A Novel 3D Culture System for High-Throughput Hepatoxicity Screening

Cells grown as three dimensional (3D) spheroids are thought to more closely mimic the in vivo physiology in terms of morphology, structural complexity and phenotype. Being more physiologically relevant, 3D cultures can be highly predictive for compound profiling and evaluation of cytotoxicity, a critical step when evaluating chemotherapeutic drug candidates.

Unfortunately, evaluation of drug cytotoxicity has traditionally relied on use of two dimensional (2D) cell culture monolayers. When grown in monolayers, cells are not exposed to soluble gradients, are forced into an apical-basal polarity, reside on a continuous layer of matrix with adhesions restricted to an x-y plane, and undergo unconstrained spreading and migration in an x-y plane. In contrast, the 3D environment replicates the presence of soluble gradients and eliminates a forced, unnatural polarity. Cells interact with discrete matrix fibrils, with adhesions distributed in all three dimensions and spreading and migration are sterically hindered. In the case of spheroids, distinct populations of cells can be observed including a proliferating rim, a middle layer of quiescent cells and a hypoxic core enriched with necrotic cells.

The difference in how cancer cells respond to chemotherapeutic drugs when grown in 2D versus 3D is profound. Figure 1 show the inhibition of primary pancreatic tumor cell growth in response to known chemotherapeutic agents, when cells were grown in monolayers or as spheroids. The difference in response from 2D to 3D can confound efforts to identify potent chemotherapeutics – whether screening for efficacy against the tumor type or off-target toxicity.¹

Figure 1.

Dose response curves vary significantly when primary pancreatic tumor cells are grown in monolayer cultures (A) as compared with 3D cystic organoids (B). Each curve represents the mean and standard deviation of 4 replicates per condition.

High Throughput Requirements

While the advantages of 3D cell culture are well-recognized, conducting cytotoxicity studies in a large scale has historically been considered either too low in throughput or too costly to make it easily amenable to high throughput screening (HTS). An early challenge in developing a high throughput assay to assess cytotoxicity was the 1536-well plate itself and the need for compatibility with a fully automated robotic platform – which off-the-shelf designs didn’t offer.

Prior to developing the hepatotoxicity assay, we implemented and validated use of a custom, ultra-low attachment 1536 round-bottom well spheroid plate to develop a 3D viability assay assessing the cytotoxic effect of drugs on HT-29 colon carcinoma cells.² The novel 3D 1536-well plate addressed the requirements outlined below and is now commercially available (Corning, cat. no. 4527):

• Simplicity of use

• Absence of the requirement for an aspiration step during the entire spheroid formation and testing process

• Uniformity and reproducibility of spheroid formation in the center of each well, both across the plate and from plate to plate

• Ability to visualize the spheroids via clear well bottoms; black sidewalls reduce cross-talk and background noise in fluorescent and luminescent-based assays

• Compatibility with widespread 1536-well plate compatible instrumentation

The cytotoxicity assay using HT-29 spheroids enabled processing of an average of 10 plates per hour. This translates to approximately 15,000 wells per hour, well within the ultra-high-throughput parameters of 100,000 test compounds per day. Applying a similar approach, we developed a 3D primary human hepatocyte culture system using the same 1536-well plates enabling high throughput, cost-effective hepatotoxicity determination.

Hepatotoxicity Determination via HTS

Drug-induced liver injury (DILI) is a significant concern for the pharmaceutical industry as it is the leading cause of failure during drug development and a major cause of drug withdrawal. More than 900 drugs, toxins, and herbs have been reported to cause liver injury, with hepatotoxicity accounting for 11–13% of acute liver failure in the U.S. Compounds can injure hepatocytes in a variety of ways including membrane disruption, blocking transport pumps and interfering with cytochrome P450 enzymes.

In addition to selecting a 1536 well-plate that would be compatible with the high throughput assay, choice of the specific cell type is critical when evaluating hepatotoxicity. While a number of in vitro approaches are used to predict the potential for drug candidates to cause hepatotoxicity, each has critical shortcomings. The utility of immortalized cell lines and primary liver cells is impacted by limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression. When cultured in monolayer, primary hepatocytes undergo changes in cell morphology, structure, polarity, gene expression, and liver-specific functions, a process referred to as de-differentiation. Liver-derived immortalized cell lines do not possess phenotypic characteristics of liver tissue and can have gene expression profiles that vary between passages or differ from primary hepatocytes.

