In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

In our group, we use the anterior chamber of the mouse eye as a transplantation site for in vivo and non-invasive imaging of liver steroids. This newly established imaging platform will provide a unique tool for researchers and expand our understanding of liver physiology and pathology in both basic and preclinical research. In biomedical research, we rely on the visualization of cellular processes to understand disease mechanisms.

And the field of liver research currently lacks methods for in vivo high resolution imaging in which liver cells can be monitored longitudinally over time. Compared to intravital liver imaging, our technique is non-invasive and the liver cells can be imaged repeatedly over numerous imaging sessions, allowing us to monitor liver function at single cell resolution in response to different challenges. With our platform, the liver steroids survive in the eye for many months, and the same recipient mouse can be imaged noninvasively over time.

This allows a reduction in the number of experimental animals we use while increasing the quality of the in vivo data. Over the past decade, our group has developed the eye imaging platform to be a powerful research tool. It was initially designed for the imaging of pancreatic eyelets, but has now been adapted to liver steroids and has the potential to be expanded to other organs and research areas.

To begin, gather all the required resources and clean the Hamilton syringe tubing and canula with 70%ethanol followed by saline. After filling them with saline, tape the Hamilton syringe to the bench horizontally. Next, prepare the anesthesia isoflurane unit and warm the heating pad to 37 degrees Celsius.

Cover the tips of the forceps attached to the solid universal joint with a piece of polyethylene tubing to form a loop. Using a 200 microliter pipette and tip, transfer the liver's PHE from the 96 well plate into a 35 millimeter cell suspension dish containing maintenance media. After anesthetizing the mouse, transfer it onto the heating pad and immobilize the head with the screws.

Then gently pop the eye out of the socket and secure it with the forceps. To prevent drying, place a drop of saline on both eyes. With the Hamilton syringe, aspirate the liver spheroids under the stereoscope.

Place the canula horizontally on a clean surface. Using a 23 gauge needle, carefully puncture the cornea and slide the needle sideways to widen the incision. Dry the seeping aqueous humor with a tissue and add saline drops to the eye to keep it moist.

Position the canula vertically to let the liver spheroids gravitate towards the tip And then gently insert it into the hole using the Hamilton syringe, slowly expuls the liver spheroids into the ACE. Using the tip of the canula, gently prod the cornea from the outside to position the liver steroid around the pupil. After five to 10 minutes, release the eye from the forceps.

To lubricate and heal the cornea, apply Vaseline eye ointment to the operated eye. Before awakening the mouse, administer the analgesic subcutaneously to avoid postoperative discomfort. To begin, anesthetize the mouse with the liver spheroids transplanted into the ACE and immobilized the head with the screws.

To prevent drying, place a drop of artificial tear gel on both the eyes. Inject the fluorescent probes intravenously through the tail vein and proceed for imaging immediately. After tilting the head, gently extrude the eye from the socket and clamp it under the imaging objective.

To fill the space between the cornea and the objective, apply a generous amount of artificial tear gel and focus on the liver spheroids through the eyepiece. Using 25x objective, acquire the in vivo images. Finally, apply Vaseline eye ointment to the imaged eye before awakening the animal.

Non-invasive intraocular in vivo imaging of engraft liver spheroids indicated the presence of blood vessels. Isle network and LDL uptake capacity. Liver spheroids expressing the fluorescent ubiquitin cell cycle indicator biosensor were monitored longitudinally at single cell resolution.