A Method for High-Throughput Robotic Assembly of Three-Dimensional Vascular Tissue

Abstract

An essential step toward commercializing engineered tissues is to scale-up and automate their production. This presents a challenge for self-assembled tissues that are fragile at early time points and difficult to handle using robotic systems. The goal of this study was to automate tissue-engineered blood vessel (TEBV) fabrication by creating a custom cell seeding and self-assembly system that is conducive to robotic manipulation, coupled with a robotic system to assemble smooth muscle cell ring units into tissue tubes. To generate self-assembled tissue ring units manually, cells are seeded at a high density into custom agarose wells that have center posts (2 mm inner diameter), around which cells aggregate and contract to form rings. Agarose is well-suited for cell seeding and ring self-assembly because it is noncell adhesive, can be autoclaved, and reproducibly cast to form wells using silicone templates. However, agarose gel is soft and makes reliable robotic manipulation challenging. To solve this problem, we designed a custom ring self-assembly plate utilizing polyetherimide (PEI) wells with MED610 three-dimensional-printed center posts and an MED610 well negative that allows the casting of individual, annular agarose cell-seeding troughs within the PEI plate. Rings cultured in the new plate system were morphologically similar to rings cultured in control agarose wells and had a slightly higher failure load. To automate tube assembly, we created a unique robotic punch system to push tissue rings out of the PEI-MED610 plate onto a stainless steel mandrel to enable tube fusion. Tubes fabricated by manual or robotic ring removal and placement demonstrated similar morphology after tube fusion, and the automated system substantially reduced the time required to assemble tubes. In summary, we developed a novel robotic assembly system to precisely manipulate self-assembled tissue rings and enable scale-up and automation of TEBV biofabrication.

Impact Statement

Self-assembled tissues have potential to serve both as implantable grafts and as tools for disease modeling and drug screening. For these applications, tissue production must ultimately be scaled-up and automated. Limited technologies exist for precisely manipulating self-assembled tissues, which are fragile early in culture. Here, we presented a method for automatically stacking self-assembled smooth muscle cell rings onto mandrels, using a custom-designed well plate and robotic punch system. Rings then fuse into tissue-engineered blood vessels (TEBVs). This is a critical step toward automating TEBV production that may be applied to other tubular tissues as well.