(L-479) Hepatocyte-fibroblast spheroid triculture with endothelial cells for the construction of vascularized hepatic tissues in a microfluidic device

Introduction:: Introduction: The liver is a highly vascularized organ that is critical in maintaining homeostasis. However, hepatocytes rapidly lose their differentiated function in a 2D environment. The diffusion limit within a tissue is a significant challenge in achieving large tissue sizes in 3D hepatic culture models. An important step of vascularization of in vitro liver tissues is the formation of new blood vessels, improving oxygen and nutrient supply, and promoting the development of functional liver tissues. Although some studies reported the use of spheroid angiogenesis models with hepatic cell lines or hepatic cancer cells [1], no model involving primary hepatocytes has been reported yet. In this study, we developed a culture model of a pro-angiogenic spheroid involving primary hepatocytes co-cultured with fibroblasts, which were introduced as supporting cells. We then utilized the secreted growth factors from the hepatocyte-fibroblast spheroid to induce the vascularization of hepatic tissue in a microfluidic device.


Materials and Methods:: Materials and Methods: In this study, primary hepatocytes were isolated from rats using the two-step collagenase perfusion method. Cell suspensions of primary hepatocytes and normal human lung fibroblasts were seeded in a non-adhesive concave plate and cultured for two days to form a spheroid. The spheroid was then suspended in a fibrin-collagen solution and introduced into a gel region in a microfluidic device (Figure 1A). After 20 minutes gelation, human umbilical vein endothelial cells were seeded in the adjacent channels for triculture, and half of the culture medium was refreshed daily. An angiogenic process was observed daily using a phase-contrast microscope. The cells were then fixed on day 14 and visualized using immunofluorescence staining. Three-dimensional analysis was performed using a confocal microscope.


Results, Conclusions, and Discussions:: Results and Discussion: Immunofluorescence staining images revealed the formation of microvascular networks with a lumen structure. The observation of microvessels approaching and extending into a hepatocyte-fibroblast spheroid indicated successful angiogenesis after 14 days of culture (Figure 1B). This finding also demonstrated that the proangiogenic growth factors secreted by hepatocytes and fibroblasts could create an environment to promote angiogenesis in the triculture.


Conclusion: This study describes a novel culture model for promoting angiogenesis in primary hepatocyte spheroids. The vascularization in the model relies on the secretion of pro-angiogenic growth factors by hepatocytes and fibroblasts, which attract endothelial cells to form microvessels extending into the hepatocyte-fibroblast spheroid.


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References (Optional): : Reference: [1] Nashimoto Y. et al., Biomaterials, 2020; 229: 119547.