Brunmaier, Laura

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

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

Engineering a Basement Membrane-Inspired Protein Therapy to Restore Endothelial Integrity and Prevent Thrombosis. Endothelial damage disrupts vascular homeostasis, leading to thrombosis, inflammation, and impaired vascular function. Current therapeutic approaches, such as anticoagulants and anti-inflammatory drugs, fail to actively restore EC integrity or prevent recurrent injury. To address this critical gap, we propose the development of a basement-membrane protein solution (BMPS) composed of laminin, nidogen-2, and perlecan, crosslinked with a biosafe crosslinker, to promote EC regeneration and barrier restoration. This project focuses on developing the BMPS formulation to enhance surface attachment and bioactivity while assessing its effectiveness in facilitating endothelial cell (EC) adhesion, migration, and function. We will systematically evaluate protein coating efficiency using ELISA and immunofluorescence staining, ensuring BMPS retention on cellularized and non-cellularized surfaces. To confirm that BMPS EC regeneration, we will conduct scratch assays to measure EC migration, followed by immunofluores- cence imaging and RNA-sequencing analysis to verify the maintenance of the EC phenotype and formation of tight junctions.The expected outcome of this research is a protein-based therapeutic that accelerates ECrepair, reduces thrombotic risk, and improves vascular stability. Successful completion of this work will provide proof-of-concept data for BMPS as a potential clinical intervention in vascular grafts, stent coatings, and regenerative therapies.

Optimization of a Chemicaly-Defined Culture System and Characterization of Endothelial Cell Iden-TITY. The development of a chemically-defined, animal-free (AF) media for EC culture is essential for advancing in vitro models, improving translational research, and mitigating the risks associated with fetal bovine serum (FBS), in- cluding batch variability and disease transmission. A key challenge in transitioning to serum-free culture conditions is ensuring that EC maintain their native phenotype and function. In this project, we focus on optimizing the AF media formulation and adaptation method that is tailored for human umbilical vein endothelial cells (HUVECs). Following opti- mization, we will utilize immunofluorescence staining and RNA-sequencing analysis to verify that the conditions do not induce unwanted loss of phenotype. Characterization includes the assessment of key EC markers such as PECAM-1 (CD31), VE-cadherin (CD144), and claudin-5. The expected outcome of this research is optimization and verification of EC function through RNA-seq and fluorescence imaging, and a foundation for future expansion into other culture systems.

Engineering an In Vitro Angiogenesis Model to Investigate Dose-Dependent Cytokine Responses in Endothelial Sprouting and Vessel Stabilization. The development of vascular microphysiological systems (MPS) has revolutionized in vitro modeling, providing biomimetic platforms to studyEC behavior under controlled con- ditions. These microfluidic models provide a platform, in conjunction with chemically-defined media, to enable precise investigation of angiogenic processes, including EC sprouting and vessel stabilization, elements that are critical for understanding vascular development, disease progression, and therapeutic responses. This project focuses on engi- neering a MPS of angiogenesis to examine cytokine significance and dose-dependent responses on EC sprouting and vessel stabilization. Using computer-aided design (CAD), 3D digital light processing (DLP) printing, and polydimethyl- siloxane (PDMS) molding, we will fabricate specialized microfluidic devices capable of sustaining the growth of cells. ECs will be seeded within the device and exposed to controlled gradients of cytokines, such as vascular endothelial growth factor and fibroblast growth factor, to quantify the effects of the cytokine on sprouting dynamics, vessel branch- ing, and barrier integrity. To systematically assess angiogenic responses, we will employ immunofluorescence imaging and RNA-sequencing to evaluate EC proliferation and lumen formation. By leveraging this engineered angiogenesis model, we aim to establish a platform for investigating therapeutic strategies and optimizing interventions for vascular regeneration.

Enhancing Inflammatory Response Detection: Local vs. Bulk Sampling in a Microphysiological Sys-TEM MPS provide a biomimetic platform for investigating EC behavior under controlled conditions, enabling precise analysis of vascular function and inflammatory responses. While bulk sampling from a reservoir fed from the outflow of the system is the standard method for quantitative analysis, this approach may fail to capture localized signaling events occurring within the cell microenvironment, and effectively dilutes cytokine concentrations to potentially undetectable limits. This project aims to determine whether local sampling conducted from the cellular environment within a microflu- idic channel improves the detection of discrete proteins compared to bulk outflow sampling. We will fabricate microfluidic devices designed for local and bulk sampling. ECs will be cultured in a vascularized hydrogel matrix and exposed to pro-inflammatory agents to induce a controlled inflammatory response. To compare sampling approaches, local sam- pling will be performed within the EC microenvironment, while bulk sampling will be taken from the reservoir outflow. Cytokine concentrations will be quantified using ELISA, and complimented by immunofluorescence imaging and RNA sequencing for inflammatory markers such as ICAM-1, VCAM-1, and E-selectin. The study will evaluate whether local- ized sampling improves the sensitivity and resolution of inflammatory response detection. The expected outcomes of this research include the development of an optimized local sampling strategy, improved understanding of inflammatory signaling gradients, and validation of microfluidic MPS as a high-resolution platform for detecting EC inflammation. This work has broad implications for disease modeling, drug screening, and vascular inflammation research.