prepared.</li>
		<li>After the 2 h required in step 1.4.14, add 50 &#181;L of Caco-2 cells suspended in DMEM to the middle of each basement membrane. Close the lid of the multi-well plate.</li>
		<li>Incubate under the sterile laminar flow hood for 10 min. Then transfer to the incubator for 30 min.</li>
		<li>Prepare a DMEM solution containing 10% FBS and 1% Pen/Strep. 
		NOTE: Prepare a sufficient solution to use a 1 mL volume per model.</li>
		<li>Add 500 &#181;L of the medium above the reconstructed model and 500 &#181;L below the filter. 
		NOTE: Care is required when adding the medium above the filter to avoid detaching or stressing the cells.</li>
		<li>Close the multi-well plate system and place it in the incubator.</li>
	</ol>
	</li>
	<li>Model preparation/formation and use (DAY 2 to DAY 6)
	<ol>
		<li>DAY 2: Carefully remove the solution from both above the reconstructed model and below the filter using a pipette. Replace with fresh 500 &#181;L of fresh DMEM (10% FBS and 1% Pen/Strep) above and below the filter, respectively.</li>
		<li>DAY 3: Repeat as above in step 1.6.1.</li>
		<li>DAY 4: Repeat as above in step 1.6.1. 
		NOTE: This intestinal model is a static cellular model; therefore, to favor the release of molecules and the growth and stimulation of cells, it is very important that the medium is changed every day.</li>
		<li>DAY 5: At this point, the model is fully formed/developed. Use these models for further studies. 
		NOTE: The best time to use the model is at 5 days. Although the cells can be maintained in an incubator for longer periods, the more time that passes, the greater the probability that the epithelial cells may grow in an uncontrolled manner, resulting in an unorganized and compact layer that is difficult to use.</li>
		<li>At 5 days, incubate the models with either toxic (gluten or lipopolysaccharide [LPS]) or beneficial components (polyphenols) of interest. Add these to the upper portion of the reconstructed model suspended in the DMEM medium. 
		NOTE: The suitable concentration of each component of interest must be calculated and suspended in a DMEM medium. Non-treated controls containing only DMEM medium must be set up for comparison to the experimental models.</li>
		<li>Incubate the control and experimental models for 24 h in an incubator.</li>
		<li>DAY 6: Remove the medium above and below the filter with a pipette. 
		&#8203;NOTE: The medium can be stored for subsequent enzyme-linked immunosorbent assay (ELISA)-type tests to measure the release of inflammatory cytokines. For this purpose, the medium must be added to sterile vials and stored at -20 &#176; C for further analysis.</li>
	</ol>
	</li>
</ol>
2. Paraffin embedding of the reconstructed intestinal mucosa models
NOTE: The entire procedure must be carried out under a chemical fume hood. Each step and respective time allocation must be strictly adhered to. For this reason, it is important to have all reagents prepared ahead of time.
<ol>
	<li>Set the paraffin machine to 58 &#176;C so that&#160;it is ready for use.</li>
	<li>Transfer the membrane inserts to clean wells using pliers in a sterile 24-well plate.</li>
	<li>Add 500 &#181;L of 37% buffered formalin in PBS above the filter and 1 mL under the filter. Close the lid and leave it under the chemical fume hood for 2 h at RT. 
	NOTE: As an alternative, 4% buffered formalin can be used at RT for 1 h.</li>
	<li>Remove the formalin and add HBSS solution both above and below the filter. Then remove the HBSS.</li>
	<li>Detach the lamina propria from the membrane insert. 
	NOTE: The cells of the intestinal mucosa usually detach very easily from the membrane insert as they are not attached to the latter. Additionally, the two sample compartments (apical and basal) remain attached, retaining the 3D structure. If, for some reason, they are not readily detached, it will be important to use a sterile disposable scalpel blade to remove the intestinal mucosa from the membrane insert. The membrane insert can be cut, thus detaching it from the plastic. Be careful never to touch the sample with the scalpel. The motive for removing the membrane insert from the collagen-embedded cells is that this filter may become detached in the subsequent fixation or paraffin embedding phases, rendering comparisons between intestinal mucosa models disproportional. Moreover, the membrane inserts within the embedded section have a different consistency which can interfere with sectioning.</li>
