Browsing by Subject "Bioengineering"

An estimated 10% of people worldwide have an enteric nervous system (ENS) related illness including irritable bowel syndrome (IBS), diabetes, colorectal cancer, fecal incontinence, and chronic constipation or diarrhea. Current drug treatments have severe side effects and often do not adequately address symptoms; a new approach is needed. ENS stimulation is a promising therapy for these patients, but a major limitation to this approach is our lack of knowledge. The human ENS is comprised of 5 million neurons and drives the digestive system, but its normal function and connections to the central nervous system (CNS) remain poorly understood. One of the major canonical signaling pathways between the ENS and the CNS is the vagus nerve, but the neural circuits involved are still under investigation. Better understanding of these circuits would provide a potential method of treatment for ENS related illness, with neurostimulation serving as an alternative to pharmaceutical treatments. Herein I describe a project which addresses these needs via development of new imaging tools to better understand the gut-brain axis, as well as demonstrating its utility as a target for treatment of gastrointestinal (GI) illness, specifically cancer-associated cachexia. Leaders in enteric neuroscience note that the continued inconsistencies in GI electrotherapies are driven by a fundamental lack of understanding of gut innervation and circuitry. New tools to directly observe colonic innervation and neuronal response, as well as a map of the whole peripheral nervous system, will reveal crucial targets for stimulation and enable more efficient targeting selection for neurostimulation or other local interventions, which will reduce off target effects and improve efficacy. To address these issues, I have developed an intravital window for direct imaging of the colon, enabling observation of colonic ENS response to stimulation in vivo for the first time. Additionally, I have developed an embryonic window, allowing visualization of embryonic GI development from E9.5 through birth. Finally, I have generated a mouse peripheral nerve map based on Diffusion Tensor Magnetic Resonance Imaging (DT MRI). Using novel scan parameters and post-processing algorithms, I identified nerve fibers throughout the body and generated quantitative tractography which specifically highlights GI innervation via the vagus nerve. Cachexia is a multi-systemic syndrome which produces weight loss, muscle atrophy, adipose wasting, fatigue, and anorexia. Affecting an estimated 1% of the global population and up to 80% of all cancer patients, cachexia is fatal in roughly 30% of cases and is incurable. Cancer-associated cachexia (CAC) is particularly devastating as in addition to resulting in decreased quality of life, CAC reduces tolerance and efficacy of cancer treatments and higher overall mortality. As many as half of all cancer deaths are attributed to CAC. There are currently no clinically meaningful treatments for CAC, despite attempts to employ dietary support, physical therapy, anti-inflammatory medication, appetite stimulants, and other supportive therapies. Herein I describe potential therapeutic approach for treatment of CAC via vagal perturbation – either by vagotomy or ultra-low frequency vagal block with an implanted stimulator. This intervention significantly attenuates weight loss, skeletal muscle atrophy, anorexia, urea cycle dysregulation, and circulating inflammatory cytokine elevation. Most importantly, it increases survival time in mice injected with tumor cells, suggesting this could be a clinically meaningful approach for treatment of CAC.

Detection and quantification of biomarkers directly from complex clinical specimens is desired and often required by healthcare professionals for the effective diagnosis and screening of disease, and for general patient care. Current methodologies to accomplish this task have critical shortcomings. Laboratory immunoassays, most notably enzyme-linked immunosorbent assay (ELISA) require extensive clinical infrastructure and complex user intervention steps to generate results and often are accompanied by a lengthy time-to-result. Conversely, available point-of-care (POC) diagnostic solutions, most notably available lateral flow immunoassays (LFIAs), often struggle with sensitivity and specificity in complex fluids, lack quantitative output and are not easily multiplexed. In this dissertation I will discuss the design, fabrication, testing, and refinement of an all-in-one fluorescence microarray integrated into a passive microfluidic fluid handling system to create a versatile and automated POC platform that can detect biomarkers from complex samples for disease management with the relative ease-of-use of an LFIA and the performance of a laboratory-grade test. The platform is driven by capillary and gravitational forces and automates all intervention steps after the addition of the sample and running buffer at the start of testing. The microfluidic cassette is built on a (poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) polymer brush which imparts two key functionalities, (1) it eliminates cellular and protein binding, and when combined with the vertical orientation of the microfluidic cassette prevents settling of debris during all assay steps. This allows for impressive sensitivities and specificities to be obtained from samples as complex as undiluted whole blood even when relying on gentle capillary and hydrostatic pressures for cassette operation. (2) Paradoxically, printed biorecognition elements can be stably and non-covalently immobilized into the POEGMA allowing for all reagents needed to conduct a sandwich immunoassay in a single step to be easily inkjet printed as spatially discrete spots into the POEGMA brush, which also stabilizes them at room temperature. Additionally, the microfluidic cassette is compatible with the “D4Scope” a handheld fluorescence detector that can quantify the output of the microfluidic cassette in seconds at the POC and is the only piece of auxiliary equipment required to operate the test.

This dissertation discusses early cassette prototypes and characterizes the performance of major device iterations (Chapter 2) before moving into three clinical applications of the cassette. First, a multiplexed serological test to detect antibodies against different proteins of the SARS-CoV-2 virus was developed (Chapter 3). Second, a multiplexed COVID-19 diagnostic test that simultaneously differentiates which variant you are infected with was developed (Chapter 4). Third, a sensitive fungal infection test for the diagnosis of talaromycosis was developed (Chapter 5). Finally, a rapidly iterative yet highly scalable injection molding fabrication process flow was created and characterized to improve performance and translatability of the cassette (Chapter 6).

