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

Summary

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.

Abstract

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, “healthy tissue” 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&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&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.

Introduction

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 body^1,2. Columnar-type enterocytes constitute the most abundant type of epithelial cells. These are responsible for maintaining epithelial barrier integrity through interactions between several barrier components, including tight junctions (TJs), playing a significant role in barrier tightening^1,3. 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 cells^3,4. 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 inflammation^5,6,7. 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 assembly^4,6,8. 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 models^8,9,10.

There has been a demand for the development of cell-based in vitro models in order to achieve the objective of the “3Rs” 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 distress^11,12,13. 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 responses^12,13. 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 tissues^8,13,14. As a result, 3D 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 toolboxes^13,14. 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. 

The 3D “healthy tissue” 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.

Protocol

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 °C in a humidified atmosphere containing 5% CO₂ (unless stated otherwise in the protocol)_.

1. Prior preparation of the cell lines used in the intestinal model system
   1. Seed L929 mouse fibroblast cells at a concentration of 5 x 10⁵ in 5 mL of Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM L-glutamine, 10% Fetal Bovine Serum (FBS), and 1% Penicillin-Streptomycin (Pen-Strep) in a 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.
   2. Seed Caco-2 cells (concentration of 2 x 10⁶) in 10 mL of DMEM medium (with 10% FBS and 1% Pen-Strep) in a 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.
   3. 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.
2. Preparation of the Transwell co-culture plate inserts
   1. Select a 24-well plate containing inserts with 0.4 µm filters.
      NOTE: The 0.4 µm filter is a standard choice in drug transport studies. The 3 µm and 8 µm filter sizes are not recommended to prevent any possible losses of the collagen-embedded cells.
   2. Using a pipette, hydrate the Transwell filters (henceforth referred to as membrane inserts) with 500 µL of Hanks' Balanced Salt Solution (HBSS) below and above the filter insert.
   3. 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.
   4. 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.
3. Preparation of the cell-free collagen lamina propria of the basic 3D intestinal model system (DAY 1)
   1. 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.
   2. Add 250 µL 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.
4. Preparation, cell counting, and addition of fibroblast (L929) and monocyte (U937) cells to the model system (DAY 1)
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. Add 1 mL of DMEM to the pellet and suspend the cells.
      NOTE: The cells must be homogeneously suspended in the solution.
   6. Remove 20 µL of the cells in DMEM and add 20 µL of Trypan blue solution. Remove 20 µL of the mix and evaluate cell density microscopically using a cell counting chamber.
   7. 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.
   8. Suspend the cells in 1 mL of RPMI. As with the L929 cells, make sure the cells are homogeneously suspended in the solution.
   9. Similarly, establish the cell density as reported in step 1.4.6.
   10. Prepare a collagen solution as described in step 1.3.1.
       NOTE: The amount of solution to be made must take into consideration 450 µL for each insert (or model).
   11. 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 µL 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 µL aliquot - a total volume of 600 µL can be prepared for the 12 filter inserts present in each 24 well-plate.
   12. Add each 50 µL aliquot containing the cells to 450 µL of collagen solution in step 1.4.10. Mix thoroughly.
   13. Overlay the pre-coated cell-free collagen lamina propria with 500 µL 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.
   14. Close the plate and place it in an incubator for 2 h to allow the solution to set.
5. Preparation, cell counting, and addition of epithelial Caco-2 cells to the intestinal model (DAY 1)
   1. 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.
   2. 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.
   3. Prepare a solution of DMEM to contain a count of 150,000 Caco-2 cells in 50 µL.
      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 µL pipette can be used to ensure homogenous distribution. Given that 12 models can be constructed at any given time, a 600 µL cell solution can be prepared.
   4. After the 2 h required in step 1.4.14, add 50 µL of Caco-2 cells suspended in DMEM to the middle of each basement membrane. Close the lid of the multi-well plate.
   5. Incubate under the sterile laminar flow hood for 10 min. Then transfer to the incubator for 30 min.
   6. Prepare a DMEM solution containing 10% FBS and 1% Pen/Strep.
      NOTE: Prepare a sufficient solution to use a 1 mL volume per model.
   7. Add 500 µL of the medium above the reconstructed model and 500 µL below the filter.
      NOTE: Care is required when adding the medium above the filter to avoid detaching or stressing the cells.
   8. Close the multi-well plate system and place it in the incubator.
6. Model preparation/formation and use (DAY 2 to DAY 6)
   1. DAY 2: Carefully remove the solution from both above the reconstructed model and below the filter using a pipette. Replace with fresh 500 µL of fresh DMEM (10% FBS and 1% Pen/Strep) above and below the filter, respectively.
   2. DAY 3: Repeat as above in step 1.6.1.
   3. 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.
   4. 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.
   5. 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.
   6. Incubate the control and experimental models for 24 h in an incubator.
   7. DAY 6: Remove the medium above and below the filter with a pipette.
      ​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 ° C for further analysis.

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.

1. Set the paraffin machine to 58 °C so that it is ready for use.
2. Transfer the membrane inserts to clean wells using pliers in a sterile 24-well plate.
3. Add 500 µL 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.
4. Remove the formalin and add HBSS solution both above and below the filter. Then remove the HBSS.
5. 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.
6. 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.
7. Replace the 35% ethanol with 25 mL of 50% ethanol and incubate for 10 min.
8. Replace the 50% ethanol with 25 mL of 70% ethanol and incubate for 10 min.
9. Replace the 70% ethanol with 25 mL of 80% ethanol and incubate for 10 min.
10. Replace the 80% ethanol with 25 mL of 95% ethanol and incubate for 10 min.
11. Replace the 95% ethanol with 25 mL of 100% ethanol and incubate for 10 min.
12. Replace the 100% ethanol with another 25 mL change of 100% ethanol and incubate for 10 min.
13. 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.
14. 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.
15. Change the paraffin and leave the samples inside the heated machine for a further 45 min.
16. 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.
17. Store the blocks at RT.
    NOTE: If the objective is to prepare sections immediately, then the blocks can be placed at 4 ° C so that they are well-cooled when used.
18. Cut 4 µm sections using a microtome.
19. Place the cut sections on slides and dry them in an oven at 37 ° C for 24 h.
20. The slides are ready for use. Store them at RT until performing either H&E histological staining or immune-histochemical reaction staining (antigen-antibody type).
    1. For immune-histochemical staining, select the antibodies of interest (either for the study of TJ proteins such as occludin or for monocyte activation, migration, and differentiation) and detect expression (via staining) using commercial kits.
    2. Similarly, measure mucus using the Alcian blue and periodic acid-Schiff (PAS) staining kit.
    3. Quantify the stained TJ proteins of interest, such as occludin, by calculating the percentage of positive pixels on micrographs taken from the microscope.
    4. Analyze pictures of the cells using image analysis software (e.g., ImageJ2 software [Wayne Rasband, version 2.9.0/1.53t]).
       1. 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.
       2. Save the selected picture as a tiff and subject it to a "clean-up" procedure to eliminate artifacts with a graphics editor (e.g., Adobe PhotoshopCC [version 20.0.4]).
       3. 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.

Results

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&E staining is first performed on an untreated control, which is cultured under the same conditions and timeframe as the experimental treatments. From visua...

Discussion

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 aim...