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 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.
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.
All procedures performed on animals were approved by the Animal Experiment Ethics Committee at Karolinska Institutet.
1. Extraction of primary mouse liver cells and generation of liver spheroids in vitro
2. Transplantation of liver spheroids into the anterior chamber of the eye (ACE)
3. In vivo imaging of engrafted liver spheroids in the ACE
Primary liver cells, enriched for hepatocytes, were isolated from the mouse liver by two-step collagenase perfusion, using a peristaltic pump to circulate warm buffers through the liver taking advantage of the organ's vasculature to deliver dissociation enzymes to all cells (Figure 1A). For this, the inferior vena cava was cannulated, and the portal vein was snipped to allow the flow-through of buffers (Figure 1B). First, an HBSS-based buffer was flushed through the liver to clear the blood. If the cannulation is successful and there are no blood clots, the liver blanches and becomes yellow within a few seconds. Secondly, a Digestion buffer containing the Liberase enzyme blend was circulated through the liver to dissociate the tissue into a single-cell suspension. The cells were manually counted and seeded into 96-well ultra-low-adherence (ULA) plates, which enable the self-assembly into spheroids within a few days. On day 5, the spheroids are formed, and the thin capsule bordering the spheroids indicates successful aggregation (Figure 1C). We wait until day 10 to transplant, at which point the spheroids are compact and have developed strong cell-cell connections. The number of seeded cells per well determined the size of the liver spheroid, with 1000, 1200, and 1500 cells/well yielding spheroids of 238 µm ± 10 µm, 248 µm ± 17 µm, and 298 µm ± 19 µm (mean ± SD) diameters, respectively (Figure 1C,D). For transplantation, we select spheroids of approximately 250 µm diameter for the following reasons: (1) the spheroids size should not be too large to avoid hypoxia and necrotic core, but should contain enough cells to support cell-cell communications and to allow graft remodeling in the eye, (2) the weight of spheroids of this size allows them to gravitate towards the iris and improve their engraftment, (3) this size is appropriate in relation to transplanting 5-10 spheroids per mouse eye.
The transplantation surgery requires a manual-threaded syringe connected to a glass cannula (Figure 2A). The glass cannula consists of a borosilicate glass capillary modified in-house to have a fine blunt tip using a micropipette puller and beveler. A simpler alternative cannula can be created using a commercially available plastic catheter connected to the syringe tubing and stabilized in a pipette tip (Figure 2B). The surgery consists of the inoculation of liver spheroids into the ACE through an incision in the cornea (Figure 2C). The spheroids were positioned on the borders of the pupil to make them better accessible for imaging and avoid them moving into the ocular angle. Albino mice were used for transplantation, as their non-pigmented iris allow the in vivo imaging of the engrafted liver spheroids. Recipient mice were transplanted into both eyes with 7-10 spheroids/eye, and stereoscopic images were taken at 3 days post-transplantation (post-Tx) as well as at 1 week and 1 month post-Tx to document the cornea healing and spheroid engraftment success (Figure 2D). Of note, the change in appearance of the liver spheroids in the ACE between when freshly transplanted and fully engrafted is due to the settlement of the graft onto the iris, as well as to the growth of a monolayer of iris cells over the spheroid. The engraftment success rate of liver spheroids in the ACE is 70% (n = 9 eyes in both male and female mice) (Figure 2E). The first days post-Tx are the most critical for survival and engraftment, likely due to the recipient animal rubbing its eyes and dislodging the spheroids before the cornea has healed. The size of the liver spheroids does not differ significantly post-Tx and changes to shape are attributed to graft remodeling and engraftment (Figure 2F). At 1 month post-Tx, all engrafted spheroids present on the iris were vascularized and innervated, as shown by immunofluorescence staining (Figure 2G).
