The authors thank Bernd Krostitz for technical assistance, Radoslaw Kedzierski for initial contributions to the project, Erik Wade for proofreading and giving helpful comments, and Prof. Heike Walles for providing the bioreactor and sharing her valuable experience with organ decellularization. We are also thankful for funding of the project and publication by the Berlin University of Technology.

NOTE: R.L. obtained ethical approval for the explantation of organs from the Landesamt f&#252;r Gesundheit und Soziales (LaGeSo). Livers were explanted from Wister rats. The inferior vena cava and the portal vein of the liver were cannulated with a 22 G cannula. Methods for the decellularization of explanted livers have been described previously.4,5 The extracellular matrices used here were obtained by excessive perfusion of a rat liver with 1% sodium deoxycholate.
1. Recellularization of Extracellular Matrix (ECM) of a Rat Liver
<ol>
	<li>Expansion of hepatic cell line HepG2
	<ol>
		<li>Culture the hepatocellular carcinoma cell line HepG2 in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 2 mM of penicillin and streptomycin, each.</li>
		<li>Seed 1.5 x 107 cells in T175 bottles and grow the cells at 37 &#176;C and 5% CO2. Harvest cells after 4 days by trypsinization for 5 min at 37 &#176;C, 5% CO2.</li>
		<li>Spin down the cells at 300 x g and resuspend them in 4 ml PBS and count the cells with a Neubauer chamber under a microscope. One T175 bottle will yield approximately 4.5 x 107 cells. 
		NOTE: Ensure the culture contains sufficient cell numbers for recellularization of the rat liver ECM with 6 x 108 HepG2 cells per ECM (10 x 175 cm2 culture flasks with 4-5 x 108 cells each).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54633/54633fig1.jpg" /> 
Figure 1. Bioreactor system. A) This bioreactor system was custom-made. It maintains the organ models at 37 &#176;C and 5% CO2. The flow rates of the peristaltic pumps can be regulated individually. B) The liver is placed in a growth chamber and connected to the perfusion circuit, which consists of a peristaltic pump, a medium reservoir, a bubble trap and a pressure sensor. <a href="https://www.jove.com/files/ftp_upload/54633/54633fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Recellularization of the ECM
	<ol>
		<li>Set up the bioreactor system, containing a liver perfusion chamber, a perfusion system, a reservoir of medium and a bubble trap. Sterilize the bioreactor system (121 &#176;C, 15 min).</li>
		<li>Connect the bioreactor system with a peristaltic pump and place it in an incubator providing appropriate conditions (37 &#176;C, 5% CO2). Place decellularized rat liver scaffolds8 in the liver perfusion chamber of the bioreactor system (Figure 1).</li>
		<li>Connect the cannulated portal vein and vena cava using tube clips to the perfusion system. Equilibrate scaffold with 150 ml RPMI medium (as described in 1.1) over 5 d with a flow rate of 1.25 ml/min.</li>
		<li>Disconnect the liver scaffold from the media circuit and inoculate the scaffold with 3 x 108 HepG2 cells (in 5 ml) via the portal vein using a 5 ml syringe, avoid air bubble formation and allow the cells to repopulate the ECM by incubating them for 1 hr in the scaffold with the pump switched off.</li>
		<li>Gradually increase the flow rate by adjusting the pump, starting with 1.25 ml/min for 10 min; 2.5 ml/min for 20 min and finally 3.75 ml/min for 30 min.</li>
		<li>Repeat steps 1.2.4-1.2.5 to reach a total cell number of 6 x 108.</li>
		<li>Run the bioreactor system at a flow rate of 3.75 ml/min. Culture the recellularized rat liver for 2 weeks. 
		NOTE: Replace one third of the medium with fresh medium (50 ml) every other day. Sample the culture medium to measure physiological parameters, such as lactate dehydrogenase activity, the pH of medium samples and the concentration of glucose and lactate.</li>
	</ol>
	</li>
</ol>
2. Transduction of the Recellularized Rat Liver
<ol start="1">
	<li>Large-Scale Production of AAV Vectors
	<ol>
		<li>Produce, purify, and quantify AAV vectors as described previously:11
		<ol>
			<li>Briefly, produce AAV vectors in roller bottles and purify them by iodixanol gradient centrifugation. Remove residual iodixanol by filtration over PD10 gel filtration columns. Determine the AAV vector concentration by qPCR using genomic AAV DNA as a standard. 
			NOTE: The self-complementary, pseudotyped AAV2/6 vector used in the present study encoded EmGFP as a reporter to demonstrate transduction efficiency and an shRNA expression cassette for the knockdown of an endogenously expressed gene (human cyclophilin B (hCycB), Figure 2). Ensure a sufficient amount of pseudotyped scAAV vectors of serotype 6 (2.7 x 1013 AAV vectors per liver model).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54633/54633fig2.jpg" /> 
Figure 2. Map of the self-complementary, pseudotyped AAV2/6 vector used in the present study. The genome consists of the inverted terminal repeats (ITR) of AAV2 and encodes EmGFP under control of the CMV promoter as well as an shRNA under control of a U6 promoter. <a href="https://www.jove.com/files/ftp_upload/54633/54633fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Transduction of Liver Model
	<ol>
		<li>Adjust the AAV vector solution to a final concentration of 2.7 x 1013 vector genomes in 5 ml by adding the respective volume of PBS.</li>
