High-Voltage Electrostatic Field Hydrogel Microsphere 3D Culture System Improves Viability and Liver-like Properties of HepG2 Cells

Abstract

Three-dimensional (3D) hepatocyte models have become a research hotspot for evaluating drug metabolism and hepatotoxicity. Compared to two-dimensional (2D) cultures, 3D cultures are better at mimicking the morphology and microenvironment of hepatocytes in vivo. However, commonly used 3D culture techniques are not suitable for high-throughput drug screening (HTS) due to their high cost, complex handling, and inability to simulate cell-extracellular matrix (ECM) interactions. This article describes a method for rapid and reproducible 3D cell cultures with ECM-cell interactions based on 3D culture instrumentation to provide more efficient HTS. We developed a microsphere preparation based on a high-voltage electrostatic (HVE) field and used sodium alginate- and collagen-based hydrogels as scaffolds for 3D cultures of HepG2 cells. The microsphere-generating device enables the rapid and reproducible preparation of bioactive hydrogel microspheres. This 3D culture system exhibited better cell viability, heterogeneity, and drug-metabolizing activity than 2D and other 3D culture models, and the long-term culture characteristics of this system make it suitable for predicting long-term liver toxicity. This system improves the overall applicability of HepG2 spheroids in safety assessment studies, and this simple and controllable high-throughput-compatible method shows potential for use in drug toxicity screening assays and mechanistic studies.

Keywords: 3D cultures; RNA-seq; hepatocyte; high-voltage electrostatic field; hydrogel; microspheres.

Figure 1

(a) Schematic diagram of the microsphere generation device, the liquid surface (D), the injection speed (S), and the voltage (V), which were changed using high-voltage power supply (HVPS) and motor; (b) the internal structure of the working chamber, including the injection needle connected to the positive terminal of the HVPS, the negative plate, and the petri dish containing the calcium chloride solution; and (c) simulation showing the electric field between the needle tip and the negative plate generated by the high-voltage power supply.

Figure 2

Size of microspheres when different parameters were used. (a) The electric field force was changed and affected the size of microspheres when varying the voltage. (b) The working distance was varied to change the electrostatic field and affected the size of microspheres. (c) Effect of injection speed on the size of microspheres. The microspheres were analyzed via light microscopy (scale bar: 200 µm).

Figure 3

Preparation of hydrogel materials. (a) Hydrogel materials were prepared with different concentrations of sodium alginate and cooling methods, and the three-dimensional structures were observed via scanning electron microscopy (scale bar: 40 µm). (b) Bioactive hydrogels were prepared by adding high (A: 1000 µL), medium (B: 500 µL), and low (C: 250 µL) doses of sodium periodate (0.25 M) to 1% sodium alginate solution and grafting 25 μg/mL and 50 μg/mL of Arg-Gly-Asp (RGD) peptide, respectively. Effects of degree of hydrogel oxidation and engrafted RGD concentration on the viability of HepG2 cells were compared. (c) Effect of adding gelatin or collagen to RGD peptide-grafted oxidized sodium alginate hydrogel (ROSAH) on hepatocyte proliferation (n = 5; ** p < 0.01 and *** p < 0.001 relative to 25C group).

Figure 4

Enhanced viability, metabolic and biotransformation competence, and lower drug sensitivity of HepG2 cells in the RCHM (Gel) culture system. (a) Proliferation of HepG2 cells in 3D versus 2D culture models. (b) EROD activity (phase I), (c) UGT activity (phase II), and (d) albumin production and (e) urea secretion in various culture models. (f) Drug sensitivity of different hepatocyte models, the purple dotted line represents 50% inhibitory rate (n = 4; * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to 2D; # p < 0.05, ## p < 0.01 and ### p < 0.001 relative to Gel).

Figure 5

Live and dead HepG2 cells were stained using Calcein-AM /PI in different 3D culture models, the viable cells were stained with Calcein-AM (green), while the dead cells were stained with PI (red), and the nuclei were stained with DAPI (blue) (scale bar: 75 µm).

Figure 6

Analysis of gene expression profiles of various HepG2 cell models. (a) Correlation heatmap of samples; (b) co-expression Venn diagram; (c) histogram showing the number of different combinations of DEGs; and (d) heatmap showing differentially expressed gene clusters (3 biological replicates per group).

Figure 7

Differential expression of marker genes among different hepatocyte models. (a) Fold change in gene expression between 3D and 2D cell models. (b) KEGG pathway enrichment analysis showing the annotation of important liver pathways and (c) fold change of the expression of DEGs in the hydrogel compared to the other 3D models. Three biological replicates per group; |log2 (fold change)| ≥ 1 p ≤ 0.05 were the thresholds indicating a significant difference between cellular models.