Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications

We present (1) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and (2) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 mu l each within 3 min at a pitch of 2.3 mm within an array of 48 x 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 x 3 HUVEC spheroids with intermediate distances of 500 mu m embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 h of incubation indicating a good viability of the deposited spheroids.