Name: Human Liver Spheroids from Peripheral Blood for Liver Disease Studies (Video) | JoVE Journal
Uploaded: 2023-01-27T13:30:00.000+00:00
Duration: 9 min 51 s
Description: Learn to generate human liver spheroids from peripheral blood mononuclear cells for toxicology and drug discovery research. This non-genetic method uses BDPC-derived hepatocytes to overcome primary hepatocyte shortages.

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5 minutes

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 main...

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 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 ...

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

Research

JoVE Journal

Bioengineering

This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

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.

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

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

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

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.

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

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.

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

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.

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

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.

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

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.

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

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.

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

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

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

Learn to generate human liver spheroids from peripheral blood mononuclear cells for toxicology and drug discovery research. This non-genetic method uses BDPC-derived hepatocytes to overcome primary hepatocyte shortages.

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

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