오가노이드 유래 상피 단층: 장 장벽 기능을 위한 임상적으로 관련된 시험관내 모델

Organoid-derived monolayers recapitulate patient’s intestinal epithelial barrier function and allow to test the effect of therapies aimed at enhancing dysfunction in each patient to identify personalized treatment strategies. Organoid monolayers provide access to the apical side of the epithelium, which is the side where the damage occurs, and it’s normal and accessible in organoids growing as 3D structures. Every organoid is different, hence the struggle.

To form tight monolayers, use organoids in proliferation phase and digest quickly to form clusters of two to four cells. Titrate the cell seeding for every model. Demonstrating the procedure will be done by Wies Van Dooremalen, a senior research associate of HUB.

Begin by coating membrane inserts with extracellular matrix or ECM. Working in a biosafety cabinet, place the membrane inserts into the support plate. Dilute the ECM 40X in ice cold DPBS with calcium and magnesium, then pipette 150 microliters of the diluted ECM into the apical compartment of each insert.

Incubate the plate at 37 degrees Celsius for at least one hour. To prepare the cells for seeding, prewarm an aliquot of cell dissociation reagent in a 37 degree Celsius water bath. Use two milliliters of the reagent for each well of a six-well plate.

Transfer the culture plate containing the organoids from the incubator to the biosafety cabinet. Process the organoids as described in the text manuscript, but do not pool multiple tubes into one tube. After harvesting the organoids, add basal medium or BM to 12 milliliters and centrifuge at 85 times G, then aspirate the supernatant.

Fill the tube containing the organoids with up to 12 milliliters of DPBS, then pipette up and down 10 times using a 10 milliliter pipette. Centrifuge at 85 times G for five minutes at eight degrees Celsius and aspirate the supernatant without disturbing the organoid pellet. Add the pre-warmed cell dissociation reagent to the organoids and resuspend them, then incubate the tubes diagonally or horizontally for five minutes in the water bath at 37 degrees Celsius.

After the incubation, pipette up and down 10 times using a five milliliter sterile plastic pipette or a P1000 pipette. Check the organoid suspension under the microscope to see if a mixture of single cells and some cell clumps consisting of two to four cells has formed. Stop cell dissociation by adding up to 12 milliliters of BM with ROCK inhibitor to the cell suspension.

Centrifuge the cells, then aspirate the supernatant without disturbing the cell pellet. When handling the same organoid culture in several tubes, pool the cell pellets before resuspending them in 12 milliliters of BM.Filter the cell suspension through a 40 micrometer strainer pre-wetted with BM and harvest the flow-through into a 50 milliliter conical tube, then wash the strainer with 10 milliliters of BM and harvest the flow-through into the same tube. Transfer the strained cell suspension into two new 15 milliliter conical tubes and repeat the centrifugation.

Aspirate the supernatant without disturbing the cell pellet and resuspend the cells in four milliliters of IEM supplemented with 10 micromolar ROCK inhibitor per full culture plate used as starting material. Mix a small amount of cell suspension in a one-to-one ratio with Trypan Blue for counting, then count the cells and calculate the total number of live cells, counting each individual cell in small clumps. Prepare a cell suspension containing 3X 10 to the sixth live cells per milliliter of IEM supplemented with 10 micromolar ROCK inhibitor.

To seed the cells, carefully aspirate DPBS from the ECM-coated inserts, keeping the plate horizontal. Pipette 800 microliters of IEM supplemented with ROCK inhibitor into each basolateral compartment and 150 microliters of the cell suspension prepared onto the ECM-coated membrane in the apical compartment dropwise. Place the plate in the incubator at 37 degrees Celsius and 5%carbon dioxide.

Once the cells have sedimented onto the membrane, measure transepithelial electrical resistance or TEER every day and image the membrane inserts using a microscope. To refresh the monolayers, remove the medium from the basolateral compartments of the plate containing the membrane inserts, then carefully aspirate the medium from the apical compartments. Add 150 microliters of fresh IEM dropwise to each apical compartment and add 800 microliters of fresh IEM to each basolateral compartment.

If using a manual TEER meter, clean the electrode with 70%ethanol and let it air dry inside the biosafety cabinet, then place the electrode in a tube containing BM.Connect the electrode to the manual TEER meter and turn the function switch to measure in ohms and the power switch on. Place the short electrode in the apical compartment of the insert and the long electrode in the basolateral compartment without touching the monolayer. Measure resistance in the blank well, then in the remaining samples.

Wash the electrode with BM between samples with different conditions. When finished, clean the electrode with demi water and with 70%ethanol, then let it air dry. This protocol was used to harvest intestinal organoids and prepare monolayers of epithelial cells.

A monolayer that was cultured for eight days in IEM is shown here. A structure can be observed when exposing monolayers to enterocyte differentiation medium or EDM, while monolayers exposed to combination medium or CDM show a smoother structure. Monolayer formation was quantitatively followed by measuring TEER.

A completely confluent monolayer had a TEER value of approximately 100 ohms centimeter squared which increased when exposed to either differentiation medium. Monolayers in all medium conditions showed a lower apparent permeability to Lucifer yellow. Lysozyme secretion by ileal monolayers cultured in IEM was higher than that of monolayers cultured in IEM until confluent and for another four days in EDM or CDM.

Monolayers cultured in IEM, IEM and subsequent EDM, or IEM and subsequent CDM show differences in morphology which was observed with H&E, Ki67, MUC2, and Alcian Blue stainings. Upon differentiation, proliferative cells decreased, while goblet cell and enterocyte marker gene expression increased in comparison to that observed under IEM conditions as shown by LGR5, MUC2, and alkaline phosphatase gene expression. When preparing monolayers, it is important to prevent anoikis in the organoids after digesting them.

Additionally, ECM in cells should be evenly distributed on the Transwell membranes. Organoid-derived monolayers allow the evaluation of molecule transports using different fluorescent substrates in a patient-specific manner.