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...

<ol>
	<li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends in Biotechnology. 31 (2), 108-115 (2013).</li><li>Ryu, N. E., Lee, S. H., Park, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+system+methods+and+applications+for+mesenchymal+stem+cells.">Spheroid culture system methods and applications for mesenchymal stem cells.</a> Cells. 8 (12), 1620 (2019).</li><li>Nevzorova, Y. A., Boyer-Diaz, Z., Cubero, F. J., Gracia-Sancho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+liver+disease+-+A+practical+approach+for+translational+research.">Animal models for liver disease - A practical approach for translational research.</a> Journal of Hepatology. 73 (2), 423-440 (2020).</li><li>Ingelman-Sundberg, M., Lauschke, V. 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+human+liver+spheroids+for+translational+pharmacology+and+toxicology.">3D human liver spheroids for translational pharmacology and toxicology.</a> Basic and Clinical Pharmacology and Toxicology. 130, 5-15 (2022).</li><li>Nelson, C. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+extracellular+matrix,+scaffolds,+and+signalling:+tissue+architecture+regulates+development,+homeostasis,+and+cancer.">Of extracellular matrix, scaffolds, and signalling: tissue architecture regulates development, homeostasis, and cancer.</a> Annual review of cell and developmental biology. 22, 287-309 (2006).</li><li>Khanna, S., Chauhan, A., Bhatt, A. N., Dwarakanath, B. S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids+as+in+vitro+models+for+studying+tumor+responses+to+anticancer+therapies.">Multicellular tumor spheroids as in vitro models for studying tumor responses to anticancer therapies.</a> Animal Biotechnology (Second Edition). , 251-268 (2020).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Riede, J., Wollmann, B. M., Molden, E., 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=Primary+human+hepatocyte+spheroids+as+an+in+vitro+tool+for+investigating+drug+compounds+with+low+clearance.">Primary human hepatocyte spheroids as an in vitro tool for investigating drug compounds with low clearance.</a> Drug metabolism and disposition: The Biological Fate of Chemicals. 49 (7), 501-508 (2021).</li><li>Soto-Gutierrez, A., 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=Differentiating+stem+cells+into+liver.">Differentiating stem cells into liver.</a> Biotechnology &amp; Genetic Engineering Reviews. 25, 149-163 (2008).</li><li>Hurrell, T., 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=Human+liver+spheroids+as+a+model+to+study+aetiology+and+treatment+of+hepatic+fibrosis.">Human liver spheroids as a model to study aetiology and treatment of hepatic fibrosis.</a> Cells. 9 (4), 964 (2020).</li><li>Chen, S., 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=Hepatic+spheroids+derived+from+human+induced+pluripotent+stem+cells+in+bio-artificial+liver+rescue+porcine+acute+liver+failure.">Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure.</a> Cell Research. 30 (1), 95-97 (2020).</li><li>Zhao, 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=Cytochrome+P450+enzymes+and+drug+metabolism+in+humans.">Cytochrome P450 enzymes and drug metabolism in humans.</a> International Journal of Molecular Sciences. 22 (23), 12808 (2021).</li><li>Becker-Koji&#263;, Z. A., Schott, A. K., Zipan&#269;i&#263;, I., Hern&#225;ndez-Rabaza, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-Free+generation+of+blood-derived+neuronal+cells.">GM-Free generation of blood-derived neuronal cells.</a> Journal of Visualized Experiments. (168), e61634 (2021).</li><li>Marchenko, S., Flanagan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemistry:+Human+neural+stem+cells.">Immunocytochemistry: Human neural stem cells.</a> Journal of Visualized Experiments. (7), e267 (2007).</li><li>Crandall, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-fetoprotein:+a+review.">Alpha-fetoprotein: a review.</a> Critical Reviews in Clinical Laboratory Sciences. 15 (2), 127-185 (1981).</li><li>Magalh&#227;es, J., Eira, J., Liz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+transthyretin+in+cell+biology:+impact+on+human+pathophysiology.">The role of transthyretin in cell biology: impact on human pathophysiology.</a> Cellular and Molecular Life Sciences 2021. 78 (17-18), 6105-6117 (2021).</li><li>Huck, I., Gunewardena, S., Espanol-Suner, R., Willenbring, H., Apte, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+nuclear+factor+4+alpha+activation+is+essential+for+termination+of+liver+regeneration+in+mice.">Hepatocyte nuclear factor 4 alpha activation is essential for termination of liver regeneration in mice.</a> Hepatology. 70 (2), 666-681 (2019).</li><li>Korver, S., 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+application+of+cytokeratin-18+as+a+biomarker+for+drug-induced+liver+injury.">The application of cytokeratin-18 as a biomarker for drug-induced liver injury.</a> Archives of Toxicology. 95 (11), 3435-3448 (2021).