Development of a valve-based cell printer for the formation of human embryonic stem cell spheroid aggregates

TLDR

The recent rise of inkjet‑based cell printing has spurred interest in biofabrication, yet achieving controllable, low‑damage processes that preserve cell viability remains a key challenge. This study develops a valve‑based cell printer that can deposit highly viable cells in programmable patterns with sub‑five‑cell droplets and evaluates how human embryonic stem cells respond to this printing process. The printer overprints opposing gradients of hESC‑laden and medium‑only bio‑inks to create uniform droplets, which are then inverted so cells aggregate by gravity into spheroids. The resulting aggregates have controllable, repeatable sizes (0.25–0.6 mm diameter for 5–140 cells) and retain stem‑cell viability and pluripotency, demonstrating the gentle, accurate nature of the valve‑based printing process.

Abstract

In recent years, the use of a simple inkjet technology for cell printing has triggered tremendous interest and established the field of biofabrication. A key challenge has been the development of printing processes which are both controllable and less harmful, in order to preserve cell and tissue viability and functions. Here, we report on the development of a valve-based cell printer that has been validated to print highly viable cells in programmable patterns from two different bio-inks with independent control of the volume of each droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human ESCs were used to make spheroids by overprinting two opposing gradients of bio-ink; one of hESCs in medium and the other of medium alone. The resulting array of uniform sized droplets with a gradient of cell concentrations was inverted to allow cells to aggregate and form spheroids via gravity. The resulting aggregates have controllable and repeatable sizes, and consequently they can be made to order for specific applications. Spheroids with between 5 and 140 dissociated cells resulted in spheroids of 0.25–0.6 mm diameter. This work demonstrates that the valve-based printing process is gentle enough to maintain stem cell viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency. This study includes the first analysis of the response of human embryonic stem cells to the printing process using this valve-based printing setup.