	<li>Place the 100 mL beakers under the chemical fume hood, each correctly labeled to include one reconstructed intestinal mucosa model system under investigation. Add 25 mL of 35% ETOH to each beaker and then add the reconstructed intestinal mucosa. Incubate for 10 min.</li>
	<li>Replace the 35% ethanol with 25 mL of 50% ethanol and incubate for 10 min.</li>
	<li>Replace the 50% ethanol with 25 mL of 70% ethanol and incubate for 10 min.</li>
	<li>Replace the 70% ethanol with 25 mL of 80% ethanol and incubate for 10 min.</li>
	<li>Replace the 80% ethanol with 25 mL of 95% ethanol and incubate for 10 min.</li>
	<li>Replace the 95% ethanol with 25 mL of 100% ethanol and incubate for 10 min.</li>
	<li>Replace the 100% ethanol with another 25 mL change of 100% ethanol and incubate for 10 min.</li>
	<li>Place 100 mL beakers under the fume hood, each correctly labeled to include one model under investigation. Add 50 mL of xylene or a histological clearing agent for 10-20 min. 
	NOTE: Histo-Clear (histological clearing agent) is recommended as the agent enhances the clarity and vibrancy of acidophilic stains. The clearing time can vary from one reconstructed intestinal mucosa sample to another and must be checked for transparency in appearance every 2-3 min.</li>
	<li>As soon as the samples are transparent, place them in a metal tissue cassette holder which is then immersed in liquid paraffin inside the heated machine for 45 min.</li>
	<li>Change the paraffin and leave the samples inside the heated machine for a further 45 min.</li>
	<li>Remove the tissue cassette holder and place on ice to cool. When the cooled sample blocks detach from the metal holder, these can be further cooled at room temperature.</li>
	<li>Store the blocks at RT. 
	NOTE: If the objective is to prepare sections immediately, then the blocks can be placed at 4 &#176; C so that they are well-cooled when used.</li>
	<li>Cut 4 &#181;m sections using a microtome.</li>
	<li>Place the cut sections on slides and dry them in an oven at 37 &#176; C for 24 h.</li>
	<li>The slides are ready for use. Store them at RT until performing either H&#38;E histological staining or immune-histochemical reaction staining (antigen-antibody type).
	<ol>
		<li>For immune-histochemical staining, select the antibodies of interest (either for the study of TJ proteins&#160;such as occludin or for monocyte activation, migration, and differentiation) and detect expression (via staining) using commercial kits.</li>
		<li>Similarly, measure mucus using the Alcian blue and periodic acid-Schiff (PAS) staining kit.</li>
		<li>Quantify the stained TJ proteins of interest, such as occludin, by calculating the percentage of positive pixels on micrographs taken from the microscope.</li>
		<li>Analyze pictures of the cells using image analysis software (e.g., ImageJ2 software [Wayne Rasband, version 2.9.0/1.53t]).
		<ol>
			<li>To perform the analysis of the pixels, process the digital images to 300 pixels/inch and convert them to 8 bits. Then, process the binary images by Color Deconvolution plugin to analyze the staining of the protein of interest, in this case, the permanent red staining of occludin.</li>
			<li>Save the selected picture as a tiff and subject it to a &#34;clean-up&#34; procedure to eliminate artifacts with a graphics editor (e.g., Adobe PhotoshopCC [version 20.0.4]).</li>
			<li>Thereafter, measure all fields of interest with the application Analyze Particle of ImageJ2, and report the data as the number of pixels. 
			NOTE: Each experiment should be performed in triplicate with three internal replicate fields analyzed for each replicate. For quantifying mucus, the same principle of measuring the pixels can be applied to the images taken of the purple-magenta-stained neutral mucins and bright blue-stained acidic mucus, respectively.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

