Name: human liver spheroids from peripheral blood for liver disease studies
Uploaded: 2023-01-27T13:30:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Human Liver Spheroids from Peripheral Blood for Liver Disease Studies at JoVE.com

The authors are especially grateful for the technical assistance provided by Oksana and John Greenacre. This work was supported by ACA CELL Biotech GmbH Heidelberg, Germany.

Ethical approval was obtained (ACA CELL Biotech GmbH/25b-5482.2-64-1) for performing these experiments and informed consent was signed by all donors before blood extraction in compliance with institutional guidelines.
1. Preparation of mononuclear cells (MNCs) from human peripheral blood (PB)
<ol>
	<li>Extract 30 mL of blood from healthy donors with the help of trained medical personnel according to the standard protocol.</li>
	<li>Isolate PBMNCs using density gradient media according to the protocol published by Becker-Koji&#263; et al.13. Isolate, by pipetting, the interphase layer between plasma and density gradient media and use sterile phosphate buffer saline (PBS) to wash the isolated cells.</li>
	<li>Use a counting chamber and count the number of cells using standard methods.</li>
</ol>
2. Dedifferentiation of MNCs upon activation with human GPI-anchored glycoprotein
<ol>
	<li>Place 6 x 106 mononuclear cells in PBS/1% bovine serum albumin (BSA) in a polystyrene tube (15 mL) and incubate with the specific antibody for 30 min at 37 &#176;C according to Becker-Koji&#263; et al.13.</li>
	<li>Centrifuge the cells at 300 x g at room temperature and replace PBS/BSA with Iscove&#39;s modified Dulbecco&#39;s medium supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Grow the cells in 15 mL polystyrene tubes in a 5% CO2 incubator at 37 &#176;C for 8-10 days as described in Becker-Koji&#263; et al.13. On day 5 (D5), add 1-2 mL of Iscove&#39;s medium supplemented with 10% FBS to each 15 mL tube.</li>
</ol>
3. Sorting of newly generated dedifferentiated cells
<ol>
	<li>Centrifuge cultured cell suspension at room temperature for 10 min at 300 x g and aspirate with a sterile pipette the resulting supernatant according to Becker-Koji&#263; et al.13.</li>
	<li>Resuspend pellet obtained by centrifugation in 90 &#181;L of cold PBS buffer (PBS pH 7.2, 0.5 % BSA, and 2 mM EDTA) and add 10 &#181;L of CD45 positive nano-sized magnetic beads and incubate on ice for 15 min.</li>
	<li>Wash the cell suspension with 2 mL of PBS buffer, centrifuge it at 300 x g for 10 min at room temperature and add 500 &#181;L of PBS buffer to the cells and resuspend thoroughly.</li>
	<li>Pipette 500 &#181;L of pre-cooled PBS buffer into the column to wash and place it in the magnetic field using a magnetic stand.</li>
	<li>Wash the cells placed on the column, twice with 500 &#181;L of PBS buffer and collect flow-through containing CD45 negative cells in Iscove&#39;s medium supplemented with 10% FBS.</li>
	<li>Use a counting chamber to determine the number of reprogrammed cells.</li>
</ol>
4. Preparation of glass coverslips for the generation of human hepatocytes
<ol>
	<li>Separate glass coverslips (14 mm) and incubate them in nonionic detergent for 10 min. Wash glass coverslips in deionized water till no bubbles remain and incubate them in 1M HCl for 30 min (adapted from Marchenko et al.14).</li>
	<li>Wash glass coverslips with deionized water at least 3x and dry them overnight at room temperature. Autoclave dried glass coverslips in a suitable container.</li>
</ol>
5. Coating cell culture plates with biolaminin for 2D hepatic differentiation of BD-PSCs
<ol>
