Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

Our organoid model is highly valuable to explore pituitary stem cell biology in homeostatic as well as pituitary remodeling conditions, such as in neonatal maturation of the gland, aging-associated functional decline, and tumor growth in the gland. To date, our pituitary organoid technique is the only available tool to reliably and robustly grow and expand primary mouse pituitary stem cells. Demonstrating the procedure will be Emma Laporte and Charlotte Nys, PhD students in my research group.

To begin, wash the mouse head with deionized water to remove the blood. Spray 70%ethanol on the head to generate a sterile environment. Then, remove the skin between the ears using sterile surgical tools.

To open the cranium, break the nose bridge with sterile scissors. Further open the cranium starting from the nose bridge toward the ears on both sides. Remove the cranium and the brain with sterile tweezers without touching the pituitary gland.

Remove the diaphragma sellae with blunt tweezers. Then, under a stereomicroscope, separate the anterior lobe from the posterior and intermediate lobes. Carefully isolate the anterior lobe with blunt tweezers and collect it in a 10-milliliter Erlenmeyer flask containing three milliliters of medium A.Add two milliliters of pre-warmed 2.5%trypsin solution.

Incubate at 37 degrees Celsius for 15 minutes. Add two milliliters of pre-warmed DNAse solution and swirl the Erlenmeyer flask 10 times. Let the pituitary sink to the bottom and remove the supernatant.

Add two milliliters of pre-warmed trypsin inhibitor solution and incubate at 37 degrees Celsius for 10 minutes. Remove the supernatant when the pituitary sinks to the bottom. Add two milliliters of pre-warmed medium B and incubate for five minutes.

Add two milliliters of pre-warmed medium C to this and incubate for 15 minutes. Let the pituitary sink to the bottom and remove the supernatant. Then, rinse it three times with pre-warmed medium C.Add two milliliters of pre-warmed medium C.Aspirate and expel the pituitary gland with a sterile, flame-polished Pasteur pipette multiple times until fragments are not visible anymore.

Transfer the suspension to a 15-milliliter tube containing 4.5 milliliters of pre-warmed DNAse solution. Rinse the flask three times with two milliliters of pre-warmed medium C and transfer the suspension to the tube. Mix the collected cell suspension and filter it through a 40-micron cell strainer into a 30-milliliter tube.

Rinse the tube and the cell strainer three times with two milliliters of medium C and transfer the suspension to the tube. Fill a glass Pasteur pipette with two milliliters of BSA. Position the tip of the pipette at the bottom of the tube and gently pipette out to form a visible density layer.

Centrifuge at 190 times G for 10 minutes at four degrees Celsius. Remove the supernatant and resuspend the cell pellet in one milliliter of ice-cold advanced DMEM/F-12. Then, quantify the cells with a cell counter.

Centrifuge the cell suspension at 190 times G for 10 minutes at four degrees Celsius and remove the supernatant. Resuspend the cell pellet in advanced DMEM/F-12 to reach a cell density of 1.1 times 10 to the 6th cells per milliliter. Add ECM to the desired volume of cell suspension at a ratio of 30:70 and mix well.

Deposit a 30-microliter drop of this mixture in each well of a pre-warmed 48-well plate. Turn the plate upside down and let the ECM solidify at 37 degrees Celsius for 20 minutes. After the incubation, carefully add 250 microliters of pre-warmed pituitary organoid medium supplemented with 10-micromolar ROCK inhibitor.

Every two to three days, change the medium without ROCK inhibitor for 10 to 14 days until the organoids are fully grown. To passage the organoids, first aspirate the medium gently and add 400 microliters of ice-cold advanced DMEM/F-12 to disintegrate the ECM. Then, collect the organoids in a microcentrifuge tube.

Wash the well with 400 microliters of ice-cold advanced DMEM/F-12. Centrifuge the tube at 200 times G for five minutes at four degrees Celsius. Remove the supernatant and add 400 microliters of pre-warmed TrypLE Express enzyme.

Mix by inverting the tube several times and incubate at 37 degrees Celsius for five minutes. Add 400 microliters of ice-cold advanced DMEM/F-12. Centrifuge at 200 times G for five minutes at four degrees Celsius and remove the supernatant.

Resuspend the pellet with 100 microliters of ice-cold advanced DMEM/F-12. Narrow a P-200 tip and use the tip to dissociate the organoids by vigorously pipetting until organoid fragments are obtained. Add 800 microliters of advanced DMEM/F-12.

Centrifuge at 190 times G for 10 minutes at four degrees Celsius and remove the supernatant. To passage the organoids, resuspend the pellet in an adequate volume of advanced DMEM/F-12 and add ECM at a ratio of 30:70. Mix well.

Continue to seed and culture the organoids as described in the text manuscript. Dissociated single cells from the anterior lobe were seeded in ECM and grown in pituitary organoid medium. 14 days after seeding, the organoids were fully developed and reached a diameter of up to 500 micrometers.

At this stage, the organoids exhibited cystic morphology with an epithelial layer enclosing a lumen. It is not recommended to use the wells in which dense structures appear after growth. For passaging, organoid fragments were used for seeding.

Seven days after passaging, favorable organoid regrowth was observed with cystic structures. Dense organoids should be discarded, as the unfavorable organoids may take over the culture. Immunofluorescence staining analysis for the epithelial markers E-cadherin and cytokeratins 8 and 18 confirmed the epithelial character of the organoids.

Expression of pituitary stem cell markers Sox2 and Trop2 demonstrated the stemness, and pituitary-specific marker LHX3 showed pituitary phenotype of the organoids. Expression of the marker Ki67 showed that the organoid-constituting cells are in a proliferative state. RTQ PCR analysis showed higher expression of stemness markers in the organoids than in the anterior lobe even after multiple passages, validating the enrichment of stem cells.

It is important to plate the appropriate number of single cells in the ECM dome at the initial seeding, and when passaging the organoids, one must remember to re-seed fragments and not single cells.