The authors would like to acknowledge that this research has received funding from the European Union&#8217;s Horizon 2020 research and innovation programme for the PATROLS project, under grant agreement No.760813

<ol>
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The authors have nothing to disclose.

Applications for 3D hepatic models vary considerably depending on the particular biochemical endpoint or adverse outcome pathway being targeted. Each model has its benefits and limitations, from interdonor variation in primary human hepatocyte (PHH) models to reduced cytochrome p450 activity in cell-line based models, but all are valuable in their own right6,12,18,19. When assessing genotoxicity there are limitations in the models compatibility with regulatory approved endpoints such as the in vitro micronucleus assay, as active proliferation is required. This is necessary, as genotoxicity assessment requires the quantification of fixed DNA damage to be assessed post cell division when there is opportunity for DNA repair to correct transient lesions. Unfortunately, highly differentiated hepatocyte (i.e., HepaRG) based spheroids or PHH microtissues, which are deemed to exhibit the most physiologically relevant liver-like characteristics form static (non-proliferative) models12,19,20. As a result, the 3D HepG2 spheroid model presented here provides a suitable, alternative model able to support genotoxicity testing. HepG2 cell-line based spheroids have sufficient actively dividing cells on the outer surface of the spheroids whilst maintaining basic liver-like characteristics, such as albumin and urea production and some CYP450 activity5,12,19. Principally this in vitro liver model has been developed to complement the micronucleus assay, as this is one of the two in vitro assays recommended in the battery for genotoxicity testing8,10,11,21. However, the model can be readily applied to DNA sequencing analysis and gene expression (RNA) technologies, while it has the potential to be further adapted and utilized for other DNA damage endpoints, such as the comet assay. Nonetheless, it is important to consider the role that ENM interference plays in some endpoint analyses. For example, flow cytometry-based analyses may not be suitable for ENM genotoxicity assessment specifically due to particle interference22.
One limiting factor of spheroid models that actively undergo cell division is their size. Optimization of seeding density is critical as there needs to be enough cells that allow the model to continue to proliferate; but not too high a cell number, which results in the spheroid becoming overly compact, leading to an increased necrotic core. The cause of this necrosis is believed to be restricted oxygen and nutrient diffusion, as the limit of this diffusion is thought to be approximately 100 &#8211; 150 &#181;m of tissue23,24. However, this does depend of the cell type, cell number, scaffold interactions and culture conditions25. Since, it has been shown that approximately 700 &#181;m diameter is the limit for avoiding premature onset of necrosis in the center of C3A spheroids, seeding 4000 HepG2 cells per spheroid ensures the diameter of the model at the time of exposure is &#8804;500 &#181;m26. Furthermore, Shah et al. established that HepG2 cells seeded above 5000 cells per spheroid exhibited a 25% reduction in viability following 7 days in culture, which could pertain to the average diameter of 680 &#181;m and limited availability of nutrients in a 20 &#181;L hanging drop5. To overcome this, the model devised in the present protocol undergoes a critical step where the hanging drop is transferred to agarose coated wells following initial formation of the spheroid. This ensures a greater volume of culture medium is present to sustain the ever-growing number of cells within the spheroids. As a result, the HepG2 spheroid model remains over 70% viable following 10 days in culture and can be utilized for long-term hazard assessment in vitro. 
Whilst the HepG2 spheroid model can support both acute and long-term exposure regimes, refreshing cell culture medium during extended culture periods is restricted for this model as complete replacement of the medium is not advised due to the potential loss of the spheroids. It is presumed that with ENM exposures, the tendency for homogenous ENM dispersions to agglomerate and sediment is high. However, it is notable that the rate at which an ENM sediments can vary depending on the particle parameters (e.g., size, shape and density) and can be determined theoretically using the in vitro sedimentation, diffusion and dosimetry (ISDD) model, or its recent derivatives, often referred to when regarding ENM (suspension) exposure approaches27,28. With this is mind, it is assumed that if only 50% of the cell culture medium is carefully removed from the surface of the cell culture, the disruption and subsequent removal of the ENM dose should in theory be minimal. However, with Brownian motion at play, this may not strictly be the case and further work into the deposition and sedimentation of each particular ENM to be tested should be undertaken to ensure the correct dosimetry is retained throughout the long-term exposure regimes27. Principally this is a potential limitation to consider when performing repeated dosing regimes as this could be critical to the final, accumulated concentration. Chemical based exposures on the other hand, whilst not without their own limitations to consider, offer a more simplistic approach in that chemical substances tend to remain in solution and thus a direct replacement of the original chemical concentration in addition to the newly added concentration ensures that any chemical lost during media refreshment is replaced accordingly29. Future applications would include evaluating the suitability of the model for repeated exposure regimes over long-term culture periods as repeated dosing strategies are crucially important for assessing the ability of a particular organ system to ameliorate or overcome the adverse effects, if any, induced by bioaccumulation of a xenobiotic substance.
In conclusion, this 3D in vitro hepatic model has the capacity to be utilized for evaluating a range of realistic exposure scenarios, thereby providing a future in vitro approach to better support both ENM and chemical hazard assessment in a routine and easily accessible manner.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61141/advanced-3d-liver-models-for-vitro-genotoxicity-testing-following">Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure</a>. The Representative Results section&#160;was updated.
Figure 6 in the Representative Results section was updated from:
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6.jpg" />
to:
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6v5.jpg" />
The fourth paragraph in the Representative Results section was updated from:
With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain &#62;70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the &#8805;14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be &#8805;0.06 mg/mL whilst urea production should be &#8805;0.4 ng/&#181;L before conducting an in vitro toxicological assessment with this model.
to:
With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain &#62;70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the &#8805;14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be &#8805;50.0&#160;ng/&#956;L whilst urea production should be &#8805;0.25&#160;ng/&#181;L before conducting an in vitro toxicological assessment with this model.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61141/advanced-3d-liver-models-for-vitro-genotoxicity-testing-following">Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure</a>. The Representative Results section&#160;was updated.

