Salvadori, A., Watanabe, M., Markovic, M., & Ovsianikov, A. (2023, November 2). Laser-Generated Vascular Structures for Liver-on-a-Chip [Poster Presentation]. LBG Meeting Innovation in Health Sciences 2023, Wien, Austria.
E308-02-3 - Forschungsgruppe 3D Printing and Biofabrication
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Date (published):
2-Nov-2023
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Event name:
LBG Meeting Innovation in Health Sciences 2023
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Event date:
2-Nov-2023 - 3-Nov-2023
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Event place:
Wien, Austria
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Keywords:
Two-photon patterning; Microvasculature; Organ-on-chip; Channels' endothelialization; Endothelial barrier function
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Abstract:
Organs-on-Chips (OoC) consist of multicellular constructs organized in a 3D manner to faithfully replicate in vitro specific functions of human organs or tissues in a highly controlled microenvironment. However, the lack of a proper and functional microvascular network remains a significant challenge, which prevents a steady supply of nutrients and oxygen and, therefore, long-term maintenance in terms of viability, morphology, and functionality. Various biofabrication methods, such as 3D bioprinting and templating strategies, have been implemented to guide and stimulate the formation of microvascular networks in a controlled and reproducible way but limited in resolution and precision (1,2). Here, we demonstrate how femtosecond laser-based bioprinting techniques could guide the endothelial cells growth and lead to perfusable and physiologically relevant microvascular structures directly on-chip.
A microfluidic device was loaded with a mixture of type I-collagen:Matrigel (3:1) prepared following a two-step gelation protocol and two-photon laser patterning was performed across the hydrogel region to create hollow cylindrical channels. HUVECs (Lonza) were seeded in both side channels, and a VEGF gradient was established across the hydrogel to stimulate their controlled proliferation and migration. At seven days, the vascular structures were perfused with FITC-Dextran to study the barrier function and stained for specific endothelial key markers, such as CD31, VE-cadherin, and ZO-1.
We demonstrated that the two-photon laser patterning led to forming perfusable cylindrical channels, faithful in size and geometry. In addition, the two-step gelation protocol minimized the cell-induced channels’ deformation, creating fully-endothelialized vascular structures stable for at least seven days of culture. High expression of CD31, VE-cadherin, and ZO-1 confirmed the presence of adherent junctions and intercellular connections. Moreover, the diffusion of FITC-Dextran demonstrated a reduced diffusional permeability in the abluminal region compared to the acellular channels, suggesting the reproduction of vascular barrier function. Future works will focus on replicating organ-specific microvasculature for OoCs in terms of viability, morphology, and functionality maintenance.