</li><li>Klyushova, L. S., Perepechaeva, M. L., Grishanova, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CYP3A+in+health+and+disease.">The role of CYP3A in health and disease.</a> Biomedicines. 10 (11), 2686 (2022).</li><li>Fujino, C., Sanoh, S., Katsura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+expression+of+cytochrome+P450+3A+isoforms+and+toxicological+effects:+endo-+and+exogenous+substances+as+regulatory+factors+and+substrates.">Variation in expression of cytochrome P450 3A isoforms and toxicological effects: endo- and exogenous substances as regulatory factors and substrates.</a> Biological &amp; Pharmaceutical Bulletin. 44 (11), 1617-1634 (2021).</li><li>Hutchinson, M. R., Menelaou, A., Foster, D. J., Coller, J. K., Somogyi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2D6+and+CYP3A4+involvement+in+the+primary+oxidative+metabolism+of+hydrocodone+by+human+liver+microsomes.">CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes.</a> British Journal of Clinical Pharmacology. 57 (3), 287-297 (2004).</li><li>Asrani, S. K., Devarbhavi, H., Eaton, J., Kamath, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+liver+diseases+in+the+world.">Burden of liver diseases in the world.</a> Journal of Hepatology. 70 (1), 151-171 (2019).</li><li>Kammerer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+liver+culture+systems+to+maintain+primary+hepatic+properties+for+toxicological+analysis+in+vitro.">Three-dimensional liver culture systems to maintain primary hepatic properties for toxicological analysis in vitro.</a> International Journal of Molecular Sciences. 22 (19), 10214 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>imaging software</td>
		</tr>
		<tr>
			<td>Centrifuge tubes 50 mL&#160;</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&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercially available detergent</td>
			<td>Procter &#38; 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&#160;</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&#160;</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&#945; 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&#160;</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 ...

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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 ...

Generation of human liver spheroids using autologous sources can contribute to various research area like toxicology, cancer, and drug discovery. The main advantage of this technique is to use BDPC-derived human hepatocytes to engineer human liver spheroids, circumventing the shortage of primary human hepatocytes or PHH. Autologous human spheroids can be extended to regenerative medicine and therapy, especially in patient with acute liver failure.Demonstrating the procedure will be Dr.Anne-Katherin Schott, project leader in my laboratory. Begin by preparing 500 milliliters of hepatoblast KnockOut serum replacement dimethyl sulfoxide or KSR DMSO medium and hepatocyte maturation medium. Aliquot the medium from the stock and add fresh hepatocyte growth factor or HGF and oncostatin M or OCM at final concentrations of 10 or 20 nanograms per milliliter for each medium change.Transfer three times 10 to the fifth blood-derived pluripotent stem cells or BD-PSCs cells into a biolaminin coated four-well plate and incubate the plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for five days. To support the endodermal differentiation, culture the cells for five days in KSR DMSO hepatoblast medium and change the medium every 48 hours. On day five, add hepatocyte maturation medium and culture the cells for an additional seven to 10 days in the incubator at 37 degrees Celsius and 5%carbon dioxide, ensuring changing of the medium every 48 hours.Count cells using a counting chamber and centrifuge BD de-differentiated cell suspension at 300 G for 10 minutes at room temperature. After removing the supernatant, resuspend BD de-differentiated cells in KSR DMSO medium at two times 10 of the six cells per milliliter concentration. After ensuring the single cell suspension, remove any additional debris by passing the cells through a 40 micrometer cell strainer.Again, count the cells using a counting chamber and prepare a sufficient volume of each cell seeding density to dispense the required volume per well. Prepare a gradient with top seeding of one million cells to a low seeding density of 4, 000 cells. Next, dispense 100 microliters of KSR DMSO medium in 96-well low attachment plates and add 100 microliters of cell seeding dilution.Incubate the low attachment plate for five days. Change 50%of the medium on the third or fourth day after seeding when the spheroids are sufficiently compact. On day five, change the medium to hepatocyte maturation medium and culture the cells in it for an additional seven to 10 days, changing the medium every 48 hours.After culturing the cells for 4, 8, 15, and 24 days using the differentiation method described earlier, remove the media. Incubate the cells with pre-warmed fixatives containing 4%paraformaldehyde in PBS for 10 minutes. Next, discard the fixative