Erratum: 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) across a plethora of human-based applications (e.g., food, cosmetics, clothing, sporting equipment, electronics, transport and medicine), it is inevitable that humans will be exposed to ENM on a regular basis. With this, there are heightened concerns that the novel, size specific physio-chemical characteristics that deem these materials advantageous in numerous applications could cause adverse effects upon human health and the environment concomitantly. Currently many&#160;international activities are in place&#160;to actively reflect more physiologically relevant exposures to these ENM and assess the potential toxicity&#160;of these materials over acute, long-term, and repeated low-dose exposure scenarios.
Hepatic toxicology is key when considering ENM exposure, as it is widely known that the liver is a major site of ENM accumulation post exposure1,2. Moreover, the liver is the primary organ system for metabolism and detoxification of substances that enter systemic circulation3. Based upon the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multicellular interactions or appropriately represent metabolic activity observed in vivo, a greater focus into developing robust and physiologically relevant in vitro 3D liver models for in vivo substitute technologies has been established4,5. Utilizing advanced 3D culture technologies improves the longevity of in vitro hepatic models allowing for long term, repeated exposure regimes to be investigated. Additionally, this advanced culture format promotes the formation of enhanced physiological, organotypic features such as bile canaliculi, active transporter processes and improved CYP450 drug metabolizing capabilities, thus improving the predictivity of the models6. Current 3D in vitro hepatic models consisting of mono-cultures (hepatocytes only) or co-cultures (hepatocytes with nonparenchymal cells) exist in several formats, ranging from microtissues or spheroids in ultralow adhesion plates, hanging drop spheroids, cells embedded in matrices and/or scaffolds and microfluidic cell culture platforms, all of which are deemed effective advanced in vitro models for hepatic toxicity assessment6,7. However, the majority of these model systems are high maintenance, require specialized equipment and are expensive. Furthermore, these models are often static (i.e., nondividing cell models) that prevents their use in the assessment of hazard endpoints, such as genotoxicity testing utilizing methods that quantify fixed DNA damage. Genotoxicity is a core prerequisite in regulatory toxicology, and it is a vital component of the risk assessment of any toxicant8. There is no single assay that can be applied to quantify all forms of DNA damage that may arise following exposure to an exogenous agent. However, a core component of the in vitro genotoxicity testing battery is the micronucleus assay, which is a reliable and multifacetted technique that measures gross chromosomal damage9. It is a gold standard technique described by the OECD Test Guideline 487, for assessing in vitro DNA damage and genotoxicity and is part of the test battery requirement for regulatory hazard assessment10,11.
The human hepatocellular carcinoma cell line, HepG2, is used widely for initial hazard assessment screening as the cells are readily available, relatively inexpensive to source, simple to culture and amenable to high throughput screening12,13. When cultured into 3D spherical structures, they have been shown to recapitulate the liver microenvironment well and offer a hepatic model with sufficient proliferative capabilities to support the micronucleus assay3. Further development of the HepG2 spheroid models was established to improve the longevity and liver-like functionality of the model in order to support genotoxicity hazard assessment over long-term, repeated exposure regimes (&#8804;14 days). Thus, in line with the principles of the 3Rs to replace, reduce and refine animal experimentation, the present protocol has been established to provide an advanced 3D in vitro hepatic model capable of reliably evaluating multiple toxicological endpoints (e.g., liver functionality, (pro-)inflammatory markers, cytotoxicity and genotoxicity) following acute, long-term and repeated chemical and ENM exposures in a routine and easily accessible manner.
Here, we present a method to establish a physiologically relevant 3D hepatocyte cell line based in vitro model system for genotoxicity hazard assessment following acute or long-term, repeated ENM exposures. The protocol can be broken down into 6 key stages: culturing cryopreserved HepG2 cells; HepG2 spheroid preparation; HepG2 spheroid transfer from hanging drop to agarose suspension; HepG2 spheroid harvest; micronucleus assay and scoring; and data analysis.