Noninvasive in vivo imaging is performed on anesthetized recipient mice using an upright confocal microscope and long-distance dipping objective (Figure 3A, Table 2). The fluorescence imaging in the ACE can be achieved through different approaches, as depicted in Figure 3B. The injection of fluorescent probes into the circulation of the recipient mouse allows the visualization of different cell types and structures within the spheroids. We used lectin to mark blood vessels (Figure 3C), CMFDA to observe the bile canaliculi network (Figure 3D) and pHrodo-LDL, which confirmed active LDL-uptake into spheroid cells (Figure 3E). Liver spheroids generated from reporter mouse models can also be used. Albumin-Cre:tdTomato spheroids allowed labeling and tracking of hepatocytes (Figure 3F), and spheroids expressing the Fluorescent Ubiquitin Cell Cycle Indicator (FUCCI) biosensor were used to visualize cell cycle dynamics at single-cell resolution (Figure 3G). Finally, liver spheroids can be genetically modified in vitro prior to transplantation, and, in the case of adeno-associated virus (AAV)-GFP transduction, the expression was observed in vivo for over 6 months (Figure 3H).
Figure 1: Isolation of primary mouse hepatocytes and generation of liver spheroids. (A) Material and equipment used for isolation of primary mouse hepatocytes: 1. Isolation buffers; 2. Water bath; 3. Peristaltic pump; 4. Petri dish; 5. Cell strainer; 6. Absorbent pad; 7. Dissection mat; 8. Cell Lifter; 9. Butterfly needle 27 G; 10. Dissection tools. (B) Abdominal cavity during surgery: the vena cava is cannulated and perfused, and the portal vein is snipped to allow flow-through of the buffers. (C) Brightfield images of the formation of hepatic spheroids in vitro at 0 (d0), 5 (d5), and 10 (d10) days post-seeding, scale bars = 200 µm. (D) Liver spheroid size at different cell-seeding concentrations, n = 21 spheroids. Please click here to view a larger version of this figure.
Figure 2: Transplantation and engraftment of liver spheroids into the ACE of mice. (A) Materials and equipment used for transplantation (Tx) of liver spheroids into the ACE: 1. Liver spheroids in culture dish; 2. Sterile saline; 3. Eye ointment; 4. Needles 23 G; 5. Cannula; 6. Hamilton syringe; 7. Anesthesia gas tube; 8. Head-holder and gas mask; 9. Heating pad; 10. Custom-made metal base plate; 11. Forceps and solid universal joint. (B) Cannula and Hamilton syringe setup: 1. Glass cannula connected to the Hamilton syringe via Portex tubing and a 27G needle; 2. Glass cannula is connected to the Portex tubing via additional segments of Silicone tubing and PharMed tubing; 3. Alternative assembled plastic cannula; 4. Parts forming the plastic cannula: 24G BD Insyte plastic catheter connected via PharMed tubing and sheathed in a cut-off 10 µl pipette tip for stability and grip. (C) Illustration of Tx surgery steps: 1. The spheroids are collected into the cannula; 2. The cornea is punctured with a needle; 3. The cannula is inserted into the incision, and the spheroids are released into the ACE; 4. From the outside of the eye, the spheroids are positioned close to the pupil and away from the incision. (D) Stereoscopic images of liver spheroids (sph) in the mouse eye on the day of surgery and at 3-, 7-, and 30-days post-Tx. Arrows indicate viable spheroids. (E) Liver spheroid (size of 1200 cells/well) engraftment rate post-Tx, n= 9 eyes in 6 recipient mice. (F) Size of liver spheroids in culture, prior to transplantation (in vitro, n= 20 spheroids from single preparation) and at 1-month post-Tx in the ACE (in vivo, n= 16 spheroids in 3 recipient mice), calculated by averaging vertical and horizontal diameters. (G) Immunofluorescence staining of engrafted liver spheroids at 2 months post-Tx, showing vascularization (CD31, pink, dashed line delineates the spheroid mass) and sympathetic innervation (tyrosine hydroxylase (TH), orange), scale bar = 100 µm. Data for panel F has been adapted with permission from Lazzeri-Barcelo et al.10. Please click here to view a larger version of this figure.
