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.