<table><tbody><tr><td>Aflotoxin B1</td><td>Sigma Aldrich, UK</td><td>A6636-5MG</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich, UK</td><td>A9539-50G</td><td></td></tr><tr><td>Autoclave Tape</td><td></td><td></td><td></td></tr><tr><td>BCG Albumin Assay</td><td>Sigma Aldrich, UK</td><td>MAK124</td><td></td></tr><tr><td>Bovine Serum Albumin Powder</td><td>Sigma Aldrich, UK</td><td>A9418</td><td></td></tr><tr><td>Cell Freezing Aid</td><td>Thermo Fisher Scientific, UK</td><td>5100-0001 - Mr Frosty</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Cytochalasin B</td><td>Merck, UK</td><td>250233</td><td></td></tr><tr><td>Cytology Metal Clips</td><td></td><td></td><td></td></tr><tr><td>Cytospin 4 Centrifuge</td><td>ThermoFisher Scientific, UK</td><td>CM00730202</td><td></td></tr><tr><td>DMEM with 4.5g/L D-Glucose, L-Glutamine</td><td>GIBCO, Paisley, UK</td><td>41965-039</td><td></td></tr><tr><td>DMEM, phenol-red free with 4.5g/L D-Glucose, L-Glutamine with Hepes</td><td>GIBCO, Paisley, UK</td><td>21063-029</td><td></td></tr><tr><td>DPX Mounting Medium</td><td>FisherScientific, UK</td><td>D/5330/05</td><td></td></tr><tr><td>Ethanol</td><td>FisherScientific, UK</td><td>10048291</td><td></td></tr><tr><td>FBS</td><td>GIBCO, Paisley, UK</td><td>10270-106</td><td></td></tr><tr><td>Filter Cards for Shandon Cytospin</td><td>FisherScientific, UK</td><td>15995742</td><td></td></tr><tr><td>Frosted Glass Slides</td><td>ThermoFisher Scientific, UK</td><td></td><td></td></tr><tr><td>Giemsa&#039;s Stain Improved R66 Solution, Gurr</td><td>VWR Chemicals, UK</td><td>MFCD00081642</td><td></td></tr><tr><td>Glass Coverslips (24 x 60)</td><td>Deckglaser, VWR</td><td>ECN631-1575</td><td></td></tr><tr><td>Haemocytometer and Coverslip</td><td></td><td></td><td></td></tr><tr><td>Immersion Oil for Microscope</td><td>Zeiss, UK</td><td>518F, ISO8034</td><td></td></tr><tr><td>Laminar Class II Tissue Culture Hood</td><td>Scanlaf Mars</td><td></td><td></td></tr><tr><td>Light Microscope</td><td>Zeiss, UK</td><td>Axiovert 40C</td><td></td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td></td></tr><tr><td>Methanol</td><td>FisherScientific, UK</td><td>10284580</td><td></td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>Non-Filtered, Sterile 200&amp;micro;l and 1000&amp;micro;l Pipette tips</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>NuncMicroWell 96-Well Microplates</td><td>ThermoFisher Scientific, Denmark</td><td>167008</td><td></td></tr><tr><td>P1000 and P200 micropipettes</td><td></td><td></td><td></td></tr><tr><td>P300 and P50 multi-channel pipettes</td><td></td><td></td><td></td></tr><tr><td>PBS pH 7.4 1X, MgCl&lt;sub&gt;2&lt;/sub&gt; and CaCl&lt;sub&gt;2&lt;/sub&gt; Free</td><td>GIBCO, Paisley, UK</td><td>14190-094</td><td></td></tr><tr><td>Pen/Strep</td><td>GIBCO, Paisley, UK</td><td>15140-122, Penicillin/Strepmyocin 100X or 10,000U/ml</td><td></td></tr><tr><td>Phosphatase Buffer Tablets</td><td>GIBCO, Paisley, UK</td><td>10582-013</td><td></td></tr><tr><td>Pipette Boy</td><td></td><td></td><td></td></tr><tr><td>Simport Scientific CytoSep Funnels for Shandon Cytospin 4 Centrifuges</td><td>FisherScientific, UK</td><td>11690581</td><td></td></tr><tr><td>Sonifier SFX 550 240V CE 1/2&quot; - Probe</td><td>Branson, USA</td><td>101-063-971R</td><td></td></tr><tr><td>T-25 and T-75 Tissue Culture Flask</td><td>Greiner-Bio-One, UK</td><td>T-25 (690175) and T-75 (660175)</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Sigma Aldrich, UK</td><td>T8154-100mL</td><td></td></tr><tr><td>Urea Assay Kit</td><td>Sigma Aldrich, UK</td><td>MAK006</td><td></td></tr><tr><td>Virkon Disinfectant</td><td>DuPont, UK</td><td>Rely+On Virkon</td><td></td></tr><tr><td>Water Bath (37?C)</td><td>Grant JBNova 18</td><td></td><td></td></tr><tr><td>Weighing Balance</td><td></td><td></td><td></td></tr><tr><td>Xylene</td><td>FisherScientific, UK</td><td>10588070</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>GIBCO, Paisley, UK</td><td>5300-054</td><td></td></tr><tr><td>0.2mL and 1.0mL Eppendorf Tubes</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>0.45&amp;micro;m Filter Unit</td><td>Millex HA, MF-Millipore, UK</td><td>SLHA033SS</td><td></td></tr><tr><td>1.0mL Syringe</td><td>BD Plastipak, FisherScientific, UK</td><td>300185</td><td></td></tr><tr><td>20mL LS Scintillation Glass Vials, 22-400 Foil Lined PP Caps</td><td>DWK Life Sciences GmbH, Germany</td><td>WHEA986581</td><td></td></tr><tr><td>37?C and 5% CO&lt;sub&gt;2&lt;/sub&gt; ISO Class 5 Hepa Filter Incubator</td><td>NUAIRE DHD Autoflow</td><td></td><td></td></tr><tr><td>3mL Pasteur Pipette</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>50mL Conical Falcon Tubes</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>50mL or 100mL Glass Bottles</td><td></td><td></td><td></td></tr><tr><td>50mL Skirted Falcon Tubes</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>5mL, 10mL and 25mL Pipettes</td><td>Greiner-Bio-One, UK</td><td></td><td></td></tr><tr><td>9.4cm Square, Petri Dish</td><td>Greiner-Bio-One, UK</td><td>688161</td><td></td></tr></tbody></table>