Figure 3: Noninvasive intraocular in vivo imaging of engrafted liver spheroids. (A) Material and equipment used for in vivo ACE imaging: 1. Upright laser scanning confocal microscope; 2. Dark box; 3. Motorized XYZ stage; 4. Dipping-objective; 5. Head-holder and gas mask; 6. Forceps and solid universal joint; 7. Heating pad; 8. Custom-made metal base plate. (B) Diagram depicting different approaches used for in vivo imaging of fluorescent readouts in liver spheroids engrafted in the eye. (C-H) Representative images of ACE-liver spheroids during in vivo imaging by confocal microscopy. The backscatter signal is used to observe the spheroid volume and structure; (C) Blood vessels labeled by i.v. injection of fluorescent lectin, scale bar = 100 µm; (D) Bile canaliculi network labeled by injection of fluorescent CMFDA, scale bar = 50 µm; (E) LDL uptake by injection of fluorescent pHrodo-LDL probe, scale bar= 100 µm; (F) Td-Tomato-expressing hepatocytes, arrowheads indicate nuclei and asterisks indicate intra-spheroid vasculature, scale bar = 50 µm; (G) Monitoring of cell cycle dynamics in FUCCI-expressing liver spheroids, scale bars = 50 µm (main image) and 20 µm (blow-up). (H) liver spheroids transduced in vitro with AAV8-GFP prior to Tx and imaged in the eye at 6 months post-Tx, scale bar = 50 µm. The image in panel G has been adapted with permission from Lazzeri-Barcelo et al.10. Please click here to view a larger version of this figure.
Table 1: Solutions used for the isolation of primary mouse hepatocytes. Composition of solutions and buffers needed for mouse hepatocyte isolation. The Digestion buffer and Gradient solution components should be mixed fresh on the day of isolation. Please click here to download this Table.
Table 2. Confocal Leica SP5 microscope settings used for intraocular in vivo imaging of liver spheroids. The table has been adapted with permission from Lazzeri et al.10. Please click here to download this Table.
This protocol describes a novel platform for intraocular in vivo imaging of liver spheroids engrafted in the ACE. The ACE has previously been used as a transplantation site of other organ-derived microtissues, such as pancreatic islets11,12, due to its unique engraftment microenvironment, being rich in vessels, nerves, and oxygen, and the access to imaging through the cornea. While intravital liver imaging enables the visualization of cells and processes in situ, longitudinal monitoring is not possible. Liver imaging through an abdominal window entails complex surgery, and the movement of the organ within the body makes single-cell tracking over time difficult. Hence, this novel imaging method enables noninvasive, longitudinal monitoring of liver cells at single-cell resolution.
This protocol is divided into three parts. First is the isolation of primary hepatocytes via two-step collagenase perfusion, adapted from Charni-Natan et al.13, with the difference that we perform the liver perfusion on the dead mouse instead of the anesthetized live animals. This variation brings certain advantages, such as fewer ethical considerations and the avoidance of anesthesia residue in the organism. In this work, we generate liver spheroids from the hepatocyte-enriched fraction of the isolation, but this does not exclude the potential to isolate other nonparenchymal cell populations using other specialized protocols to make coculture spheroids of diverse composition14,15.
The second part of this protocol involves the transplantation of the liver spheroids into the ACE of recipient mice. This is a quick (under 10 min) and simple surgery performed in anesthetized mice and does not require any post-operative treatment. The cornea puncture self-seals and heals over 3-5 days. Occasionally, during the healing process, some hazing is observed around the incision, but this clears within a few days. We have not experienced cases of anterior synechia in the eyes of operated animals. We perform the transplantation procedures in a clean but open-air laboratory and without issues with infections in the operated eyes. The inoculation and engraftment of spheroids in the eye do not compromise the vision or alter the behavior of the recipient animal. In this protocol, we use isoflurane anesthesia for both transplantation surgery and in vivo imaging, which is well tolerated in mice. Due to its dose-dependent effect, it can be easily adjusted throughout the procedures and brings the advantage of reducing sleeping and awakening times. However, alternative injectable anesthetics can be used. After transplantation, we generally allow 1 month for the spheroids to fully engraft, become vascularized, and innervated, before performing treatment interventions and in vivo imaging. We have also shown that transplantation and engraftment is possible using human liver spheroids and immunocompromised recipient mice10.
