Limitations of conventional hepatocyte culture
Hepatocytes are commonly used to assess compounds for drug-induced liver injury (DILI). However, conventional hepatocyte monolayers and suspensions are prone to losing phenotype and function over long-term culture, such as cytochrome P450 (CYP) metabolism. This limits their utility for CYP-mediated drug metabolism and induction assays. Three-dimensional (3D) hepatocyte cultures have been shown to improve on these cultures in maintaining phenotype, such as drug clearance and CYP activity. Moreover, spheroids can help to recreate native tissue environments better than monolayers and suspension cultures.
Primary hepatocytes are considered the gold standard for screening hepatotoxicity but suffer from high lot-to-lot variability, limited quantity and genetic diversity due to the small number of donors. Induced pluripotent stem cell-derived (iPS) hepatocytes may provide a solution to each of these problems, promising a theoretically unlimited source with greater reproducibility and a broader selection of possible donors.
Magnetic 3D Bioprinting for Hepatocyte Culture
Magnetic 3D bioprinting provides a rapid, easy-to-use platform for 3D cell culture. In magnetic 3D bioprinting, the cells are magnetized by adhering a biocompatible nanoparticle assembly, NanoShuttle™, to their membrane, and then aggregated into spheroids using a mild magnetic field.
This method is especially suited for hepatocyte culture. It allows spheroid aggregation within hours, greatly reducing time needed for formation compared to other 3D methods. Magnetic forces also allow the aggregation of smaller cell numbers to sufficient density, compared to the larger numbers needed to achieve confluence in monolayer cultures. Moreover, as magnetized spheroids can be held down with magnetic forces, sample retention is greatly increased. This is especially advantageous when using non-lytic assays in standard microplate systems, which require repeated exchange of medium via pipette while maintaining the culture for future assays. Magnetic 3D bioprinting can also be used to develop co-culture models with fine spatial organization, which is of interest to those who seek to incorporate non-parenchymal cells (stellate, Kupffer) with hepatocytes. Co-culture with magnetic 3D bioprinting has been previously demonstrated with aortic valve cells, lung cells and white adipose tissue.
Finally, magnetic 3D bioprinting only requires magnets and NanoShuttle™, and easily fits into existing cell culture workflows. Altogether, magnetic 3D bioprinting holds advantages over other 3D methods to support the formation and culture of hepatocyte spheroids.
In this study, we generated hepatocyte spheroids with magnetic 3D bioprinting.
An additional challenge to bioprinting hepatocytes is that they are non-adherent, and even “plateable” populations tend to have <50% plating efficiencies, namely after thaw. Rather than use previous methods of magnetization, which required plating cells, we developed a new method to magnetize non-adherent cells (Figure 1). As a result, spheroids can be bioprinted without plating, allowing for bioprinting within hours of thawing or harvesting. NanoShuttle™ non-specifically binds to adherent and unadherent cells, allowing greater output from the same hepatocyte populations.
Assays for Hepatocyte Spheroids
We used the P450-Glo™ CYP3A4 Assay (Cat.#V9001) and CellTiter-Glo® Luminescent Cell Viability Assay (Cat.#G7570) to evaluate CYP activity and viability of our bioprinted hepatocyte spheroids, respectively.
The P450-Glo™ Assay assesses CYP450 activity of cultures by providing a CYP-specific proluciferin substrate to cells and quantifying the amount of luciferin cleaved from this substrate by the CYP450 enzymes. The substrates and reaction products are cell-permeable. The luciferin, a stable by-product, diffuses into the culture medium, and the luciferase reaction can be performed in a separate microplate with aliquots of media from the original cell culture plate. This allows repeat assays without compromising the culture and multiplexing with cell viability assays.
The CellTiter-Glo® Luminescent Cell Viability Assay is an endpoint viability assay that quantifies the amount of ATP in metabolically active cells. Cells are lysed and luminescence is read from the medium. It is useful when measuring viability at the end of culture, when an intact culture may not be needed.
A challenge to these assays is the diffusion of reagents into dense hepatocyte spheroids. To overcome this limitation, the protocols of P450-Glo™ and CellTiter-Glo® Assays were adjusted to include extended incubation times to ensure diffusion of proluciferin or luciferin, and to allow thorough lysis of viable cells to more accurately measure ATP, respectively.
These assays provide simple, inexpensive methods for assessing viability and liver-specific functions of magnetically bioprinted hepatocyte spheroids.
Materials and Methods
Magnetization and bioprinting of hepatocyte spheroids
Induced pluripotent stem cell-derived (iPS) -hepatocytes (ReproHepato™, ReproCELL) were thawed as per vendor’s protocols, using thawing medium. To magnetize cells for spheroid culture, the cells were resuspended in thawing medium to 1 x 106 cells/ml in a centrifuge tube. NanoShuttle™-PL was added at 1µl/10,000 cells and mixed into solution. Tube was placed on an orbital shaker at room temperature for 2 hours to shake at 175rpm. The cells were then seeded into cell-repellent 384-well plates (CELLSTAR® plates, Greiner Bio-One) at 10,000 cells/well and placed on a 384-well magnetic drive consisting of 384 cylindrical magnets (outside dimension = 0.0625 inches) positioned under each well. The magnetic drive aggregates the magnetized cells to the bottom of the well, promoting cell contact and adhesion to allow for cells to form and maintain a coherent structure, even after the magnetic field is removed (Figure 1).