		<li>Disconnect the liver from the media circuit by removing the tubing from the cannula. Connect a 5 ml syringe to the cannula of the portal vein and inject the complete AAV vector solution (5 ml). 
		NOTE: Optionally, add phenol red (5 &#181;g/ml) to follow the distribution of the AAV vector solution throughout the liver.</li>
		<li>Incubate for 1 hr without pumping. Gradually increase flow rate, starting with 1.25 ml/min for 10 min; 2.5 ml/min for 20 min and 3.75 ml/min for 30 min. Culture the recellularized rat liver over 6 days. 
		NOTE: Replace one third of the medium as given in NOTE under 1.2.7</li>
	</ol>
	</li>
</ol>
3. Assessment of the Recellularized Transduced Rat Liver
<ol start="1">
	<li>Hematoxylin and eosin (HE) staining and Immunohistochemical Analysis
	<ol>
		<li>Take samples (0.5 x 0.5 x 1.5-2 cm) from each liver lobe using a scalpel.</li>
		<li>Incubate samples (from 3.1.1) in 4% paraformaldehyde (PFA) + 4% sucrose solution for 1.5 hr at 4 &#176;C. (Caution: PFA is toxic and carcinogenic. Always keep PFA within a fume hood and wear appropriate protective clothing.) Wash three times with PBS (1 min per wash step) and incubate in 8% sucrose overnight at 4 &#176;C.</li>
		<li>Pour fixing medium into plastic cryomolds, place the samples into fixing medium free of air bubbles. Add fixing medium until sample is well covered. Store embedded samples at -80 &#176;C until further use.</li>
		<li>Prepare cryo-sections (10 &#181;m) with a cryotome12. Assess recellularization by hematoxylin + eosin staining8. Assess transduction efficiency by immunohistochemical staining for the gene-of-interest (here: EmGFP).</li>
	</ol>
	</li>
	<li>Molecular biological sampling
	<ol>
		<li>Sample each liver lobe with a biopsy punch (4 mm in diameter). Isolate total RNA, DNA and proteins according to the manufacturer&#39;s instructions for the assessment of transgene expression of EmGFP, and hCycB knock down8.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chang, T. T., Hughes-Fulford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+and+spheroid+culture+of+human+liver+hepatocellular+carcinoma+cell+line+cells+demonstrate+distinct+global+gene+expression+patterns+and+functional+phenotypes.">Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes.</a> Tissue Eng. Part A. 15 (3), 559-567 (2009).</li><li>Butler, D., Callaway, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientists+in+the+dark+after+French+clinical+trial+proves+fatal.">Scientists in the dark after French clinical trial proves fatal.</a> Nature. 529 (7586), 263-264 (2016).</li><li>Enger, P. O., Thorsen, F., Lonning, P. E., Bjerkvig, R., Hoover, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+vectors+penetrate+human+solid+tumor+tissue+in+vivo+more+effectively+than+adenoviral+vectors.">Adeno-associated viral vectors penetrate human solid tumor tissue in vivo more effectively than adenoviral vectors.</a> Hum. Gene Ther. 13 (9), 1115-1125 (2002).</li><li>Uygun, B. E., 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=Decellularization+and+recellularization+of+whole+livers.">Decellularization and recellularization of whole livers.</a> J. Vis. Exp. (48), e2394 (2011).</li><li>Hillebrandt, K., 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=Procedure+for+Decellularization+of+Rat+Livers+in+an+Oscillating-pressure+Perfusion+Device.">Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device.</a> J. Vis. Exp. (102), e53029 (2015).</li><li>Baptista, P. 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=The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid.">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53 (2), 604-617 (2011).</li><li>Yagi, H., 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-scale+whole-organ+bioengineering+for+liver+transplantation:+a+regenerative+medicine+approach.">Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.</a> Cell Transplant. 22 (2), 231-242 (2013).</li><li>Wagner, 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=Use+of+a+three-dimensional+humanized+liver+model+for+the+study+of+viral+gene+vectors.">Use of a three-dimensional humanized liver model for the study of viral gene vectors.</a> J. Biotechnol. 212, 134-143 (2015).</li><li>Kay, 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=State-of-the-art+gene-based+therapies:+the+road+ahead.">State-of-the-art gene-based therapies: the road ahead.</a> Nat. Rev. Genet. 12 (5), 316-328 (2011).</li><li>Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endgame:+glybera+finally+recommended+for+approval+as+the+first+gene+therapy+drug+in+the+European+union.">Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union.</a> Mol. Ther. 20 (10), 1831-1832 (2012).</li><li>Wagner, A., R&#246;hrs, V., Kedzierski, R., Fechner, H., Kurreck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+quantification+of+adeno-associated+virus+vectors+for+RNA+interference+applications+using+quantitative+polymerase+chain+reaction+and+purified+genomic+adeno-associated+virus+DNA+as+a+standard.">A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard.</a> Hum. Gene Ther. Methods. 24 (6), 355-363 (2013).</li><li>Takagi, H., 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=Microdissected+region-specific+gene+expression+analysis+with+methacarn-fixed,+paraffin-embedded+tissues+by+real-time+RT-PCR.">Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.</a> J. Histochem. Cytochem. 52 (7), 903-913 (2004).</li></ol>