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.

Suitability of this cell-line based 3D liver spheroid model for long-term culture and genotoxic hazard assessment was evaluated by conducting baseline characterization to determine the viability and liver-like functionality of the model over the duration of 14 days in culture as well as its applicability for the micronucleus assay.
Baseline Characterization of the 3D HepG2 Liver Spheroid Model 
Prior to any in vitro toxicological assessment, it is important to check that the 3D HepG2 spheroids have formed properly before performing the agarose transfer or chemical/ENM treatment. HepG2 spheroids produced using the hanging drop method usually take 2 - 3 days post seeding (4000 cells/spheroid) to form compact, spherical shaped spheroids with an average diameter of 495.52 &#181;m W x 482.69 &#181;m H as shown in Figure 5A-5C. HepG2 spheroids that have formed correctly and are acceptable to be used for in vitro toxicological assessment must have a compact, spherical shaped structure with a smooth surface and no visual projections. Figure 5 provides examples of good quality (Figure 5D-F) and a poor quality (Figure 5G-I) spheroids. The latter of which should be discarded. Typically, 90-95% of spheroids formed per plate will form correctly and be viable for further experimentation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61141/61141fig5.jpg" /> 
Figure 5: Light microscopy images displaying the natural morphology of the HepG2 spheroids formed via the hanging drop method. (A-C) show Day 2 and (D-I) Day 4 HepG2 liver spheroids post seeding. (D-F) are examples of good quality HepG2 spheroids whilst (G-I) shows poorly formed spheroids. All images were taken on a X20 objective using a microscope. The scale bar represents 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61141/61141fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To further confirm HepG2 spheroid viability, a basic colorimetric Bromocresol Green Albumin (BCG) Assay or Urea Assay can be performed to assess their liver-like functionality. Liver-like functionality was assessed in line with viability using the Trypan Blue Exclusion Assay over a 14 day culture period to determine the longevity of the liver spheroid model and establish if it could support long-term or repeated ENM/chemical based hazard assessment (Figure 6). Albumin concentration remained consistent over the duration of the culture period. Urea production displays an increase in the concentration of urea produced per spheroid over a week in culture before reaching a plateau by day 7. It is important to note that the levels of albumin and urea produced in the 3D HepG2 spheroids are substantially higher than that observed in the same cell line cultured in a 2D format. Indeed, 2D cultures of HepG2 cells, peak albumin and urea levels were 0.001 mg/mL and 0.010 ng/&#181;L respectively. Furthermore, in previous work published by Shah et al. using an almost identical HepG2 spheroid system, the authors highlight a notable improvement in metabolic activity (CYP1A1 and CYP1A2) in the 3D HepG2 in vitro model systems when compared to the 2D cultured HepG2 cells5.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6v5.jpg" /> 
Figure 6: 14-Day baseline characterisation data for HepG2 liver spheroids. Following transfer from hanging drop, (A) highlights the viability of the HepG2 spheroid model over a 14 day period whilst (B) and (C) highlight the liver-like albumin and urea functionality respectively. Mean data &#177; SEM presented, n = 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61141/61141fig6v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain &#62;70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the &#8805;14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be &#8805;20.0&#160;ng/&#181;L whilst urea production should be &#8805;0.25 ng/&#181;L before conducting an in vitro toxicological assessment with this model.
Genotoxicity Assessment of Engineered Nanomaterials 
