To produce TEBVs with complex geometry and to improve cell seeding along the scaffolds, we developed a rotary system with a random rotation movement allowing effective and uniform cell distribution. Previously, the production of self-assembled linear small-caliber blood vessels seeded on polyethylene terephthalate glycol (PETG) pretreated with ultraviolet-C rays (UV-C) has been shown to ensure proper cellular attachment and optimized extracellular matrix (ECM) secretion/assembly 14. However, these are not ideal for production of more complex geometry tri-layered TEBV composed of an adventitia, a media and an intima tunica 4, 14, 15, 16. The current state of dynamic cell seeding allows an easy production of linear TEBV with the use of roll bottles and the perfused seeding of endothelial cells in a tubular construct. Furthermore, in a tri-dimensional (3D) environment, a uniformly monitored cell distribution is needed to foster homogeneous tissue remodeling and to avoid competition for nutrients in areas with higher cell densities 10, 11, 12, 13. Consequently, dynamic cell seeding techniques have established themselves over simpler static approaches 7, 8, 9. One of the challenges in vascular tissue engineering is still, however, to improve cellular seeding, distribution, and organization to homogeneously incorporate cells onto a tubular structure. Different techniques to generate TEBVs have been developed over the years, each showcasing pros and cons, and can be classified in three main categories: (1) vascular conduits made of cells seeded on manufactured scaffolds, (2) vascular conduits made by cell-sheet engineering and (3) bioprinting 5, 6. Through model refinement, it is now possible to produce patient-derived tissue-engineered blood vessels (TEBV) with defined genetic backgrounds to better understand the pathobiology behind vascular diseases 3, 4. The advancement of tissue engineered vascular grafts in recent years presents a promising clinical option for the treatment of vascular diseases or to provide alternative in vitro models to study these complex disorders 1, 2. The production of patient-derived small-caliber TEBVs with complex geometry and optimized cellular distribution all along the vascular reconstructed may be an innovative way to model various vascular diseases such as intracranial aneurysms. With this simple to use spherical system, fully biological branched TEBV constructs were also produced by seeding human fibroblasts directly on custom-made complex geometry PETG mandrels. This spheric seeding method was compared to other approaches, such as dynamic and static seeding, and clearly shows uniform cell distribution on PETG scaffolds. The seeding conditions, such as cell concentration, seeding speed and incubation time were optimized via count of cells adhered on the PETG scaffolds. Custom made seeding chambers are placed inside the system and hold Y-shaped polyethylene terephthalate glycol (PETG) scaffolds. In this report, the design and fabrication of an innovative seeding system with random spherical 360° rotation is described. The use of a novel spherical rotary cell seeding system allows effective and uniform dynamic cell seeding for a viable in vitro tissue-engineered model. The main goal of the work reported in this article was to produce an entirely human branched small-caliber TEBV. Moreover, there is a need for complex geometry TEBV for study of multifactorial vascular pathologies, such as intracranial aneurysms. Tissue-engineered models have also proven to be valuable tools in disease modelling. Entirely biological human tissue-engineered blood vessels (TEBV) were previously developed for clinical use.
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