**4. Discussion**

In this study, we were able to construct a novel and a ffordable bioprinting device with the ability to produce sca ffold-free tissues through the utilization of layer-by-layer printing of cellular spheroids. This device could allow the enhanced production of viable tissues to be used in drug testing, translational research, and various clinics.

With the design and development of the layer-by-layer print head, e fficiently picking up the spheroids from a reservoir was paramount for the overall success of the bioprinter. Through testing with beads of size similar to that of the spheroids, we were able to achieve nearly 98% e fficiency when picking up the beads in a single 4 × 4 layer. Although the print head designed in this project could only transfer a maximum of 16 beads or spheroids, changes can easily be made to the number and arrangemen<sup>t</sup> of holes in the bottom of the print head. This would allow for the construction of both larger tissues as well as custom arrangements to be used in di fferent applications where 3D models of the native tissue microenvironment are needed [30,31]

In addition, we were able to show the reliability of the bioprinter to print in a localized position repeatedly. The lowest overlap coe fficient calculated between any two layers was 0.997. The closer to 1.0 this value is, the more colocalized the beads of di fferent layers are. This corresponded to the precise placement of the spheroids on the needles when testing with hiPSC spheroids. This precision will allow for the proper alignment of layers in the construction of native tissue microenvironments as well as any cellular gradients found within tissues. The reproducibility and speed of printing viable tissues are critical for the success of bioprinting in the clinical environment [32,33]. The only commercially available spheroid printing device, Regenova by Cyfuse Biomedical, was used to generate a tubular, vessel-like structure that was made from approximately 500 spheroids [26]. Itoh et al. noted that to place so many spheroids onto the needles, it took the bioprinter approximately 1.3 h (78 min). This corresponds to approximately six spheroids printed per minute. The bioprinter we present here can print 16 spheroids per minute, that is, an over 250% increase in the printing rate of spheroids. Besides, given the success and e fficiency of the print head in picking up and transferring a full layer of spheroids to the printing surface, we can quickly increase the number of holes in the bottom of the print head to accommodate more spheroids and thus produce larger tissues.

Compared to other methods which utilize single-spheroid printing, a process requiring a multitude of precise positioning controls and visual analysis, this layer-by-layer method allows to straightforwardly pick up and place the spheroids [22]. This not only leads to a faster printing speed but also decreases the overall cost of our device, which totaled approximately \$2000, a significant reduction in price compared to the only commercially available option.

Despite the advancements in sca ffold-free spheroid printing that have been shown here, there remain a few limitations in this presented design. One in particular is the inability to customize the size and shape of the tissues. Future iterations will need to have the ability to print di fferent tissue shapes (e.g., vessel, round, etc.) to expand the capabilities to other fields of tissue engineering. Also, the current design is limited to holding only one type of spheroid for printing. Having the choice to print layers of di fferent types of spheroids would increase the customization potential. Further design changes could allow for the placement of multiple spheroid reservoirs on the spheroid bath stage of the bioprinter.
