Three-Dimensional Printing of Cell Exclusion Spacers (CES) for Use in Motility Assays
Migration and invasion (migration/invasion) assays are common methods used to study cell motility, development, inflammation, wound healing and invasion of malignant cells in cancer.
In migration/invasion assays, several approaches including restrictive barrier (cloning wells), inserts or specialized equipment (scratch tools) are used to create defects in monolayers which can be used to monitor the influence of drug treatments on the rate and completeness of cells ‘in-filling which may provide information about therapeutic effectiveness of different agents.
Some authors have described ‘ad hoc’ solutions for producing cell patterning, such as using parafilm inserts. Such solutions are inexpensive but not highly reproducible, and more cost-effective durable inserts which can restrict cell adhesion to create cell patterns for migration/invasion assays would be a useful tool. 3D printing is one potential low cost solution that could be used to accomplish this goal.
3D printing is projected to be a disruptive manufacturing technology, which may enable labs around the world to fabricate custom assays in house and on-demand. 3D printing technologies also have ability to easily incorporate many bioactive test substances including antibiotics, chemotherapeutics, and hormones either as a pre-print additive or a post-print binder. These compositional and surface modifications allow highly complex models to be created which can even incorporate bioprinting.
Purpose
Cell migration/invasion assays are widely used in commercial drug discovery screening. 3D printing enables the creation of diverse geometric restrictive barrier designs for use in cell motility studies, permitting on-demand assays. Here, the utility of 3D printed cell exclusion spacers (CES) was validated as a cell motility assay.
Methods
A novel CES fit was fabricated using 3D printing and customized to the size and contour of 12 cell culture plates including 6 well plates of basal human brain vascular endothelial (D3) cell migration cells compared with 6 well plates with D3 cells challenged with 1uM cytochalasin D (Cyto-D), an F-actin anti-motility drug. Control and Cyto-D treated cells were monitored over 3 days under optical microscopy.
Results
Day 3 cell migration distance for untreated D3 cells was 1515.943μm ± 10.346μm compared to 356.909μm ± 38.562μm for the Cyt-D treated D3 cells (p < 0.0001). By day 3, untreated D3 cells reached confluency and completely filled the original voided spacer regions, while the Cyt-D treated D3 cells remained significantly less motile.
Conclusions
Cell migration distances were significantly reduced by Cyto-D, supporting the use of 3D printing for cell exclusion assays. 3D printed CES have great potential for studying cell motility, migration/invasion, and complex multi-cell interactions.
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