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 &#38; 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.

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&#160;&#160;in vitro
<ol>
	<li>Preparation
	<ol>
		<li>For cannulation, liver resection, and isolation of primary liver cells, prepare the following sterile or single-use materials in addition to serological pipettes and centrifuge tubes (Figure 1A): peristaltic pump, swing-bucket centrifuge, absorbent pad, dissection mat, two curved-tip forceps, surgical scissors, one 27 G butterfly needle, one 70 &#181;m cell strainer, one cell lifter, one 100 mm Petri dish, cell-counting chamber, and 96-Well, U-shaped-bottom microplates.</li>
		<li>Prepare the solutions used in the liver perfusion, isolation of primary liver cells, and generation of liver spheroids as listed in Table 1. 
		NOTE: The Digestion buffer and the Gradient solution should be prepared fresh.</li>
		<li>Set up the perfusion system consisting of a peristaltic pump, which conducts solutions from the 42 &#176;C water bath to the liver (Figure 1A). The higher temperature of the water bath ensures that the buffers reach the liver at the optimal temperature of 37 &#176;C. Customize this depending on the tube length and the room temperature (RT).</li>
		<li>For the cannulation, fit a 27 G butterfly needle to the end of the tube. Keep the plating media used in the final isolation steps at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Pre-heat the following solutions in the 42 &#176;C water bath in 50 mL centrifuge tubes: 40 mL of PBS, 20 mL of perfusion buffer, and 12 mL of digestion buffer.</li>
		<li>To clean and warm the pump tubes, circulate approximately 20 mL of the pre-heated PBS.</li>
		<li>Change the tubing to the perfusion buffer, prime the tube and butterfly needle, and set the flow rate to 4 mL/min. Ensure no bubbles in the tubing during buffer changes throughout the protocol.</li>
		<li>Euthanize the mouse by cervical dislocation and use needles to fix the limbs to the dissecting board.</li>
		<li>Wet the abdomen fur with 70% ethanol and dissect it to access the digestive organs.</li>
		<li>Move the intestines to the right to expose the portal vein and inferior vena cava (Figure 1B).</li>
		<li>Cannulate the vena cava at approximately halfway of its length with the needle in a horizontal position, ensure it is stable, and start the pump.</li>
		<li>When the liver starts to inflate, or white dots appear in the closer lobes, cut the portal vein to allow the blood and perfusion buffer to drain out.</li>
		<li>The liver should start blanching immediately. Encourage clearing using curved forceps to clamp the portal vein at 5 s intervals.</li>
		<li>Repeat step 1.2.9 until the liver is yellow and cleared of blood (approx. 15-20 mL of perfusion buffer).</li>
		<li>Stop the peristaltic pump to change the tubing to the digestion buffer and restart the pump. When the digestion buffer reaches the liver, reduce the flow rate to 2.5 mL/min. 
		NOTE: The phenol red in the digestion buffer allows to discriminate its arrival to the liver and enables the adjustment of pump parameters during the treatment.</li>
		<li>To encourage the buffer to reach all hepatic lobes and ensure proper digestion, repeat step 1.2.9 several times.</li>
		<li>Stop the flow of the peristaltic pump when the digestion buffer is depleted or the liver looks sufficiently digested. 
		NOTE: The degree of digestion can be visually monitored by gently pinching the liver lobes with forceps and checking if small marks appear on the tissue. The liver will also become flimsy.</li>
		<li>To extract the liver, cut the hepatic ligaments and connections within the abdominal cavity, aiming to remove it entirely, and place it in the Petri dish containing 10 mL of cold plating medium (Table 1).</li>
		<li>After removing the gallbladder, make small pinches on the lobes using forceps, lightly tearing the hepatic capsule. By shaking the liver in the dish, observe cells pouring out into the medium.</li>
		<li>Holding the liver steady with forceps, gently drag the cell lifter along the lobes to release the cells. 
		NOTE: Correct interlobular digestion will lead to cell suspension in the media, not tissue fragments.</li>
		<li>Using a serological pipette, collect the cell suspension from the Petri dish and filter through the 70 &#181;m cell strainer placed on a 50 mL centrifuge tube. Use fresh plating media to wash the dish of digested liver cells and transfer them to the filter.</li>
		<li>Centrifuge at 50 x g for 5 min at 4 &#176;C to pellet the cells.</li>