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disease that causes loss of ambulation and premature death in affected boys. In the 1980s, the cause of the disease was attributed to mutations in the DMD gene, yet almost 40 years later there is still no cure. The large size of the gene, diversity of patient mutations, and delivery challenge of modifying skeletal muscle systemically have all limited the application of therapies to correct the disease-causing mutations. Gene editing with CRISPR-Cas technology is poised to revolutionize our ability to treat genetic diseases. For DMD, several landmark studies have demonstrated the strong therapeutic potential of using CRISPR to restore dystrophin protein in DMD models. Yet many challenges remain in converting proof-of-concept editing to a safe and effective therapy. Here, we focus on optimizing and developing methods to evaluate the two most promising strategies for using gene editing to treat DMD: exon deletion and exon skipping.In aim 1, we tackle the problem of searching for highly efficient gRNAs in the vast sequence space of DMD introns by applying high-throughput screening techniques to measure the relative deletion efficiency of gRNA pairs. We discover novel gRNA pairs for the deletion of DMD exon 51 and demonstrate an improvement over previous methods of gRNA pair discovery. In aim 2, we evaluate the potential of “CRISPR 2.0” editing tools that provide next-generation control over DNA editing to produce DMD exon skipping with reduced disruption to the genome. We discover a base editor and gRNA design that efficiently skips exon 45 and restores dystrophin expression and apply unbiased characterization methods to interrogate the impact of such editing.

Wound healing is a vastly complicated process. While this can be said about many biological functions in the body, wounds present a particularly difficult problem due to their inherent irregularity or uniqueness. Because different wounds behave and heal differently, or not at all, different therapies must be developed to treat them effectively. The research presented here details several approaches to progress not only the entire field of wound healing research, but also focuses on hydrogel technology improvements. Using titanium 3D printing, cap-able splints were constructed to not only ease the surgical process but also enable efficient daily wound access for treatment administration or wound tracking over time without the need to completely undress and redress the wound. The titanium splints did prove effective for daily monitoring but did still require some surgical prowess. To remove the need for surgical skills, an adhesive wound splint was developed by incorporating ethoxylated polyethyleneimine (EO-PEI) into the traditional polydimethylsiloxane (PDMS) polymer recipe resulting in adhesive PDMS (aPDMS). The aPDMS splints drastically reduced surgery time per animal without compromising wound splinting performance. Traditional bulk hydrogels have been used in wound healing research but have yet to be clinically implemented in a widespread manner due in part to their resistance to cellular infiltration or integration with the host. Using hyaluronidase (HAase) on a hyaluronic acid (HA) based hydrogels to partially degrade the surface of bulk gels yielded a looser nano-scale mesh size that enhanced cellular infiltration into the gel and granted better access to nanoparticle therapy loaded within. Finally, a biologically active viscous salve loaded with heavy chains (HC) of the serum protein Inter-α Inhibitor (IαI) was designed to leverage HC’s ability to mitigate the inflammatory response such that normal wound healing regeneration could ensue.

Malignant gliomas are the most common primary brain tumor in adults, with an incidence of five cases per 100,000 persons per year. Grade IV glioblastoma is the most aggressive form and prognosis remains poor despite the current gold-standard first-line treatment – maximal safe resection and combination of radiotherapy with temozolomide chemotherapy – resulting in a median survival of approximately 20 months. Tumor recurrence occurs in virtually all glioblastoma patients, and there currently exists no accepted treatment for these patients. Recent advances in novel directed therapeutics are showing efficacy and have entered clinical trials. This work spans the pre-clinical and clinical development of a bispecific antibody – EGFRvIII:CD3 bi-scFv – for the treatment of malignant gliomas.

Chapter 1 reviews current front-line immunotherapy research in the fields of antibodies, including BiTEs and checkpoint inhibitors, and tumor vaccinations, including peptide and dendritic cell vaccinations. Furthermore, challenges specific to high-grade gliomas as well as opportunities for combination therapies are discussed. Chapter 2 introduces the architecture of the novel bispecific antibody EGFRvIII:CD3 bi-scFv and provides an overview of the molecule’s efficacy in various models. EGFRvIII:CD3 bi-scFv is a truncated antibody with dual specificity. One arm targets the epidermal growth factor receptor mutation variant III (EGFRvIII), a tumor-specific antigen found on glioblastoma. The other arm targets the human CD3 receptor on T cells. As an obligate bispecific antibody, simultaneous binding of both receptors by multiple EGFRvIII:CD3 bi-scFv’s results in the crosslinking of CD3 receptor, activation of T cells, and release of perforin/granzyme which lyses the proximal EGFRvIII-expressing tumor cells. EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma.

Chapter 3 outlines the in-house development of a scalable clinical production process using a WAVE (GE) bioreactor and describes the cGMP-compliant clinical production of EGFRvIII:CD3 bi-scFv. The 250-liter cGMP-production run yielded more than four grams of clinical drug material.

Chapter 4 demonstrates that EGFRvIII:CD3 bi-scFv produced using the cGMP development process is efficacious in both in vitro and in vivo models of glioblastoma. The chapter also describes the approach used to calculate the starting dose for the upcoming first-in-human clinical trial. First-in-human clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. In order to establish a first-in-human dose, as advised by the FDA for bispecific antibodies, this work uses a MABEL approach to select a safe starting dose for EGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng EGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a first-in-human clinical study.

