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 ...

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.

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

Research

JoVE Journal

Bioengineering

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of the mouse liver. A mouse is injected with a dye that can stain blood vessels. To observe the mouse liver in vivo, it is exposed and fixed in a frame. Two and three-dimensional images of the blood vessels in the liver tissue are obtained using a multiphoton microscope. Images of the tissues at the selected sites are continuously acquired to observe long-term changes; the dynamic changes of blood vessels in the liver tissues are also observed. Multiphoton microscopy is a method for observing cell and cell function in deep tissue sections or organs. Multiphoton microscopy has sensitivity to tissue microstructure and enables imaging of biological tissues at high spatial resolution in vivo, providing the ability to capture the biochemical information of the organization. Multiphoton microscopy is used to observe part of the liver but fixing the liver to make the image more stable is problematic. In this experiment, a special vacuum suction cup is used to fix the liver and obtain a more stable image of the liver under the microscope. In addition, this method can be used to observe dynamic changes of specific substances in the liver by marking such substances with dyes.

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate&#8211;dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

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 ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...