		<li>Remove the supernatant, leaving approximately 1 mL to cover the cell pellet, swirl the tube to resuspend the cells, and then gradually add 10 mL of cold plating medium.</li>
		<li>Add the 10 mL of gradient solution to the cell suspension and gently invert the tube 10 times.</li>
		<li>Centrifuge at 200 x g for 10 min at 4 &#176;C.</li>
		<li>The pellet contains viable liver cells enriched for hepatocytes, while the supernatant contains dead cells and debris. Discard the supernatant using a serological pipette, leaving approximately 1 mL, and resuspend the pellet by gentle swirling.</li>
		<li>Add 20 mL of cold plating medium to the cell suspension and centrifuge at 50 x g for 5 min at 4 &#176;C to wash off the gradient solution.</li>
		<li>Remove the supernatant, leaving approximately 1 mL above the pellet, and resuspend the cells in 20 mL of cold plating medium. 
		NOTE: Here, the cell pellet can be compacted, so if needed, use a 10 mL serological pipette to gently dissociate the cells.</li>
		<li>Manually determine the cell number and viability using a cell counting chamber and Trypan Blue. 
		NOTE: The hepatocyte cells will quickly precipitate in the tube; to resuspend them gently invert the tube a few times.</li>
		<li>Seed the liver cells in 200 &#181;L/well of plating medium at 1200 cells/well into 96-well ultra-low adherence plates. 
		NOTE: The optimal volume of media per well is 200 &#181;L; however, it is possible to seed cells in 100 &#181;L/well.</li>
		<li>Spin the plates at 200 x g for 3 min to gather the cells in the center of the wells.</li>
		<li>Incubate (37 &#176;C, 5% CO2) the cells and leave them to form spheroids for 5 days (Figure 1C) naturally.</li>
		<li>On day 5, carefully remove half of the media in the well and replace it with serum-free maintenance media (Table 1). Repeat this step every 48 h until day 10, when the liver spheroids are ready to be transplanted. 
		NOTE: The formation of a capsule-like structure in cultured liver spheroids shows good aggregation and viability.</li>
	</ol>
	</li>
</ol>
2. Transplantation of liver spheroids into the anterior chamber of the eye (ACE)
<ol>
	<li>Preparation
	<ol>
		<li>For transplantation of liver spheroids into the ACE, ensure to arrange the following resources (Figure 2A): stereomicroscope, isoflurane anesthesia unit, induction chamber, isoflurane, heating pad, custom-made metal base plate, mouse head-holder and gas mask, forceps attached to the solid universal joint, Hamilton 500 &#181;L threaded plunger syringe, silicone, polyethylene and pump tubing, custom-made blunt glass cannula or 24 G catheter, ethanol 70%, sterile saline, sterile 23 G needles, eye ointment (liquid paraffin and Vaseline at 1:1 ratio), disposable 1 mL syringe, and cell suspension dish 35 mm.</li>
		<li>Clean the Hamilton syringe, tubing, and cannula by passing 70% ethanol and saline.</li>
		<li>Fill the Hamilton syringe, tubing, and glass cannula with saline and fix the Hamilton syringe to the bench in a horizontal position with tape (Figure 2A).</li>
		<li>Use a custom-made blunt glass cannula.
		<ol>
			<li>Elongate a borosilicate glass capillary using a dual-stage micropipette puller to an inner diameter of &#62;300 &#181;m to allow the aspiration of the spheroids.</li>
			<li>Bevel and blunt the tip using a microelectrode beveler and expose the cannula tip to a flame for a few seconds to soften the edges. 
			NOTE: The microelectrode beveler consists of a rotating sanding stone that is operated manually; therefore, specific settings are non-applicable.</li>
		</ol>
		</li>
		<li>Alternatively, construct a cannula using the plastic portion of a 24 G catheter (Figure 2B).</li>
		<li>Prepare the anesthesia isoflurane unit and warm the heating pad to 37 &#176;C.</li>
		<li>Cover the tips of the forceps attached to the solid universal joint with a piece of polyethylene tubing to form a loop, which helps to stabilize the eye.</li>
		<li>Transfer the liver spheroids from the 96-well plate into a 35 mm cell suspension dish with maintenance media using a pipette and 200 &#181;L tip.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Anesthetize the mouse in the induction chamber using a dose of 2.5% isoflurane and 280 mL/min air.</li>
		<li>When the mouse is unconscious, lower the anesthesia to 1.8% isoflurane and 280 mL/min air, connect the anesthesia tube to the head-holder, and quickly transfer the animal onto the heating pad, positioning the nose inside the head-holder.</li>
		<li>Immobilize the head with the screws, gently pop the eye out of the socket and secure it with the forceps and place a drop of saline on both eyes to prevent drying.</li>
