Microfluidic wound healing assay using a pneumatically actuated membrane

Abstract

Cell migration is an essential aspect of many physiological and pathological processes in the human body including wound healing, inflammation-based recruitment of cells as well as cell growth and differentiation. Since movement along surfaces is an universal cellular process, migration and wound healing assays have therefore been routinely applied in biomedical research, pharmaceutical studies, and toxicological screenings. The ability of cells to move into cell free areas provides information on cell viability, cell movement and cellular mechanisms in the absence and presence of different chemical stimuli, thus elucidating the health status of a cell culture. Although several in-vitro well plate solutions exist, to date there is no satisfactory microfluidic solution that offers an automated, miniaturized and integrated induction of defined wounds. The need of a reproducible, automated and cost-effective method that offers high throughput capabilities is further given by an increasing demand of regulatory agencies to reduce and replace animal testing methods. To bridge this technological gap the aim of this thesis was the development of a microfluidic cell culture device for wound healing assays that allows the reproducible induction of circular wounds using a pneumatically-actuated flexible membrane. Soft lithography as a rapid prototyping method was employed for the fabrication of the two layer architecture using a photosensitive thermoset OSTEmer. The induction of mechanical wounds using pneumatically actuated membranes resulted in the creation of reproducible wounds (RSD 4\%) with the possibility of providing repeated wounding without destroying the underlying surface coating. Furthermore, cell debris are almost entirely removed using the laminar flow in the microfluidic channels leading to the absence of injured cells at the wound edge after the mechanical creation of the wound. All these aspects are considered key parameters for conducting reliable cell migration assays. The biological application was demonstrated using human umbilical vein endothelial cells (HUVECs) as a model, since these cells play an important role in the healing of wounds. The healing advancement and cell migration was studied in the presence and absence of an inflammatory cytokine TNF-$\alpha$ and a cell proliferation inhibitor mitomycin-C. Overall, the developed technology platform combines the advantages of microfluidics including low cell numbers and reduction of reagents with the simultaneous and reproducible creation of identical wounds on-chip. Furthermore, the system allows to mimic in-vivo endothelial cell environment that adequately recreates physiological relevant shear force conditions, which are critical for the maintenance of endothelial functions. The presented technology platform further enables automated and up-scalable pharmaceutical migration assays as well as wound healing screenings with well-defined and precise wound areas. Compared to the golden standard, the Scratch Assay, assay time also dramatically decreased. This fully automated, miniaturized, integrated lab-on-a-chip solution enables a highly controlled and reproducible mechanical wound healing assay realization for high throughput investigations.