BACKGROUND: The cornea is the outer portion of the eye and protects the eye from infection or debris. When the cornea becomes compromised due to age and disease (specifically Diabetes Mellitus), it becomes impaired and can have profound impacts on an individual’s quality of life by leading to vision loss or blindness. The different layers of the cornea all contain many proteins and collagen, and have varying degrees of thickness and biomechanical properties. Stiffness in the cornea has either been measured via the use of AFM (Atomic Force Microscopy) which involves removing a slice of the cornea and adhering to the surface, as a function of IOP (Intraocular Pressure), or tensile testing. Previous research has also used the nanoindenter to measure the stiffness of different layers in the intact globe (eyeball) within the mouse head or by adhering to PEG submerged in PBS. However, no studies to our knowledge have used the intact globe exposed to air and placed on a 3D printed model to measure different corneal layers via the use of nanoindentation. METHODS: 6 C57BL/6J mice were obtained between 8-12 and 27 weeks of age, had the eyes extracted, and half remained with intact epithelium while the other half had the epithelium abraded with a 1.5 mm trephine. The eyes were placed in keratinocyte solution (KCM) for preservation while they were transported to the site with a nanoindenter. The globes were then placed on a 3D printed holder, cornea facing up, and irrigated with KCM solution in between indentation measurements. The PIUMA Optics 11 Nanoindenter was used to measure the Effective Young’s Modulus of the epithelium, basement membrane, and stroma. The Oliver & Pharr modeling was used as opposed to the Hertzian Model due to the biomechanical and adhesion properties of the eye. RESULTS: A comparison of control mice at 9 weeks shows an average Effective Young’s Modulus of 30.73 kPa, and an average Effective Young’s Modulus for 15 week old mice of 62.50 kPa for the epithelium. The average Effective Young’s Modulus of the basement membrane for 9 week control mice was ~6.2 kPa and for older 27 week mice was ~6 kPa. The Effective Young’s modulus for the stroma of 9 week old mice was ~68.3 kPa and for 27 week old mice was ~ 222.7 kPa. CONCLUSION: It was observed that stiffer substrates (in this instance, stiffer layers) require stiffer probes. The opposite is true of softer substrates, which require softer probes. It is beneficial in either instance to use a larger tip radius as there will be more contact and surface area measurement, so the probe has less recoil due to the adhesion from the corneal layers. The values observed in this study correlated with the values seen in the study conducted by Xu et al. However, the basement membrane values were different and could be due to probe specifications or layer thinness. Additional studies are needed to observe changes in Young’s Modulus based on probe characteristics with diseases such as Diabetes Mellitus (DM).

Precision medicine involves a personalized approach to healthcare acknowledging an individual-to-individual variability in genetics, environment, and lifestyle. Research in precision medicine can be pulled from basic, clinical or epidemiological sciences from which imaging, omics, or other types of data can be mined to contribute to the ‘information commons’ from which patient-specific patterns are drawn. Advancement in basic sciences that focus on imaging and other high content information processing for precision medicine is of particular importance in fields like oncology and nephrology where, diseases like focal segmented glomerular sclerosis have neither seen drug development in the past two decades, nor have an affirmative treatment plan. One reason is the lack of high-content high-throughput platforms for drug testing with a physiologically appropriate microenvironment. This work aims to build a high-content platform for podocyte-based drug discovery. Having demonstrated that cells in patterns demonstrate a phenotypic change representative of the in vivo condition, we first start by building a robust 96-well plate with our microfabricated platform as its base. We demonstrate a relevant increase in expression of podocyte specific proteins like synaptopodin in our patterned podocytes along with a decrease in cell-to-cell variability when compared with their unpatterned counterparts. Next, we demonstrate the use of our platform as a tool for drug discovery by showing that we achieve a reproducible actin-based dose response curve using human podocyte cell lines, something that has never been done to our knowledge. We see that our platform pushes immortalized podocytes towards cell cycle arrest at a much earlier timepoint during differentiation with improved functional performance metrics, such as lower motility and increased cytoskeletal segregation. We show that our platform may be able to extend its versatility by synergizing the effects of substrate shape and stiffness, and we show a potential application in studying the effect of this platform on the expression of Yes associated protein (YAP). We demonstrate the flexibility of this platform using another case study, this time with cancer cells. It is well known that the progression of neoplastic cells to metastasis is a major contributing factor to poor prognosis. The metastatic cascade involves invasion and migration coupled by angiogenesis and intravasation. The subsequent cells that survive circulation and attach to the endothelium extravasate and colonize in a distal location. Current techniques, such as Transwell and scratch assays that attempt to quantify metastatic potential are difficult to scale and consequently may be challenging to use in large-scale drug testing. We show that using our platform, we are able to rapidly quantify the metastatic potential of cancer cells in situ with high sensitivity, independent of cell seeding density. By changing the dimensions of our microfabricated patterns, we are able to vary the mechanical resistance that the cell experiences traversing and use this to mimic cell invasion across different microenvironments. Importantly, we show that we can quantify the effect of the metastatic potential in response to a pharmacological intervention and thus demonstrate that we can use this platform for drug testing. In conclusion, we present a novel multifaceted platform and demonstrate its versatility with two different applications. In the context of drug discovery, we show that the platform serves as a superior model for podocyte injury. The reproducible Puromycin aminonucleocide (PAN) actin-based dose response curves obtained using this platform, opens avenues to investigate the effect of various targeted therapies for podocyte-based kidney diseases. In the context of screening for metastatic potential of cancer cells, we show that our platform exhibits a superior sensitivity to existing screening techniques. Additionally, the potential of using a patient’s own cells in the future for either application presents exciting avenues for precision medicine.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.