in vivo fluorescence imaging of engrafted liver spheroids in murine ace

Imagem intraocular in vivo não invasiva de esferoides hepáticos enxertados em camundongos

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&#160;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.

Autor em destaque: Aproveitando as câmaras oculares do camundongo para estudos não invasivos de esferóides hepáticos

Imagens in vivo de esferoides hepáticos enxertados na câmara anterior do olho do camundongo

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.

Imagem de fluorescência in vivo de esferoides hepáticos enxertados na ECA murina 

Para começar, anestesie o camundongo com os esferóides hepáticos transplantados para a ECA e imobilize a cabeça com os parafusos. Para evitar o ressecamento, coloque uma gota de gel lacrimal artificial em ambos os olhos, injete as sondas fluorescentes por via intravenosa através da veia da cauda e prossiga para a imagem imediatamente. Depois de inclinar a cabeça, expulse suavemente o olho do encaixe e prenda-o sob a objetiva de imagem.Para preencher o espaço entre a córnea e a objetiva, aplique uma quantidade generosa de gel lacrimal artificial e concentre-se nos esferóides do fígado através da ocular. Usando a objetiva 25x, adquira as imagens in vivo. Por fim, aplique pomada de vaselina no olho fotografado antes de acordar o animal.Imagens intraoculares in vivo não invasivas de esferoides hepáticos engraft indicaram a presença de vasos sanguíneos, rede de canalículos biliares e capacidade de captação de LDL. Os esferoides hepáticos que expressam o biossensor indicador do ciclo celular da ubiquitina fluorescente foram monitorados longitudinalmente com resolução de célula única.

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Research

JoVE Journal

Biology

In Vivo Fluorescence Imaging of Engrafted Liver Spheroids in Murine ACE

201 visualizações.  Karolinska University Hospital. Para começar, anestesie o camundongo com os esferóides hepáticos transplantados para a ECA e imobilize a cabeça com os parafusos. Para evitar o ressecamento, coloque uma gota de gel lacrimal artificial em ambos os olhos, injete as sondas fluorescentes por via intravenosa através da veia da cauda e prossiga para a imagem imediatamente. Depois de inclinar a cabeça, expulse suavemente o olho do encaixe e prenda-o sob a objetiva de imagem.Para preencher o espaço entre a córnea e a ...

Vídeo: Imagem de fluorescência in vivo de esferoides hepáticos enxertados na ECA murina 

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

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

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.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...