The model system presented here conserves liver phenotype and prolongs viable culture time, two essential requirements for a robust screening method. The system uses HepatoCells and companion medium (Corning, cat no. 354881 and 354882). HepatoCells were developed to retain the morphology and physiological properties of primary human hepatocytes but overcome the inherent shortcomings such as large inter-individual variability and finite supply. The single-use, cryopreserved immortalized cells are derived from primary human hepatocytes, have low lot-to-lot variability, high cell purity (>99% hepatocyte-like cells) and robust activity induction response for major CYP enzymes. With a virtually unlimited supply and consistent performance, HepatoCells offer a reliable in vitro hepatic model for ADME/Tox characterizations and other liver research applications.

The culture system was first developed and evaluated in 384-well format (Figure 2A). Cells were plated at 50 μL at varying densities to understand hepatocyte spheroid formation and to assess whether the response was linear and determine the Z-factor (Z’), a tool for comparing and evaluating the quality of high throughput assays.³ Bright field images of the wells show uniform, homogeneously rounded spheroids (Figure 2B).

Figure 2.

384-well plate assay protocol (A) and appearance of spheroids (B).

Following incubation of the HepatoCells with test compounds and controls, cell viability was determined using the CellTiter-Glo® 3D cell viability assay (Promega, cat no. G9681). The assay reagent penetrates large spheroids and measures ATP as an indicator of viability, generating a luminescent readout. Figure 3 shows the concentration response curves (A) and Z’ values at different time intervals following addition of the CellTiter-Glo® (B). Across all time intervals, the Z’ values were excellent, ranging from 0.79 at 15 minutes to 0.90 at 60 and 90 minutes, respectively. A Z’ value of 0.5 or higher is indicative of compliancy with HTS requirements.

Figure 3.

Concentration response curves for drugs tested on HepatoCells in 384-well format. Assay statistics over time show Z’ values greater, and in some cases, significantly greater than 0.5. 12 replicates per condition was used to determine the statistics. The curve represents the mean and standard deviation of 8 replicates per each concentration

The model system was then converted to a 1536-well format (Figure 4A). Plates were monitored over the course of 7 days to ensure appropriate formation of spheroids (4B). By day 4, rounded spheroids had formed at lower initial seeding densities; at higher densities, spheroids are more amorphous. Bright field images, combined with Z-stacking images, confirmed the wells contained 3D spheroids. Additional studies demonstrated that luminescence readings and spheroid volumes were linear across a range of cell concentrations and that spheroids were homogenous across the wells and centered in each well, important considerations for high throughput screening (data not shown). Z’ values confirmed the robustness of the assay, now in a 1536-well format. A cell seeding density of 1000 cells/well was used in subsequent assays.

Figure 4.

Assay protocol adapted for 1536-well plates and assessment of HepatoCells spheroids using 10x objective. Bar scale in lower right is 100 μm.

Screening NCI and FDA Libraries

National Cancer Institute (NCI) and Food and Drug Administration (FDA) libraries of approved oncology drugs were screened via this high throughput assay to assess hepatotoxicity. With a nominal starting concentration of 24.8 μM, the NCI compounds were tested in triplicate as a 10-point, 3-fold serial dilution. A compound was qualified as a hit if it had a cytotoxic concentration of 50% (CC50) less than 10 μM. A total of 11 hits were found among this library of 99 compounds. A similar assessment was completed for the FDA library in which compounds were tested at a nominal single concentration of 10 μM against ~2820 oncology drugs approved in the US, Canada, EU and Japan and resulted in a 3.7% hit rate, which is considered high compared to other 3D cytotoxicity assays.¹ Hit rates vary between assays and is very dependent on the assay sensitivity but we typically observe a hit rate around 0.5–1%.

Active hits from the FDA library screening were then tested in a dose dependent manner for confirmation and potency analysis. The report shown in Table 1 summarizes the dose response results along with compound information, promiscuity, response data and confirmation screening for the five most potent hits from the FDA library. The first compound listed, dactinomycin, is quite potent in terms of cytotoxic profile with an IC50 of 8.5 nM. This drug has reported clinical cases of severe liver injury including sinusoidal obstruction syndrome and acute toxic injury to hepatocytes.⁴

Table 1.

Sample of results from screening of FDA library versus Hepatocells in 1536-well plate assay along with dose response curves

Conclusion

Having the right model for drug screening is essential for predicting compounds that may cause drug-induced liver injury. The 3D hepatocellular model system described here may offer investigators significant advantages over traditional 2D monolayer cell culture in terms of maintaining morphological and functional characteristics of tissue and providing a better representation of in vitro drug toxicity. This high throughput assay effectively identifies hepatocellular toxic compounds and can provide insight into marketed drugs that may not have initially appeared to be hepatotoxic.