Our research goal is to establish robust techniques to generate staple intestinal monolayer interfaces derived from adult bovine small and large intestinal organoids. We employed a multimodal analytical approach to confirm the functional epithelial barrier formation within the established monolayers. Unlike three-dimensional models, 2D monolayers offer an exposed, accessible luminal surface crucial for studying host pathogen interactions.
These monolayers reflecting the in vivo gut with multiple cell lineages now provide tailored protocols for investigating pathogens in specific gut sections such as small and large intestinal niches. Our research identified optimized culture conditions to create and sustain 2D monolayers from cells of bovine small and large intestinal organoids. We confirmed the formation of functional epithelial barriers within these monolayers using transepithelial electrical registrants and paracellular permeability assays as well as immunocytochemistry staining techniques.
Our protocol addressed the research gap by focusing on the lack of intestinal organoid-derived monolayer models of bovines. These models have been well studied in mice and humans, however, bovines which are major reservable for intestinal pathogens with serious public health implications, haven't been adequately explored. Our research establishes and characterizes a 2D bovine monolayer culture system.
This innovative tool allows for the exploration of normal and disease states of bovine intestinal physiology with potential applications extending into biomedical and translational research of public health significance. Begin by coding the extracellular matrix on cell culture inserts for organoid-derived 2D monolayer culture. Apply 100 microliters of extracellular matrix coating to the apical chamber of each prepared cell culture insert and replace the lid.
Then incubate the cell culture plate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for one hour. For rectal organoid cells, after the incubation, replace the extracellular matrix coating with rectal monolayer culture medium and incubate overnight. After enzymatic digestion of the organoids and filtration to single cells, centrifuge the cell suspension at 200 G for five minutes at four degrees Celsius and then discard the supernatant.
Next, prepare 200 microliters of the cell suspensions per cell culture insert in monolayer culture media. Retrieve the 24-well cell culture plate, containing extracellular matrix-coated cell culture inserts. Using vacuum suction, carefully empty the apical chamber of each cell culture insert to avoid disrupting the coating.
Carefully apply 200 microliters of the single cell suspension to the apical chamber of each cell culture insert. Add 500 microliters of the appropriately supplemented monolayer culture medium to the basolateral chamber of each well. Then incubate the plate in a humidified incubator to promote cell adhesion and growth, forming a confluent 2D monolayer on the cell culture insert.
Change the culture media in both the apical and basolateral chambers every other day, starting 48 hours after cell seeding. For immunofluorescent staining of organoid derived 2D monolayer, carefully cut out the cell culture insert membrane with a scalpel blade and place the cell culture insert on a glass slide. Add mounting media on a cover slip and place it on the glass slide before observing the cells.
One day after seeding dissociated mature organoids onto a cell culture insert, a 2D monolayer appeared to form. The scanning electron microscopy revealed well-developed microvilli and cellular demarcation on the apical surface of the 2D monolayers five days after seeding. Immunofluorescent staining of the 2D monolayers confirmed the presence of apical brush border, basolateral adherence junctions, and mucus-producing goblet cells in both ileal and rectal organoid-derived 2D monolayers.
To begin, retrieve the plate containing cell culture inserts with organoid-derived 2D monolayers from the incubator. Remove the monolayer culture media from both the apical and basolateral chambers of the cell culture inserts to be assessed. Gently wash the apical and basal chambers two times with 200 and 500 microliters of prewarmed PBS.
Remove the wash solution from the apical chamber and apply 200 microliters of 0.5 milligrams per milliliter, 4 kDa FITC-dextran tracer in PBS to the apical chamber of the cell culture insert. Incubate the plate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for 20 minutes. Then sample 50 microliters from the basal lateral chamber of the incubated 24-well plate and transfer it to a 96-well plate compatible with a microplate reader.
Replace 50 microliters of fresh PBS in the basal lateral chamber of the sampled well. Using a pre-calibrated microplate reader, immediately measure the fluorescence intensity at an excitation wavelength of 495 nanometers and an emission wavelength of 535 nanometers.