		<li>Under the stereoscope, use the Hamilton syringe to aspirate and collect the liver spheroids into the tip of the cannula and leave them to rest horizontally on a clean surface. 
		NOTE: Aspirating media along with the liver spheroids helps to prevent them from sticking to the cannula walls.</li>
		<li>Carefully puncture the cornea using a 23 G needle and dry the seeping aqueous humor with a tissue. If needed, to make the incision wider, carefully slide the needle sideways to slice the cornea. 
		NOTE: A single-use sterile needle is used to perform the corneal puncture, so the cornea is not disinfected prior to incision.</li>
		<li>Add saline drops to the eye to avoid drying.</li>
		<li>Take the cannula containing the liver spheroids and hold it vertically to allow the spheroids to gravitate toward the tip of the cannula.</li>
		<li>Gently insert the cannula into the hole, and with the bevel directed towards the pupil, use the Hamilton syringe to slowly expulse the liver spheroids into the ACE (Figure 2C). 
		NOTE: Before removing the cannula, waiting a few seconds for the liquid pressures inside and outside the eye to readjust and avoid the spheroids escaping back out of the eye is recommended.</li>
		<li>From outside the cornea, position the liver spheroids around the pupil and away from the incision by gently prodding the cornea with the tip of the cannula (Figure 2C).</li>
		<li>Wait ~5-10 min for the liver spheroids to settle onto the iris before releasing the eye from the forceps.</li>
		<li>Apply vaseline eye ointment to the operated eye, which helps lubricate and heal the cornea.</li>
		<li>If desired, proceed to operate on the second eye following the same method.</li>
		<li>Before awakening the mouse, administer an analgesic to avoid post-operative discomfort, for example, 0.1 mg/kg buprenorphine in sterile saline, administered subcutaneously. 
		NOTE: Only one dose of analgesics was administered to the mice as they recovered quickly from this small procedure and did not show any signs of pain or altered behavior. Since this procedure is very quick (taking under 10 min) and causes only minor discomfort, the mice require no post-operative care, other than the post-operative analgesia administered before awakening the animal.</li>
	</ol>
	</li>
</ol>
3. In vivo imaging of engrafted liver spheroids in the ACE
<ol>
	<li>Preparation
	<ol>
		<li>Prepare the following materials and instruments for noninvasive in vivo imaging of liver spheroids engrafted in ACE (Figure 3A): upright confocal microscope, long working-distance water-dipping objective, isoflurane anesthesia unit, induction chamber, isoflurane, heating pad, custom-made metal base plate, mouse head-holder and gas mask, forceps attached to the solid universal joint, artificial-tear gel, eye ointment (liquid paraffin and Vaseline at 1:1 ratio).</li>
		<li>Optional materials include injectable fluorescent probes, disposable syringes, and 27 G needles for tail intravenous injection.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Anesthetize the mouse in the induction chamber using a dose of 2.5% isoflurane and 280 mL/min air.</li>
		<li>When the mouse is unconscious, lower the anesthesia to 1.8% isoflurane and 280 mL/min air, connect the anesthesia tube to the head-holder, and quickly transfer the animal onto the heating pad, positioning the nose inside the head-holder.</li>
		<li>Immobilize the head in the head-holder by using the screws.</li>
		<li>Place a drop of artificial tear gel on both eyes to prevent drying.</li>
		<li>At this point, intravenously inject fluorescent probes through the tail vein and image immediately after.</li>
		<li>Tilt the head, gently pop the eye out of the socket, and secure it with forceps and into position under the objective.</li>
		<li>Apply a generous amount of artificial tear gel to fill the space between the cornea and the objective and focus on the liver spheroids through the eyepiece. 
		NOTE: If possible, remove the eyepiece of one of the oculars to gain non-magnified vision and more easily locate the spheroids on the iris.</li>
		<li>In order to obtain high-resolution imaging despite the breathing movements of the animal, use 25x objectives and the following imaging settings: format 512 x 512 pixels, scan speed of 600 Hz, and a Z-stack thickness of 3 &#181;m. See detailed imaging settings in Table 2. 
		NOTE: Throughout the imaging, the anesthesia concentration is adjusted from 1.6-2.2 mL/h isoflurane to achieve a shallow, controlled breathing rhythm and thereby minimize the movement of the animal.</li>
		<li>At the end of the imaging session, treat the imaged eyes with vaseline eye ointment before removing isoflurane and awakening the animal.</li>
	</ol>
	</li>
</ol>