Chapter 5 describes the pharmacokinetic properties of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, the chapter first describes the development of a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, was used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. These results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.

Finally, Chapter 6 provides an outlook of the future development of cancer therapeutics for treating malignant gliomas.

Osteoarthritis (OA) is one of the leading causes of disability in the United States, afflicting over 27 million Americans and imposing an economic burden of more than $128 billion each year (1, 2). OA is characterized by progressive degeneration of articular cartilage together with sub-chondral bone remodeling and synovial joint inflammation. Currently, OA treatments are limited, and inadequate to restore the joint to its full functionality.

Over the years, progresses have been made to create biologic cartilage substitutes. However, the repair of degenerated cartilage remains challenging due to its complex architecture and limited capability to integrate with surrounding tissues. Hence, there exists a need to create not only functional chondral constructs, but functional osteochondral constructs, which could potentially enhance affixing properties of cartilage implants utilizing the underlying bone. Furthermore, the molecular mechanisms driving chondrogenesis are still not fully understood. Therefore, detailed transcriptomic profiling would bring forth the progression of not only genes, but gene entities and networks that orchestrate this process.

Bone-marrow derived mesenchymal stem cells (MSCs) are routinely utilized to create cartilage constructs in vitro for the study of chondrogenesis. In this work, we set out to examine the underlying mechanisms of these cells, as well as the intricate gene correlation networks over the time course of lineage development. We first asked the question of how transforming growth factors are determining MSC differentiation, and subsequently utilized genetic engineering to manipulate this pathway to create an osteochondral construct. Next, we performed high-throughput next-generation sequencing to profile the dynamics of MSC transcriptomes over the time course of chondrogenesis. Bioinformatics analyses of these big data have yielded a multitude of information: the chondrogenic functional module, the associated gene ontologies, and finally the elucidation of GRASLND and its crucial function in chondrogenesis. We extended our results with a detailed molecular characterization of GRASLND and its underlying mechanisms. We showed that GRASLND could enhance chondrogenesis, and thus proposed its therapeutic use in cartilage tissue engineering as well as in the treatment of OA.

Traumatic brain injuries (TBIs) are a major public health concern and socioeconomic burden worldwide. In recent years, brain injuries in US service personnel have focused attention on TBI affecting the military population (Bass et al., 2012). Blast injuries have become the most common cause of mortality and morbidity in soldiers returning from Iraq and Afghanistan (Owens et al., 2008, Warden, 2006). The frequency of blast-related sequelae found in allied forces has led some to call it the ‘signature wound’ of the wars abroad.

The growing incidence of TBI has spurred an increase in research efforts within the neurotrauma community to define TBI etiology. Identification of the critical injury mechanisms underlying TBI is an area of greatest need. Our understanding of TBI etiology, physical damaging mechanisms, and pathophysiology remains inadequate. The ability to design specific countermeasures and targeted prevention strategies is restricted by an incomplete understanding of the underlying damaging mechanisms.

Cavitation, the formation of vapor filled cavities in a liquid medium, has been proposed as a damaging mechanism of TBI in both blunt impacts (Ward et al., 1948, Gross, 1958) and blast-induced neurotrauma (Moore et al., 2008, Panzer et al., 2012c). The cavitation hypothesis of TBI centers on observation that high energy events such as high-explosive blast impingement onto the head generate large pressure transients in and around the brain. Localized areas of low pressure may surpass the tensile limits of the cerebrospinal fluid vaporizing the fluid and forming cavitation bubbles. These voids grow, potentially displacing surrounding tissue. When the bubbles collapse, perhaps violently, jets of liquid with potentially large localized pressures and temperatures may be created, damaging surrounding tissue.

The main objective of this dissertation was to develop an experimental foundation and provide empirical evidence for cavitation as a damaging mechanism of blast-induced TBI. This dissertation uses biofidelic surrogate head models of blast and in vivo animal models of blast injury to address the unanswered questions surrounding cavitation and blast neurotrauma. Foremost, cavitation response was observed in the surrogate head form exposed to blast conditions associated with injury. The 50% risk of cavitation occurs at a blast level of 262 kPa incident overpressure and 1.96 ms duration. This blast dosage represents a 62% chance of mild intracranial bleeding from scaled ferret experiments (Rafaels et al., 2012). Cavitation onsert, growth, and collapse were confirmed through high-speed imaging of the fluid layers of the contrecoup, while strong acoustic emission signatures associated with cavity collapse were captured and time matched with the video. Near-harmonic frequencies at 64 kHz, 126 kHz, and 267 kHz were associated with the energetic collapse of the bubbles. Our results provide compelling evidence that primary blast alone may induce cavitation that leads to TBI.

Evidence of cavitation was recorded in live porcine specimen exposed to blast. Acoustic sensors mounted to the skull of each specimen recorded acoustic emissions during blast exposure. Scaled spectral analysis revealed acoustic energy in higher frequencies bands with peaks at 64 kHz, 139 kHz, and 251 kHz, closely matching the spectral peaks associated with void collapse in surrogate experiments. To our knowledge, this study is the first to present evidence of blast-induced cavitation in a live animal model in the field of cavitation TBI research.

The results presented in this dissertation also greatly improve our understanding of how mechanical loads are imparted onto the head during a blast exposure and how this loading leads to cavitation onset. Strain analysis of the surrogate head indicates wall compliance from skull deformation and shear wave propagation through the skull as significant physical factors driving the tensile fluid responses in the head. Future design considerations for preventative measures should account for these physical mechanisms.

