Fabrication of Angiogenic Sprouting Coculture of Cell Clusteroids Using an Aqueous Two-Phase Pickering Emulsion System

Abstract

Tumor cell spheroids and 3D cell culture have generated a lot of interest in the past decade due to their relative ease of production and biomedical research applications. To date, the frontier in tumor 3D models has been pushed to the level of personalized cancer treatment and customized tissue engineering applications. However, without vascularization, the central parts of these artificial constructs cannot survive without an adequate oxygen and nutrient supply. The formation of a necrotic core into in vitro 3D cell models still serves as the major obstacle in their wider practical application. Here, we propose a rapid formation protocol based on using a water-in-water (w/w) Pickering emulsion template to generate phenotypically endothelial/hepatic (ECV304/Hep-G2) coculture cell clusteroids with angiogenic capability. The w/w Pickering emulsion template was based on a dextran/poly­(ethylene oxide) aqueous two-phase system stabilized by whey protein particles. The initial cell proportion in the coculture clusteroids can easily be manipulated for optimal performance. The cocultured pattern of the endothelial/hepatic cells could significantly promote the production of angiogenesis-related proteins. Our study confirmed that cocultured clusteroids can stimulate cell sprouting without the addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers at a 1:2 ratio of Hep-G2/ECV304. Angiogenesis gene production in the coculture clusteroids was enhanced with VEGF, urea, and insulin-like growth factor-binding protein along with angiogenesis-related marker CD34 levels, also indicating angiogenesis progress. Our aqueous two-phase Pickering emulsion templates provided a convenient approach to vascularize a target cell type in 3D cell coculture without additional stimulating factors, which could potentially apply to either cell lines or biopsy tissues, expanding the clusteroids downstream applications.

Engineering pre-vascularized 3D tissue and rapid vascular integration with host blood vessels via co-cultured spheroids-laden hydrogel

“…In a hydrogel, the ability of co-cultured cells to remodel the ECM allows them to make cells sprout out of the initially loaded pores of the hydrogel by reconstructing the hydrogel network, and the cells can also interact with neighboring cells [42]. For example, human dermal fibroblasts and microvascular ECs (1:1 ratio) encapsulated within a polyethylene glycol hydrogel and a co-cultured spheroid composed of Hep-G2 and ECs (1:1 ratio) within an alginate hydrogel achieved significantly enhanced expression of MMP2, and MMP9, respectively, compared with a mono-cultured group [43,44]. Similarly, the co-cultured spheroids (3:1 ratio of hADSCs/HUVECs) revealed that the ECM remodeling ability was greater than that of mono-cultured spheroids within a GelMA hydrogel (figures 3(b) and S3).…”