The scope of the work is to use these in vitro three-dimensional human cell line models to investigate the fact of beneficial as well as helpful compounds on intestinal tissue. The models can be used to further research understanding, and also as a basic drug models. Using the plant-derived compounds, spermidine and eugenol, both separately and together in supplement form, we measure beneficial increases in autophagy and increases in inflammation using these models.
Then we were also able to show the potentially harmful effect of gluten extracted from certain wheat varieties on intestinal tissues. We are currently using economically priced, standardized, an easily repeatable in vitro system that better reflect the physiology of the human intestine as an alternative to animal models. Since we also use commercial human cell line rather than plant-derived cells, the responses from the study are more applicable to the general population.
The protocol is based entirely on the use of three different lines. These render the model more standardized and reproducible. Furthermore, the most simultaneous cultivation of three different cell lines, enterocytes, fibroblasts, and immune system cells, allows for cellular responses that are closer to what happens in the patient.
To begin, select a 24-well plate containing inserts with 0.4 micron filters Using a pipette, add 500 microliters of HBSS below and above the filter inserts. Then close the multi-well plate, and place it in an incubator for two hours. After incubation, carefully aspirate the HBSS from the plate.
Prepare a cell-free collagen solution in a sterile 50-milliliter tube in DMEM on ice. Add 250 microliters of collagen above the membrane filter, and place the lid on the plate. Allow the solution to polymerize that room temperature.
Next, remove the L929 fibroblast cell culture from the incubator. Add two milliliters of a prewarmed Trypsin-EDTA solution to the flask, and place it in an incubator for three to five minutes. Transfer the cell suspension to a sterile 15-milliliter tube, and centrifuge at 645 g for five minutes.
Using a vacuum pump, aspirate the supernatant, and resuspend the pellet in one milliliter of DMEM. Add 20 microliters of cell suspension into a tube containing 20 microliters of Trypan Blue solution, and use a cell counting chamber to evaluate the cell density. Next, remove the U937 monocyte cell culture from the incubator.
Centrifuge the cell suspension, and resuspend the pellet in one milliliter of RPMI. After ensuring cells are homogenously suspended, count them using a cell counting chamber. Next, prepare cell suspensions containing 50, 000 L929 and 15, 000 U937 cells, respectively, in 50 microliters of DMEM.
Add each 50 microliter cell suspension to 450 microliters of collagen and mix thoroughly. Overlay the pre-coated cell-free lamina propria with 500 microliters of the collagen cell solution. Close the plate and place it in an incubator for two hours to allow the solution to set.
Next, remove the Caco-2 cell culture from the incubator. Using a vacuum pump, aspirate the medium, and rinse the cells with 10 milliliters of PBS. Add five milliliters of PBS Trypsin-EDTA solution, and place it in an incubator for five to eight minutes.
Then centrifuge the cell suspension, and resuspend the pellet in one milliliter of DMEM. After counting, prepare a cell suspension containing 150, 000 Caco-2 cells and 50 microliters of DMEM. Place the plate in a laminar flow hood for 10 minutes before transferring it to the incubator.
After 30 minutes, add 500 microliters of the DMEM above the reconstructed model and 500 microliters below the filter, and return the plate to the incubator. The next day, carefully replace the medium with 500 microliters of fresh DMEM above and below the filter, respectively. For paraffin embedding of cells, set the paraffin machine to 58 degrees Celsius.
Using pliers, transfer the membrane inserts to clean wells of a sterile 24-well plate. Add 500 microliters of 37%Buffered Formalin and PBS above the filter and one milliliter below the filter. Close the lid, and leave the plate in a fume hood for two hours.
Once the lamina propria is detached from the membrane insert, transfer the intestinal mucosa into a beaker containing 25 milliliters of 35%ethanol, and incubate for 10 minutes. After dehydration and increasing ethanol concentrations, place the samples in 50 milliliters of xylene or a histological clearing agent for 10 to 20 minutes. Once the samples are transparent, place them in a metal tissue cassette holder, and submerge them in liquid paraffin inside a heated machine.
After 45 minutes, use a microtome to cut four micron sections. Place the cut section on slides, and dry them in an oven at 37 degrees Celsius for 24 hours. An acceptable 3D intestinal equivalent mucosa model consists of Caco-2 cells formed in a tight and regular monolayer above the extracellular matrix-rich lamina propria.
Unacceptable models include those showing excessive growth, disorganized epithelial layers, malformation, or lack of epithelial layer formation. The pro-inflammatory effect of wheat protein with a high gluten content disrupted the cellular monolayer and reduced the thickness of the epithelial layer compared to the control group The occludin protein content was significantly higher in the control group than in the group exposed to gluten protein. CD14 and CD11b staining of U937 monocytes showed that wheat protein with a high gluten content activated the monocytes and induced their migration and differentiation into macrophages.
Under the lipopolysaccharide challenge, Caco-2 cells increased mucus production and the release of the inflammatory marker midkine.
