Brunmaier, Laura

• Ph.D., Candidate, Biomedical Engineering, South Dakota School of Mines & Technology

• B.S., Applied Biological Sciences, South Dakota School of Mines & Technology

Project 1: Optimization of an Animal-Free Culturing Method and Verification of Sustained Endothelial Cell Type
The successful development of a defined media for cell culture presents a host of advantages including reduction of disease transmission, facilitation of translational research, and enablement of bioassays that are finely tuned. The current standard for pre-clinical trial testing of drugs and devices is the animal model, however, a critical need exists for the development of biomimetic models composed of human constituents. Fetal bovine serum (FBS) has a nearly universal use in cell culture, but it presents consequential drawbacks, such as significant variability from batch to batch, serious risk of disease transmission, and concerns regarding ethical collection practices. As interest continues to grow around the development of biomimetic in vitro assays and translational biomedical research, the need for full identification of components in cell media and stimulating factors become increasingly important. Here, we present our formulations for an animal-free (AF) media and our adaptation procedures for human umbilical vein endothelial cell (HU-VEC). Our goal is to optimize our AF media formulation and procedures then proceed to verification that the conditions did not induce differentiation of the HUVEC. This effort is intended to preserve sufficient passages for experimentation providing the foundation for controlled drug screening and device evaluation assays.

Project 2: Optimization of an Animal-Free Culturing Method and Verification of Sustained Endothelial Cell Type
Epidemiological studies have demonstrated strong causal evidence to link the inhalation of particulate matter to the exacerbation of pathology in the cardiovascular system, ranging from myocardial infarction and atherosclerosis to direct cytotoxicity and inflammation. Ultrafine particles are ubiquitous in ambient air, in industrial sites, and in air pollution. When particles are inhaled, deposition can occur in the lungs, and the mechanisms of pathology have been well studied. However, particulate matter on the nano scale can translocate from the lungs into the bloodstream to circulate throughout the body. Evidence exists of oxidative stress and cytotoxicity that is caused from nanoparticle exposure to the endothelium, but this evidence does not support the extent of cardiovascular pathology that is found in large epidemiological studies. Sub-micron particulate matter has been shown to stimulate endothelial cells to illicit an inflammatory response. Currently, studies that control the particle size exposure to cells and examining the transcellular transport of the particles are limited. No studies have been done in vitro that include physiological conditions to better identify this response. We are proposing to advance our in vitro model of nanoparticle exposure to investigate cellular uptake and the resulting magnitude and breadth of the inflammatory response in endothelial cells.

Project 3: Investigating the Deleterious Effects of Radiation in a Human Microphysiological Vascular Platform
The chronic exposure to ionizing radiation (IR) that is experienced by astronauts in space poses major cardiovascular health risks. IR-induced activation of the inflammasome complex in endothelial cells may lead to further systemic inflammation and the onset of cardiovascular disease. To combat the deleterious effects of IR, human biomimetic models must be developed and exposed to doses of IR that emulate the conditions that astronauts experience. Here, we propose such a model and method. We will develop a microfluidic device were we culture endothelial cells in a 3D environment that mimics the in vivo physiological conditions to grow a capillary bed. The device will then be exposed to IR with an overall dose of 0.5 Gy to 2.0 Gy for up to 14 d, and we will evaluate the subsequent inflammatory response that is indicative of inflammasome activation. Specifically, we will use ELISA to measure levels of caspase-1, IL-18, and IL-1B, collect output media from the device to test for the presence  and quantity of cells that may have undergone pyroptosis, and then harvest the cells from the hydrogel channel to collect rt-PCR data on NLR protein expression. At the completion of this project, we expect to have developed an in vitro microvasculature device and acquired data to determine if low dose IR leads to inflammasome activation. Future studies may include the implementation of drugs or materials that can block or reverse inflammasome activation.

Project 4: Development and Characterization of a Tissue Engineered Vascular Graft
Cardiovascular disease is among the most prevalent pathologies worldwide. Coronary artery disease is a form of cardiovascular disease that is targeted to the vessels that supply the heart with blood. Occlusion of one of these arteries can lead to a heart attack, where partial occlusion generally displays in symptoms like shortness of breath and chest pains, among others. When a patient exhibits multiple diseased coronary vessels, they may be a candidate for coronary artery bypass grafting (CABG), where a surgeon will harvest autologous vessel and suture it into the vessels on or around the heart to bypass the occlusion. However, multiple issues may arise, including unsuitable or previously harvested vessels. If vessels are available for harvesting, the sutured vessels may experience failure as a result of thrombosis, occlusion, or restenosis. These complicated states that are prevalent among CABG patients have motivated the development of tissue engineered vascular grafts (TEVG). To construct a mechanically viable TEVG, we hypothesize that an acellular and multilayer tube with intentional fiber orientation can both withstand cardiac blood pressure while mimicking tissue compliance. The proper formation of a scaffold requires an understanding of how the polymers forming the scaffold behave under different forces and manipulations. The goals of this project is to characterize a biomaterial using various imaging and rheological techniques to determine an appropriate processing method for the material. Processing methods include electrospinning, dip-coating, centrifugal spinning, or extrusion. Following processing, the material will be mechanically characterized to develop further relationships between innate material properties, processing method and the resulting mechanical output.