Precision bioprinting-based extrusion of tumour spheroids on pre-matured in vitro tissue models on demand

Abstract Tissue-engineered tumour models utilizing organ-specific three-dimensional (3D) tumour spheroids are crucial for developing and validating novel imaging systems targeted to their detection. This study explores the use of 3D bioprinting to develop and standardize the precise placement of tumour spheroids on prefabricated tissue models. These models can be used as a validation platform for novel biomedical diagnostic devices. Such integrated systems enable rapid, minimally invasive detection and characterization of cancer cells, reducing reliance on traditional biopsy-based methods. The standardized deposition of spheroids onto prefabricated tissue models required 1. The development of a spring-loaded bracket to keep tissue samples in a defined position during 3D printing; 2. A custom-made, Python-based graphical user interface to facilitate the determination of spheroid printing positions within a standard 12-well cell culture plate. The program digitally reproduces the plate layout within a Cartesian coordinate system centred at the plate ’ s geometric midpoint, ensuring compatibility with standard 3D bioprinter coordinate systems; 3. A standardized workflow to define the time-point of extrusion of spheroids and placement onto tissue, which is predetermined accurately by suspending the spheroids in density-matched methylcellulose solution. The accurate placement of individual tumour spheroids (∼500 µ m) onto prefabricated tissue models was validated using a custom-made positioning analysis plate, yielding results of translational offset at x =21.50 µ m and y = 18.63 µ m. This work utilizes simple-to-follow and readily available lab instruments and customized 3D bioprinters, rather than acquiring specialized equipment. Future studies will focus on shortening the workflow presented here and assessing its adaptability to other particle-based tissue models, e.g. microgels.