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The conventional method of culturing primary human hepatocytes (PHH) in a 2-dimensional (2D) monolayer presents limitations in the study of hepatic biology, liver function, and drug-induced hepatotoxicity. Traditional 2D hepatocyte cultures dedifferentiate, resulting in the loss of specific hepatic function in approximately 5 days. We have developed and characterized a PHH 3-dimensional (3D) spheroid culture system that preserves hepatic function and promotes culture longevity.
Gibco PHH can easily be assembled into a 3D spheroid culture in 5 days using Thermo Scientific Nunclon Sphera low-attachment U-bottom 96-well microplates and Gibco plating medium and plating supplements. The 3D spheroid hepatocyte culture requires a significantly lower number of cells than its 2D counterpart, allowing this system to better support high-throughput assays. Moreover, the PHH in the 3D spheroid culture are functionally viable for at least 3 weeks, enabling long-term studies of hepatocyte function.
Figure 1. Workflow of assembly and characterization of primary hepatocytes in 3D spheroid culture.(A) Spheroids were imaged in phase at 10x magnification. These images show spheroid formation by day 5 of culture. (B) Spheroid size is directly proportional to the number of cells seeded. Spheroids were imaged using the Invitrogen EVOS FL Auto 2 Cell Imaging System (Cat. No AMAFD2000) at 4x. (C) Plating of hepatocyte spheroids in a Nunclon Sphera 96-well U-bottom microplate shows consistency in spheroid formation across the plate.
Figure 2. Evaluation of formation of bile canaliculi in hepatic spheroids. HepG2 spheroids during week 2 (left) and hepatic spheroids during week 1 (right) were stained with 5-CFDA and DAPI and imaged using the Thermo Scientific CellInsight CX7 platform at 10x magnification. Hepatic spheroids show clear formation of bile canaliculi in comparison to the HepG2 spheroids (used as the negative control).
Figure 3. Albumin secretion in 2D and 3D spheroid hepatic cultures. The concentration of albumin secreted is normalized to the total number of cells per well.
Figure 4. CYP3A4 activity in 2D and 3D spheroid hepatic cultures. CYP3A4 activity was measured using the Promega P450-Glo CYP3A4 Assay with Luciferin-IPA. CYP3A4 activity was found to be significantly higher in the 3D spheroids than in the 2D culture. The data presented are the mean ± SEM (n = 3 for the 2D culture, n = 8 for the 3D spheroids).
Figure 5. ATP synthesis by 3D spheroid hepatic cultures is proportional to the number of cells. Using the CellTiter-Glo 3D Cell Viability Assay in 3 replicates, ATP synthesis by individual spheroids was measured during week 1.
Figure 6. Drug-induced cytotoxicity assayed using 3D spheroid hepatic cultures.
Drug | IC50 (2D culture) | IC50 (3D culture) |
---|---|---|
Chlorpromazine | 34 μM | 14 μM |
Diclofenac | 331 μM | 396 μM |
Table 1. IC50 of drug-induced cytotoxicity for 2D hepatocytes and 3D spheroid hepatic cultures.
Collectively, these data confirm that cultures of 3D spheroid–qualified human hepatocytes have been characterized to show stable morphology, viability, and hepatocyte-specific functions for at least 3 weeks. We have demonstrated that our 3D spheroid–qualified hepatic cultures are functional, as indicated by formation of bile canaliculi as well as sustained albumin secretion. In comparing CYP3A4 activity on day 5 of 2D hepatic cultures and during week 1 of 3D spheroid hepatic cultures, we have shown that 3D spheroid cultures have significantly higher activity (Figure 4). We also show that this 3D spheroid hepatic culture system can be used to analyze drug-induced cytotoxicity in hepatocytes. Ultimately, these data indicate that the reduced number of cells required for 3D spheroid formation as well as the sustained longevity of these cultures may better support high-throughput assays and long-term studies of hepatocyte functions.
Find out more at thermofisher.com/admetox and thermofisher.com/spheroid
P450-Glo and CellTiter-Glo are a trademarks of Promega Corporation. GraphPad Prism is a trademark of GraphPad Software, Inc.
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