Screening of Additive Manufactured Scaffolds Designs for Triple Negative Breast Cancer 3D Cell Culture and Stem-Like Expansion

Abstract

Breast cancer stem cells (BCSCs) are tumor-initiating cells responsible for metastasis and tumor reappearance, but their research is limited by the impossibility to cultivate them in a monolayer culture. Scaffolds are three-dimensional (3D) cell culture systems which avoid problems related with culturing BCSC. However, a standardized scaffold for enhancing a BCSC population is still an open issue. The main aim of this study is to establish a suitable poly (lactic acid) (PLA) scaffold which will produce BCSC enrichment, thus allowing them to be studied. Different 3D printing parameters were analyzed using Taguchi experimental design methods. Several PLA scaffold architectures were manufactured using a Fused Filament Fabrication (FFF) 3D printer. They were then evaluated by cell proliferation assay and the configurations with the highest growth rates were subjected to BCSC quantification by ALDH activity. The design SS1 (0.2 mm layer height, 70% infill density, Zigzag infill pattern, 45° infill direction, and 100% flow) obtained the highest proliferation rate and was capable of enhancing a ALDH+ cell population compared to 2D cell culture. In conclusion, the data obtained endorse the PLA porous scaffold as useful for culturing breast cancer cells in a microenvironment similar to in vivo and increasing the numbers of BCSCs.

Keywords: 3D printing; PLA; TNBC; breast cancer stem cells; scaffolds; three-dimensional cell culture.

Figure 1

Microscopic characterization of PLA scaffold configurations. Top side was visualized under an optical microscope and images were used to calculate pore area and filament diameter. (Scale bar: 2 mm).

Figure 2

MDA-MB-231 cell proliferation for each PLA scaffold configuration (3D) as compared to a two-dimensional surface (2D) (n = 3).

Figure 3

Main effect plots for each parameter on cell proliferation rate obtained through Quantum XL software. (A) Layer height. Samples printed with 60% infill density, grid infill pattern, 60° infill direction and 90% flow. (B) Infill density. Samples printed with 0.15 mm layer height, grid infill pattern, 60° infill direction, and 90% flow. The value of 70% significantly increased cell proliferation. (C) Infill pattern. Samples printed with 0.15 mm layer height, 60% infill density, 60° infill direction, and 90% flow. Zigzag pattern showed a light trend to obtain a higher proliferation rate. (D) Infill direction. Samples printed with 0.15 mm layer height, 60% infill density, grid infill pattern, and 90% flow. The value of 45° significantly increased cell proliferation. (E) Flow. Samples printed with 0.15 mm layer height, 60% infill density, grid infill pattern, and 60° infill direction. Significant differences are indicated as * (p < 0.05).

Figure 4

MDA-MB-231 cell proliferation assay of PLA scaffold configurations SS1, SS2 and 27/SS3 (3D) compared to two-dimensional surface (2D) (n = 3). Significant differences are indicated as * (p < 0.05).

Figure 5

Optical microscope images of MDA-MB-231 cells attached to scaffold walls. (A) MDA-MB-231 cells on a 2D cell culture. (B) Cells attached to the SS1 configuration scaffold. (C) MDA-MB-231 cells attached to scaffold configuration 18. (D) Cells attached to scaffold configuration 25. White arrows indicate cells adhered to PLA filaments. Images from optical microscopy 100×. Scale bar: 0.25 mm.

Figure 6

ALDEFLUO^TM assay results for three scaffold selected architectures and 2D control for 3 and 6 days. (*) Denotes significant (p < 0.05) differences between scaffold culture and 2D sample (n = 3).