<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>

The authors declare no conflict of interest.

The basic reconstructed intestinal mucosa model system presented here (Figure 6) combines physiological complexity (more physiologically relevant 3D cell cultures containing a Caco-2 monolayer with an ECM-rich lamina propria support containing fibroblasts and monocytes) with experimental simplicity (using commercial human cell lines to produce standardized and easily repeatable system)13. As such, this model system is deemed a suitable alternative to murine models aimed at evaluating the effect of potential medicinal drugs/food components on intestinal barrier integrity. In this regard, previous articles substantiate the efficacy of the present cell culture model in testing both potentially beneficial compounds (plus dose dependency) as well as harmful food components7,16,17. Moreover, LPS or alternative pro-inflammatory reagents (2,4,6-trinitrobenzene sulfonic acid or dextran sulfate sodium) can be added to the present intestinal mucosa model to simulate IBD&#160;symptoms. In so doing, it is potentially possible to study the etiology of intestinal diseases from a structural aspect as well as from the expression of cytokines, such as pro-inflammatory MDK, which is a marker of IBD15. This basic model system also has eventual drug screening potential for the broader population. Although the model system is physiologically more complex than 2D models13,14, the limitations in reproducing complex human physiology include the absence of both a microfluidic system (fluidic perfusion of a continuous supply of nutrients and removal of waste products as well as mechanical stimuli)8,14 and the microbiome (evaluating host-microbiome interactions in intestinal diseases)8,14.
The advantage of using the present basic 3D intestinal mucosa model in combination with paraffin embedding for light microscopic evaluation of epithelial barrier integrity is that several tissue sections can made from a single experiment, thereby permitting the analysis of numerous parameters. Transepithelial electrical resistance (TEER)3 is a commonly used method to assess intestinal barrier integrity and has been used in conjunction with macromolecular permeability studies3,18, as well as widely reported studies on Western blot evaluation of TJ protein expression.&#160; Western blot evaluation provides quantitative insight into overall changes in protein expression, and TEER serves as a quantitative measure of barrier integrity. Instead, microscopic evaluation provides a valuable tool for the qualitative visualization and assessment of local protein changes and interactions18.
Microscopic evaluation of a model with an immune component permits the visualization of immune responses, such as the differentiation of monocytes into macrophages and the migration of activated monocytes in the ECM, as demonstrated here by CD14 and CD11b staining. Identification can be made via immunohistochemistry and/or immunofluorescence staining on individual tissue sections, as was shown for CD14 and CD11b in Figure 3. Immunohistochemistry permits the structural visualization of the reconstructed intestinal mucosa tissue as a whole. Moreover, it is possible to identify both the nuclei and the cytoplasm in cells. In contrast, confocal immunofluorescence does not allow the tissue to be seen as a whole (only the markers of interest and DAPI-stained nuclei). However, it provides a very clean image where the background color is eliminated. The advantage of this is that it permits greater precision in evidencing the positivity or negativity of the markers and as such, the calculation of any differences in color intensity (for example, a marker that is more expressed in one cell than in another). Potentially the extent of migration of the CD14 monocytes from within the lamina propria to the Caco-2 monolayer could also be estimated from multiple replicate images taken at various time points. Nonetheless, evidence of inflammation was evident from the presence of both CD14 and CD11b stained cells.
A&#160;range of 20&#8211;40 &#956;g total protein is used by the majority of laboratories for western blotting19. The quantification of numerous gap junction proteins (occludin, claudin, zonulin, and E-caderin) can be performed by immunohistochemical and/or fluorescent staining on individual tissue sections, respectively necessitating significantly less protein. Concurrently, from the same experiment, the integrity of the epithelial layer in response to compounds can also be visualized from the pattern, shape, and structure of cells with H&#38;E staining.
The efficacy of a basic 3D reconstructed intestinal mucosa for use in staining mucus production, TJ proteins, and proteins produced from immune responses is dependent on overcoming critical steps, particularly in the construction of the model system and&#160;during tissue fixation.&#160; Regarding model construction, using the correct confluence and seeding the correct number of Caco-2 cells (indicated by the notes in the protocol), respectively, is fundamental. Too many cells or an incorrect confluence would result in a thick and disorganized epithelial layer. Instead,&#160;too few cells would result in either a reduced or a lack of epithelial layer formation. Equally fundamental at seeding is the correct cell confluence and number of L929 and U937 cells in the ECM, as well as the correct handling of the collagen during preparation. Excess cells or rapid polymerization of the collagen would result in a compact and malformed ECM, in turn impacting on the structure of the epithelial cells above. Similarly, too few collagen-forming L929 cells would result in an incorrect consistency of the ECM, whereas&#160;too few U937 monocytes would not favor an immune response. Also important is that cells be used for experimentation after 5 days, as a prolonged incubation would result in excessive cell growth resulting in a compact epithelial layer. Moreover, given that the present design lacks a microfluidic system and is representative of a static intestinal cellular model, to favor the release of molecules and the growth and stimulation of cells, it is very important that the medium be changed every day. Regarding the fixation protocol, the clearing phase is critical and given that the time required for the tissues to become transparent in paraffin may vary, each intestinal mucosa system must be closely observed during this phase. Throughout the fixation procedure, care must be taken in handling these models in order to avoid damaging&#160;them.
In conclusion, provided strict adherence is paid to the critical points in the construction and fixation of the basic 3D intestinal equivalents, the models can provide numerous tissue sections for the evaluation of multiple parameters in pharmaceutical screening and toxicity studies.