	<li>Place autoclaved glass coverslips with a sterile pair of tweezers in 4 well plates and turn UV lights on for 30 min to ensure sterile conditions.</li>
	<li>Thaw aliquots of biolaminin and add 120 &#181;L of 5 &#181;g/mL biolaminin to each glass cover slip. Leave the coated glass coverslips overnight at 4 &#176;C.</li>
	<li>Remove the excess of biolaminin and add 200 &#181;L of hepatocyte differentiation medium as described below.</li>
</ol>
6. Preparation of hepatocyte differentiation media
<ol>
	<li>Make 500 mL of hepatoblast knockout serum replacement (KSR)/dimethyl sulfoxide (DMSO) medium consisting of 76.4% knockout DMEM (KO-DMEM), 20% KSR, 0.5% L-glutamine, 1% non-essential amino acids (NEAA), 0.1% &#946;-mercaptoethanol, 1% DMSO, and 1% penicillin-streptomycin (Pen/Strep).</li>
	<li>Prepare 500 mL of hepatocyte maturation medium containing 1% L-glutamine, 10 &#181;M hydrocortisone 21-hemisuccinate sodium salt (HCC), and 1% Pen/Strep.</li>
	<li>Aliquot medium from the stock and add fresh hepatocyte growth factor (HGF) and oncostatin M (OSM) at final concentrations of 10 ng/mL and/or 20 ng/mL for each medium change. 
	&#8203;NOTE: Oncostatin M is a cytokine belonging to the interleukin 6 group of cytokines, important for hematopoiesis as well as for liver development.</li>
</ol>
7. Culturing hepatic cells differentiated from BD-PSCs
<ol>
	<li>Put 3 x 105 BD-PSCS cells into each well of a 4-well plate coated with biolaminin.</li>
	<li>Place the 4-well plates in the incubator at 37 &#176;C and 5% CO2.&#160;Culture the cells for 5 days in KSR/DMSO hepatoblast medium that supports endodermal differentiation and change the medium every second day.</li>
	<li>Switch to hepatocyte maturation medium on day 5 and culture the cells for an additional 7-10 days in the incubator at 37 &#176;C and 5% CO2. Change the medium every 48 h.</li>
</ol>
8. 3D spheroid hepatic differentiation
<ol>
	<li>Count cells using a counting chamber.</li>
	<li>Centrifuge BD-dedifferentiated cell suspension at 300 x g for 10 min at room temperature. Remove supernatant and resuspend BD-dedifferentiated cells in KSR/DMSO medium at 2 x 106 cells/mL.</li>
	<li>Pass the cells through a 40 &#181;m cell strainer to ensure a single-cell suspension and to remove any additional debris.</li>
	<li>Count cells using a counting chamber and prepare a sufficient volume for each cell seeding density in order to dispense the required volume per well. Prepare a gradient with top seeding of 1 x 106 cells to a low seeding density of 4000 cells.</li>
	<li>Dispense 100 &#181;L of KSR/DMSO medium in 96 well-low attachment plates and add 100 &#181;L of cell seeding dilution.</li>
	<li>Place low attachment plate in the incubator at 37 &#176;C and 5% CO2 and culture them for 5 days.</li>
	<li>Change 50% of the medium from days 3-4 after seeding when the spheroids are sufficiently compact.</li>
	<li>On day 5, change the medium to hepatocyte maturation medium and culture the cells for an additional 7-10 days for further maturation. Change the medium every second day.</li>
</ol>
9. Immunofluorescence analysis of newly generated 2D liver cell cultures
<ol>
	<li>Culture the cells according to the differentiation method described above for 4, 8, 15, and 24 days and remove media.</li>
	<li>Incubate cells with pre-warmed fixative consisting of 4% paraformaldehyde in PBS for 10 min. Discard the fixative and wash the cells 2x with PBS for 5 min each. Immediately add 0.1% triton X-100 solution and permeabilize the cells for 5 min. Wash 2x with PBS.</li>
	<li>Add blocking solution made of PBS and 5% BSA and place on rocker plate for 1 h at room temperature.</li>
