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


Alan Faulkner-Jones1, Sebastian Greenhough2, Jason A King2, John Gardner2, Aidan Courtney2 and Wenmiao Shu1

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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.


87.85.Lf Tissue engineering

87.15.N- Properties of solutions of macromolecules

87.17.Ee Growth and division


Biological physics


Issue 1 (March 2013)

Received 15 November 2012, accepted for publication 16 January 2013

Published 4 February 2013


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