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.

<table><tbody><tr><td>Alcian Blue/PAS kit</td><td>ScyTek Laboratories, Inc.</td><td>APS-1, APS-2</td><td>kit for immunohistochemical staining</td></tr><tr><td>Blue Trypan solution</td><td>Thermo Fisher</td><td>15250061</td><td>cell count analyses</td></tr><tr><td>Caco-2 colorectal adenocarcinoma cells</td><td>ATCC</td><td>ATCC HTB-37</td><td>cell line</td></tr><tr><td>Citro-Histo-Clear Limonene based</td><td>Histoline laboratories</td><td>R0050CITRO</td><td>reagent for paraffin embedding</td></tr><tr><td>Dulbecco&amp;#39;s Modified Eagle Medium (DMEM)</td><td>GIBCO</td><td>11965092</td><td>cell colture reagent</td></tr><tr><td>Embedding Center</td><td>Histoline laboratories</td><td>TEC2900</td><td>instrument for paraffin embedding</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GIBCO</td><td>A5256701</td><td>cell colture reagent</td></tr><tr><td>Hanks&amp;#39; Balanced Salt Solution (HBSS)</td><td>GIBCO</td><td>12350039</td><td>cell colture reagent</td></tr><tr><td>Human Midkine ELISA kit</td><td>Cohesion Biosciences</td><td>CEK1270</td><td>kit ELISA</td></tr><tr><td>Inverted microscope</td><td>Eclipse Ts2, Nikon</td><td>MFA34100</td><td>microscope</td></tr><tr><td>L929 mouse fibroblasts</td><td>ATCC</td><td>ATCC &amp;#174;-CCL1</td><td>cell line</td></tr><tr><td>LC3-II</td><td>Novus Biologicals</td><td>NB910-40752SuperNovusPack</td><td>antibody</td></tr><tr><td>L-Glutamine</td><td>GIBCO</td><td>A2916801</td><td>cell colture reagent</td></tr><tr><td>Occludin</td><td>Novus Biologicals</td><td>NBP1-87402</td><td>antibody</td></tr><tr><td>Paraffin Lab-O-Wax PLUS 56 &amp;#176;C&amp;#8211;58 &amp;#176;C</td><td>Histoline laboratories</td><td>R0040 PLUS</td><td>instrument for paraffin embedding</td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>15070063</td><td>cell colture reagent</td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>15070063</td><td>cell colture reagent</td></tr><tr><td>rat tail collagen type I</td><td>GIBCO</td><td>A1048301</td><td>cell colture reagent</td></tr><tr><td>Roswell Park Memorial Institute Medium (RPMI)</td><td>GIBCO</td><td>21870076</td><td>cell colture reagent</td></tr><tr><td>Sodium pyruvate</td><td>GIBCO</td><td>11360070</td><td>cell colture reagent</td></tr><tr><td>Thermo Fisher Countess II FL Automated Cell Counter</td><td>Thermo Fisher</td><td>TF-CACC2FL</td><td>cell counting instrument</td></tr><tr><td>Transwell</td><td>Costar Corning</td><td>3413</td><td>plastic for cell colture</td></tr><tr><td>Trypsin</td><td>GIBCO</td><td>15090046</td><td>cell colture reagent</td></tr><tr><td>U937 a pro-monocytic, human myeloid leukemia cell line</td><td>ATCC</td><td>ATCC CRL-1593.2</td><td>cell line</td></tr><tr><td>UltraTek Alk-Phos Anti-Polyvalent (permanent red) Stain Kit</td><td>ScyTek Laboratories, Inc.</td><td>AMH080</td><td>kit for immunohistochemical staining</td></tr></tbody></table>

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.