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

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	<li>Bell, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+human+hepatocyte+spheroids+as+a+model+system+for+drug-induced+liver+injury,+liver+function+and+disease.">Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.</a> Sci Rep. 6, 25187 (2016).</li><li>Lauschke, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+rearrangements+of+cellular+MicroRNA+signatures+are+key+drivers+of+hepatocyte+dedifferentiation.">Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation.</a> Hepatology. 64 (5), 1743-1756 (2016).</li><li>Oliva-Vilarnau, N., Vorrink, S. U., Ingelman-Sundberg, M., Lauschke, V. 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M., Shafagh, R. Z., Hendriks, D. F. G., Ingelman-Sundberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+primary+hepatocyte+culture+systems+for+analyses+of+liver+diseases,+drug+metabolism,+and+toxicity:+Emerging+culture+paradigms+and+applications.">3D primary hepatocyte culture systems for analyses of liver diseases, drug metabolism, and toxicity: Emerging culture paradigms and applications.</a> Biotechnol J. 14 (7), e1800347 (2019).</li><li>Shibuya, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+the+hepatocyte+spheroids+for+hepatocyte+transplantation.">The efficacy of the hepatocyte spheroids for hepatocyte transplantation.</a> Cell Transplant. 30, 9636897211000014 (2021).</li><li>Hamazaki, K., Doi, Y., Koide, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulated+multicellular+spheroid+of+rat+hepatocytes+transplanted+intraperitoneally+after+90%+hepatectomy.">Microencapsulated multicellular spheroid of rat hepatocytes transplanted intraperitoneally after 90% hepatectomy.</a> Hepatogastroenterology. 49 (48), 1514-1516 (2002).</li><li>Marques, P. 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B., Berggren, P. 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P-OB is the cofounder and CEO of Biocrine AB, IBL, and BL are consultants for Biocrine AB.

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 &#181;m. 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 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.

