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.
Tests showed that 80-90% of the embryonic stem cells stayed viable.
a) Schematic drawing of the cell printer system. (b) Detailed schematic of the two dispensing systems.
The rapidly developing field of regenerative medicine promises to repair, replace and regenerate damaged cells, tissues or organs through stem cell therapy. Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to self-renew indefinitely and the potential to differentiate into any cell type. Totipotent stem cells can differentiate into all cell types found in an organism, whereas pluripotent stem cells can only differentiate into those cells which are found in an adult. These unique potency characteristics make hESCs ideal for use in a number of applications such as modelling early embryonic development. The potentially limitless numbers of differentiated hESC progeny can also be used for clinical tissue engineering/replacement applications such as novel drug discovery and testing for the pharmaceutical industry.
Although human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs) have been printed in the past, until now, there have been no reports of attempts to print hESCs. hESCs are known to be more sensitive to physical manipulation, more demanding in terms of their requirement for extracellular matrix coatings for routine cell culture and are more difficult to transfect with plasmid DNA. However, they do have a greater potential to generate a wider variety of differentiated cell types than hMSCs and tissues generated would be expected to yield better models of human biology than those using mESCs as precursors. Here, we report the first investigation into the response of hESCs to the printing process and associated spheroid creation procedure. A new cell printing platform has been developed, capable of depositing hESCs with precise quantity and high cellular viability, while maintaining the pluripotency of the stem cells. The combined methods of hanging-drop spheroid formation with valve-based cell deposition systems were used for the controllable and repeatable creation of uniform hESC spheroids of specific sizes. The combination of a single valve-based deposition system and the hanging-drop technique has recently been shown to be effective in producing spheroids from mESCs using a single nozzle system. However, in this paper we present a dual nozzle system which enables combinatorial printing of hESCs and results in a system with increased throughput as multiple bio-inks can be printed simultaneously. In addition we used microwell plates for aggregate creation by printing cells directly into the wells. This allowed the spheroids to form in situ without the need to transfer them to a well plate, after they have formed, therefore lowering the amount of stress applied to the cells during the aggregation procedure.
The cell printer successfully dispensed droplets in the repeating square grid pattern, dispensing a droplet at each corner of the square and then overprinting the existing droplets on each subsequent pass. This demonstrates that the cell printer has high spatial accuracy and is capable of returning to previous locations using the same coordinates. By factoring in the linear offset between the two nozzles, droplets can be overprinted, either of different materials or by the same material, in order to increase or decrease the concentration. By overprinting droplets encapsulating cells in medium with additional medium, the cell concentration of that droplet will decrease.
We developed a valve-based bioprinter and reported the first study on the response of hESCs to the printing process. This work demonstrates that the valve-based printing process is gentle enough to maintain hESC viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency. Due to the dual nozzle setup, the system is also able to create gradients of cells and other bio-inks which, when used in conjunction with the hanging droplet technique, yields gradients of cellular aggregates. The resulting aggregates are uniform and have repeatable size or size ranges which means that they can be made for specific applications (e.g. production of macrophages or other blood cells) that require going through an EB phase en-route to the eventual terminally differentiated cell type. Differing from previous studies, we printed directly onto the microwell plate allowing us to create and culture spheroid aggregates without the need to transfer them after formation therefore lowering the amount of stress applied to the cells during the aggregation procedure. The ability to print hESCs for the generation of 3D structures will allow us to create more accurate human tissue model, which is essential to the in vitro drug development and toxicity-testing. Additionally, this may also pave the way for human stem cells to be incorporated into clinical protocols either for patient implantation of in-vitro regenerated organ or direct in-vivo cell printing for tissue regeneration.