The third part of this method is the in vivo imaging of the engrafted liver spheroids in the ACE. This protocol describes the in vivo imaging setup, which uses microscopy equipment commonly found in research imaging facilities. Moreover, the specialized materials, such as the mouse head-holder and the plastic cannula, are now commercially available. With this imaging setup, we are able to capture z-sections and obtain a three-dimensional reconstruction of the spheroid architecture, depending on the depth of laser penetration and fluorescence detection. Monitoring cellular function in the engrafted liver spheroids relies on the visualization of fluorescent proteins that report on cell types, cellular functions, and dynamics. Thus, this imaging platform can be exploited using different modalities, alone or in combination: (1) Fluorescent probes can be administered intravenously, e.g., antibodies to label and track cells as well as functional dyes; (2) Liver spheroids can be generated from cells isolated from reporter mouse models that express liver-specific fluorescent proteins, e.g., FUCCI liver spheroids that report on cell cycle dynamics; (3) The formation of liver spheroids in vitro can be combined with transfection or transduction, to equip the spheroids with fluorescent proteins and biosensors. e.g., adeno-associated viruses. In our experimental settings and by using a single photon for excitation, the imaging depth that is possible to achieve is roughly 60-100 µm. However, this is dependent on the laser power and multiphoton imaging availability, fluorescence probe emission characteristics and sensitivity of the detectors, as well as the angle of the eye in which the spheroid is engrafted. After imaging acquisition, the downstream image analysis can be performed using popular programs such as Image J and Imaris. For example, in the case of the FUCCI reporter, cell-cycle active cells in green can be counted and contrasted to the number of total red cells to assess cell-cycle activity within the engrafted spheroid. Additionally, the ACE-imaging platform allows substances to be applied to the eye (in the form of eye drops) or injected directly into the ACE to treat the graft and monitor its reaction. Post-mortem, the transplanted spheroids can easily be retrieved by manual microdissection and can provide valuable information by ex vivo techniques, such as immunofluorescence staining, transcriptomic analysis, etc.10.
This technique has certain limitations. The first is that from our experience, the recipient mice must be albino, i.e., have non-pigmented iris. Upon engraftment, the liver spheroids become covered by a monolayer of iris cells, which does not affect the viability or function of the spheroids, but the pigment in the iris cells prevents imaging. A second consideration is the stability during intraocular imaging in anesthetized mice. During the in vivo imaging sessions, the anesthesia concentration and breathing of the animal must be closely monitored to minimize movement. Nevertheless, using the imaging settings indicated here, we are able to achieve high-resolution imaging at the single-cell level.
To summarize, this protocol describes the implementation of a noninvasive in vivo imaging platform of liver-like tissue engrafted in the eyes of mice. We use easy procedures, common equipment, and affordable materials, making it an attainable approach for many investigators. This model combines the advantages of in vitro 3D-liver spheroids with the in vivo milieu and optical accessibility provided by the ACE to create a valuable platform for studying liver physiology and pathology in basic research and pre-clinical settings.
The authors have nothing to disclose.
This work was supported by the Swedish Diabetes Association, Funds of Karolinska Institutet, The Swedish Research Council, Novo Nordisk Foundation, The Family Erling-Persson Foundation, Strategic Research Program in Diabetes at Karolinska Institutet, The Family Knut and Alice Wallenberg Foundation, The Jonas & Christina af Jochnick Foundation, Swedish Association for Diabetology and ERC-2018-AdG 834860-EYELETS. The figure drawings were created by FL-B using BioRender.com.
