Traditional in vitro scratch assays lack standardization due to poor control over wound geometry and fail to account for cell proliferation. Here, we developed a novel scratch assay that enables precise control over wound geometry using CAD/CAM laser photoablation and takes cell proliferation into consideration using a simple reaction-diffusion based mathematical model. We demonstrated that diffusivity in precisely photoablated cell layers serves as a more accurate measure of cell motility than the rate of gap closure. Further, we biologically validated this assay using cells harvested from patients and patient-derived xenografts to gain insights into the influence of the presence stromal cells on metaplastic and non-metaplastic triple negative breast cancer metastasis.

Conventional wound healing assays are inadequate for evaluating the influence of symmetry on wound healing as they either offer poor geometrical control of the wounds created or fail to trigger a wound healing response in cell layers. To address these issues, herein we developed a novel CAD/CAM laser direct write photoablation assay that enables the generation of wounds with precisely defined and highly customizable geometries. As proof of principle, we biologically validated this assay to study the influence of symmetry and glycemic condition on dermal wound healing. We demonstrated that dermal wounds with symmetrical geometries tend to heal faster than those with asymmetrical geometries regardless of the glycemic conditions. Further, we elucidated the relative strength of the effects of symmetry and glycemic condition on healing rates in the context of the dermal wound size.

The most enriched cell types in the breast tumor microenvironment are cancer cells, cancer-associated fibroblasts, and tumor-associated macrophages. To recapitulate the cellular dynamics of the breast tumor microenvironment in vitro, the most abundant cell types need to be incorporated. Laser direct write bioprinting offers a precise, gentle, and reproducible method to print disparate cell types in user-defined geometries. Herein, we develop novel laser direct write cell printing protocols -- first as a customizable generalized framework, which is then adapted to print homotypic and heterotypic cancer-stromal arrays, and human macrophages. We demonstrate the ability to fabricate in vitro heterocellular constructs for studying cell-cell signaling in healthy and diseased microenvironments, as well as the capability to print human immune cells with high fidelity to pave the way for bioprinting immunocompetent tissue models going forward.