This dissertation also makes important contributions to blast injury research by presenting a clinically relevant murine model of blast TBI. Murine blast lethality risk and functional behavior outcomes before and after blast injury are presented. We provide guidelines for small animal blast testing, along with methodological recommendations for benchtop shock tube design and specimen placement in relation to the shock tube.

The contributions of this dissertation further serve as an important methodological guide to the neurotrauma and biomechanics community studying blast-related TBI and cavitation as a damaging mechanism. The developed surrogate head system and cavitation detection techniques provide a research template and are a springboard to future research efforts elucidating the damaging effects of cavitation during TBI.

Malonyl-CoA (malonyl-CoA) is a platform chemical that serves as a precursor for a wide range of commercial products and pharmaceutical intermediates. In E. coli, malonyl-CoA levels are tightly regulated to remain at low levels. Two Stage Dynamic Metabolic Control (DMC) is a tool previously demonstrated to improve desired metabolite flux for several products. This work leverages DMC to improve malonyl-CoA fluxes. Specifically, we demonstrate coordinated dynamic reductions in the activities of fabI (enoyl-ACP reductase), gltA (citrate synthase), zwf (glucose-6-phosphate dehydrogenase) and glnB (nitrogen regulatory protein PII-1), during stationary phase lead to synergistic improvements in malonyl-CoA flux and the production of malonyl-CoA dependent products, 1,3,6,8-tetrahydroxynaphthalene (THN), Triacetic Lactone (TAL), and Phloroglucinol (PG). We also discuss the unique set of limitations that were observed for both TAL and PG biosynthesis as well as the strategies that were tested to overcome them. Additionally, we provide a historical review of the challenges associated with the production of Phloroglucinol. Finally, we end with a critical review focused on the bioproduction of an Acetyl-CoA and Succinyl-CoA derived product, Adipic Acid, to give perspective to common challenges associated with biobased product development.

Bone grafting is one of the most commonly used surgical methods to augment bone regeneration in orthopedic procedure. While using natural bones, such as autograft and allograft are considered as the gold standard techniques, they suffer from numerous drawbacks including scarcity, donor site complications, and potential disease transmission. To overcome these limitations, mineralized poly (ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid (PEGDA-co-A6ACA) composed of an organic phase and an inorganic, biomineralized phase that recapitulates certain aspects of dynamic mineral environment of native has been developed. The real-world application this biomineralized material in treating bone defects in vivo depends upon a myriad of parameters including scaffold structural parameters (e.g. pore size), mechanical properties (e.g. strength and toughness), and host environments (e.g. age of the recipient). In this dissertation, I explored these biomaterial and biological parameters for biomaterials mediated bone regeneration through leveraging endogenous healing mechanism. First of all, I evaluated the potential of mineralized biomaterials to induce bone repair of a critical-sized cranial defect in the absence of exogenous cells and growth factors. I demonstrated that the mineralized biomaterial alone can support complete bone formation within critical-sized bone defects through recruitment of endogenous cells and neo-bone tissue formation in mice. By providing a bone-specific mineral environment, these biomaterials induce osteogenic commitment of recruited host progenitor cells and support the maintenance of cells relevant for the formation and function of bone tissues, including vascularization of the implant during repair. Based on these findings, I further investigated the effect of the scaffold pore size on in vivo ectopic bone formation. Biomineralized PEGDA-co-A6ACA hydrogels were made to have an interconnected macroporous network with different pore size ranges (45-53 μm, 90-106 μm, 160-180 μm, 212-250 μm or 300-355 μm) and similar overall porosity between 65% to 70%. Using these scaffolds, I evaluated their abilities to promote ectopic bone formation upon subcutaneous implantation in wild-type mice as a function of time. I found that scaffolds with pore sizes larger than 100 μm showed similar bone formation abilities, whereas in scaffolds with pore sizes 45-53 μm, cell infiltration only happened at the peripheral region of the scaffolds. Results from this study revealed that pore size of the scaffolds had a prominent influence on the extent of cell infiltration and bone ingrowth. While such biomaterial-mediated in situ tissue engineering is highly attractive, success of this approach relies largely on the regenerative potential of the recruited endogenous cells, which is anticipated to vary with age of the host. To this end, I investigated the effect of the age of the host on mineralized biomaterial-mediated bone tissue repair using critical-sized cranial defects as a model system. Mice of varying ages, 1-month-old (juvenile), 2-month-old (young-adult), 6-month-old (middle-aged), and 14-month-old (elderly), were used as recipients. I showed that the biomineralized scaffolds support bone tissue formation by recruiting endogenous cells for all groups albeit with differences in an age-related manner. The age of the recipient mice had a significant influence on the quantity and quality of the neo-bone tissues characterized in terms of bone mineral deposition and bone tissue-specific markers, where delayed bone formation and decreased quantity of neo-bone tissue formation were observed in older mice. The real-world applications of the biomineralized materials for aiding bone tissue regeneration are greatly limited by the lack of mechanical strength and toughness of the materials. To enhance the mechanical property of the biomineralized scaffold, I further proposed a double network (DN) hydrogel system with an asymmetric network structure, where the first network is tightly cross-linked by A6ACA with crosslinker N, N'-Methylenebisacrylamide (bisacrylamide), and the second network is loosely crosslinked PEGDA. The effects of bisacrylamide crosslinker concentration (2 mol.%, 4 mol.% and 6 mol.%), and molecular weight (Mn: 3.4 kDa, 6 kDa, 10 kDa, and 20 kDa) of 20 w/v % PEGDA on mechanical properties of the resultant DN-hydrogels were investigated and compared to those of single network (SN) hydrogels of the same composition. Findings from this study showed that increase in crosslinker concentration of the first network was correlated with lower ultimate compressive strain, higher compressive strength, toughness and elastic modulus. Furthermore, DN-hydrogels prepared in this work displayed swelling ratios ranging from 569 ± 20% to 1948 ± 12%. Among all compositions, DN-hydrogel with 6 mol.% bisacrylamide and PEGDA 10 kDa demonstrated the highest compressive strength (3.47 ± 0.35 MPa), highest toughness (0.60 ± 0.03 MJ/m3), and elastic modulus (1.04 ± 0.09 MPa). Using this composition, porous DN-hydrogels with interconnected pore architecture were fabricated through polymethylmethacrylate (PMMA) bead leaching method. Resultant porous hydrogels demonstrated potent biomineralization capabilities, and the matrix-bounded CaP minerals were able to undergo dissolutions. Given the high strength and biomineralization capacity, DN-hydrogels reported here could be useful for developments of tissue engineering scaffolds for bone tissue regenerations. Overall, this dissertation explores different biomaterial designs and biological factors in biomaterial-mediated in vivo bone tissue repair, providing materials insights that are useful to researchers and engineers in designs of biomaterials to leverage endogenous healing mechanism for tissue regeneration and repair.

