**1. Introduction**

With the development of cutting-edge strategies and techniques for generating complex, functional tissues, bioprinting, or three-dimensional (3D) printing of biological materials, has become a critical tool in tissue engineering [1–7]. Bioprinted and fabricated tissues have immense clinical potential including organ and tissue regeneration, drug testing, organ models for surgical practices, and organ replacement. The conventional method of bioprinting is to print cells with or into a scaffold material. Scaffold printing, with either a synthetic or natural polymer, can provide the necessary structure and microenvironment for proper cell growth and survival [8–10]. This method can be used to dictate how structures are arranged, where cells are positioned in this scaffold, and what sort of factors are present to optimize the growth and function of the cells. Polymers can also be designed to degrade depending on a specific set of environmental conditions or with the introduction of a particular solution. However, with synthetic polymers, biocompatibility could pose problems if their use is not appropriately addressed in in vivo studies, leading to an increased use of natural polymers and hydrogels for bioprinting [11–15]. These types of materials possess the same ability to provide an adequate structure for the cells and can be designed to mimic the in vivo environment better, decreasing the chances of rejection if used in the body.

Cells, however, possess the innate ability to produce their sca ffold material in the form of extracellular matrix (ECM) as they grow and interact with one another [16–19]. Determining how to harness this ability and become completely sca ffold-free e ffectively has led to the use of 3D spheroids as building blocks for larger tissues [20]. Spheroids can easily fuse together when in close proximity to or touching another spheroid. The e fficacy of spheroids as building blocks for larger tissues has been demonstrated using sca ffold materials to provide a structure or a particular shape for the proper fusing of spheroids [21]. However, this method still relies on additional materials for tissue generation. This led to the invention of the Kenzan bioprinting method.

The Kenzan method utilizes an array of stainless steel microneedles to provide a structure that the spheroids can be punctured onto to facilitate the fusion process without additional materials [22]. This method allows an actual scaffold-free tissue to be generated from various cell types for a custom-designed tissue. Regenova (Cyfuse Biomedical, Tokyo, Japan) is the only bioprinter on the tissue engineering market which utilizes the Kenzan method. Despite its young age and high cost, it has already been used to generate functional tissues from a multitude of cell types and lineages [23–26]. This device, however, places one spheroid at a time on the needles, requiring a longer printing process as the size of the tissue increases. Having the ability to build larger, more clinically relevant tissues in a shorter length of time using this method would be very beneficial for various fields of medicine and clinical research.

We hypothesize that a device which can print spheroids with a layer-by-layer method will not only reduce the manufacturing time of clinically relevant-sized tissues but also assists in advancing the field of tissue engineering by introducing an a ffordable alternative for printing sca ffold-free, spheroid tissues. The research described here is focused on the design, construction, and testing of such a bioprinter. We also show that we were able to build a working and a ffordable prototype which e fficiently and accurately transfers cellular spheroids to a needle array, one 3 mm × 3 mm × 1 mm layer at a time.

### **2. Materials and Methods**

### *2.1. Preparation of Needle Arrays*

The printing method used by the utilized bioprinter relies on the accurate and precise placement of needles for the proper fusion of spheroids into a single tissue. Needle arrays (Figure 1Aii,Fi) were made using sterile stainless steel needles that were 180 μm in diameter and ~15 mm in length. To aid in the precise placement of the needles, a custom stainless steel plate (Figure 1C,Fii) that possessed an array of through holes, machined using a laser drilling technique, was used. With this plate, needles were placed into a 4 × 4 array, each with a pitch of 800 μm. Once the needles were loaded into the plate, the exposed blunt ends were placed in a silicone solution (Sylgard 184, Dow Corning, Midland, MI, USA) and cured at 70 ◦C for 2 h to hold the needles in place during the printing process e ffectively. After curing, the needle array assembly was autoclaved and then kept until ready for use with the bioprinter.

### *2.2. Bioprinter Design and Construction*

The bioprinter (Figure 1A) was designed to fit easily into a biosafety cabinet and operate under sterile conditions. It is 10 inches tall and 8 inches wide, with a depth of 13 inches. The bioprinter was entirely modeled in Fusion 360 (Autodesk) before manufacturing the required pieces for the device. The model of the bioprinter as well as the built bioprinter are shown in Figure 1. The bioprinter consists of three main parts: a spheroid print head (Figure 1D), a spheroid bath stage (Figure 1E), and a needle array bath (Figure 1F). The spheroid print head (Figure 1Ai,Di) was designed to hold a 4 × 4 array of spheroids in a single layer. Each spheroid is approximately 800 μm apart when held on the end of the print head. The print head was machined from 316 stainless steel, and through holes were cut into the bottom of the print head with a diameter of approximately 550 μm, allowing the needles to pass through but preventing spheroid aspiration (Figure 1B). The beads were picked up by a vacuum pump (12V vacuum pump, SparkFun, Niwot, CO, USA) connected to the print head. The spheroid bath stage functioned to hold the reservoir/container of beads during the printing process. The reservoir consisted of a watch glass made from polytetrafluoroethylene (PTFE), whose concave bottom allowed for the beads to collect in the center after each layer was picked up. The beads were printed onto the needles which were held inside the needle array bath. Each piece of the printer can either be sterilized by autoclave or ethylene oxide.

**Figure 1.** Design and construction of the bioprinter and its components. (**A**) A model of the bioprinter was first made using the CAD modeling software. The crucial components of the bioprinter are (i) the layer-by-layer print head and (ii) the needle array. (**B**) The print head has approximately 550 μm through holes cut into the bottom in a 4 × 4 arrangement, with a space of 800 μm between each hole. (**C**) The needle array is made up of 180 μm-diameter stainless steel needles placed 800 μm apart in a matching 4 × 4 arrangement. (**D**–**F**) The other components were 3D printed and are as follows: (**D**) (i) spheroid print head, (**E**) spheroid bath stage, and (**F**) needle array bath with (i,ii) needle array. Scale bars: (**D**)(i) and (**F**)(i) 5 mm; (**F**)(ii) 2 mm.