	<li>Dilute primary antibodies in dilution buffer 1% BSA/PBS as follows: albumin (ALB) 1:50, alpha-1 fetoprotein (AFP) 1:250, cytokeratin 18 (CK18) 1:50, hepatocyte nuclear factor 4 alpha (HNF4&#945;) 1:1000 and transthyretin (TTR) 1:50. Use 50 &#181;L of antibody dilution per well.</li>
	<li>Incubate the cells for 1 h at room temperature. Then discard the antibody solution, wash the cells for 5 min, and repeat the wash step 3x.</li>
	<li>Prepare the following secondary antibodies in dilution buffer: rabbit anti-chicken IgG (Texas red) 1:1000, goat anti-mouse IgG (488) 1:1000, and goat anti-rabbit (488) 1:500. Use 50 &#181;L of antibody dilution per well and incubate the cells for 30 min at room temperature.</li>
	<li>Wash the cells 3x with PBS for 5 min each and mount the coverslips with mounting media containing DAPI for microscopic analysis.</li>
</ol>
10. Live staining of newly formed liver spheroids
<ol>
	<li>Carefully discard culture media while not touching the spheroids, add freshly made PBS with 0.1% triton X-100 solution, and incubate for 5 min to permeabilize the cells.</li>
	<li>Wash the spheroids with the medium by slowly pipetting for 5 min, repeat 2x.</li>
	<li>Incubate the spheroids with the primary antibodies ALB (1:50), AFP (1:250), CK18 (1:50), CYP2E1 (1:200), and CYP3A4 (1:200) prepared in PBS with 1% BSA for 1 h in 5% CO2 incubator at 37 &#176;C. Use 50 &#181;L of antibody dilution per well.</li>
	<li>Carefully remove the excess antibody solution and wash the spheroids with medium 3x.</li>
	<li>Prepare the corresponding secondary antibodies goat anti-mouse IgG (Cy3), goat anti-mouse IgG (488), and rabbit anti-chicken IgG (Texas red) at a dilution of 1:1000 and 1:500 for goat anti-rabbit (488) in PBS in 1% BSA. Use 50 &#181;L of antibody dilution per well and incubate for 20 min in the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Carefully wash 3x with medium and leave the plate for 30 min in the incubator at 37 &#176;C and 5% CO2 before performing fluorescence microscopy.</li>
</ol>
11. Examination of spheroids using a fluorescence microscope
<ol>
	<li>Switch on the fluorescence light source 10 min before use, turn on the computer and open the imaging software.</li>
	<li>Use an objective with 4x magnification, click on button 4x in the toolbar to have the correct scale bar selected, then place the 96-well plate on the stage center plate.</li>
	<li>Turn the LED light source on, use the brightfield filter, and position the plate to the well of interest using the x-y-axis stage adjustment knob.</li>
	<li>Change to camera light path, click the button Live in imaging software to visualize the image on the screen, and ensure the spheroid is centered using the x-y-axis knobs and focus using the coarse/fine focus knob. 
	NOTE: The shape of the spheroids remains constant after applying live staining.</li>
	<li>Choose the Brightfield Observation method in the toolbar, put exposure settings to automatic, and click the button Snapshot in the camera control panel to take a picture. Then save the picture as .vsi file using the appropriate name in the folder of interest.</li>
	<li>Place ambient light shielding plate to turn off LED light, change to filter for B-excitation, choose 488 Observation method in the toolbar, open shutter, take a picture by clicking button Snapshot, close shutter, then save file as described above.</li>
	<li>Repeat this with a filter for G-excitation using the appropriate observation method (Texas red or CY3). Then reiterate the whole procedure for each well of interest.</li>
	<li>Return the plate to the 5% CO2 incubator at 37 &#176;C and culture it as described above.</li>
</ol>

