Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Cancer Technologies

Cancer Technologies - Poster Session B

Poster H6 - Development of a 3D-Printed Millifluidic Device to Study How Bacteria Alters the 3D Migration of Breast Cancer Cells

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The breast tumor microenvironment (TME) is a complex, heterogeneous system consisting of several different cell types including, cancer, stromal, and immune cells. Recent work has identified the presence of tissue-resident bacteria making up the breast tumor microbiome which has been suggested to alter the behavior of cancer cells driving metastatic spread and drug resistance. The first step in metastasis is the migration of cancer cells away from the primary tumor towards the vasculature which is accomplished through chemotaxis, the directed migration of cells to chemical concentration gradients. While many studies have explored cancer cell chemotaxis to specific chemical gradients, few studies have attempted to look at the chemotactic response due to soluble factors released by bacteria in the TME. This is due to limited experimental techniques capable of co-culturing these two vastly different cell types. The goal of this work is to investigate this through the development of a 3D printed millifluidic device to analyze cancer cell chemotaxis due to the presence of bacteria (Fig. 1A). The device contains three parallel fluidic chambers connected by small gaps with the center channel designed to contain a cancer cell laden hydrogel and one outer channel to contain bacteria and the other containing only media to induce a chemical gradient across the device via passive diffusion (Fig. 1B).

Materials and

Methods:  The millifluidic device was designed in SOLIDWORKS, converted to gcode in Simplify3D, and printed using PLA (polylactic acid) filament on an Ender3 Series 3D printer. A second, circular 3D printed imaging chamber (18 mm in diameter with an interior lip of 4 mm and wall thickness of 1 mm) was similarly designed, printed, and coupled with a 25mm x 25 mm glass coverslip with vacuum grease (Fig. 2). The devices were designed to contain a previously developed thiol-acrylate (TA) hydrogel scaffold to support the 3D migration of two different cell lines (NIH3T3 fibroblasts and MDA-MB-231 triple negative breast cancer cells). Cell spreading and migration were evaluated in three different formulations of the TA hydrogel containing increasing weight percentages (wt%) from 7.5 to 8.5 wt% at two different seeding densities (1x105 and 5x105 cells/mL). The wt% of the hydrogels were controlled by the amount of ETTMP and PEGDA added to the hydrogel solution. Cells were counted, centrifuged, and re-suspended the pre-mixed TA hydrogel and then seeding into the circular imaging chamber to visualize cell spreading using phase contrast light microscopy on a Leica DMi1. The cell-laden imaging chambers were incubated for 24 and 48 h in a 37°C CO2 incubator followed by imaging via light microscopy. Cell shape was quantified using ImageJ to approximate the size and circularity of the cells to evaluate how TA hydrogel wt% affects cell spreading. Terminal live/dead staining was performed using fluorescence microscopy and Calcein-AM (green, live) and Ethidium homodimer-1 (red, dead).

Results, Conclusions, and Discussions: Prior work with the TA hydrogel focused on its application as a scaffold to support 3D culture and required a stiffer environment to support the formation of 3D spheroids. Hydrogels that are too stiff will prevent the spreading and 3D migration of the cells. Initial experiments assessed the role of hydrogel stiffness, wt.%, and cell seeding density on cell spreading and migration using NIH3T3 fibroblasts due to their inherent migratory tendencies. The first experiments used a 96 well plate to culture the cells; however the small volume increased hydrogel degradation and the well shape induced cell settling and adhesion to the bottom of the wells before hydrogel polymerization. This led to the design of imaging chambers, providing a higher-throughput method compared to the millifluidic device. The seeding protocol was altered by pre-culturing cell-laden hydrogel in a tube before transferring to the imaging chamber, reducing cell settling. The NIH3T3 cells were suspended in TA hydrogels of 7.5 wt.% (Fig. 3A,D), 8 wt.% (Fig. 3B,E), and 8.5 wt.% (Fig. 3C,F). Two cell densities, 5x105 cells/mL (Fig. A-C) and 1x105 cells/mL (Fig. D-F), were tested to determine if cell number affected their ability to spread and migrate. Results showed that cells suspended in the 7.5 wt% gel failed to spread and tended to aggregate, with the gel degrading after 24 hours (Fig. 3A,D). Cells in 8.0 and 8.5 wt%, (Fig. 3B-C, E-F) struggled to move, but these hydrogels resisted degradation, suspending the cells in a 3D environment. The fibroblast line may not be suited for 3D migration studies, so ongoing work will focus on the hydrogel wt.% effects on cell spreading and migration in the MDA-MB-231 cell line. Additionally, ongoing work will explore alternative hydrogels, such as collagen-I, as the scaffold to support the 3D migration of the cell lines. This work will start with the circular imaging chambers and then the millifluidic device. After validating these methods, cells will be seeded within the hydrogel in the millifluidic device’s center channel and a bacterial strain (E. coli) will be introduced.

Acknowledgements (Optional):