*2.4. Process Parameter Optimization*

The fabrication parameters and design feature values used for the experimental setup are shown in Table 1. A wide range of characteristics and parameter values were selected from the literature as the screening values with which to start. A wide range of processing parameters were selected based on the research work focused on tissue engineering with similar goals to ours, i.e., the enrichment or treatment of cells of the applicability for drug delivery. Thus, previous screening experiments were carried out to adjust the range of the processing parameters for the scaffolds. By following a sequential flowchart (Figure 3), the optimal tested value to be selected for each parameter was determined. Optimization was first performed using a generic geometrical form. A fixed circular scaffold design was used as the control pattern: 0.4 mm in diameter and layer height extruded filament, 1 mm distance between filaments, 90◦ deposition angle, 19 mm in diameter scaffold, and eight scaffold layers. As optimization progressed, design feature values were replaced by the optimal ones, resulting in a final scaffold design suitable for three-dimensional cancer cell culture. Furthermore, the cancer cell culture is now more like real physiological conditions, including an enrichment of the CSCs' subpopulation. Each step on the flowchart presented in Figure 3 included parameter testing and a physical scaffold variable measurement to assess the quality of the printing. Thus, optimal parameter values were sequentially determined and considering the final application as the optimal function to be reached. Physical variables, such as printed filament diameter, first layer height, and real distance between filaments, were measured using an inverted optical microscope (Nikon, Tokyo, Japan). Printed structures, as well as a nanometric ruler, were placed on the stage. Binomial variables (material adhesion, adhesion of contiguous filaments, printing quality such as the absence of blobs etc.) were assessed by sight. Finally, the cell efficiency of the different deposition angles was evaluated through a three-dimensional breast cancer cell culture to validate the parameters selected. Breast CSCs were used because their expansion would represent a new opportunity to develop new treatments against cancer stem features related to cancer relapse and metastasis.

#### *2.5. Cell Line*

MCF-7 breast carcinoma cells (ATCC® HTB-22™) and NIH/3T3 murine fibroblasts cell lines (ATCC® CRL-1658™) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-7 and NIH/3T3 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (Gibco, Walthman, MA, USA) supplemented with 10% fetal bovine serum, 1% L-glutamine (which means 2 mM L-glutamine), 1% sodium pyruvate (which means 1 mM sodium pyruvate), 50 U/mL penicillin and 50 μg/mL streptomycin (HyClone, Logan, UT, USA). Cells were maintained at 37 ◦C and in a 5% CO2 atmosphere.

#### *2.6. Scaffold Sterilization*

Scaffolds were sterilized following a previously-described methodology [2,24]. Meshes were submerged in a 70% ethanol/water solution overnight, washed with PBS (Gibco, Walthman, MA, USA), and finally exposed to UV light for 30 min. Only the top side was irradiated because PCL has a semi-transparent behavior when exposed to UV wavelengths [25]. This sterilization method was followed to avoid any changes in the stents' final properties [18].
