**5. Conclusions**

In conclusion, we present here a custom device for printing spheroids using a novel layer-by-layer method. This bioprinter can e fficiently pick up and transfer spheroids to the printing stage in a single, complete layer, decreasing the printing time of large tissue constructs. With this precise and accurate transfer of spheroid-like beads to the printing surface, custom tissues can easily be constructed on the needles in future experiments. Also, we were able to prove, using hiPSC spheroids, that we can successfully print a single layer of spheroids onto the needle array printing surface. In addition, we were able to build and manufacture this device at an a ffordable price, making it easier for other researchers

to use the technology if brought to the market. Although there are additional design considerations that can be incorporated into future iterations, this device helps to advance and improve the growing area of scaffold-free bioprinting and could one day become a go-to method in tissue engineering.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-666X/10/9/570/s1, Video S1: Layer-by-layer bioprinting process. Video S2: Alginate bead synthesis. Video S3: Printing of alginate beads onto the needle array. Video S4: Printing of two layers of alginate beads onto the needle array. Spreadsheet S1: Major data for figures including efficiency data, colocalization data, and alginate bead diameter data. Image S1: Immunolabeling of hiPSC spheroids.

**Author Contributions:** Conceptualization, J.Z.; Methodology, W.L., A.M., A.M.K.-K., and J.Z.; Software, W.L.; Validation, W.L., A.M., D.P., and A.M.K.-K.; Formal Analysis, W.L.; Investigation, W.L., A.M., D.P., and A.M.K.-K.; Resources, J.Z.; Data Curation, W.L.; Writing—Original Draft Preparation, W.L.; Writing—Review & Editing, W.L., A.M., D.P., A.M.K.-K., R.K. and J.Z.; Visualization, W.L., D.P., A.M.K.-K., R.K., and J.Z.; Supervision, R.K. and J.Z.; Project Administration, J.Z.; Funding Acquisition, J.Z.

**Funding:** This research was funded by the National Heart, Lung, and Blood Institute (NHLBI); gran<sup>t</sup> numbers 5R01HL114120, 5R01HL131017, 5U01HL134764 to J.Z. Also, additional funding was provided by the American Heart Association through the Predoctoral Fellowship; gran<sup>t</sup> number: 19PRE34380484 to W.L. and the Scientist Development Grant; gran<sup>t</sup> number: 17SDG33670677 to R.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
