Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Our protocol provides tools to study the pig intestinal epithelium for veterinary and biomedical research. Intestinal organoids from pigs can be used to study nutrient transport, barrier function, and host-microbe interactions. Through preservation of pig intestinal epithelial crypts allow to create a large stock of stem cell that can later be used to culture organoid in 3D or 2D monolayers.

This protocol is detailed for the jejunum, but it can also be used for other intestinal segments such as duodenum, ileum, and colon. To begin, quickly thaw a vial containing 900 frozen crypts in a water bath at 37 degrees Celsius. Transfer the crypt solution into a 15 milliliter conical tube.

Centrifuge at 300 x g for five minutes at room temperature and remove the supernatant. Add 150 microliters of the extracellular matrix or ECM with a cooled tip to obtain a final concentration of 150 crypts per 25 microliters of ECM. Pipet up and down 10 times to obtain a homogenous suspension of crypts in the ECM.

Seed six wells with a 25 microliter drop per well in a pre-warmed 48-well plate. Incubate for 30 minutes at 37 degrees Celsius and 5%carbon dioxide for polymerization of the ECM. Add a 250 microliter per well of culture of medium at room temperature and incubate at 37 degrees Celsius and 5%carbon dioxide.

Change the culture medium every two to three days. To passage 3D organoids derived from frozen crypts 10 days after seeding, remove the culture media and add 250 microliters of pre-warmed PBS at 37 degrees Celsius. After incubation, replace the PBS with 250 microliters of pre-warmed enzyme dissociation reagent supplemented with 10 micromolar rock inhibitor at 37 degrees Celsius in each well.

Detach the organoids in the ECM by scraping with a P-1000 pipette and homogenized carefully by pipetting five times. Incubate for five minutes at 37 degrees Celsius and 5%carbon dioxide before dissociating the cells by pipetting up and down 10 times. Add 500 microliters of DMEMc in each well containing dissociated cells and pull up to 12 wells in a 15 milliliter conical tube containing three milliliters of cold DMEMc.

Centrifuge at 500 x g for five minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in one milliliter cold DMEMc. Count the cells with a dilution of one to two in trypan blue with a cell counter.

Centrifuge the necessary volume of the cell solution to have 3000 live cells per dome. Resuspend the pellet with 17 microliters of cold DMEMc per 3000 live cells on ice. Adjust the volume to the required number of wells.

Slowly add 33 microliters of cold ECM per 3000 live cells. Adjust the volume to the required number of wells and homogenize without making bubbles. Then see the wells with 50 microliters of the ECM cell suspension per well in a pre-warmed 24-well plate and incubate for 30 minutes for polymerization of the ECM.

Add 500 microliters of the culture medium per well. Then incubate and change the culture medium every two to three days. To coat the culture insert, prepare the coating solution containing collagen IV diluted at 50 micrograms per milliliter in cold PBS.

Add 150 microliters of the diluted coating solution to each cell culture insert and incubate overnight or for three hours. Five days after splitting, detach the organoids with the ECM by scraping them with a P-1000 pipette. Homogenized carefully by pipetting in the culture medium and transfer to a 15 milliliter conical tube containing five milliliters of cold DMEMc on ice.

Centrifuge the collected organoids at 500 x g for five minutes at four degrees Celsius. Carefully aspirate the supernatant and resuspend the cell pellet in one milliliter of pre-warmed enzyme dissociation reagent supplemented with 10 micromolar ROCK inhibitor. Pipet up and down 10 times to initiate dissociation of the organoids.

Incubate for five minutes in a 37 degree Celsius water bath for enzymatic digestion. Mechanically disrupt the organoids by pipetting 10 times and add four milliliters of ice cold DMEMc. Centrifuge and discard the supernatant before resuspending the organoid cell pellet in one milliliter of DMEMc.

Count the cells in trypan blue with a cell counter and calculate the necessary volume to seed 2.5 times 10 to the fifth cells per culture insert. Centrifuge the necessary volume of the cell suspension corresponding to 2.5 times 10 of the fifth cells per culture insert. During centrifugation, carefully aspirate the coating solution from the culture inserts and allow it to dry under the hood without the lid for five minutes.

After centrifugation, discard the supernatant and resuspend the pellet in the necessary volume of 2D medium supplemented with 10 micromolar ROCK inhibitor. Seed 200 microliters of the cell suspension onto the coated permeable membrane. Add 500 microliters of the 2D medium supplemented with 10 micromolar ROCK inhibitor to the lower compartment and incubate.

A high density of cells should be observed on the membrane. One day after seeding, replace the epical and basal media with fresh 2D media without the ROCK inhibitor. Change the media each day.

The monolayer becomes confluent one day after seeding and can be used for its experiments. In this study, epithelial crypts were obtained from the pig intestine and cryopreserved for long-term storage in liquid nitrogen. After thawing, the crypt stem cells were seeded in the ECM.

The organoids were observed within three to four days and rapidly grew and developed budding structures. Approximately 10 days after thawing, the passage of organoids was performed to expand the culture. For optimal maintenance of the culture, the organoids used for packaging must present a clear and empty lumen and well-defined edges with no black debris in the lumen as observed in mature organoids.

Cells attach and form a fully confluent monolayer within one day which is confirmed by the high transepithelial electrical resistance, or TEER, of around 700 domes square centimeters. The TEER remains high for three days. Actin staining indicates that the apical side of the epithelial cells is oriented toward the lumen in 3D organoids.

Organoid cells seeded in culture inserts form a confluent single layer of epithelial cells with the apical side oriented toward the upper compartment. Occludin staining reveals the presence of tight junctions at the apical side of the epithelial cells in 3D organoids and in cell monolayers. It's important to remember that organoid used to seed cell monolayers should appear clear with an empty lumen without black debris corresponding to an accumulation of dead cells.

Monolayers from pig intestinal organoid can be used to test the effects of metabolites or microbes on the epithelial barrier function by using functional assays, imaging, and gene expression analysis.