The first important aspect is to determine the acceptability of the basic 3D intestinal equivalent mucosa for experimental purposes. This is performed with the most widely used stain in histology and histopathology laboratories, namely hematoxylin (stains nuclear material deep blue-purple color) and eosin (stains cytoplasmic material varying shades of pink). The H&#38;E staining is first performed on an untreated control, which is cultured under the same conditions and timeframe as the experimental treatments. From visualizing the pattern, shape, and structure of cells, the success of this intestinal cell model system is determined by the Caco-2 cells forming a tight and regular monolayer above the extracellular matrix (ECM)-rich lamina propria after 5 days (Figure 1A). Examples of model systems that would be deemed unacceptable for further analyses include those showing excessive growth with disorganized epithelial layers, as shown after 10 days (Figure 1B). An additional example is an epithelial layer that is too thick and disorganized (Figure 1C), caused either by the seeding of excess Caco-2 cells or an excessive incubation. Malformation (Figure 1D), attributable either to seeding cells that were not in an active phase of proliferation, or problems in the preparation of the lamina propria or during fixation with paraffin (cells became detached /ruined), is also an example of an unacceptable reconstructed intestinal mucosa. Similarly unacceptable is the lack of epithelial layer formation (Figure 1E), indicative of the seeding of too few Caco-2 cells or problems in the preparation of the lamina propria. Using acceptable 3D intestinal equivalents, from which multiple sections can be generated, various parameters can then be investigated from a single experiment.
Of the various parameters that can be analyzed, barrier integrity in response to pro-inflammatory inducers can be visualized, and TJ proteins stained and quantified and compared to untreated control model systems. Here we demonstrate the pro-inflammatory effect of 40 &#181;g/mL total wheat protein from a durum wheat source with a high gluten content7, on both cellular integrity as well as TJ occludin protein content (Figure 2A). Using H&#38;E histological staining, the control model system shows the Caco-2 columnar cells forming a tight and regular monolayer. In contrast, the effect of the gluten-containing protein sample was shown to cause disruption of the monolayer, with more nonspecific eosin staining (Figure 2A). Moreover, the thickness of the epithelial layer can be measured and is shown to be significantly reduced after exposure to pro-inflammatory gluten (Figure 2A,B). Using an antibody to occludin (conjugated to red chromogen staining), occludin protein could be visualized and quantified. Significantly higher occludin protein content was evident in the control group compared to the intestinal mucosa exposed to gluten protein for 24 h (Figure 2A,C).
The intestinal equivalents comprised of an immune component (represented by U937 monocytes) are also ideal in investigating the effects of pro-inflammatory inducers on monocyte activation, migration, and differentiation into macrophages. Once again, using 40 &#181;g/mL total wheat protein extracted from a wheat source with a high gluten content, pro-inflammatory activation of U937 monocytes in the lamina propria was evident from fast red coupled CD14 staining (Figure 3A). Activation is detected from the increased expression of this red-stained membrane glycoprotein on monocytes and macrophages. Increased CD14 protein was also detected with fluorescein isothiocyanate (FITC), which stains green under immunofluorescence. Migration was evidenced from the presence of activated U937 cells close to the Caco-2 monolayer. Where the monolayer had ruptured, the U937 cells were shown to have entered within the Caco-2 cell monolayer (Figure 3A). There was no evidence of CD14-stained monocyte migration in the control group (Figure 3A). The differentiation of monocytes into macrophages is identifiable from the marker CD11b. No CD11b staining was evident in the control group with either red chromogen or FITC (Figure 3B). Instead, in the tissues exposed to gluten, CD11b macrophages were observed both in the ECM-rich lamina propria and within the ruptured Caco-2 monolayer (Figure 3B).
Using LPS to simulate intestinal inflammation, here we show that it is also possible to use this model system to investigate acidic mucin and mucopolysaccharides produced by the Caco-2 cells in the untreated control and the LPS treatment, respectively (Figure 4A). Using Alcian blue and PAS staining, acidic mucus is stained bright blue, and neutral mucins are stained purple-magenta, respectively. The extent of mucus staining can similarly be quantified and is shown to be significantly induced under 1 ng/mL LPS challenge (Figure 4B). Moreover, prior to fixation, the medium can be removed to investigate pro-inflammatory cytokine production. Although numerous excretory cytokines can be measured, in this particular instance, midkine (MDK) was selected. MDK is an endogenous inflammatory marker induced by the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cell (NF-&#954;B) pathway, and is also associated with inflammatory diseases of intestinal cells15. Under the LPS challenge, MDK is significantly induced in comparison to the control (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65484/65484fig01.jpg" /> 
