Poster T15 - Development of a microfabrication pipeline to pattern ECM proteins and guide EndMT mechanobiology

Introduction: Endothelial-to-mesenchymal transition, or EndMT, is the process where endothelial cells undergo molecular changes that shift their phenotype from that of endothelial cells to that of mesenchymal-like cells. This biological process is implicated in many physiological and pathological conditions, such as cancers and fibrotic diseases, and has been recognized as a mediator in atherosclerosis and vascular calcification, leading to research to determine whether the EndMT process is a suitable therapeutic target for the treatment of these pathologies. Studies in recent years have provided an improved understanding of how to assess EndMT; however, the marker expression signatures of EndMT are highly variable, and better methods are needed to characterize the EndMT continuum. It has also been shown that endothelial cells can undergo EndMT in response to mechanobiological forces, and while research has been done to control EndMT via proteins and culturing, little is known about the effect of micropatterns on EndMT mechanobiology and, by extension, its differentiation. Furthermore, studying the effects of micropatterns on any cell’s biology is both time and resource-intensive due to the costs of micropatterning, causing it to be out-of-reach for many research projects. We aimed to develop a novel and cost-effective protein-stamp fabrication, cell culture, and image analysis pipeline to study and control the EndMT process to aid in the identification of therapeutic targets for EndMT-related diseases, as well as for further applications in tissue engineering and regenerative medicine.

Materials and

Methods: Our pipeline consists of three unique phases; the fabrication of a PDMS stamp, the culturing of the endothelial cells, and the analysis of subsequent images that are taken. To fabricate the stamp, a resin mold of the desired micropattern was created in CAD and printed using a FormLabs Form 2 SDS resin printer. The molds were then cleaned twice for five minutes in an IPA solution. A PDMS stamp was then created using a mixture with a 10:1 ratio of base to curing agent that was poured into the resin mold and vacuum-desiccated for 30 minutes. The stamp was then cured in a 60C oven for 16 hours before being carefully removed from the mold and cut to create stamps of the desired size. To induce hydrophilicity in the stamp before protein seeding, plasma cleaning and a 1% PEG treatment were used. The stamps were then seeded with fibronectin and stamped onto a cell culture plate. After further washes and treatment, the endothelial cells were added to the plate and adhered to the proteins. Washing excess cells off produced a plate with the cells growing in the shape of the desired micropattern. TNF-α was then added to induce EndMT in the cells before further washing and staining to prepare for imaging. Microscopy was performed at 4x magnification. Our custom CellProfiler pipeline was then used to count and determine the orientation of the cells in the images to see the effects EndMT and the micropattern had on cell chirality.

Results, Conclusions, and Discussions: Overall, we developed a cost-effective and time-efficient pipeline that can be used to study the effects of different micropatterns on the mechanobiology of EndMT. With micropatterning, we developed a cheap and accessible method for developing molds to produce PDMS stamps thanks to the driving motivation of the project timeline and constrained budget. At first, many microfabrication methods were studied and attempted. The best method given our constraints was resin printing, as it has a higher resolution than standard, plastic 3D printing and is readily accessible to us. Using the Formlabs Form 2 SDS resin printer, custom CAD molds were time-efficiently printed, with print duration averaging between 4-8 hours per mold depending on the size and pattern being printed. This time represents the upper range of printing duration due to the print settings being set to the highest resolution. After printing, molds were easily cleaned and prepared using the FormLabs cleaning protocol and an IPA solution. The effective resolution of these molds was calculated to be around 100 microns as the theoretical resolution of the printer is not necessarily the absolute resolution. The production of the PDMS stamps took another day due to the long curing time needed to make the stamps less sticky. Overall, the microfabrication process generally allows a user to create a PDMS stamp within two days from the start of the print to the stamp being complete and ready for use. After coating the stamps with protein, we were able to successfully create the micropatterned matrix to seed the cells onto, and our cell seeding protocol was optimized through trial and error, allowing us to efficiently seed cells onto our stamped micropatterns as well as induce EndMT through TNF-α. The custom image analysis software we developed allows us to quickly take an image of these cells and analyze the chirality to determine the effects that the micropattern had on the mechanobiology of the cell and the induction of EndMT. This technique allows us to design any custom micropattern to seed cells onto then automatically analyze the images taken to determine the effects on EndMT.

Acknowledgements (Optional): Jeffrey Hsu, MD, PhD