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.
This Hep G2 model provides a relevant alternative in vitro testing system to more reliably assess the potential induction of fixed DNA damage following long-term exposure to nanomaterial with an aim to minimize the need for testing in animals. The Hep G2 spheroid model possesses the ability to remain viable and functional over a 14 day period, as well as sustain a suitable level of proliferation. This supports the testing of a range of biochemical and genotoxicity endpoints, including the micronucleus assay.
Before attempting this protocol, I suggest you do a few practice runs first. Take care when cell seeding or changing media, as these requires a delicate approach. Begin by adding 100 microliters of sterile room temperature PBS to the wells of a 96 well culture plate.
Invert the lid of the plate and carefully pipette 20 microliter drops of the cell suspension into the center of each well groove of the lid. Use a multi-channel pipette, adding two to four drops at a time, to ensure accuracy of placement. Only seed four steroids at a time and ensure that the pipette tips are all aligned before beginning.
The angle at which you hold the multi-channel will make a difference to the way the spheroids are formed. Make sure that the drops are centered within the grooves of the wells on the lid, then gently flip the lid on top of the plate so that the drops hang over the wells with PBS. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three days prior to spheroid transfer onto agarose.
After the incubation, remove the plate from the incubator and carefully lift the lid off the plate, discard the PBS, tap the plate to remove any residual liquid, and allow the plates to air dry for two to three minutes. After melting the agarose, gently swirl it to remove any bubbles and add 50 microliters into the base of each well. Leave the plate at room temperature for two minutes, then add 100 microliters of pre-warmed DMEM on top of the solid agarose layer in each well.
Place the lid with this spheroid droplets back on top of the plate so that the spheroids are again hanging, then centrifuge the plate for three minutes at 200 times G to transfer the spheroids into the individual wells of the plate. Incubate the plate for another 24 hours to allow the spheroids to settle. Following dispersion of the engineered nanomaterials, or ENMs, dilute them to the final desired concentration with pre-warmed DMEM.
Aspirate 50 microliters of medium from each well with the spheroids and replace it with 50 microliters of medium with the ENM. To harvest the spheroids, use a 200 microliter pipette to aspirate the 100 microliters of cell culture medium with the spheroid tissue from each well, taking care to avoid contact with the agarose. Collect the spheroids in a sterile 15 milliliter centrifuge tube.
Centrifuge the spheroid suspension at 230 times G for five minutes, then remove the supernatant and store it at minus 80 degrees Celsius until further analysis. Wash the pellet of spheroids in one milliliter of sterile, room temperature PBS, then centrifuge them at 230 times G for three minutes and discard the supernatant. Resuspend the spheroids in 500 microliters of 0.05%Trypsin-EDTA solution and incubate them for six to eight minutes at 37 degrees Celsius and 5%carbon dioxide.
After the incubation, gently pipette the trypsinzed Hep G2 cells up and down to fully dissociate and resuspend them prior to neutralizing with one milliliter of DMEM. Centrifuge the cell suspension at 230 times G for five minutes, discard the supernatant, and resuspend the cell pellet in two milliliters of PBS. To create a cuvette funnel set up, place the prepared microscope slide into the metal support, place a filter card on top of the slide, then secure the cuvette funnel on top.
Arrange the cuvette funnels in the cytocentrifuge with the funnel facing up so that 100 microliters of cell suspension can be directly added into each one. Then proceed with fixing the slides according to manuscript directions. Prepare a 20%Giemsa staining solution diluted in phosphatase buffer.
Gently pipette the solution up and down to mix it, then filter it with folded filter paper in a funnel. Use a Pasteur pipette to add three to five drops of filtered Giemsa solution to the cytodot on each slide and leave it for eight to 10 minutes at room temperature. Wash the slides in two successive phosphatase buffer washes.
Then briefly rinse them under cold water to remove any excess stain and leave them to air-dry. Prior to any in vitro toxicological assessment, it is important to check that the 3D HepG2 spheroids have formed properly. Four days post seeding, compact spherical-shaped spheroids with a smooth surface and no visual projections should form.
Good quality and poor quality spheroids four days post seeding are demonstrated here. Typically, 90 to 95%of spheroids formed per plate will form correctly and be viable for further experimentation. Viability and liver-like functionality was assessed over a 14 day culture period to determine the longevity of the liver spheroid model and establish if it could support long-term ENM or chemical-based hazard assessment.
Albumin concentration remained consistent over the duration of the culture period, while urea production per spheroid increased before beginning to fall at day 14. For genotoxicity assessment, the micronucleus assay was used to determine the presence of micronuclei following acute and long-term ENM exposures. The spheroids were exposed to two ENMs, titanium dioxide and silver.
A similar trend for genotoxicity was observed after acute exposure to both ENMs, but the elevated genotoxicity response was not evident after a long-term, five day exposure. Following this method, both the supernatant and the spheroids can be harvested for a multitude of biochemical endpoints. These include cell viability, liver function assays, SID 450 analysis, poor inflammatory markers, and gene expression.
This technique is currently being tested in a number of contract research labs who wants to apply it to routine geotoxicity testing of both chemicals and nanomaterials. This has the potential to reduce the reliance on in vivo testing approaches.
