Name: human liver spheroids from peripheral blood for liver disease studies
Uploaded: 2023-01-27T13:30:00
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...

<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. 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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 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 ...

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 ...

Research

JoVE Journal

Bioengineering

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