In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye
March 29th, 2024
Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.
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.
Overview
This study presents a noninvasive imaging platform for liver spheroids engrafted in the anterior chamber of the mouse eye. The approach allows for longitudinal monitoring of liver function at single-cell resolution.
Key Study Components
Area of Science
- Neuroscience
- Biomedical Imaging
- Liver Physiology
Background
- Current liver research lacks non-invasive imaging techniques.
- In vivo imaging is crucial for understanding liver physiology and pathology.
- Existing methods do not allow for repeated imaging of liver cells over time.
- The anterior chamber of the mouse eye serves as a novel transplantation site.
Purpose of Study
- To develop a platform for non-invasive imaging of liver spheroids.
- To monitor liver function longitudinally in a single animal.
- To reduce the number of experimental animals used in research.
Methods Used
- Preparation of liver spheroids from primary liver cells.
- Transplantation of spheroids into the mouse eye.
- In vivo imaging using confocal microscopy.
- Monitoring of liver spheroids for several months post-transplantation.
Main Results
- Successful engraftment of liver spheroids in the mouse eye.
- Longitudinal imaging revealed cellular processes and blood vessel presence.
- Demonstrated the potential for repeated imaging sessions.
- Enhanced understanding of liver function in response to various challenges.
Conclusions
- The developed platform is a powerful tool for liver research.
- It allows for non-invasive, high-resolution imaging of liver cells.
- The technique can potentially be adapted for other organs.
Frequently Asked Questions
Executive Industry Relevance
Noninvasive, longitudinal imaging of liver spheroids engrafted in the mouse eye enables repeated, high-resolution monitoring of liver cell function and pathology in vivo. This platform addresses a critical gap in discovery-stage liver research by allowing dynamic, single-cell analysis over time within the same animal. The approach supports predictive confidence in disease modeling and functional target validation, enhancing translational continuity and portfolio decision-making.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct visualization of liver cell responses to stimuli at single-cell resolution over time.
- Supports mechanistic de-risking by allowing repeated interrogation of cellular processes in a living system.
- Facilitates functional target validation through dynamic, longitudinal observation of engrafted tissue.
Screening & Assay Development
- Provides a validated, reproducible in vivo system for quantitative imaging-based assays.
- Enables standardization of imaging protocols for consistent, cross-study data generation.
- Supports screening of pharmacological or genetic interventions with real-time, noninvasive readouts.
Translational & Preclinical Research
- Aligns with disease-relevant modeling by enabling chronic monitoring of liver function and pathology.
- Facilitates translational biomarker discovery through repeated, quantitative imaging in the same animal.
- Improves risk-adjusted advancement by reducing animal use and increasing data quality per subject.
Pipeline & Workflow Integration
This imaging platform bridges early discovery and preclinical research by enabling hypothesis testing, pathway analysis, and functional validation in a longitudinal, in vivo context.
- Discovery Biology: Supports hypothesis-driven interrogation of liver cell behavior and response to interventions.
- Screening: Delivers reproducible, quantitative imaging outputs suitable for compound or genetic screening.
- Analytics: Provides high-content, single-cell data for robust statistical comparison across conditions and timepoints.
- Translational Research: Maintains continuity from discovery through preclinical validation by modeling chronic liver processes.
- Enterprise Reuse: Offers a reusable, adaptable platform for diverse liver and potentially other organ studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in liver research.
- Operational Value: Enhances standardization, reproducibility, and scalability of in vivo imaging workflows.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by maximizing data from each animal.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of liver-targeted programs.
Implementation Considerations
- Requires expertise in microsurgery, confocal imaging, and animal handling.
- Needs access to confocal microscopy and appropriate fluorescent probes.
- Demands cross-team standardization of imaging and analysis protocols.
- Adaptation to other organoid or tissue types may require protocol optimization.
- Potential limitations include model-specific physiological differences and imaging depth constraints.