The authors have nothing to disclose.

The reconstituted 3D livers described here provide a model to study viral vectors in a humanized system. Repopulation of the ECM of a rat liver with a human hepatocellular carcinoma cell line generates a vascularized system which permits the study of large biologics. These results provide a proof-of-concept that the reconstituted liver model can be efficiently transduced with a viral vector.
For the experiments shown here, each and every lobe of the liver was transduced by the AAV vector. However, in some preliminary experiments, single lobes were not repopulated with cells. It is therefore important to prevent cell debris or other components from occluding the vascular system. To test whether all lobes can be perfused, a non-toxic dye such as phenol red can be flushed through the liver model.
Another crucial matter is keeping the liver scaffold sterile. While treatment with ethanol or antibiotics is disadvantageous for the vascular system, irradiation of the extracellular matrix with &#947;-radiation preserved the vessels and sterilized the sample.
Furthermore, the size of explanted livers will differ, so that the number of cells used for the recellularization procedure and the repopulation time may have to be adjusted to obtain reproducible results.
The rat livers used in the present study are comparatively large and require large amounts of cells and test reagents (e.g., AAV vectors). In addition, the repopulation procedure took more than two weeks. This limits the number of replicates which can be done with reasonable effort. We are currently establishing the model for mouse livers, which are only approximately one fifth of the volume of rat livers, allowing the use of fewer cells and less test reagents. Although proportional scale-down of the cell number seems reasonable, the exact amounts need to be determined in further experiments.
Another shortcoming of the present model is the use of the hepatocellular HepG2 cell line. Experiments are ongoing to develop the use of hepatocytes differentiated from induced pluripotent stem cells, which will provide a physiologically more relevant model. Furthermore, the liver consists of multiple cell types in addition to the hepatocytes, e.g., Kupffer cells and sinosoids. We assume that the different cell types will repopulate their natural environments when an ECM is recellularized with multiple cell types.
The 3D liver model combines several advantages. A major disadvantage of conventional in vivo models is that animal physiology differs substantially from human physiology. Toxic side effects of the treatment of a human patient may therefore remain undetected. This shortcoming can be overcome by reconstituting the 3D liver model with human cells that more closely reflect the biology of human patients.
The second advantage of the liver model is its contribution to animal welfare. Although animal components are required for the reconstitution experiments, the approach still follows the aims of the 3R principle (replacement, reduction, refinement), as surplus animals can be used that were sacrificed for other animal experiments, i.e., no additional animals are needed and the approach completely avoids suffering of animals which is frequently associated with in vivo experiments. Spheroids are an alternative tool to study cellular processes in a 3D system. However, spheroids are not vascularized so that large substances and biologics do not penetrate deeply into the inner parts of the structure. These problems have been overcome with the vascularized 3D liver model.
In the experiments described here, AAV vectors were investigated, since they are among the most promising candidates for gene therapeutic applications. As numerous gene therapeutic approaches aim at targeting the liver, e.g., for the treatment of infections with hepatitis viruses or of alpha-1-antitrypsin deficiency, the 3D liver can be used in the process of these AAV vector development. It is, of course, also suitable for the study of other hepatotropic viral vectors, e.g., adenoviral vectors. In addition, it can be used to study infectious hepatitis viruses such as hepatitis B or C virus. It can, for example, be employed to design new antiviral strategies. Moreover, 3D organ models represent promising tools to develop new cytostatic therapeutics to treat cancer and to carry out toxicological studies. On the long run, artificial livers may be used in regenerative medicine as transplants. Taken together, the 3D liver model offers a wide range of applications in infection biology and other fields of biomedical research.