Muscle fibrosis is caused by muscle injury, dystrophy, sarcopenia, and rheumatoid arthritis. This condition is characterized by hardening and scarring, which impairs contractile muscle function. To understand how fibrotic disease affects muscular function, we created a model of human skeletal muscle fibrosis using three-dimensional engineered skeletal muscle (myobundles). Furthermore, to investigate the effect of skeletal muscle fibrosis on the vascular system, we integrated the fibrotic skeletal muscle with tissue engineered blood vessels. Treating myobundles with Transforming Growth Factor β1 (TGF-β1) reproduced key characteristics of fibrotic skeletal muscle including reduced contractile force, disrupted contractile protein organization, increased stiffness, and expression of profibrotic genes. Treatment with a selective inhibitor (SB525334) of TGF-β1 receptor (ALK5, TGF-βRI) increased contractile function and decreased ECM deposition, consistent with animal studies in the literature. We also observed endogenous secretion of TGF-β1 in our myobundles which is of novel biological significance. siRNA knockdown of TGF-β1 increased contractile force. Testing anti-fibrotic drug Nintedanib in this model, showed an increase in tetanus force production in 2 out of 3 donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Western blot analysis of Nintedanib also providence evidence of its inhibition of TGF-β1 signaling by the reduction of phosphorylated Smad2/3. Repositioned anti-fibrotic drug Suramin treatment of fibrotic myobundles resulted in increase of tetanus force production in all three donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Suramin’s influence on TGF-β1 signaling in our system was found not to be as targeted as Nintedanib as there was only reduction in Smad3 phosphorylation and not Smad2 phosphorylation. Anti-fibrotic drug testing in our model was also able to wean out donor specific sensitivity to the drugs with donor 3. Skeletal myobundles were integrated with Tissue Engineered Blood Vessels (TEBVs) to identify the effect of skeletal muscle fibrosis on blood vessels or the human vasculature. Integrated TEBVs with 5 ng/ml TGF-β1 dosed myobundles showed reduced function, increased mesenchymal markers such as vimentin and alpha smooth muscle actin, and increased endothelial cell inflammation. Our results suggest a detrimental effect of skeletal muscle fibrosis on blood vessels and show an interaction between the skeletal muscle fibrosis and the human vasculature This model provides a platform to study skeletal muscle fibrosis alone or its effect on the vasculature and allows for testing anti-fibrotic drugs and assessing myobundle function along with disease influence on human vasculature.

Important physiological processes, including migration, morphogenesis, and differentiation, and pathophysiological processes, including cancer and fibrosis, have been increasingly tied to cell’s abilities to sense mechanical stimuli from the extracellular matrix (ECM) and either generate or respond to mechanical loads in turn. Mechanical stimuli from the ECM is integrated at focal adhesions (FAs), a subcellular structure consisting of hundreds of interacting proteins that mediate physical connections between the ECM and force-generating actin cytoskeleton. At the molecular level, underlying this integration process is mechanotransduction, where the mechanical deformation of load-bearing proteins alters protein function to regulate signaling. This process is thought to expose cryptic protein binding domains that lead to the downstream recruitment and formation of mechanosensitive protein complexes. However, an incomplete understanding of mechanotransduction, and the relevant molecular players involved, prevents a mechanistic understanding of all mechanosensitive processes. In turn, this has hindered advancements in the development of therapies to combat mechanosensitive diseases, as well as efforts to manipulate cell response through the design of bio-instructive scaffolds in tissue engineering and regenerative medicine. To address this issue, the central goal of this dissertation is to develop and utilize molecular-scale tools to probe the role of molecular-scale forces on protein function and elucidate relevant molecular players in mechanotransduction. To date, available techniques for studying the role of molecular-scale forces on protein function remain technically challenging and low throughout. Thus, we sought to develop novel imaging- and biochemical-based assays that were capable of probing protein response to molecular tension within cellular contexts where both spatiotemporal control of cellular force generation and signaling networks were maintained. More specifically, we developed two separate assays that work in concert to first, characterize the specificity of the protein’s molecular tension-sensitive recruitment to FAs, and then unbiasedly uncover all molecular tension-sensitive protein interactions. We developed an imaging-based assay, termed Fluorescence Tension Co-localization (FTC), that integrates immunofluorescence labeling, molecular tension sensors, and machine learning to determine the sensitivity, specificity, and context-dependence of molecular tension sensitive protein recruitment mechanisms. When we applied FTC to study the mechanical linker protein, vinculin, we found constitutive and context specific molecular tension-sensitive protein recruitment mechanisms that varied with adhesion maturation. More specifically, we found that in immature FAs, vinculin tension specifically recruits integrin-associated proteins while in mature FAs, vinculin tension specifically recruits actin-associated proteins. We also developed a separate biochemical based assay, that integrates proximity-dependent biotin labeling techniques with biophysical knowledge of key residues required for protein loading to determine the mechanosensitive binding interactions of key FA proteins. Using streptavidin pulldown assays to isolate the interacting proteins, we found that vinculin forms proximal protein interactions with an FA protein, migfilin, that has not been previously identified as a vinculin binding partner. In summary, this dissertation focuses on developing novel molecular-scale assays for studying mechanosensitive protein recruitment and interaction mechanisms. Using these tools, we identified multifaceted tension sensitive protein recruitment mechanisms associated with vinculin during adhesion maturation, as well as identified a novel proximal binding partner, migfilin. Overall, this establishes the importance of molecular loads across single proteins in regulating other protein activity. Widespread use of these developed assays will help elucidate a more mechanistic understanding of mechanotransduction through the identification and study of relevant molecular players.

The zebra finch, an Australian songbird, is a uniquely powerful model organism for the study of vocal production learning, and its song system shares behavioral, anatomical, and genetic properties with the human spoken language circuit. However, research in zebra finches are disadvantaged by the lack of proper tools and techniques for tractable investigation of the molecular underpinnings of vocal learning. Here, I worked to close the gap in three areas. First, I induced a continuous zebra finch cell line capable of monoclonal cell line generation for in vitro characterization and testing in zebra finch cells. Second, I utilized advanced methods to improve the descriptive cellular resolution of several genes with specialized expression in the song system that are convergent with humans and I tested genome editing tools in vivo to demonstrate the potential for their gene ablation in the zebra finch. Third, I modified transgenic techniques used in poultry toward the more efficient and versatile generation of transgenic songbirds. Finally, I used an in situ hybridization method I modified on the NR4A2 gene to validate avian brain organization hypothesis of Jarvis et al., 2013. This work provides new avenues for exploring avian biology and progress towards a more genetically tractable model system for songbird neuroscience.

The extracellular environment is a key regulator of cell behavior, providing both biochemical factors and mechanical signals to influence the form and function of cells. The process by which cells sense and respond to environmental mechanical signals is often mediated through force-dependent changes in protein structure and function through a poorly understood process known as mechanotransduction. Towards elucidating the molecular processes underlying mechanosensitive regulation, molecular tension sensors (MTSs) have been created to measure forces experienced by specific proteins inside cells. However, an incomplete understanding of the effects of intracellular forces on fluorescent protein (FP) function within the context of MTs limits sensor application and interpretation. To advance our understanding of the molecular events mediating mechanotransduction, it is necessary to improve on existing approaches as well as to develop new technologies for probing mechanical consequences inside cells. In this dissertation we aim to address this limitation by creating a generalizable assay for probing the effects of cell-generated forces on FP function towards improving the use and interpretation of MTSs. Additionally, we describe the development of a new “synthetic” actin crosslinking sensor which leverages FP mechanosensitivity to provide new insights into mechanical processes inside cells.In our initial efforts, we focused on investigating the effects of cell generated forces on FP function within vinculin-based MTSs. We chose vinculin as our model system as vinculin is a well-studied mechanosensitive protein, known to play a critical role in force transmission inside cells. Additionally, the vinculin tension sensor (VinTS) has been extensively characterized, validated, and utilized in a broad array of applications. Leveraging the relationship between FRET measurements and fluorophore stoichiometry with vinculin MTSs, we developed a generalizable assay for evaluating changes in ensemble MTS measurements in terms of fluorophore contributions. Furthermore, we validated this new method on an extensive MTS data set containing over 2000 cells expressing vinculin sensors. Our analysis revealed that FP stoichiometry within VinTS was modulated significantly within individual focal adhesions (FAs) in an actomyosin-dependent manner, and that both load magnitude and load duration likely play a role. Additionally, we found that this force-mediated loss of FP function, or “mechanical quenching,” is a reversible process, consistent with nonequilibrium transitions in protein structure. To investigate FP mechanosensitivity further, we developed an engineered FRET-based actin crosslinking (ABD) sensor to serve as an improved experimental platform, within which FPs would be subjected to higher loads in a manner free of endogenous biochemical regulation. Within this new system, higher tensile loading and FP mechanical quenching was observed at dynamic actin networks. Furthermore, we found that FP mechanical quenching within these sensors was mediated by non-muscle myosin II (NMII) activity and appears to be reversible. In addition, we found that FPs exhibit different sensitivities to intracellular mechanical loads. To probe the molecular origins of ABD sensors loading within cells, we manipulated the organization and dynamics of actin structures by tuning substrate stiffness within engineered in vitro culture systems. Using this approach, we found that ABD sensors reported increased loads and FP mechanical quenching at dynamic actin networks in response to softer substrates. By coupling FRET-based MTSs with the tunability of in vitro culture models, we demonstrated the application of ABD sensors to probe changes in tensile loading in response to environmental mechanical cues. In summary, this dissertation describes the development of novel tools for studying the effects of intracellular forces on FP function within the context of FRET-based tension sensors. Using these tools, we found that FPs, like mechanosensitive signaling proteins, can undergo nonequilibrium transitions in response to cell-generated forces. Based on these observations, we propose that FP mechanical quenching within MTSs could potentially serve as an entirely new way to visualize and probe mechanical consequences within force-sensitive proteins. By exploiting the mechanosensitivity of FPs as a mechanical consequence, new insights into molecular force-sensitive processes inside cells may be obtained.

Upgrading raw biogas (~60% CH4, 40% CO2, 1000-5000 ppmv H2S) to renewable natural gas (RNG) (> 97% CH4, < 2% CO2, < 4 ppmv H2S) for injection into the grid is a desirable endeavor. RNG would allow for a clean alternative to natural gas derived from fossil origin, and it also have a versatile use as a transportation fuel and source of heating energy. Current physical-chemical technologies, such as pressure swing absorption and organic chemical scrubbing, can successfully upgrade raw biogas to meet RNG standards (1,2). However, they are energy intensive, costly, and can remove fractions of methane gas along with the impurities. Recently, biological biogas upgrading technologies have emerged as a promising solution for converting raw biogas to RNG. The method relies on hydrogenotrophic methanogens to reduce the CO2 fraction of raw biogas to CH4 using H2 as the electron donor. This method is advantageous compared to traditional biogas upgrading methods because is sequesters carbon emissions while increasing the volumetric production of methane. While early studies on biological biogas upgrading in continuously stirred tank reactors were conceptually validating, hydrogen mass transfer resistance from the gas-to-liquid phase prevented fast upgrading capacities from being realized. Slow biogas upgrading rates hinder the economic feasibility of the process. Furthermore, these studies only focused on CO2 removal when in reality, other impurities, such as corrosive H2S, must also be removed before RNG injection into the natural gas pipeline.

The overall objective of this thesis research is to develop a biological trickling filter reactor that can upgrade biogas to RNG standards at fast upgrading capacities while biologically co-removing H2S. A biological trickling filter was chosen for this investigation because they are characterized by a high specific surface area for biofilm growth, high biomass density, and are known for their high overall mass transfer coefficients; all factors that contribute to high conversion rates. A proof-of-concept study validated that this approach could achieve upgrading rates that were 5 – 30 times faster than other bioreactor configurations. This finding supported further studies that aimed to investigate hydrogen mass transfer resistance specifically in a biological trickling filter reactor. This was accomplished using a highly sensitive dissolved hydrogen sensor, which collected concentrations in real-time. Using this sensor, experiments were conducted to assess mass transfer resistance in the gas and liquid films. It was discovered that there was no external resistance in the gas-film. Furthermore, the liquid phase was a main barrier for mass transfer and reducing the liquid film thickness can significantly improve biogas upgrading capacities by 20%.

In addition to laboratory experiments, a robust and conceptually correct mathematical model was developed for a biogas upgrading biological trickling filter. The model was used to provide deeper insight into process fundamental and identify biological versus mass transfer limitations in the bioreactor. The model successfully replicated complex experimental findings and confirmed that liquid transport through the bioreactor bed was faster than the rates of mass transfer and biological conversion. A sensitivity analysis revealed that the model was most sensitive to the empty bed contact time and the maximum rate of reaction. Interestingly, the mass transfer coefficient for the liquid film (kLa) did not significantly improve the biogas upgrading rate for the bioreactor. This is because the model predicts that the bulk of hydrogen mass transfer occurs from the gas to non-wetted biofilm phase.

Concluding mass transfer resistance testing and process optimization, it was demonstrated that the engineered bioreactor could successfully upgrade various biogas compositions to RNG standards. The rates achieved for these experiments (10 – 20 m3CH4 m-3 d-1) were 1.5 – 25 times faster than other comparable research studies. To determine the economic feasibility of this technology, a paper scale-up cost analysis was conducted to estimate the investment and operation costs of a biological trickling filter upgrading raw biogas (60% CH4, 40% CO2) to RNG (> 97% CH4 < 2% CO2). This was accomplished by using experimental findings to scale the dimensions and determine heating and cooling requirements based on seasonal temperatures. Cost estimates for parts were acquired through vendor quotes. The cost analysis showed that the bioreactor is economically feasible however, the H2 acquisition cost was ~ 650% of the bioreactor investment cost. This is because H2 was acquired from the electrolysis of excess wind and solar energy and the cost of the hydrolyzer was ~ $1,000,000. Despite this significant cost, the total amortized cost of the biological biogas upgrading system was comparable to current physical-chemical upgrading technologies.