For genotoxicity assessment, the micronucleus assay was used to determine the presence of micronuclei following both acute (24 h) and long-term (120 h) ENM exposures. Aflatoxin B1 is a known liver carcinogen16,17 and is a recommended positive control for the micronucleus assay. Optimization experiments have shown that 0.1 &#181;M of Alfatoxin B1 induces a significant positive (&#8805;2.0 fold increase) genotoxic response in 3D HepG2 liver spheroids and thus is used in every micronucleus assay conducted with this model. To ensure the validity of the micronucleus assay results using the HepG2 spheroid model, the background micronucleus frequency for HepG2 cells used in this 3D in vitro model should lie within a range of 0.6% &#8211; 1.2%. As a result, Alfatoxin B1 should induce a genotoxic response of at least two-fold higher than that seen with the negative control; thus, 0.1 &#181;M of Alfatoxin B1 should induce a micronuclei frequency between 1.5% &#8211; 3.0%. Using these control parameters, ENM associated genotoxicity in vitro can then be reliably assessed. Based on OECD Test Guideline 487, it is important to note that when testing an ENM or chemical, the concentrations selected should not induce more than 55% &#177; 5% cytotoxicity (indicated by a reduction in CPBI or RVCC values in relation to the negative control)11. Figure 7 illustrates the data generated when Aflatoxin B1 and two ENMs (titanium dioxide (TiO2) and sliver (Ag)) were evaluated following both acute and long-term exposures in the HepG2 spheroids, and subsequent genotoxic potential was analyzed using the micronucleus assay. Both ENMs assessed were tested at a noncytotoxic, low dose of 5.00 &#181;g/mL over an acute (24 h) exposure and long-term (120 h) exposure regime. A similar trend for genotoxicity across both TiO2 and Ag ENMs can be observed, whereby the elevated genotoxicity response that resulted following 24 h exposure was not evident after a long-term 5 day exposure. This was despite sustained genotoxicity induced by the Aflatoxin B1 positive control at both time-points.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61141/61141fig7.jpg" /> 
Figure 7: Genotoxicity assessment following TiO2 and Ag ENM exposure on HepG2 liver spheroids. Genotoxicity (micronucleus frequency) assessment using the micronucleus assay post (A) acute (24 hour) and (B) long-term (120 hour) exposure to 5.00 &#181;g/mL of TiO2 and Ag ENM. Negative control is a media only, whilst the positive control is 0.1 &#181;M of Aflatoxin B1. Mean data (n=2) presented &#177; SD. Significance indicated in relation to the negative control: * = p&#8804; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61141/61141fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure at JoVE.com

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

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 ...

advanced-3d-liver-models-for-vitro-genotoxicity-testing-following

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.7K Views.  Swansea University Medical School. Deze procedure werd vastgesteld om te worden gebruikt voor de ontwikkeling van geavanceerde 3D-leverculturen in vitro, die een meer fysiologisch relevante beoordeling kunnen bieden van de genotoxische gevaren die gepaard gaan met blootstelling aan nanomaterialen gedurende zowel acute als langdurige, herhaalde dosisschema's.

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

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.

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 ...

Cancer Research

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 ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Medicine

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.

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 ...

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.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

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

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