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The corresponding author declares that she is a patent holder related to Novel human GPI-linked protein. She co-founded and works with ACA CELL Biotech GmbH. The other authors declare there are no conflicts of interest.

The liver is a major organ in the human body with many essential biological functions, such as the detoxification of metabolites. Due to severe liver failures like cirrhosis and/or viral hepatitis, there are nearly 2 million deaths per year worldwide. Liver transplantations rank second in solid organ transplantations worldwide, but only about 10% of the current need is met22.
Primary human hepatocytes (PHH) are often used to study liver toxicity. These cells can be maintained in culture for a short time retaining their specific functions. Also, the number of cells available from a single donor is limited, in addition, these cells cannot be expanded in the culture therefore, the shortage of donor PHH remains the main obstacle for hepatotoxicity studies. PSCs represent a renewal source of human tissues and can be used for the generation of 3D hepatic cultures11.
Liver 3D culture systems show multiple advantages when compared to 2D. Shorter differentiation time and accurate mimicking of in vivo processes enable for more precise studies on drug-induced toxicity, better prediction of liver liability, and are more cost-effective23. The liver spheroid cultures due to their autologous feature could be a great advantage over primary human hepatocytes (PHH) circumventing the disadvantages related to their use and may become a gold standard for application in testing drug toxicity and has a potential future application in regenerative medicine.
We have demonstrated here that BD-PSCs generated from steady-state peripheral blood can be successfully differentiated into endodermal/hepatocyte progenitors/mature hepatocytes with steady albumin secretion and phenotypic stability expressing hepatocyte markers. Moreover, the engineered 3D human hepatocyte spheroid cultures demonstrate the potential functional activity by expressing the enzymes that belong to cytochrome P450, like CYP3A4 and CYP2E1.
The most important step in the protocol is to obtain good quality and number of fresh human MNCs for the reprogramming process. The use of frozen MNCs results in a reduced number of reprogrammed cells.
We have engineered human liver spheroids using activated PBMNCs cultures with and without applying immunomagnetic sorting that separates reprogrammed cells from mature blood cells. The slight difference in using these two methods relies on the higher density of 3D structure when using purified reprogrammed cells. The expression of hepatocyte markers remains consistent in both cell culture preparations.
Due to the limited availability of PHH, the method potentially represents the biologically relevant closest alternative to autologous fresh hepatocytes to study hepatic function in vitro, like xenobiotic metabolisms and liver toxicity, host-pathogen intervention, and cell biology in general. The possibility of using BD-PSCs in regenerative medicine while being autologous and non-teratogenic is the subject matter of further studies in our laboratory.

In recent years three-dimensional (3D) spheroid culture systems have become an important tool to study various areas of cancer research, drug discovery, and toxicology. Such cultures raise great interest because they bridge the gap between two-dimensional (2D) cell culture monolayers and complex organs1.
In the absence of an adhesive surface, compared to the 2D cell culture, the formation of spheroids is based on the natural affinity of these cells to cluster in 3D form. These cells organize themselves into groups consisting of one or more types of mature cells. Free of foreign materials, these cells interact with each other like in their original microenvironment. The cells in 3D culture are much closer and have a proper orientation toward each other, with higher extracellular matrix production than 2D cultures, and constitute a close to natural environment 2.
Animal models have been used for a long time to study human biology and diseases3. In this regard, there are intrinsic differences between humans and animals, which makes these models not entirely suitable for extrapolative studies. 3D culture spheroids and organoids represent a promising tool to study tissue-like architecture, interaction, and crosstalk between different cell types that occur in vivo and can contribute to reducing or even replacing animal models. They are of particular interest for studying the pathogenesis of liver diseases as well as drug screening platforms4.
3D spheroid culture is of particular importance for cancer research as it can eliminate the discontinuity between the cells and their environment by reducing the need for trypsinization or collagenase treatment needed for preparing the tumor cell monolayers for 2D cultures. Tumor spheroids enable the study of how the normal versus malignant cells receive and respond to signals from their surroundings5 and are an important part of tumor biology studies.
Compared to the monolayer, 3D cultures consisting of various cell types resemble tumor tissues in their structural and functional properties and therefore are suitable for studying metastasis and invasion of tumor cells. That is why such spheroid models are contributing to accelerating cancer research6.
Spheroids are also helping to develop the technology to create human organoids because tissue and organ biology are very challenging to study, particularly in humans. Progress in stem cell culture makes it possible to develop 3D cultures like organoids consisting of stem cells and tissue progenitors as well as different types of mature (tissue) cells from an organ with some functional characteristics like a real organ that can be used to model organ development, diseases, but they also can be considered useful in regenerative medicine7.
Primary human hepatocytes are usually used for studying in vitro biology of human hepatocytes, liver function, and drug-induced toxicity. Cultures of human hepatocytes have two main drawbacks, firstly, the limited availability of primary tissue like human hepatocytes, and secondly, the tendency of hepatocytes to rapidly dedifferentiate in 2D culture thereby losing their specific hepatocyte function8. 3D hepatic cultures are superior in this regard and have recently been made from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs)9. Bioengineered hepatic 3D spheroids are of particular interest to study development, toxicity, genetic and infectious diseases of the liver, as well as in drug discovery for the treatment of liver diseases10. Lastly, they also have the potential to be used clinically, knowing that acute liver diseases have a mortality rate of nearly 80%, bio-artificial liver and/or hepatic spheroids could potentially rescue these patients by providing partial liver function until a suitable donor can be found11.
We have established a protocol for the generation of human hepatic spheroids using blood-derived pluripotent stem cells (BD-PSCs) to prepare differently sized spheroids containing 4000 to 1 x 106 cells and analyzed them by means of light microscopy and immunofluorescence. We also tested the capability of hepatocyte-specific function, assessing the expression of cytochrome P450 3A4 (CYP3A4) and 2E1 (CYP2E1) enzymes that belong to the cytochrome P450 family that have important roles in cellular and drug metabolism through the process of detoxification12.