and wash the cells with PBS two times for five minutes each.Immediately add 0.1%Triton X-100 solution and permeabilize the cells for five minutes before two washes of PBS. Add a blocking solution made of PBS and 5%bovine serum albumin or BSA before placing the cells on a rocker plate for one hour at room temperature. Dilute primary antibodies in dilution buffer 1%BSA PBS and add 50 microliters of the antibody dilution per well.Discard the antibody solution after a one-hour incubation at room temperature and give three washes of five minutes each using PBS. Add 50 microliters of secondary antibodies diluted in dilution buffer in each well and incubate the cells for 30 minutes at room temperature. After washing the cells three times with PBS for five minutes each, mount the coverslips with mounting media containing DAPI for microscopic analysis.Carefully discard culture media without touching the spheroids and add freshly prepared PBS with 0.1%Triton X-100 solution and incubate for five minutes to permeabilize the cells. Wash the spheroids two times with the medium by slowly pipetting the medium. Next, incubate the spheroids with 50 microliters of the primary antibodies for one hour.Once done, carefully remove the excess antibody solution and give three washes of medium. Prepare the corresponding secondary antibodies like goat anti-mouse IgG Cy3, goat anti-mouse IgG 488, and rabbit anti-chicken IgG Texas Red in PBS with 1%BSA and add 50 microliters of antibody dilution per well before 20 minutes of incubation in the carbon dioxide incubator. Again, wash thrice with the medium before 30 minutes of incubation in the incubator.Switch on the fluorescence light source 10 minutes before use. Turn on the computer and open the imaging software. Use 4X magnification objective by clicking on the 4X button in the toolbar to select the correct scale bar.Then place the 96-well plate on the stage center plate. Turn on the LED light source, use the brightfield filter, and position the plate to the well of interest using the XY axis stage adjustment knob. Change to camera light path and click the live button in the imaging software to visualize the image on the screen.Ensure the spheroid is centered using the XY axis knobs and focus using the coarse or fine focus knob. Next, choose the brightfield observation method in the toolbar, put exposure settings to automatic, and click the snapshot button 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.Place ambient light shielding plate to turn off the LED light and change the filter for B excitation. Then choose 488 observation method. Open the shutter, take a picture by clicking the snapshot button and close the shutter and save the file as a vsi.Repeat the process with a filter for G excitation to reiterate the process for each well of interest. Morphological changes during the hepatic differentiation process showed that the BD-PSCs differentiated into hepatocytes through three stages. The first stage represented the differentiation into endodermal cells.The second differentiation into hepatic progenitor cells exhibiting a typical polygonal morphology. And the third, the maturation to hepatocytes. Immunofluorescence analysis showed the strong expression of endoderm human liver progenitor markers like the alpha fetoprotein or APF and transthyretin or TTR in the cells at the first stage of hepatic differentiation process at L4 to L8 day.However, their expression decreased at L15 day. The expression of albumin or ALB and hepatocyte nuclear factor four alpha or HNF4 alpha appeared firstly at L4, increased throughout the differentiation time L4 to L15, and reached a strong and stable expression during the maturation time L15 to L24. Spontaneous aggregation of cells was observed in low attachment plates containing hepatocyte induction or maturation medium initiated spheroid formation.A consistent correlation was observed between steroid spheroid and the variable number of cells. The spheroids formed and differentiated at L14 and live stained with antibodies revealed the potential hepatic functional activity of BD-PSCs derived spheroids. The preparation of mononuclear cells for reprogramming is the most important step in this procedure.Generating human liver spheroids is the first step to creating organoids in vitro simplified version of the organ in 3D with micro anatomy similar to that in vivo. Organoids created in the lab are used to study organogenesis, disease development, and nutrients.

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1.4K Views.  ACA CELL Biotech GmbH. Generation of human liver spheroids using autologous sources can contribute to various research area like toxicology, cancer, and drug discovery. The main advantage of this technique is to use BDPC-derived human hepatocytes to engineer human liver spheroids, circumventing the shortage of primary human hepatocytes or PHH. Autologous human spheroids can be extended to regenerative medicine and therapy, especially in patient with acute liver failure.Demonstrating the procedure will be Dr....