Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Our lab focuses on examining host-pathogen interactions and the immune cell dynamics that occur following infection. By using a robust cell culture model, we hope to more closely mirror the complex interactions occurring in vivo at a tissue level. Some of the published literature describing intergeneration in cattle lacks the necessary details for the protocol to be easily reproducible, making its application by inexperienced researchers challenging.

The benefit of our model is that we provide an extremely detailed protocol that can be used successfully by novice researchers. Creating Transwell monolayer enables co-culture with immune cells, revealing insights into tissue-pathogen interaction. while maintaining in vivo like organization.

This versatile model is valuable for studying numerous cattle diseases with a complexity beyond single-cell models. Our lab will employ this model to investigate the immune factors influencing susceptibility to Cryptosporidium infection. It will facilitate comparison between adult cattle and calves, while preserving tissue architecture and the immune cell microenvironment.

The findings from this work have direct translational potential to human health. To begin, place cell culture inserts into a 24-well tissue culture adapter blade. Precoat the apical side of the inserts with 100 microliters of Basement Membrane Extracellular matrix, or BME in enteroid growth medium.

Load an extra insert for use as a control during barrier integrity measurements. Next, incubate the coated insert at 37 degrees Celsius under 5%carbon dioxide for one hour. Once incubation is complete, aspirate the 3D enteroid culture medium.

Harvest the bovine ileal enteroids in one milliliter of ice cold wash media. Use a cell scraper to detach the domes, and collect them into a 15-milliliter conical tube. With a one-milliliter pipette tip, titrate 30 times to generate enteroid fragments.

Next, use a 200-microliter pipette tip to further titrate about 40 times to break up the enteroid fragments. Make up the volume of the tube to 10 milliliters with ice cold wash media. Then centrifuge the tube at 300 g for five minutes at room temperature.

Carefully aspirate the supernatant, including the BME layer. Next, for every four domes, resuspend the pellet in one milliliter of pre warmed TrypLE express enzyme. Transfer the mixture to a 24-well plate, and incubate at 37 degrees Celsius under 5%carbon dioxide for 10 minutes.

Gently pipette the mixture with a one-milliliter pipette to further break down the enteroids. Then pipette the mixture with a 200 microliter pipette to break the fragments into single cells. Use a three-milliliter syringe fitted with a sterile 22-gauge needle to aspirate and dispense the cell suspension four times to achieve a single-cell suspension.

With a microscope, monitor cell dissociation until 80%of the enteroids are broken down into single cells. Now collect the cell suspension into a 15-milliliter conical tube. Add four volumes of FBS wash media to quench the reaction.

Then filter the enteroids through a pre-coated 40-micrometer cell strainer twice into a 50-milliliter conical tube. Centrifuge the suspension at 300 g for five minutes to pellet out the cells. Aspirate the supernatant of a centrifuge tube of dissociated bovine ileal enteroid cell suspensendium.

Resuspend the pellet in a small volume of organoid growth media. Next, use the Trypan Blue dye exclusion method and the hemocytometer to determine the cell viability. Carefully remove any excess coating solution from the cell culture insert.

Seed 200 microliters of the single cell suspension on the apical surface of a pre-coated culture insert. Now, add 700 microliters of FBS complete media to the basal lateral side of the insert. Move the plate about 10 times in the shape of the number eight to evenly distribute the cells over the insert.

Then place the plate on the plate warmer in the biosafety cabinet for 10 minutes. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for 48 hours. After incubation, replace the media on the apical and basal components with fresh enteroid growth media.

The next day, remove the media from the apical and basal lateral compartments. Carefully wash the insert with PBS, and replace it with enteroid differentiation media, supplemented only with inhibitors. Enterosphere formation was observed a few hours after culture of the plated bovine crypts.

After two days, the lumen of the spheres was well-defined with budding structures observed at Day 4. Mature enteroids were observed by Day 7. Immunostaining of the seven-day old, 3D enteroids showed the presence of different cell lineages.

E-cadherin protein was localized at the adherence junction. The enteroids were positive for enteroendocrine cells and lysozyme-producing paneth cells. Confluent 2D monolayers were observed in less than one week of culture.

To begin, remove the Transwell culture plate containing the 2D bovine ileal enteroid monolayer from the incubator. Allow the plate to equilibrate at room temperature in the biosafety cabinet for a few minutes. Ensure the STX2 electrodes have been preconditioned, and the volt-ohm-meter calibrated to 1, 000 ohms, as per the manufacturer's instructions.

Insert the longer end of the probe into the basal lateral compartment and the shorter end into the apical compartment of the Transwell epithelial cell culture. With the setup stabilized, make three transepithelial electrical resistance measurements per Transwell insert, including the insert devoid of cells. Compute the average of the measurements for each respective insert.

Subtract the average measurement of the blank wells from the experimental well then multiply it by the surface area of the insert to determine the resistance of the epithelial barrier. Confluent 2D monolayers were observed in less than one week of culture. TEER measurements showed a steady increase in values over seven days before declining on Day 12.

Confocal microscopy of the stained 2D monolayer demonstrates localization of DAPI nuclear stain, E-cadherin, and F-actin staining. The cells appear to be differentiated with enteroendocrine cells, paneth cells, and enterocyte cell lineages. Apical stimulation of the monolayer resulted in increased cytokine production.