The monitoring of liver function in mammals during health and disease is limited by the lack of high-resolution, noninvasive in vivo imaging techniques. The visualization of this organ is hindered by its inaccessible location, and in order to piece together cellular processes, in vivo studies rely on the sacrifice of animals at different time points. To circumvent this imaging limitation, much work relies on in vitro models, in which liver-like microtissues are visualized and studied in a controlled environment.

In recent years, the development of three-dimensional culture systems, such as liver spheroids, has aided and advanced liver research. Liver spheroids are multicellular aggregates that mimic the microenvironment and complex cell-cell interactions of liver tissue to a certain extent¹ and offer clear advantages over traditional monolayer cultures²^,³. Liver spheroids are also used as models for different liver diseases⁴^,⁵^,⁶ and have been instrumental to understanding disease mechanisms. Still, key limitations of the current in vitro liver models are the lack of a physiological in vivo milieu and the limited utilization time in culture (around 20 days)³. Liver spheroids have been previously transplanted to different sites in vivo, such as under the kidney capsule⁷ or intraperitoneally⁸, which are not accessible for optical imaging. Intravital liver imaging is a state-of-the-art technique offering cell-resolution real-time imaging. Currently, this in situ liver imaging is only possible on the exteriorized organ, which is highly invasive and often terminal⁹. Although the fitting of an abdominal window would allow repeated liver imaging sessions, it entails complex surgery and after-care.

To perform longitudinal monitoring at cellular resolution, we explored transplantation of liver spheroids into the anterior chamber of the eye (ACE) of mice, where the liver-like tissue is engrafted in a physiological milieu, connected to the body stimuli, and accessible for optical imaging. The cornea is a transparent tissue and acts as a window through which microtissues engrafted on the iris can be imaged non-invasively and longitudinally by confocal microscopy. Here, we present a workflow of this newly developed platform for in vivo imaging of liver spheroids¹⁰. This protocol is a step-by-step guide for its implementation, divided into (1) the extraction of primary mouse liver cells and in vitro formation of liver spheroids, (2) the transplantation of liver spheroids into the ACE of recipient mice, and (3) the in vivo imaging of engrafted liver spheroids in anesthetized mice. Furthermore, we will showcase some of the possibilities and applications of this imaging platform.

In Vivo Fluorescence Imaging of Engrafted Liver Spheroids in Murine ACE