Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

With lacrimal gland organoids, one can look into how certain genes control lacrimal gland homeostasis. Additionally, the organoids can be used for regenerative medicine. The main advantage of adult stem cell-derived lacrimal gland organoid technology is that it allows the growth and study of healthy and non-transformed lacrimal gland epithelial cells.

These organoids could eventually be used to treat patients with dry eye disease. Begin by preparing the medium and material required for the experiment, followed by combining all the components of the digestion medium before the experiment. Pre-wet the scalpels in this medium to avoid tissue pieces sticking to them.

Retrieve the mouse lacrimal gland tissue from the medium, place it in the Petri dish, and mince it using pre-wetted scalpels. Once the tissue pieces are less than 0.5 cubic millimeters, scrape them off the Petri dish with the scalpel, and transfer them to a 15-milliliter tube containing the digestion medium. For a very small human biopsy, place it directly in the digestion medium without mincing.

Incubate the 15-milliliter tube having tissue pieces for up to 15 minutes in a 37-degree Celsius water bath. In the meantime, narrow a Pasteur pipette tip in a flame, and pre-wet it in the base medium. To facilitate the dissociation process, pipette the minced mixture up and down every five minutes with the pre-wetted, narrowed Pasteur pipette.

When many single cells and small clumps are visible under the microscope, stop the dissociation by adding 10 milliliters of the base medium. After spinning down at 400g for five minutes, remove the supernatant, and resuspend the pellet in 10 milliliters of the base medium to repeat the wash and pellet down the cells. Remove the large, undigested tissue pieces and remaining collagen fibers by filtering the minced mixture through a 70-micrometer strainer.

Spin the eluate at 400g for five minutes. After removing the supernatant, resuspend the cell pellet in 100 microliters of cold extracellular matrix, or ECM, for a single mouse lacrimal gland and 50 microliters of cold ECM for a human biopsy, without creating the bubbles. Seed up to 100 microliters of cells per well of a 12-well suspension plate using a P200 to make approximately 20-microliter droplets in the well.

Place the plate upside down in a humidified incubator at 37 degrees Celsius for 20 to 30 minutes to allow ECM solidification. Once the ECM is solidified, add approximately one milliliter of room temperature mouse expansion medium per well of a 12-well plate. Freshly prepare one milliliter of human differentiation medium containing individual components that induce secretion by the lacrimal gland organoids.

At an automated brightfield time-lapse microscope, set up the positions to be imaged in the plate, the time interval of five minutes, and the duration of four hours. Ensure an entire ECM droplet is visible at each position. Before starting to image, remove the culture medium from the wells to be imaged without moving the plate from the microscope, and replace it with the freshly prepared, well-resuspended human differentiation medium containing compounds, including forskolin and noradrenaline.

As a negative control, include a well with refreshed differentiation medium. Click the run button on the microscope, and analyze the results after four hours. After dissociating the organoids as mentioned in the text, discard the supernatant.

Resuspend the cells in 80 microliters of the electroporation buffer. In a 1.5-milliliter tube, prepare the plasmids required for the experiment. Add the plasmids to the cells, and mix well by pipetting up and down.

Set up the electroporator with the parameters for poring pulse and transfer pulse. Place the cells with the plasmids in an electroporation cuvette. Immediately measure the resistance, which should be between 0.3 amperes and 0.55 amperes, and electroporate immediately.

Once done, transfer the cells to a 1.5-milliliter tube, and add 400 microliters of electroporation buffer supplemented with a Rho-kinase inhibitor. Allow the cells to recover at room temperature for 30 minutes. Next, pellet the cells at 500g for five minutes.

After discarding the supernatant, plate the cells in 200 microliters of ECM. After solidification, add the mouse expansion medium. For organoid preparation, split the human lacrimal gland organoids approximately three days before the transplantation day.

To extract the organoids from the ECM, add dispase to the culture medium to reach a final concentration of 0.125 units per milliliter, and thoroughly resuspend the ECM droplets to disrupt them using a P1000. Place the plate back in the 37-degree Celsius incubator for 30 minutes. Resuspend the organoids in 10 milliliters of base medium to wash out the enzyme.

After pelleting the cells at 400g for five minutes, resuspend them in 50 microliters of cold human expansion medium supplemented with 5%ECM. Place the organoid suspension on ice, and proceed immediately to the transplantation. For orthotopic transplantation in mice, aspirate the organoid suspension in an insulin needle.

When the mouse is asleep, quickly place it on its side with the main lacrimal gland accessible. Inject five microliters of the organoid suspension directly through the skin into the lacrimal gland. Allow the mouse to recover, and assess the presence of any transplantation-related discomfort daily, especially in the eye.

Following the dissection of the mouse lacrimal gland, the enzymatic and mechanical digestion generated small tissue fragments, including the acini and the ducts. In the successful mouse lacrimal gland organoid derivation, cystic organoids of approximately 500-micrometer diameter were found after seven days. Human lacrimal gland organoids grew as cysts within three to four days and reached a full-grown size in 10 to 14 days after tissue isolation.

After five days and seven days, respectively, in the differentiation medium, the mouse and human lacrimal gland organoids became denser. Applying the cyclic AMP activator forskolin or the neurotransmitter norepinephrine resulted in organoid swelling within less than three hours. In successful electroporation, organoids resistant to hygromycin grew out.

The growing organoid clones larger than 300 micrometers were picked before their differentiation. PCR amplification of the Pax6 locus targeted by guide RNA yielded a 367 base pair band for all the selected clones. One homozygous knockout mouse lacrimal gland clone out of six sequenced was obtained using the guide RNA targeting Pax6.

Some clones grew out well, but some organoid clones were lost after picking or began to differentiate. Out of the 10 organoid clones picked, seven grew out well. Organoid engraftment was confirmed one month after injecting the organoids into the mouse lacrimal gland by staining for a human-specific marker, the human nucleolar antigen.

One thing to pay attention to is not to over-digest the tissue, nor the organoids. Otherwise, the cells might die, and this would reduce the organoid establishment rate. Lacrimal gland organoids can be analyzed by histology.

By doing so, one can get insight in their cellular composition.