<table><tbody><tr><td>27 G butterfly needle</td><td>Venofix</td><td>4056388</td><td></td></tr><tr><td>AAV8-CAG-GFP</td><td>Charles River</td><td>CV17169-AV9</td><td>Incubated with isolated hepatocytes at 1 &amp;micro;L/mL during liver spheroid formation</td></tr><tr><td>Absolute and 70% ethanol</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Absorbent pad</td><td>Attends</td><td>203903</td><td></td></tr><tr><td>Albumin-Cre;RCL-tdTomato (B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J ; B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J)</td><td>Jackson</td><td>#003574 and #007914</td><td>Mice obtained from in-house breeding</td></tr><tr><td>B6 albino mice (B6(Cg)-Tyrc-2J/J)</td><td>Jackson</td><td>#000058</td><td>Mice obtained from in-house breeding</td></tr><tr><td>B6;129P2-Gt(ROSA)26Sor[tm1(CAG-Venus/GMNN,-Cherry/CDT1)Jkn]/JknH</td><td>INFRAFRONTIER/EMMA</td><td>EM:08395</td><td>Mice obtained from in-house breeding</td></tr><tr><td>BD Insyte IV Catheter 24 G x 0.75 in</td><td>BD Medical</td><td>381212</td><td></td></tr><tr><td>Borosillicate standard glass cappilaries&amp;nbsp;</td><td>World Precision Instruments</td><td>1B150-4</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell strainer, 70 &amp;micro;m</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Custom-made metal plate</td><td>Hardware store</td><td>N/A</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Dual-Stage Glass Micropipette Puller</td><td>Narshige</td><td>Model PC-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>Electric heating pad</td><td>Hardware store</td><td>N/A</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>N/A</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Green CMFDA</td><td>Abcam</td><td>ab145459</td><td>Reconstituted in DMSO, administered at 100 &amp;micro;g/mouse in PBS 10% FBS</td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>81242</td><td>Model 1750 Luer Tip Threaded Plunger Syringe, 500 &amp;micro;L</td></tr><tr><td>HBSS; no calcium, no magnesium and no phenol red</td><td>Gibco</td><td>14175095</td><td></td></tr><tr><td>HCX IRAPO L 25x/0.95 W objective</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Induction chamber 0.8 L</td><td>Univentor</td><td>8329001</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS-G)</td><td>Gibco</td><td>41400045</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>N/A</td><td></td></tr><tr><td>Lectin DyLight-649</td><td>Invitrogen</td><td>L32472</td><td>Administered at 1 mg/mL and 100 &amp;micro;L/mouse</td></tr><tr><td>Liberase TM Research Grade</td><td>Sigma-Aldrich</td><td>5401127001</td><td></td></tr><tr><td>Microelectrode beveler</td><td>World Precision Instruments</td><td>Model BV-10</td><td></td></tr><tr><td>Mouse head-holder and gas mask</td><td>Narshige</td><td>Model SGM-4</td><td></td></tr><tr><td>Nunclon Sphera 96-Well, U-Shaped-Bottom Microplate</td><td>Thermo Fisher</td><td>174929</td><td></td></tr><tr><td>Oculentum simplex</td><td>APL</td><td>N/A</td><td></td></tr><tr><td>PBS 10x</td><td>Gibco</td><td>14080055</td><td></td></tr><tr><td>PBS 1x; no calcium, no magnesium</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644</td><td></td></tr><tr><td>Peristaltic pump</td><td>Ismatec</td><td>Model ISM795</td><td></td></tr><tr><td>PharMed BPT Pump Tubing</td><td>VWR</td><td>VERN070540-07</td><td>Inner diameter 0.76 mm, outer diameter 2.46 mm</td></tr><tr><td>pHrodo Red-LDL</td><td>Invitrogen</td><td>L34356</td><td>Administered at 1 mg/mL and 100 &amp;micro;L/mouse</td></tr><tr><td>Portex Fine Bore Polyethylene Tubing</td><td>Smiths Medical</td><td>800/100/140</td><td>Inner diameter 0.4 mm, outer diameter 0.8 mm</td></tr><tr><td>Silicone dissection mat</td><td>Hardware store</td><td>N/A</td><td></td></tr><tr><td>Sodium chloride 0.9%</td><td>Braun</td><td>N/A</td><td></td></tr><tr><td>Solid Universal Joint</td><td>Narshige</td><td>Model UST-2</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>Model M80</td><td></td></tr><tr><td>Suspension culture dish 35 mm</td><td>Sarstedt</td><td>833900500</td><td></td></tr><tr><td>Temgesic</td><td>Indivor</td><td>N/A</td><td>Administered s.c. at 0.05 mg/mL and 2 &amp;micro;L/g mouse</td></tr><tr><td>Translucent Silicone Tubing</td><td>VWR</td><td>228-1450</td><td>Inner diameter 1.5 mm, outer diameter 3 mm</td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>Univentor 400 Anesthesia unit&amp;nbsp;</td><td>Univentor</td><td>8323001</td><td></td></tr><tr><td>Upright laser scanning confocal microscope</td><td>Leica</td><td>Model TCS SP5 II</td><td></td></tr><tr><td>Viscotears</td><td>Novartis</td><td>N/A</td><td></td></tr><tr><td>William&#039;s E Medium; no glutamine, phenol red</td><td>Gibco</td><td>22551089</td><td></td></tr></tbody></table>