Figure 1: Untreated control 3D intestinal equivalents. Hematoxylin and eosin (H&#38;E) staining of untreated control 3D intestinal equivalents (Caco-2 /U937/L929 co-cultures) for 24 h as a verification of the efficacy of the basic 3D intestinal mucosa model for experimental purposes. (A) An acceptable experimental model is compared to unacceptable models, showing excessive (B) epithelial growth and disorganized layers after 10 days, (C) excessive epithelial thickness and disorganization, (D) malformation, and (E) no epithelial cell layer. The scale bar in each image equals 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65484/65484fig02.jpg" /> 
Figure 2:&#160;Treated 3D intestinal equivalents. (A) Hematoxylin and eosin (H&#38;E) staining, and occludin expression of 3D intestinal equivalents (Caco-2 /U937/L929 co-cultures) exposed for 24 h to 40 &#181;g/mL total protein from a high gluten containing modern wheat mix compared to the control (CTRL). The scale bar in each image equals 50 &#181;m. (B) Quantification of Caco-2 cell thickness and (C) occludin-red chromogen staining. Significant differences were determined by one-way variance (ANOVA) and represented as *** 0.0001&#60; P &#60; 0.001. The black dots represent the number of replicates. This figure has been adapted with permission from Truzzi et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65484/65484fig03.jpg" /> 
Figure 3: CD14 and CD11B staining. CD14 staining of U937 monocytes and CD11B U937 differentiated macrophages in 3D intestinal equivalents (Caco-2/U937/L929 co-cultures) exposed to no added protein (control [CTRL]) or 40 &#181;g/mL total protein from a high gluten containing modern wheat mix after a period of 24 h. For CD14 and CD11b staining, fast red was used as a chromogen and nuclei were counterstained with eosin. The scale bar in each image equals 20 &#181;m. CD14 and CD11b staining with fluorescein isothiocyanate (green) was also shown under immunofluorescence. The nuclei were counterstained with 4&#39;,6-diamidino-2-phenylindole (DAPI). The scale bar in each image equals 50 &#181;m. Arrows indicate the presence of monocytes (CD14 positive cells) and macrophages (CD11b positive cells). This figure has been adapted with permission from Truzzi et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65484/65484fig04.jpg" /> 
Figure 4: Hematoxylin and eosin (H&#38;E) staining, and mucus expression from Alcian Blue/Periodic Acid-Schiff Stain (PAS) staining. (A) Caco-2 cells in basic 3D intestinal equivalents (Caco-2 /U937/L929 co-cultures) after 24 h in the untreated equivalent and reconstructed intestinal mucosa model systems exposed to 1 ng/mL lipopolysaccharide. The scale bar in each image equals 50 &#181;m. (B) Quantification of mucus expression was determined by one-way variance (ANOVA) with significance reported as **** P &#60; 0.0001. The black dots represent the number of replicates. This figure has been adapted with permission from Truzzi et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65484/65484fig05.jpg" /> 
Figure 5: Midkine expression. Midkine expression in the medium of 3D intestinal equivalents (Caco-2 /U937/L929 co-cultures) after 24 h in the untreated control (CTRL) and intestinal mucosa model systems exposed to 1ng/mL lipopolysaccharide. Quantification of midkine expression was determined by one-way variance (ANOVA) with significance reported as **** P &#60; 0.0001. This figure has been adapted with permission from Truzzi et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65484/65484fig06.jpg" /> 
Figure 6: Schematic diagram representing the construction and experimental use of intestinal equivalents, as well as the fixation and microscopic analyses of parameters from tissue sections. The schematic diagram sums up the construction of the intestinal equivalents and possible experimental treatments that can be administered to these models. The generation of multiple tissue sections from a single experiment permits the analyses of numerous structural and immune aspects of the intestinal mucosa, as illustrated in Figure 1, Figure 2, Figure 3, and Figure 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65484/65484fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.0K Views.  Alma Mater Studiorum-University of Bologna. 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.

Video: Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Bioengineering

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Immunology and Infection

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Enhanced Intestine-on-Chip Platform for Immunological Insight

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Medicine

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

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Chapters in this video

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome