Molecular, Cellular, and Tissue Engineering Commons^™

The Impact Of Dipeptides On Product Quality In Chinese Hamster Ovary Cells, Louise C. Herman

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Biopharmaceuticals are medicines produced in cells which are used to treat diseases such as cancer. Monoclonal antibodies (mAbs) represent the majority of approved biotherapeutics. MAbs are mostly produce using Chinese hamster ovary (CHO) cells. CHO cells are favored for resiliency, scalability, and established safety, and are considered the “workhorse” organism for biopharmaceuticals.  Amino acids are a key component of cell culture media, however several amino acids have solubility limits, such as cysteine, cystine, and tyrosine.  At neutral pH, these amino acids are poorly soluble.  However, dipeptides, such as N,N'-di-L-Alanyl-L-Cystine (ACCA) have been reported to increase cysteine and cystine availability. Further, …

Fluorescence-Based Quantification Of Hepatic Ldl Uptake And Donor Hepatocyte Engraftment Using Atto 700–Labeled Ldl, Victor Agustin Cortes Flores

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Familial hypercholesterolemia (FH) is an inherited disorder characterized by elevated low-density lipoprotein cholesterol (LDL-C) that increases the risk of premature atherosclerotic cardiovascular disease. In severe FH, including low-density lipoprotein receptor (LDLR), hepatic LDL clearance is profoundly impaired and many lipid lowering treatment strategies have limited effectiveness. Current advances in ex vivo CRISPR–Cas9 hepatocyte gene editing in vivo and ex vivo approaches for treatment of FH are under development.

However, a central barrier to translation is the ability to quantitatively measure the percentage of healthy gen edited hepatocytes in the liver while simultaneously assessing functional hepatic LDL handling. This thesis establishes …

Diabetes-Induced Mitral Valve Degeneration: Tissue-Engineered Solutions And Antioxidant Therapy, Samantha Hahn

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Mitral valve (MV) fibrosis is a progressive pathological condition characterized by leaflet thickening and stiffening, ultimately compromising valve function. This process is accelerated in diabetes, where chronic hyperglycemia elevates reactive oxygen species (ROS) and alters the mechanics of the extracellular matrix (ECM), collectively promoting the activation of valvular interstitial cells (VICs). Under healthy conditions, VICs maintain a quiescent, fibroblast-like phenotype responsible for sustaining ECM homeostasis. However, pathological biochemical cues or mechanical stress drive VICs toward a myofibroblast phenotype. While this transformation is essential for short-term repair, its persistence contributes to maladaptive MV remodeling and fibrosis. Diabetic stressors may be mitigated …

Mitral Annular Calcification In Diabetes: Valvular Interstitial Cells, Antioxidant Therapy, And Tissue Engineering Approaches, Raleigh J. Keenan

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Mitral annular calcification (MAC) is a chronic, progressive, and degenerative condition that impairs mitral valve function and increases the risk of cardiovascular disease. The mitral annulus is a saddle-shaped, fibrous structure that is primarily composed of collagen and elastin. The annulus also contains interspersed lipids that are involved in cellular metabolism, as well as inflammatory and osteogenic signaling. These lipids increase the annulus’ susceptibility to calcification, under conditions that increase mechanical stress (hypertension) and promote atherosclerosis (diabetes mellitus). These conditions intensify calcification by promoting the activation of valvular interstitial cells (VICs), increasing fibrosis, and inducing osteogenic differentiation. Although it is …

The Effects Of Cigarette Smoke Extract On Osteoarthritis In A Primary Joint Tissue Explant Model, Emily Sawvell

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Osteoarthritis (OA) is the most common degenerative joint disease in the world with an economic burden of nearly $140 billion annually. Factors identified to increase the risk of developing OA include age, obesity, sex, race, genetics, and previous joint injury. However, one risk factor that has been scarcely studied in OA etiology is cigarette smoking. While some studies have reported smoking to protect against OA, increasing evidence suggests that cigarette smoking results in more severe and painful OA, increases the rate of developing OA, and reduces the efficacy of pain medications and arthritis-targeting therapies. Despite this, the impact of cigarette …

Development Of Translational Microscale Systems To Interrogate How Biophysical And Biochemical Cues Alter The Phenotype Of Hormone Positive Breast Cancer, Braulio Ortega Quesada

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Cancer is a complicated disease and one in every three diagnoses are women with breast cancer. Recent research shows that the progression of cancer depends strongly on interactions with other non-cancerous cells and non-cellular components. Metastasis, the spread of cancer to other parts of the body, is a key step in cancer progression and makes treatment harder, leading to poor response to treatments culminating in death in ~90% of patients. This dissertation focused on creating a series of microdevices as novel pre-clinical models of breast cancer. Two different devices were fabricated to (1) study how exposing cancer cells to the …

Ex Vivo Multiplex Knockdown Of Angptl3 And Cypor In Hepatocytes As A Novel Cell Therapy For Familial Hypercholesterolemia, Callie D. Clark

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Inherited metabolic diseases (IMDs) affect approximately 1 in 800 individuals. The only curative treatment for IMDs is liver transplantation. However, transplantation carries significant risks, including organ rejection and a lifelong need for immunosuppression. Therefore, gene editing has emerged as a promising alternative to liver transplantation for treating IMDs.

Familial hypercholesterolemia (FH) is and IMD characterized by elevated LDL-C levels resulting in premature cardiovascular disease. CRISPR-Cas9-mediated gene editing to disrupt the gene encoding angiopoietin-like 3 (ANGPTL3) in the liver represents a novel therapeutic approach for FH. Nonviral delivery performed ex vivo is safer than systemic delivery because gene editing is in …

Multifunctional Peptides To Improve Nucleic Acid Delivery For Ovarian Cancer Treatment, Kharimat Lora Alatise

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RNA interference (RNAi) therapies, such as small interfering RNA (siRNA), offer a high-precision approach to reducing the expression of disease-promoting genes with minimal off-target effects. For aggressive diseases like ovarian cancer, RNAi therapy can serve as a robust treatment modality if delivered successfully. Over 70% of ovarian cancer patients are diagnosed in stages three and four, where the disease becomes more difficult to treat. High relapse rates and drug resistance in these stages demonstrate the need for novel strategies to sensitize tumors to chemotherapy, making siRNA a promising avenue for targeting resistance-associated oncogenes.

However, the delivery of siRNA remains a …

The Development Of A Novel Cartilage Analog Scaffold For Chondral Regeneration, Vishal Thomas

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Focal Chondral Defects (FCDs) are cartilage injuries that affect about 900,000 Americans every year. Left untreated, these may lead to osteoarthritis and eventually necessitate painful knee replacements. Current treatment options are limited in terms of durability, cost, and availability of grafts. Tissue engineering, using natural scaffolds, are a highly attractive alternative promising cost efficiency, abundance, and long-term benefits. However, their low mechanical strength and dense matrices are impediments that could lead to sub-optimal repair.

In this work, a natural biomaterial, the bovine nucleus pulposus (bNP), derived from intervertebral disc tissue, was identified as a candidate scaffold for cartilage regeneration. Mechanical …

Light-Induced Phase Separation Purification For Monoclonal Antibodies, Xuyang Chen

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Novel Nucleus Pulposus Biomaterial Development And Low Field Mri Assessment For Intervertebral Disc Degeneration, Mario Krussig

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Intervertebral disc degeneration (IVDD) is a prominent pathology in the modern world that causes debilitating pain for many patients. The onset of IVDD occurs in the nucleus pulposus (NP). NP biomaterials will play a vital role in mitigating the progression of IVDD. Within this work, we sought to synthesize a solubilized extracellular matrix (ECM)-based NP biomaterial. Utilizing pepsin or urea extraction, bovine NP ECM was successfully solubilized (SBNPs). SBNP formulations were characterized biochemically, evaluated for cytotoxicity, and down-selected for front-running formulations. Leading candidate SBNPs were incorporated into an alginate bead biomaterial model to determine the potential bioactivity with human adipose-derived …

An In Vitro Platform To Characterize Myocardial Wound Remodeling, Jonathan Heywood

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Heart Failure, sometimes called Congestive Heart Failure, is a collection of pathological disruptions in which the heart is unable to pump blood properly. It affects over 6 million people in the United States, with the cost-per-patient at approximately $30,000 and the total cost estimated to be $160 billion by 2030. There is no single cause of heart failure, as it can manifest as a varying array of diseases, disorders, and syndromes. However, many of these underlying diseases result specifically from dysregulation of mechanically active cells called cardiac fibroblasts, which play a critical role in the remodeling of the extracellular matrix …

Engineering Next-Generation Cardiovascular Therapies, Ryan Walker Barrs

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Cardiovascular diseases are the leading cause of death worldwide. Heart disease, in particular, accounted for 1 in every 5 deaths in the United States in 2021, representing a substantial public health burden that will require more effective treatment options to alleviate. Heart transplant surgery is currently the only option to completely restore cardiac function, but many patients die while waiting for a transplant due to a longstanding organ donor shortage. Thus, there is a dire need for accessible treatment options that can repair damaged hearts. Recent advances in tissue engineering and regenerative medicine have accelerated the development of therapies for …

Experimental And Computational Platforms For Studying Systems Mechanobiology, Brendyn Miller

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Mechanical stimulation through physical activity has been shown to play an important role in treating and preventing several non-communicable diseases such as hypertension, lower back pain (LBP), type-2 diabetes mellitus, and several cancers. This is accomplished through the regulation of cellular behavior and tissue remodeling within the body at both the micro- and macro-scale levels. The goal of mechanobiology research is to gain in-depth knowledge and understanding of how cells sense physical forces in conjunction with other biochemical cues and translate those factors into important biological functions that either maintain tissue homeostasis or lead to pathological states. Understanding these processes …

Tandem Peptides Targeting Her2 For Delivery Of Cd44 Sirna Into Her2+ Breast Cancer Cells In Vitro, James Kalogeros

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More than 300,000 women will be diagnosed with breast cancer, and approximately 48,000 women will die this year due to the disease in the United States. HER2+ breast cancer accounts for 20-25% of invasive breast cancer, and it has a worse prognosis and higher early-stage mortality rate than other breast cancer subtypes. Common treatments for HER2+ breast cancer involve surgical removal of the breast or systemic therapy with chemotherapeutic drugs, both of which cause damage to healthy tissue. Due to the lack of a natural ligand for the HER2 receptor, there is a need for improved targeting strategies for the …

Prevention And Reversal Of Elastin-Associated And Phosphate-Dependent Arterial Calcification Using Targeted Nanotherapeutics In Pre-Clinical Models, Fatema Tuj Zohora

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Vascular calcification can be life-threatening, involving calcium phosphate crystal deposits in the blood vessels. The calcification in the intimal layer of the arterial wall is usually associated with atherosclerotic plaque build-up that, in the advanced stage, harbors ‘dispersed’ and ‘spotty’ deposits of calcium phosphate crystals in the necrotic core of the plaque. In contrast, medial arterial calcification (MAC) can form independently of atherosclerosis and appears as ‘Railroad-like’ calcific deposits along degraded elastin lamellae in the medial layer of arteries. The mechanisms of such elastin-associated medial arterial calcification and potential therapies are the key concerns of this dissertation. MAC is common …

Investigating The Potential Of A Cell-Based Gene Editing Therapy For Inherited Metabolic Liver Disease, Ilayda Ates

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Inherited metabolic diseases (IMDs) affecting the liver are relatively rare but collectively have a prevalence of 1 in 800 live births. These diseases result from autosomal recessive single-gene mutations, leading to organ dysfunction and potentially fatal consequences if left untreated. One potential therapeutic strategy for IMDs of the liver involves using CRISPR-Cas9-induced loss of function mutations. However, translating this approach into the clinic is limited by the need for safe and effective CRISPR delivery methods. Adeno-associated viral vectors (AAVs), commonly used for CRISPR delivery, are associated with significant safety and efficacy concerns, including risks for immunogenicity, off-target mutagenesis, and genotoxicity …