For monolayer culture, cells were seeded into cell-adherent 96-well plates (CELLSTAR® plates, Greiner Bio-One,) pre-coated with collagen type I (Sigma-Aldrich) at 75,000 cells/well.
Figure 1. Magnetic 3D bioprinting and flexible methods for magnetization of cells. Cells can plated or left in suspension for magnetization, then seeded into multiwell plates with cell-repellent coatings. Magnets under the well bottoms aggregate the cells in a matter of hours or even minutes.
iPS-hepatocytes were cultured for 24 hours in thawing medium, then another 6 days in maintenance medium to allow for phenotypic maturation, which was exchanged at days 3 and 5. Spheroid cultures remained on the magnet during this time to ensure cell-cell contact and establish the integrity of structures. With spheroids, all media exchange was performed utilizing a magnetic drive, a “holding magnet”, with a larger cylindrical magnet (outside dimension = 0.125 inches) for holding down spheroids and preventing sample loss.
CYP Induction Assay
CYP3A4 induction was measured after exposure to rifampicin using the P450-Glo™ CYP3A4 Assay with Luciferin-IPA. At day 7, maintenance medium was replaced with a serum-free induction medium. Rifampicin was added at 0–200µM in 1% DMSO. After 72 hours of exposure, the induction medium with rifampicin was replaced with 3µM Luciferin-IPA in William’s E media. Plates were incubated for 2 hours before transferring 50% of medium into a separate white microplate (Greiner Bio-One) with Luciferin Detection Reagent. CellTiter-Glo® Reagent was added to the original culture plate and incubated for 20 minutes. All plates were read for luminescence on a Synergy 4 (Biotek).
In this study, we validate magnetic 3D bioprinting as a means to quickly assemble and assay hepatocyte spheroids. iPS-hepatocytes formed competent spheroids within 24 hours of thaw and maintained their structure and viability over the culture period, even after repeated medium exchanges (Figure 2).
Figure 2. (Top) Brightfield image of cells cultured in 2D just prior to being detached for magnetic 3D bioprinting. (Middle) Hepatocyte spheroid (5,000 cells/well) formation in 96-well cell-repellent plates after 24, 36 and 48-hour incubations. Brightfield images used 2.5X objective. (Bottom) CYP3A4 activity and cell viability of 3D hepatocyte cultures. Raw RLU and normalized percentage of initial P450-Glo™ CYP3A4 (left) and CellTiter-Glo® 3D signal (right) after 0, 1, 3, 5 and 7 days of incubation at 37°C/5% CO2;
n=6 for each condition and timepoint tested.
After rifampicin was added at day 7 for 72 hours of exposure, iPS-hepatocytes showed a significantly higher induction of CYP3A4 activity (p < 0.05), as well as a higher baseline, than monolayers (Figure 3). With the rifampicin concentration of 200µM, CYP3A4 activity increased 8.87 ± 1.22 times in spheroids and 4.45 ± 2.11 times in monolayers. Both models showed a similar cytotoxic sensitivity to rifampicin, with IC50 of 41.94µM in 3D and 51.04µM in 2D. CYP3A4 activity does not show an overall decrease with seemingly cytotoxic doses of rifampicin but only a decrease in its rate increase. The increased induction at the higher dose likely overcomes any fall due to reduced viability. When normalized for changes in viability, the spheroid culture shows a 55-fold induction over control with 200µM of rifampicin.
In addition, the unadhered magnetization method used here clearly provides a viable alternative to the typical plated magnetization method and allows manipulation of suspension cell types as well as 3D culture of adherent cell types without intermediate 2D culture.
Figure 3. CYP3A4 activity and viability of iPS-hepatocytes after 72 hour exposure to rifampicin (0–200µM). Panel A. CYP3A4 activity as measured by the P450-Glo™ CYP3A4 Assay and normalized by luciferin standards and cell number. A significant difference in CYP3A4 induction between monolayers (red) and spheroids (blue) was found (p < 0.05), as well as a higher baseline. Panel B. Viability as measured by CellTiter-Glo® assay. No significant difference was found between viability in 2D and 3D. Error bars represent standard error.
Magnetic 3D bioprinting presents a viable method for assembling and culturing hepatocyte spheroids. Magnetically bioprinted hepatocyte spheroids are easy to form for high-throughput testing, maintain phenotype in long-term culture, are easy to handle with high sample retention and fit within existing cell culture workflows. The Promega luminescent assays provide simple, effective methods for measuring hepatocyte function and viability at multiple time points in culture. Overall, we demonstrate that magnetic 3D bioprinting can meet the need for high-throughput hepatocyte testing amongst many needs in cell culture and cell-based assays.
This work was supported by a National Institutes of Health (NIH) Small Business Innovation Research (SBIR) Phase I and Phase II grant (R43ES024644) administered by the National Institute of Environmental Health Sciences (NIEHS).