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.

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&#39;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 &#181;m &#177; 10 &#181;m, 248 &#181;m &#177; 17 &#181;m, and 298 &#181;m &#177; 19 &#181;m (mean &#177; SD) diameters, respectively (Figure 1C,D). For transplantation, we select spheroids of approximately 250 &#181;m 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66234/66234fig01.jpg" /> 
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 &#181;m. (D) Liver spheroid size at different cell-seeding concentrations, n = 21 spheroids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66234/66234fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66234/66234fig02.jpg" /> 
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 &#181;l 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 &#181;m. Data for panel F has been adapted with permission from Lazzeri-Barcelo et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66234/66234fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66234/66234fig03.jpg" /> 
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 &#181;m; (D) Bile canaliculi network labeled by injection of fluorescent CMFDA, scale bar = 50 &#181;m; (E) LDL uptake by injection of fluorescent pHrodo-LDL probe, scale bar= 100 &#181;m; (F) Td-Tomato-expressing hepatocytes, arrowheads indicate nuclei and asterisks indicate intra-spheroid vasculature, scale bar = 50 &#181;m; (G) Monitoring of cell cycle dynamics in FUCCI-expressing liver spheroids, scale bars = 50 &#181;m (main image) and 20 &#181;m (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 &#181;m. The image in panel G has been adapted with permission from Lazzeri-Barcelo et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66234/66234fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66234/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66234/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies at JoVE.com

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

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 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 ...

in-vivo-imaging-liver-spheroids-engrafted-anterior-chamber-mouse

Nanyang Technological University

Research

JoVE Journal

Biology

1.1K Views.  Karolinska University Hospital. In onze groep gebruiken we de voorste oogkamer van de muis als transplantatieplaats voor in vivo en niet-invasieve beeldvorming van leversteroïden. Dit nieuw opgerichte beeldvormingsplatform zal een uniek hulpmiddel bieden voor onderzoekers en ons begrip van leverfysiologie en pathologie in zowel fundamenteel als preklinisch onderzoek vergroten. In biomedisch onderzoek vertrouwen we op de visualisatie van cellulaire processen om ziektemechanismen te begrijpen, en op het gebied van leveronder...

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Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Cancer Research

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Medicine

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Immunology and Infection

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Imaging Fixture Supported Mouse Liver Staging for Multiphoton Microscopy Based Vascular Visualization: A Stabilization Device-assisted Imaging Technique to Obtain Multi-dimensional Images of Blood Vessels in Mouse Liver

Source: Rongrong, W. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60932" target="_blank">Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue</a>. J. Vis. Exp. (2021)
This video describes an imaging procedure to observe and study intravascular dynamics of a mouse liver tissue using multiphoton microscopy following stabilization of the organ. Multiphoton microscopy enables the imaging of biological tissue at high spatial resolution in vivo.

Source: Rongrong, W. et al. Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue. J. Vis. Exp. (2021)
This video describes an imaging ...

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

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