27 G butterfly needle | Venofix | 4056388 | |
AAV8-CAG-GFP | Charles River | CV17169-AV9 | Incubated with isolated hepatocytes at 1 µL/mL during liver spheroid formation |
Absolute and 70% ethanol | N/A | N/A | |
Absorbent pad | Attends | 203903 | |
Albumin-Cre;RCL-tdTomato (B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J ; B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) | Jackson | #003574 and #007914 | Mice obtained from in-house breeding |
B6 albino mice (B6(Cg)-Tyrc-2J/J) | Jackson | #000058 | Mice obtained from in-house breeding |
B6;129P2-Gt(ROSA)26Sor[tm1(CAG-Venus/GMNN,-Cherry/CDT1)Jkn]/JknH | INFRAFRONTIER/EMMA | EM:08395 | Mice obtained from in-house breeding |
BD Insyte IV Catheter 24 G x 0.75 in | BD Medical | 381212 | |
Borosillicate standard glass cappilaries | World Precision Instruments | 1B150-4 | |
Cell lifter | Corning | 3008 | |
Cell strainer, 70 µm | Falcon | 352350 | |
Custom-made metal plate | Hardware store | N/A | |
Dexamethasone | Sigma-Aldrich | D4902 | |
Dual-Stage Glass Micropipette Puller | Narshige | Model PC-100 | |
EDTA | Sigma-Aldrich | E9884 | |
Electric heating pad | Hardware store | N/A | |
FBS | Gibco | N/A | |
GlutaMAX | Gibco | 35050061 | |
Green CMFDA | Abcam | ab145459 | Reconstituted in DMSO, administered at 100 µg/mouse in PBS 10% FBS |
Hamilton syringe | Hamilton | 81242 | Model 1750 Luer Tip Threaded Plunger Syringe, 500 µL |
HBSS; no calcium, no magnesium and no phenol red | Gibco | 14175095 | |
HCX IRAPO L 25x/0.95 W objective | Leica | N/A | |
HEPES | Gibco | 15630080 | |
Induction chamber 0.8 L | Univentor | 8329001 | |
Insulin-Transferrin-Selenium (ITS-G) | Gibco | 41400045 | |
Isoflurane | Baxter | N/A | |
Lectin DyLight-649 | Invitrogen | L32472 | Administered at 1 mg/mL and 100 µL/mouse |
Liberase TM Research Grade | Sigma-Aldrich | 5401127001 | |
Microelectrode beveler | World Precision Instruments | Model BV-10 | |
Mouse head-holder and gas mask | Narshige | Model SGM-4 | |
Nunclon Sphera 96-Well, U-Shaped-Bottom Microplate | Thermo Fisher | 174929 | |
Oculentum simplex | APL | N/A | |
PBS 10x | Gibco | 14080055 | |
PBS 1x; no calcium, no magnesium | Gibco | 14190144 | |
Penicillin-Streptomycin | Gibco | 15140122 | |
Percoll | Sigma-Aldrich | P1644 | |
Peristaltic pump | Ismatec | Model ISM795 | |
PharMed BPT Pump Tubing | VWR | VERN070540-07 | Inner diameter 0.76 mm, outer diameter 2.46 mm |
pHrodo Red-LDL | Invitrogen | L34356 | Administered at 1 mg/mL and 100 µL/mouse |
Portex Fine Bore Polyethylene Tubing | Smiths Medical | 800/100/140 | Inner diameter 0.4 mm, outer diameter 0.8 mm |
Silicone dissection mat | Hardware store | N/A | |
Sodium chloride 0.9% | Braun | N/A | |
Solid Universal Joint | Narshige | Model UST-2 | |
Stereomicroscope | Leica | Model M80 | |
Suspension culture dish 35 mm | Sarstedt | 833900500 | |
Temgesic | Indivor | N/A | Administered s.c. at 0.05 mg/mL and 2 µL/g mouse |
Translucent Silicone Tubing | VWR | 228-1450 | Inner diameter 1.5 mm, outer diameter 3 mm |
Trypan Blue | Sigma-Aldrich | T8154 | |
Univentor 400 Anesthesia unit | Univentor | 8323001 | |
Upright laser scanning confocal microscope | Leica | Model TCS SP5 II | |
Viscotears | Novartis | N/A | |
William's E Medium; no glutamine, phenol red | Gibco | 22551089 |