*3.9. Optimal Process Parameters Values*

After the optimization experiments and basic cell culture tests had been completed, the optimal processing parameters for PCL scaffold printing were established (see Table 3) once the methodology had been applied to set each optimal parameter for cell cultures and for future experiments with CSCs culture enrichment using PCL scaffolds.


**Table 3.** Optimal processing parameter values to be used for PCL scaffold printing.

#### **4. Discussion**

In this work, a methodology to optimize the processing parameters for PCL scaffold production using a RepRap 3D printer has been developed. By using an optimization flowchart, PCL scaffolds suitable for cell culture were manufactured (Figure 3). The optimal processing parameters determined are closely related to those defined in other studies using the same technology and material. Domingos et al., (2013) set up an 80 ◦C printing temperature, 10 mm/s velocity, an approximately 0.3 mm filament diameter and a layer height of 0.28 mm [15]. A previous study by the same research group used an extrusion temperature of 70 ◦C and a speed of 8 mm/s [1]. These small variations support the idea of using a single, common methodology (Figure 3) to optimize the processing parameters. Compared with previous work in the literature, the flowchart provided here makes it

easier to adjust scaffold design features and processing parameters according to cell line characteristics. Several case studies were run to validate the flowchart depicted in Figure 3. Results show how cell culture is improved by using scaffolds which allow cell cultures to be created in 3D conditions and optimized based on the cells' features. In addition, process parameters were also evaluated using cell culture experiments. All scaffold culture experiments presented sterility resulting from the sterilization procedure described here. Both 60◦ and 45◦ scaffolds showed adequate design parameters for the MCF-7 cell cultures. In particular, the 60◦ scaffold design displayed the highest percentage of cell attachment, and exhibited good biocompatibility for the MCF-7 breast cancer cells. In contrast, the NIH/3T3 fibroblast cells presented a more homogeneous growth along the three scaffold designs. However, the 90◦ scaffold showed the highest cell proliferation value. Therefore, different kinds of cells may prefer different scaffold architectures, further demonstrating the need of a common procedure to find the optimal values. Moreover, a tumor and a non-tumor cell line were tested, showing the flexibility of the flowchart described here.

Three-dimensional cell culture on scaffolds may also be improved by other fabricationindependent parameters such as polarity of cell culture plates, culture media and time [26], as well as different cell culture types, including a dynamic model [27]. This optimization will be the focus of further studies as we attempt to improve cell attachment percentages. Furthermore, CSC population enrichment by cell culture on scaffolds will be evaluated using different approaches.

To date, most of the work related to scaffold production focuses on optimizing design features and forgets about improving fabrication parameters [1,9,16]. In this work, a flowchart to optimize the parameters of the whole process has been proposed (Figure 3) to help with their selection. In addition, this methodology may be further used to set up scaffold manufacturing (both the design features and the fabrication parameters) when using a RepRap 3D printer or any other AM technologies and/or materials.

#### **5. Conclusions**

In this work, the design features and fabrication parameters of scaffolds and the RepRap 3D printer were optimized to produce PCL scaffolds suitable for three-dimensional cell cultures. The optimization was performed following a detailed and unidirectional flowchart, thus providing some procedural guidelines with great potential for other popular manufacturing technologies and materials. The contribution of this paper is for scaffolds made with PCL materials. However, this experiment was only carried out to validate the methodology developed as a valuable method for future cell cultures. Often, physicians work with 2D cell cultures, but, as seen here, 3D cell cultures appear to be good method of improving cell culture enrichment. Furthermore, as the design features and manufacturing parameters need to be set for the different cell lines used each time, this methodology will help physicians and other operators to do just that.

Moreover, the scaffolds produced were proven to allow cell attachment and cell growth. The 60◦ scaffold design mainly worked for the MCF-7 cells and the 90◦ for the NIH/3T3 fibroblasts. Three-dimensional cell cultures with PCL scaffolds fabricated with a 3D printer offer both researchers and clinics a set of novel applications for the future. The flowchart developed represents a new tool with which to quickly manufacture scaffolds for a wide range of applications, including cell cultures and tissue engineering. For instance, the use of 3D cell cultures can boost CSC populations to study new therapeutic treatment.

**Author Contributions:** Conceptualization, J.C. and T.P.; Methodology, J.C.; Validation, A.J.G. and M.R.; Formal Analysis, M.R., J.M. and E.P.; Writing-Original Draft Preparation, J.C.; Writing-Review & Editing, M.R., E.P. and A.J.G.; Supervision, T.P. and J.C.

**Funding:** This research was funded by Spanish Grants: Fundación Ramón Areces, Instituto de Salud Carlos III (PI1400329) and Ministerio de Economía y Competitividad (DPI2016-77156-R), and through the support of the Catalan Government (2014SGR00868) and the University of Girona (MPCUdG2016/036).

**Conflicts of Interest:** The authors declare no conflicts of interest. The sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