In Vitro And In Vivo Diabetic Models For Assessment Of Tissue Engineered Vascular Grafts, Juan Carlos Carrillo Garcia

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Diabetes has become one of the leading causes of lower-limb loss worldwide. Every 30 seconds, a person loses a limb due to diabetic-related vascular complications. About one-third of patients needing lower-limb bypass surgery have debilitated autologous vessels unsuitable for use, and no other good long-term options are available. These detrimental effects on the vasculature are caused mainly by the hyperglycemic and hyperlipidemic conditions derived from diabetes. Under these conditions, an increase in advanced glycation end products (AGEs) and reactive oxygen species leads to irreversible crosslinks of extracellular matrix proteins, accelerating vascular pathology through vascular stiffening, endothelial dysfunction, inflammation, atherosclerosis, fibrosis, …

Assesment Of Structure, Function, And Microevolutionary Dynamics Of Extrachromosomal Circular Dna In Chinese Hamster Ovary Cells, Dylan Chitwood

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Chinese hamster ovary (CHO) cell lines are among the most popular expression hosts used in biopharmaceutical manufacturing due to relative ease of culture, capacity to perform human-like post-translational modifications, and non-susceptibility to viruses. However, the intrinsic plasticity of the CHO genome can lead to undesired genetic rearrangements, phenotypic shifts, reduced product quality, and early culture termination that prevents continuous biomanufacturing. A characteristic of plastic and unstable genomes that is poorly understood in CHO cells is extrachromosomal circular DNA (eccDNA). EccDNAs are focal amplifications of the genome that reside in the extranuclear space. These plasmid-like entities are structurally complex and are …

In Vitro Bioreactor For Mechanical Control And Characterization Of Tissue Constructs, Samuel Coeyman

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Heart failure (HF) currently affects over 6 million Americans, 50% of whom die within 5 years of their initial diagnosis. A major contributor to the onset of HF is cardiac fibrosis in the myocardium, which arises when fibroblasts (FBs) are activated in response to heightened mechanical stress from overload conditions like hypertension. Activated FBs remodel the extracellular matrix (ECM) and secrete ECM proteins including collagen. FB remodeling has been studied in the past by applying forces and/or deformations to three-dimensional, cell-seeded gels and tissue constructs in vitro. Unfortunately, previous stretching platforms have traditionally not enabled mechanical property assessment to be …

Development Of A Tissue Engineered Cardiac Patch, Howard Herbert

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Cardiovascular Disease(CVD) is the leading cause of mortality in the developed world. CVD is most commonly manifested as atherosclerosis of the coronary arteries leading to Myocardial Infarction(MI). After MI, fibrosis of the ventricular wall leads to heart failure(HF), a pandemic affecting 26 million people globally. While therapies are continuously developed to combat HF, the treatment of choice, whole heart transplant, is limited by the availability of donor hearts. It is clear that there is a need to develop a long-term solution to combat HF and its enormous economic burden. Tissue Engineering and Regenerative Medicine holds promise as a possible solution …

Investigating Spatial Heterogeneity In Myocardial Wound Environments To Improve Therapy, Michael Potter

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Heart failure is a broad pathology manifestation categorized by an inability of the heart to successfully pump blood throughout the vast vessel network of the body. Within the United States, heart failure is projected to increase by approximately 46% from 2012 to 2030. Modalities of heart failure are generally related to wall mechanics that are impacted following myocardial infarction events. Interplay exists between the wall mechanics, responding cell populations, and the spatial heterogeneities in the resultant scar. This interplay directs the myocardium towards heart failure modalities governed by overly stiff or compliant states. It is essential to elucidate details underlying …

Optimizing Crispr/Cas9-Mediated Knockdown Of Angptl3 In Liver Cell Lines And Mouse Hepatocytes, Meredith Reeves

