Screening of electrospun PS/PCL scaffolds for three-dimensional triple negative breast cancer cell culture: impact of solvent, hydrophobicity, and setup orientation

Abstract

Triple-Negative Breast Cancer (TNBC) presents a significant challenge due to its aggressiveness and lack of targeted therapies. Understanding the interaction between TNBC cells and the extracellular matrix (ECM) in three-dimensional (3D) culture systems is vital for developing accurate in vitro models. This study explores the impact of electrospinning setup orientation, solvent selection, and polymer composition on scaffold design and TNBC cell culture. Various polystyrene (PS) and poly-ε-caprolactone (PCL) combinations were electrospun using different solvent combinations (dichloromethane/dimethylformamide (DCM/DMF), tetrahydrofuran/dimethylformamide (THF/DMF), chloroform/dichloromethane (Chl/DCM) and acetone) and setup orientations (vertical, horizontal). Scaffolds were characterized using Scanning Electron Microscopy (SEM) to assess fiber diameter and pore size. Cell proliferation and morphology were analyzed through MTT assay, SEM and Confocal Laser Scanning Microscopy (CLSM). Pore area and fiber diameter were influenced by solvent combination (THF/DMF < DCM/DMF < acetone < Chl/DCM) and orientation setup (horizontal < vertical). Sterilization assay revealed that 1-hour immersion in 70% ethanol followed by 30 min ultra-violet (UV) light exposure achieved sterilization with minimal scaffold degradation. High proliferation with no significant reduction compared to monolayer culture was found in some scaffolds and variability in cell morphology between scaffolds was also detected. Results highlight the critical role of scaffold printing parameters for 3D TNBC cell culture. Electrospun PS/PCL 40/60 scaffolds dissolved in DCM/DMF are promising in vitro models, providing a valuable tool for cancer research.

Keywords: Electrospinning; Fiber morphology; Poly-ε-caprolactone; Polystyrene; Scaffolds; Three-dimensional cell culture.

Fig. 1

Scanning electronic microscopy (SEM) images from (a) A1 scaffolds (b) A2 scaffolds, (c) A3 scaffolds, (d) A4 scaffolds, (e) A5 scaffolds, (f) A6 scaffolds, (g) B1 scaffolds, (h) B2 scaffolds, (i) B3 scaffolds, (j) B4 scaffolds, (k) B5 scaffolds, (l) B6 scaffolds, (m) C1 scaffolds, (n) C2 scaffolds, (o) C3 scaffolds and (p) D1 scaffolds spun in vertical (upper panel) and horizontal (lower panel) setup orientation. Beads are indicated by arrows. Scale bar: 10 μm. Images are representative of three independent experiments in triplicate (n = 3).

Fig. 2

Sterilization effect on PS/PCL scaffolds weight. Data are represented as mean ± SEM of all the scaffolds from three independent experiments in triplicate (n = 3). Levels of statistically significance are indicated as *(p < 0.050), **(p < 0.010), and ***(p < 0.001). Setup orientation is represented by symbol shape (circle: vertical; square: horizontal).

Fig. 3

Scanning electronic microscopy (SEM) images from (a) A1 scaffolds (b) A2 scaffolds, (c) A3 scaffolds, (d) A4 scaffolds, (e) A5 scaffolds, (f) C1 scaffolds, (g) C2 scaffolds, and (h) D1 scaffolds spun in vertical (upper panel) and horizontal (lower panel) setup orientation. Scale bar: 10 μm. NA = not available because the scaffold presented with beads. Images are representative of three independent experiments in triplicate (n = 3).

Fig. 4

Cell proliferation of MDA-MB-231 cells cultured in monolayer (2D) or three-dimensional scaffolds. Data are represented as mean ± SEM of all the scaffolds from three independent experiments in duplicate (n = 3). Levels of statistically significance are indicated as *(p < 0.050), **(p < 0.010), and ***(p < 0.001) compared to 2D conditions. Setup orientation is represented by symbol shape (circle: vertical; square: horizontal).

Fig. 5

(a) Confocal laser scanning microscopy (CLSM) images (upper panel) and scanning electronic microscopy (SEM) images (lower panel) from A2, A3 and C2 scaffolds spun in vertical or horizontal. Scale bar: 20 μm (SEM); 100 μm (CLSM) and enlarged picture (x3). Cells for CLSM imaging were fixed and immunostained using rhodamine-phalloidin (red) and DAPI (blue). Images are representative of four independent experiments in duplicate (n = 4). (b) Cytoplasm and (c) nucleus circularity of MDA-MB-231 cells cultured on A2, A3 and C2 scaffolds. Value equal to 1 means a circle and 0 no-circle. Data are represented as mean ± SEM of all the scaffolds from four independent experiments in duplicate (n = 4). Levels of statistically significance are indicated as *(p < 0.050), **(p < 0.010), and ***(p < 0.001).

Fig. 6

(a) Water Contact Angle (WCA) measurements of A2, A3 and C2 scaffolds spun in both vertical and horizontal orientations. Data are represented as mean ± SEM of all the scaffolds from three independent experiments in duplicate (n = 3). (b) Storage modulus (E’) as a function of displacement for A2, A3 and C2 scaffolds, obtained from Dynamic Mechanical Analysis (DMA). Data are represented as mean ± SEM of all the scaffolds from three independent experiments in duplicate (n = 3). Setup orientation is represented by symbol shape (circle: vertical; square: horizontal).