Most current biomedical research relies on one of two approaches, either two-dimensional (2D) cell culture experiments or animal models, which are three-dimensional (3D) by their very nature. However, these approaches have some severe drawbacks. Cells grown in 2D culture have been shown to differ in gene expression patterns and cell physiology from those cultivated under 3D conditions.1 Animal models, in addition to being associated with ethical concerns, often do not model human physiology well. Although the lack of obvious toxic effects of a compound must be confirmed in animal models prior to the first dosing in humans, multiple cases have been documented in which severe, sometimes fatal, adverse effects have occurred in clinical trials.2
To overcome these shortcomings, humanized 3D ex vivo organ models have become important research tools. When cultivated under suitable conditions, cells self-assemble into 3D structures known as spheroids. However, these spheroids lack a vascular system, which limits the distribution of small molecular compounds, large biologics and viral vectors alike. For example, adenoviral vectors only transduced the outer cell layers of spheroids prepared from human glioblastomas.3 A solution to this problem is the use of an organ model containing a vascular system. To this end, the organ of interest can be explanted from an animal, and the animal cells can be replaced by human cells. Various methods for decellularization of animal livers by treatment with detergents or sodium cholate have been described.4-6 The resulting extracellular matrix (ECM) harbors cytokines and growth factors which regulate various cellular processes.7 It can be used as a scaffold for recellularization with human cells to obtain a functional organ model.
In a recent study, we used a humanized 3D liver model to study distribution and transgene expression of an adeno-associated virus (AAV) vector.8 AAV vectors belong to the most promising viral vectors for gene therapeutic applications.9 The first, and to date only, approved gene therapeutic intervention in the Western world uses an AAV vector for the transfer of lipoprotein lipase.10

<table><tbody><tr><td>Incubator</td><td>Fraunhofer</td><td>/</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Fraunhofer</td><td>/</td><td></td></tr><tr><td>Flange with groove</td><td>Duran</td><td>2439454</td><td>modified by gaffer</td></tr><tr><td>O-Ring Transparent</td><td>Duran</td><td>2922551</td><td></td></tr><tr><td>Quick Release Clamp</td><td>Duran</td><td>2907151</td><td></td></tr><tr><td>Flat Flange Lid</td><td>Duran</td><td>2429857</td><td>modified by gaffer</td></tr><tr><td>Screw thread Tube</td><td>Duran</td><td>2483802</td><td>modified by gaffer</td></tr><tr><td>Screw thread Tube</td><td>Duran</td><td>2483602</td><td>modified by gaffer</td></tr><tr><td>Silicone sealing Ring</td><td>Duran</td><td>2862012</td><td></td></tr><tr><td>Screw Cap</td><td>Duran</td><td>2924013</td><td></td></tr><tr><td>Screw Cap</td><td>Duran</td><td>2924008</td><td></td></tr><tr><td>Screw Cap with aperture</td><td>Duran</td><td>2922709</td><td></td></tr><tr><td>Screw Cap with aperture</td><td>Duran</td><td>2922705</td><td></td></tr><tr><td>Filter</td><td>Sarstedt</td><td>831,826,001</td><td></td></tr><tr><td>Silicone Tubing</td><td>VWR</td><td>228-1500</td><td></td></tr><tr><td>Tube connector</td><td>Ismatec</td><td>ISM556A</td><td></td></tr><tr><td>Biocompatible Tubing</td><td>Ismatec</td><td>SC0736</td><td></td></tr><tr><td>T175 culture flasks</td><td>Greiner bio-one</td><td>660 160</td><td></td></tr><tr><td>RPMI 1640</td><td>BioWest SAS (Th. Geyer)</td><td>L0501-500</td><td></td></tr><tr><td>glutamine</td><td>BioWest SAS (Th. Geyer)</td><td>X0551-100</td><td></td></tr><tr><td>Trypsin</td><td>BioWest SAS (Th. Geyer)</td><td>L0940-100</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>BioWest SAS (Th. Geyer)</td><td>L0022-100</td><td></td></tr><tr><td>fetal calf serum</td><td>cc pro</td><td>S-10-M</td><td></td></tr><tr><td>Tissue-Tek O.C.T.</td><td>Weckert-Labortechnik</td><td>600001</td><td></td></tr><tr><td>HepG2</td><td>DSMZ</td><td>ACC 180</td><td></td></tr><tr><td>Cryomold 15 x 15 x 5 mm</td><td>Sakura</td><td>4566</td><td></td></tr><tr><td>Biopsy punch 4 mm</td><td>pfm medical</td><td>48401</td><td></td></tr><tr><td>Nucleospin miRNA</td><td>Macherey &amp;amp; Nagel</td><td>740971.10</td><td></td></tr><tr><td>Nucleospin RNA/DNA Buffer Set</td><td>Macherey &amp;amp; Nagel</td><td>740944</td><td></td></tr></tbody></table>

