Vascularized Co-Culture Clusteroids of Primary Endothelial and Hep-G2 Cells Based on Aqueous Two-Phase Pickering Emulsions

Abstract

Three-dimensional cell culture has been extensively involved in biomedical applications due to its high availability and relatively mature biochemical properties. However, single 3D cell culture models based on hydrogel or various scaffolds do not meet the more in-depth requirements of in vitro models. The necrotic core formation inhibits the utilization of the 3D cell culture ex vivo as oxygen permeation is impaired in the absence of blood vessels. We report a simple method to facilitate the formation of angiogenic HUVEC (human umbilical vein endothelial cells) and Hep-G2 (hepatocyte carcinoma model) co-culture 3D clusteroids in a water-in-water (w/w) Pickering emulsions template which can overcome this limitation. This method enabled us to manipulate the cells proportion in order to achieve the optimal condition for stimulating the production of various angiogenic protein markers in the co-cultured clusteroids. The HUVEC cells respond to the presence of Hep-G2 cells and their byproducts by forming endothelial cell sprouts in Matrigel without the exogenous addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers. This culture method can be easily replicated to produce other types of cell co-culture spheroids. The w/w Pickering emulsion template can facilitate the fabrication of 3D co-culture models to a great extent and be further utilized in drug testing and tissue engineering applications.

Keywords: 3D cell culture; angiogenesis; co-culture; water-in-water Pickering emulsions.

Figure 1

Schematic illustration of the HUVEC and Hep-G2 co-culture clusteroids in the w/w Pickering emulsion template and clusteroids angiogenesis. Created with BioRender.com.

Figure 2

Bright-field (A,E) and fluorescence microscope observation of mixed Hep-G2 and HUVEC (cell ratio = 5:1) cells encapsulated by 5.5 wt% DEX in 5.5 wt% PEO emulsions before (A–D) and after shrinking by 11 wt% PEO (E–H) set at FITC (B,F), CFSE far-red set at TRITC (C,G), and dual fluorescence FITC and TRITC channels (D,H). Clusteroids in the emulsions (A–D) and clusteroids taken out from the emulsions (E–H) were taken for imaging immediately after their formation. The Hep-G2 cells and HUVEC cells were pre-stained with CFSE and CFSE far-red, respectively. The bar is 50 μm.

Figure 3

Three-dimensional Z-stacked image of co-cultured Hep-G2 and HUVEC clusteroids at a cell ratio of (A–C) 1:1 and (D–F) 5:1 set at different fluorescence channels: FITC (A,D), CFSE far-red (B,E), and dual fluorescence (C,F). The box size is 400 μm × 400 μm.

Figure 4

(A–D): Bright-field observation of the co-culture clusteroids sprouting after 7 days of culture with a cell ratio of 5:1 (Hep-G2:HUVEC) in 5 mg mL⁻¹ Matrigel. The corresponding Wimsprouts analysis of (A,B) is shown in (C,D). The bar is 50 μm.

Figure 5

SEM observation of the: (A) individual Hep-G2 clusteroids, (B) individual HUVEC clusteroids, and (C) co-culture of Hep-G2 and HUVEC clusteroids at a cell ratio of 5:1. The cells kept growing in the Matrigel before the gels were degraded.

Figure 6

(A) Angiogenesis array membrane of individual Hep-G2 clusteroids, HUVEC clusteroids, and the co-culture Hep-G2 and HUVEC clusteroids at a Hep-G2 to HUVEC cell ratio of 5:1 after 21 days of proliferation in the Matrigel. The initial total cell number was 1 × 10⁶ mL⁻¹. (B) Angiogenesis-related protein production in the individual Hep-G2 clusteroids, HUVEC clusteroids, and the co-culture Hep-G2 and HUVEC clusteroids. Data were plotted as mean ± standard deviation of at least three independent experiments. Statistically significant differences between each region are denoted by * (p < 0.05) or ** (p < 0.01).

Figure 7

(A): VEGF; (B): MMP-2; (C): HIF1-α; and (D): IGFBP-1. Production of the individual Hep-G2 clusteroids, HUVEC clusteroids, and co-culture clusteroids at a cell ratio of 5:1 (Hep-G2: HUVEC) during 21 days of proliferation in 5 mg mL⁻¹ Matrigel. The initial total cell number was 1 × 10⁶ mL⁻¹ for all three sets. Data were plotted as mean ± standard deviation of at least three independent experiments. Statistically significant differences between each region are denoted by * (p < 0.05) or ** (p < 0.01).