Innovative 3D Tri-Culture Systems for Advanced in vitro Tissue Interface Engineering

Abstract

3D tissue engineering has enhanced the study of complex structures like the tendon-to-bone interface; it consists of tendons, uncalcified fibrocaltogenous and calcified fibrocartigoues, and bone cells. The unique gradual arrangement allows for smooth transfers of mechanical force during mobility without compromising anatomical integrity. In vitro, studies require complex models to imitate such an environment. This study investigates the fabrication of a novel design with two compartments for different cell-encapsulating hydrogels, separated by a 2mm gap. The gap can be left empty, filled with hydrogel, or populated with a third cell type to suit various experimental needs. A 3D-printed resin lid with six inferiorly projected positive rectangular impressions was designed to fit 6-well plates using computer-aided design software. 7ml polydimethylsiloxane (PDMS) mixture was cast inside each well around the rectangular shapes, leaving six cured elastomeric rectangular wells, which were extracted and mounted on a glass slide utilising PDMS crosslinker as a bonding agent. Additionally, a 35mm plate's lid was utilised as a cover. The system's integrity was tested with a manual peeling test and daily microscopic observation. Fluorescent confocal microscopy assessed Compartmentalization by individually labelling osteoblast and fibroblast rat cells before encapsulating them separately in fibrin gel and cell viability, which was assessed using Calcein AM and Propidium Iodide staining. The well-on-glass slide system successfully fabricated and passed integrity and sterility tests with the ability of containing up to 7.14 ml working solution, it also supported more than 98% of cell viability at day 7, proliferation, while both cells miniating their 3D form with in the fibrin gel up to day 10 of the experiment. In conclusion, our novel 3D cell culture system promises more in-depth investigations of cellular behaviours, migration and differentiation in the formation of tissue interfaces. To aid in advancing our understanding of more complex anatomical structures such as interfaces through in vitro studies to ultimately, advance the clinical application.