GFP Reporter Cell Line - HUVEC

The construction of vascular networks within microfluidic chips is crucial for the long-term culture of three-dimensional (3D) cellular aggregates-such as spheroids, organoids, tumor organoids, or tissue explants. Although microvascular network systems and organoid technologies have advanced rapidly, achieving vascularization within "organoids-on-chips" models remains a significant challenge in the field of tissue engineering. Most existing microfluidic devices struggle to accurately mimic the complex fluid dynamics found in vivo and often require cumbersome technical setups. In light of these limitations, researchers have developed an innovative platform for constructing and monitoring vascular endothelial networks in real time. These networks can be formed around mesenchymal spheroids, pancreatic islet spheroids, and vascular organoids derived from pluripotent stem cells; these 3D biological structures can be continuously cultured on-chip for up to 30 days. The results demonstrate that these vascular endothelial networks establish functional connections with the endothelial cell-rich spheroids and vascular organoids, successfully providing effective intravascular perfusion to these 3D structures. Furthermore, the researchers found that the vascularized culture method proposed in this study significantly promotes the growth, maturation, and functional performance of organoids on-chip. This microphysiological system offers a viable "organ-on-chip" model for achieving the vascularization of various 3D biological tissues, thereby laying a solid foundation for realizing the perfusion culture of organoids using advanced microfluidic technologies.

Here, researchers constructed cellular aggregates composed of human fibroblasts and GFP-labeled HUVECs (hereinafter referred to as "mesenchymal spheroids") and seeded them into microfluidic channels. They first investigated the effects of fluid flow by culturing these mesenchymal spheroids either individually or under static versus flow conditions (Figure 1a). The results demonstrated that under dynamic perfusion conditions, the formation of endothelial networks was significantly enhanced. Compared to static conditions, the number of vascular junctions, meshes, and vascular segments, as well as the total segment length, increased significantly by 4.4-fold, 6.5-fold, 5.0-fold, and 4.8-fold, respectively. These findings indicate that flow conditions can directly drive the differentiation of these mesenchymal spheroids toward vascular-like structures (Figure 1a-c).

Figure 1. Generation of anastomosed endothelial networks through functional vascularization of mesenchymal spheroids. (Quintard C, et al, 2024)

Subsequently, the researchers incorporated HUVECs into a hydrogel mixture with the aim of establishing functional connections between the HUVECs lining the inner walls of the microchannels and the entrapped mesenchymal spheroids. The HUVECs within the spheroids expressed GFP, whereas the HUVECs suspended within the gel prior to injection expressed RFP; this design enabled the real-time visualization and distinction of the different cell populations and their interactions (Figure 1d). On Day 0, the mesenchymal spheroids were introduced into the microchannels and precisely captured at predetermined positions, where they maintained their original spherical morphology. By Day 3, preliminary tissue organization of the endothelial cells was observed; by Day 7, a network-like structure with a three-dimensional configuration had formed, remaining stable through Day 13 (Figure 1d). Spontaneous anastomosis occurred between the layer of RFP-labeled HUVECs and the vascular structures within the mesenchymal spheroids (Figure 1d, inset), thereby establishing an interconnected network at the capture sites.