study of viral vectors in a three-dimensional liver model repopulated with the human hepatocellular carcinoma cell line hepg2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

For the assessment of the extent of recellularization, cryo-sections were prepared from each lobe of each recellularized liver model. The sections were then analyzed by hematoxylin and eosin staining. As can be seen in Figure 3, each lobe of the three liver models, denoted as TLM1, TLM2 (transduced liver models 1 +2) and Ctrl (control liver model that was recellularized, but not transduced), were repopulated with HepG2 cells. This hepatocellular carcinoma cell line was established from the tumor tissue of a 15-year-old Argentine boy. Some areas of the liver models were more intensively recellularized than others. The reasons for these variations remain to be determined.
<img alt="Figure 3" src="/files/ftp_upload/54633/54633fig3.jpg" /> 
Figure 3. Hematoxylin and eosin stain of each lobe of the three analyzed liver models. TLM1+2: transduced liver models 1 and 2; Ctrl.: control liver model that was recellularized but not transduced. Scale bar: 200 &#181;m. (Re-printed with permission from Ref. 8.) <a href="https://www.jove.com/files/ftp_upload/54633/54633fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the next step, the transduction of the liver models was assessed. To this end, DNA was prepared from 28 punch biopsies of each of the liver models and the vector titer was quantified by qPCR. On average, 55 and 90 internalized vector genomes per cell (VG/cell) were measured for the two transduced liver models. Experiments in 2D cell culture have shown 30 VG/cell are sufficient for strong transgene expression and RNAi-mediated silencing. Production of EmGFP was analyzed by RT-PCR and Western blotting. In fact, expression of the reporter was detected in 80-90% of the biopsies on the mRNA and protein levels, respectively.8 To obtain a comprehensive picture of the transduction efficiency, cryo-sections were immunohistologically analyzed. As can be seen in Figure 4, areas of the liver model that were successfully recellularized, as visualized by DAPI staining, also gave strong fluorescent signals in the immunohistochemical analysis of EmGFP expression. As expected, the control liver model, not treated with AAV vectors, did not show any EmGFP expression.
<img alt="Figure 4" src="/files/ftp_upload/54633/54633fig4.jpg" /> 
Figure 4. Immunohistochemical analysis of EmGFP expression. Cells that repopulated the liver models were visualized by DAPI staining (blue); EmGFP expression was visualized by immunochemical staining (red). TLM1+2: transduced liver models 1 and 2; Ctrl.: control liver model that was recellularized but not transduced. Scale bar: 200 &#181;m. (Re-printed with permission from Ref. 8.) <a href="https://www.jove.com/files/ftp_upload/54633/54633fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In a final test, AAV-mediated knockdown of hCycB was analyzed by qRT-PCR. The knockdown of hCycB was found to be between 70 and 90%, averaged over all lobes of the two transduced liver models (Figure 5).
<img alt="Figure 4" src="/files/ftp_upload/54633/54633fig5.jpg" /> 
Figure 5. RNAi-mediated knockdown of hCycB in AAV-transduced 3D liver models. Silencing of hCycB was determined by qRT-PCR. Mean values and standard deviations (SD) were calculated for all samples of each transduced 3D liver model (TLM1 and 2, respectively) and normalized to the mean value of the non-transduced control (Ctrl.). (Re-printed with permission from Ref. 8.) <a href="https://www.jove.com/files/ftp_upload/54633/54633fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2 at JoVE.com

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

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8.0K Views.  Institute of Biotechnology, Berlin University of Technology. The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

Video: Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Medicine

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

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

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

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

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

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

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

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

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

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

Immunology and Infection

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

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

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

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

Bioengineering

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Assembloid Technology: Pioneering Liver Development Research

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

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

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

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

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