<table><tbody><tr><td>Albumin antibody</td><td>Sigma-Aldrich</td><td>SAB3500217</td><td>produced in chicken</td></tr><tr><td>Albumin Fraction V</td><td>Carl Roth GmbH+Co. KG</td><td>T8444.4</td><td></td></tr><tr><td>Alpha-1 Fetoprotein</td><td>Proteintech Germany GmbH</td><td>14550-1-AP</td><td>rabbit polyclonal IgG</td></tr><tr><td>Biolaminin 111 LN</td><td>BioLamina&amp;nbsp;</td><td>LN111-02</td><td>human recombinant</td></tr><tr><td>CD45 MicroBeads</td><td>Miltenyi</td><td>130-045-801</td><td>nano-sized magnetic beads</td></tr><tr><td>Cell Strainer</td><td>pluriSelect</td><td>43-10040-40</td><td></td></tr><tr><td>CellSens&amp;nbsp;</td><td>Olympus</td><td></td><td>imaging software</td></tr><tr><td>Centrifuge tubes 50 mL&amp;nbsp;</td><td>Greiner Bio-One</td><td>210270</td><td></td></tr><tr><td>CEROplate 96 well</td><td>OLS OMNI Life Science</td><td>2800-109-96</td><td></td></tr><tr><td>CKX53&amp;nbsp;</td><td>Olympus</td><td></td><td></td></tr><tr><td>Commercially available detergent</td><td>Procter &amp;amp; Gamble</td><td></td><td>nonionic detergent</td></tr><tr><td>CYP2E1-specific antibody</td><td>Proteintech Germany GmbH</td><td>19937-1-AP</td><td>rabbit polyclonal antibody IgG</td></tr><tr><td>CYP3A4&amp;nbsp;</td><td>Proteintech Germany GmbH</td><td>67110-1-lg</td><td>mouse monoclonal antibody IgG1</td></tr><tr><td>Cytokeratin 18</td><td>DakoCytomation</td><td>M7010</td><td>mouse monoclonal antibody IgG1</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418-50ML</td><td></td></tr><tr><td>DPBS</td><td>Thermo Fisher Scientific</td><td>14040091</td><td></td></tr><tr><td>FBS</td><td>Merck Millipore</td><td>S0115/1030B</td><td>Discontinued. Available under: TMS-013-B</td></tr><tr><td>Glass cover slips 14 mm</td><td>R. Langenbrinck</td><td>01-0014/1</td><td></td></tr><tr><td>GlutaMax 100x Gibco</td><td>Thermo Fisher Scientific</td><td>35050038</td><td>L-glutamine</td></tr><tr><td>Glutaraldehyde 25%</td><td>Sigma-Aldrich</td><td>G588.2-50ML</td><td></td></tr><tr><td>Goat anti-mouse IgG Cy3</td><td>Antibodies online</td><td>ABIN1673767</td><td>polyclonal</td></tr><tr><td>Goat anti-mouse IgG DyLight 488</td><td>Antibodies online</td><td>ABIN1889284</td><td>polyclonal</td></tr><tr><td>Goat anti-rabbit IgG Alexa Fluor 488</td><td>Life Technologies</td><td>A-11008</td><td></td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>30721-1LGL</td><td></td></tr><tr><td>HepatoZYME-SFM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17705021</td><td>hepatocyte maturation medium</td></tr><tr><td>HGF</td><td>Thermo Fisher Scientific</td><td>PHG0324</td><td>human recombinant</td></tr><tr><td>HNF4&amp;alpha; antibody</td><td>Sigma-Aldrich</td><td>ZRB1457-25UL</td><td>clone 4C19 ZooMAb Rbmono</td></tr><tr><td>Hydrocortisone 21-hemisuccinate (sodium salt)</td><td>Biomol</td><td>Cay18226-100</td><td></td></tr><tr><td>Knock out Serum Replacement - Multi Species Gibco</td><td>Fisher Scientific</td><td>A3181501</td><td>KSR</td></tr><tr><td>KnockOut DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>12660012</td><td>Discontinued. Available under Catalog No. 10-828-010</td></tr><tr><td>MACS Buffer</td><td>Miltenyi</td><td>130-091-221</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi</td><td>130-042-303</td><td>magnetic stand</td></tr><tr><td>MEM NEAA 100x Gibco</td><td>Thermo Fisher Scientific</td><td>11140035</td><td></td></tr><tr><td>Mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>31350010</td><td>50mM</td></tr><tr><td>MiniMACS columns</td><td>Miltenyi</td><td>130-042-201</td><td></td></tr><tr><td>Nunclon Multidishes</td><td>Sigma-Aldrich</td><td>D6789</td><td>4 well plates</td></tr><tr><td>Oncostatin M</td><td>Thermo Fisher Scientific</td><td>PHC5015</td><td>human recombinant</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>PBS sterile</td><td>Carl Roth GmbH+Co. KG</td><td>9143.2</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Biochrom GmbH</td><td>A2213</td><td>10000 U/ml</td></tr><tr><td>PS 15ml tubes sterile</td><td>Greiner Bio-One</td><td>188171</td><td></td></tr><tr><td>Rabbit anti-chicken IgG Texas red</td><td>Antibodies online</td><td>ABIN637943</td><td></td></tr><tr><td>Roti Cell Iscoves MDM</td><td>Carl Roth GmbH+Co. KG</td><td>9033.1</td><td></td></tr><tr><td>Roti Mount FluorCare DAPI</td><td>Carl Roth GmbH+Co. KG</td><td>HP20.1</td><td></td></tr><tr><td>Roti Sep 1077 human</td><td>Carl Roth GmbH+Co. KG</td><td>0642.2</td><td></td></tr><tr><td>Transthyretin antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SAB3500378</td><td>produced in chicken</td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>HFH10</td><td>1%</td></tr></tbody></table>

