(J-379) Construction of microvessels with designed shape and size in laser-fabricated hydrogel for the study of vascular remodeling in vitro

Introduction:: In the field of tissue engineering, it is crucial to understand the mechanism of vascular remodeling to engineer microvessels in vitro [1]. It is widely accepted that flow-induced mechanical stimuli, especially shear stress, are important factors in vascular remodeling [2]. However, few in vitro models can precisely evaluate the effect of flow on microvessels. Microvascular networks are developed by endothelial cells in a self-organizing manner, making it difficult to construct simplified microvessels in vitro. In this study, we established an in vitro model of simplified microvessels in a microfluidic device with laser-fabricated hydrogel, which allows us to investigate the impact of flow on vascular remodeling.


Materials and Methods:: A femtosecond laser was utilized to generate multiphoton absorption in a gel region of a microfluidic device. Two types of hydrogel, collagen gel and a mixture of collagen and fibrin gel, were fabricated to create hollow structures by scanning the laser. Human umbilical vein endothelial cells (HUVECs) were then seeded in the luminal space of the hollow structures and cultured to form microvessels along the hollow structures. Flow was introduced to allow the culture medium to pass through the hollow structures, and the vascular formation was observed by phase-contrast microscopy until day 7. On day 7, the cells were fixed and stained to visualize actin filaments and cell nuclei, which were analyzed in three-dimensions using a confocal microscope.


Results, Conclusions, and Discussions:: Results and Discussion: First, reflection images of a gel region in a microfluidic device were obtained by confocal microscopy. We confirmed that hollow structures with the designed shape and size, such as a straight line in the scale of microvessels, were successfully fabricated within the gel. Next, we investigated whether the HUVECs seeded into the luminal space of the hollow structures could form microvessels. Fluorescence staining images of the cells on day 7 revealed the formation of microvessels along the hollow structures (Figure 1). Microvessel formation was observed both in collagen and fibrin-collagen gel. In addition, we found that vascular formation was promoted by the flow in a magnitude-dependent manner compared to static conditions. Furthermore, we found that expansion of HUVECs, especially during the first three days of the culture, is crucial for the stable construction of microvessels along the hollow structures.



Conclusions: In this study, we successfully constructed in vitro microvessels with designed shape and size both in collagen and fibrin-collagen gel. This culture model will enable us to further investigate the microvessel remodeling processes in reaction to hemodynamic cues.


Acknowledgements (Optional): :

References (Optional): : [1] Sano H. et al., Biofabrication, 2020; 12; 045008.

[2] Santamaria R. et al., Front. Physiol., 2020; 11: 1-21.