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Familial hypercholesterolemia (FH) is a genetic condition characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C) that leads to an increased risk of developing cardiac disease early in life (Shah et al., 2020). Current treatments such as statins and PCSK9 inhibitors have helped lower LDL-C levels, however they require repeated administration every 4-6 weeks to remain effective (Raal et al., 2018). Angiopoietin-like 3 (ANGPTL3) is an inhibitor of plasma lipid metabolism that has become a promising molecular target for the treatment of FH. Individuals with non-functional copies of ANGPTL3 demonstrate low levels of plasma LDL-C and triglycerides, indicating a protective …

A 3d Tissue Engineering Model To Study Mitral Valve Annulus Calcification Under Diabetic Conditions, Erin James

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The most complex heart valve is the mitral valve (MV). Many pathologies can affect the MV, including stenosis, regurgitation, prolapse, and mitral annulus calcification (MAC). MAC is chronic degeneration of the annulus, which is the fibrous, saddle-shaped “ring” that can contract and relax with the myocardium. The prevalence of MAC is around 15% but increases in patients with other cardiovascular diseases and risk factors. It is also thought to increase in patients with type 2 diabetes, but MAC has not been properly characterized within this population because of confounding factors such as cardiac disease and kidney disease. The goal of …

Mitral Valve Tissue Engineering - A Dynamic Model For Investigating The Mechanism Of Valvular Pathology, Collin Owens

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Heart valve disease affects an average of 2.5% of the population in the United States. The mitral valve (MV) is the most complex of the heart’s four valves and is most associated with the disease by exhibiting altered extracellular matrix (ECM) which translates into stenosis or regurgitation. These diseases are typically degenerative in nature and can be accelerated by risk factors such as diabetes and hypertension. With diabetes and hypertension affecting 425 million and 1.39 billion people worldwide, further investigation into these risk factors is warranted. This study aims to develop and test an in vitro model of MV disease. …

Nonviral Approaches For Delivery Of Crispr-Cas9 Into Hepatocytes For Treatment Of Inherited Metabolic Disease, Tanner R. Rathbone

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Inherited metabolic diseases (IMDs) of the liver have a collective occurrence of about 1 in 800 births. Current therapies for IMDs of the liver are limited, and the only curative option for patients is orthotopic liver transplantation. Due to shortages of organ donors, immunosuppression following the transplantation, and mortality risks associated with the procedure, alternative treatment options are necessary for curing IMDs of the liver. Therapeutic gene editing has been proposed as a potential strategy for treating IMDs of the liver. Adeno-associated viral vectors (AAVs) are the most widely used delivery method for liver-targeted gene therapies. Although widely used for …

Design Of A Four Channel Pulsatile Perfusion Bioreactor For Ex-Vivo Study Of Vascular Grafts, Thomas M. Fair

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Cardiovascular diseases are the leading cause of death in the United States. Atherosclerosis in peripheral arteries is a major contributing factor. Autologous saphenous vein grafts are the most common bypass grafts for treating peripheral artery diseases. A quarter of these grafts fail within a year, and around half have failed within 10 years of the initial surgery. Graft failure is attributed to the development of intimal hyperplasia indicated by the migration and proliferation of vascular smooth muscle cells, fibroblasts, and the deposition of extracellular proteins.

Increases in flow, pressure, the pulse frequency, and the differential of pressure that model an …

Investigation On Effects Of Lactate Adaptation Using Metabolic Indicators And Amino Acids To Explore Cell Metabolism, Lisa Uy

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Biopharmaceutical products are drugs that are made from living cells. One protein type of high importance to biopharmaceuticals is monoclonal antibodies. Monoclonal antibodies allow for highly specific, targeted treatment of diseases and detection of molecules related to diseases. With major roles in detecting and/or treating diseases like human immunodeficiency virus (HIV), coronavirus disease 2019 (COVID-19), and some cancers, monoclonal antibodies are in demand. This demand has led to a need for efficient cell culture systems to produce high yields of protein with minimal waste. The most commonly used cell culture system for these high-value proteins is Chinese hamster ovary (CHO) …