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.

We successfully differentiated human BD-PSCs into endoderm/hepatic progenitor cells and hepatocytes by applying a two-step protocol. Morphological changes during the hepatic differentiation process are shown in Figure 1. BD-PSCs differentiate into hepatocytes going through three different stages. The first stage represents the differentiation into endodermal cells L4, the second, differentiation to hepatic progenitor cells (hepatoblast) L8, exhibiting a typical polygonal morphology, and the third, the maturation to hepatocytes L15-L24.
Immunofluorescence analysis was performed to confirm the hepatic differentiation of BD-PSCs as presented in Figure 2. Strong expression of endoderm/human liver progenitor marker, like alpha-fetoprotein (AFP), a major plasma protein in fetal serum whose concentration is very low in adult organisms and is therefore considered as a marker for hepatocytes&#39; precursor15 and transthyretin (TTR), a major thyroid-hormone binding protein involved in transporting thyroxine from the bloodstream to the brain16 are found in the cells at the first stage of the hepatic differentiation process at L4 to L8. However, their expression decreases at L15, while the expression of albumin (ALB), the most abundant plasma protein produced mainly by the liver and utterly critical for hepatic differentiation, as well as hepatocyte nuclear factor 4 alpha (HNF-4&#945;), a hepatocytes transcription factor that is involved in the expression of liver-specific genes17&#160;appears firstly at L4, rises throughout the differentiation time L4-L15 reaching a strong and stable expression during the maturation time L15-L24.
Cytokeratin 18 (CK18) is a cytoskeletal protein, one of the major components of intermediate filament expressed in the liver18. The results show that, as expected, CK18 expression correlates with mature hepatocytes (L15-L24), and it is not expressed in hepatocyte progenitor cells.
The well-defined protocol for hepatocyte differentiation in 2D cultures enables the engineering of hepatic 3D cultures starting with BD-PSCs.
We demonstrate here that spontaneous aggregation of these cells in low attachment plates containing hepatocyte induction/maturation medium initiates spheroid formation. The growth track was followed by imaging cells at L2, L4, and L7. (Figure 3A) There is a consistent correlation between spheroid volume and the variable number of cells, as presented in Figure 3B.
The liver is an organ in which most of the drugs in the human body get metabolized. Cytochrome P450 is a superfamily of enzymes (monooxygenases) that are of pivotal importance in the processes of drug and cellular metabolism, detoxification of xenobiotics, and homeostasis. To assess the potential functional activity of BD-PSCs derived hepatic spheroids, we analyzed the expression of drug-metabolizing enzymes like CYP3A4 and CYP2E1, members of CYP3 and CYP2 families19.
Most of the drugs that are used today including codeine, cyclosporin A, erythromycin, acetaminophen, and diazepam as well as many steroids and carcinogens, are metabolized due to the activity of CY3A4 enzyme20. CYP2E1 is involved in the metabolism of endogenous substrates like ethylene glycol, benzene, carbon tetrachloride, and particularly the most important highly mutagenic compound like nitrosamine21.
The spheroids that are formed and differentiated according to the protocol at D14, live stained with antibodies to these two enzymes, reveal the potential hepatic functional activity of BD-PSCs derived spheroids (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64703/64703fig01.jpg" /> 
Figure 1: Differentiation of BD-PSCs to hepatic-like cells. Representative micrographs of morphological changes throughout hepatic differentiation of BD-PSCs showing endodermal L4, or polygonal shape L8 morphology finally reaching maturation state at L15 to L24. Scale bars: upper row 50 &#181;m, lower row 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64703/64703fig01large.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/64703/64703fig02.jpg" /> 
Figure 2: Immunofluorescence analysis of BD-PSCs re-differentiation towards hepatic cells. Endoderm/hepatocytes progenitor and hepatocytes specific markers are expressed during liver differentiation of BD-PSCs in 2D cultures. On days L4 to L8, micrographs show decreased expression of endoderm/hepatic progenitor AFP and TTR while their expression disappeared from L8-L24. Expression of hepatocytes ALB and HNF&#945; markers arise at L4 and increase during maturation, whereas the expression of CK18 appeared first at L15, reaching the maximum at L24. Scale bars for graphs L4-L15: 50 &#181;m and for L24: 20 &#181;m. Control is presented in Supplementary Figure 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64703/64703fig02large.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/64703/64703fig03.jpg" /> 
Figure 3: Formation of 3D spheroids upon hepatic differentiation of BD-PSCs. (A) Variable cell numbers of BD-PSCs starting with 1 x 106 to 4000 cells were seeded into low attachment plates, and differentiation was performed according to the two-stage procedure as stated in the Protocol. The generation of 3D human liver spheroids was imaged at different time points, shown are representative phase contrast images at each time during the culture time period. Scale bar: 200 &#181;m. (B) Diameters of at least 4 hepatic spheroids for each size were measured at L4 using microscope imaging software and volumes were calculated. Error bars show standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64703/64703fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64703/64703fig04.jpg" /> 
Figure 4: Hepatocyte functional markers are expressed in BD-PSCs-derived liver spheroids. BD-PSCs were differentiated into hepatocytes. Direct immunofluorescence analysis was performed on live cells at L14 using antibodies to ALB, AFP, CK18, and CYP2E1 and CYP3A4, members of the cytochrome P450 family. Scale bar: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64703/64703fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Negative control for immunofluorescence analysis of BD-PSCs re-differentiation towards hepatic cells. Endoderm/hepatocytes progenitor and hepatocytes specific markers are expressed during liver differentiation of BD-PSCs in 2D cultures. Scale bar: 100&#160;&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64703/Supp Figure 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Human Liver Spheroids from Peripheral Blood for Liver Disease Studies at JoVE.com

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

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 ...

human-liver-spheroids-from-peripheral-blood-for-liver-disease-studies

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Pharmacokinetics: Drug Excretion and Clearance

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1.6K Views.  ACA CELL Biotech GmbH. Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Video: Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Cancer Research

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Chemistry

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Developmental Biology

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Immunology and Infection

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Biology

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

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.

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.

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

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Summary

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Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

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