The final study of this thesis investigated the potential to biologically co-treat CO2 and H2S using nitrate as the terminal electron donor. Since the addition of nitrate favored undesired oxidation-reduction reaction pathways with hydrogen, a method was developed to map electron transfers. The effect of nitrate on methanogensis was tested with and without sulfur oxidizing bacteria. Under both conditions, nitrate had a negative impact on methanogenesis and ultimately, prevented co-treatment from being achieved. While attempting to co-treat H2S and CO2, it was discovered that dissimilatory nitrate reduction to ammonium was favored over denitrification. The electron balance confirmed that a competition for electrons from hydrogen did exist. This competition required N:S feeding ratios upwards of 16:1, which far exceeded the theoretical ratios of (4:1) for denitrifying bacteria. While the high nitrate loading rates allowed for high H2S removal efficiencies (98%), they inhibited methanogenesis so that carbon dioxide removal efficiencies did not meet RNG standards. Thus, future work should focus on alternative electron donors for sulfur oxidation and quantifying methanogenesis inhibition caused by sulfur-oxidation/denitrification pathways.

In this thesis, a self-focusing multi-spark (SFMS) shock wave generator is developed to provide flexibility in controlling the beam size and shape in an electrohydraulic shock wave lithotripter. Such a device will allow us to better distribute the shock wave energy to match the anatomic features in the urinary collecting system or respiration movement of the stone to improve stone fragmentation efficiency while reducing tissue injury. In this study, we present the design, fabrication and evaluation of the multi-pin titanium electrodes by 3D printing, integration of the SFMS shock wave generator, acoustic field characterization based on hydrophone measurements, and stone fragmentation tests using stone phantoms confined within a polyurethane rubber holder of elliptical shape. The effects of pin number on pressure output and electrode degradation are evaluated in order to produce a consistent pressure waveform with increased electrode lifespan.

Experiments were conducted using two transducer configurations: case 1 (axisymmetric) – with all transducers connected, and case 2 (non-axisymmetric) - with transducers on the two side sections disconnected. A fiber optic probe hydrophone was mounted on a 3D computer controlled translational stage to perform acoustic field characterization. Stone fragmentation test was conducted with stone phantoms placed inside a stone holder made of soft tissue mimicking material polyurethane rubber to evaluate the stone comminution efficiency. To assess the effect of multi-pin design on electrode damage and output pressure variations, transducer lifespan experiments were performed. Specifically, individual transducers used in the SFMS, but with different pin numbers (1, 10, and 45) were fired up to 2000 shocks.

The FOPH measurement results show that the SFMS can generate an axisymmetric focal zone with the -6 dB focal width of 16 mm, or a non-axisymmetric focal zone with the -6 dB focal width elongated to 28 mm in the side-section direction, while in the perpendicular direction the -6 dB focal width is 16 mm, accompanying a peak positive pressure of 39.0±1.8 MPa at an input electric pulse energy of 600 J. Such a focal zone has stone comminution efficiency of 37.68±5.11% and 69.58±8.29% for stone fragment smaller than 2.0 mm and 2.8 mm, respectively, after 150 pulses. After more than 2000 pulses, the pressure output drops only by 2%, and lifespan of the transducer (defined by a peak output pressure drop less than 10%) is expected to exceed 6000 pulses. Altogether, we have demonstrated that the SFMS can generate an elongated non-axisymmetric focal zone with higher stone comminution efficiency, and has significantly increased electrode lifespan. The SFMS shock wave generator may provide a flexible and versatile design to achieve accurate, stable, and safe lithotripsy for kidney stone treatment.

Genome-wide association studies have identified thousands of DNA variants associated with specific phenotypes, yet it remains unknown which variants are causal. Additionally, more than 90 percent of common variants occur in noncoding genomic regions, further complicating efforts to predict their function. Large consortia efforts have leveraged functional genomics assays to characterize the genome-wide and epigenome-wide features of noncoding regions and common candidate cis-regulatory elements. However, these predictions are largely based on DNA sequence or correlation of epigenome marks alone, and do not provide information for which genes and broader regulatory networks are controlled by these candidate cis-regulatory elements. Advancements in CRISPR/Cas9-based genome and epigenome editing tools and multiplexed screening assays have enabled systematic perturbation of candidate cis-regulatory elements. We first sought to establish principles for performing noncoding CRISPR screens. We next applied these principles to investigate a subclass of cis-regulatory elements that respond to mechanical stimuli. Finally, we combined noncoding CRISPR screening approaches with single cell transcriptome profiling to clarify the regulatory landscape in diverse cell types in the Major Histocompatibility (MHC) Locus, one of the most complex regions of the human genome. Through these efforts, we establish guidelines for noncoding CRISPR screen design, execution, and analysis, identify mechanosensitive cis-regulatory elements and their role in complex cellular processes, and reveal cell-type specific and shared regulatory mechanisms governing gene expression in the MHC locus. Collectively, these studies provide experimental and computational frameworks for future investigation of cis-regulatory element function and will enable further dissection of variant-gene-phenotype relationships.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human CSPG4 antibodies or antibody fragments. In addition, the efficacy of cytotoxic monotherapies, such as the CSPG4-targeting immunotoxins (ITs), is limited by hyperactive anti-apoptotic pathways prevalent in tumor cells. Therefore, there is a need to discover novel, fully human antibodies for CSPG4-targeting immunotherapies and to develop new strategies that sensitize resistant CSPG4-expressing tumor cells to IT therapies.

To discover fully human antibodies that can be developed into potential CSPG4-targeting therapeutics, my first aim is to develop novel human single-chain variable fragments (scFvs) with high binding affinity and specificity to the CSPG4 antigen. Affinity maturation was performed on a novel, fully human anti-CSPG4 scFv using the random mutagenesis approach. A yeast display library was constructed for the mutant clones, and screened using a modified whole-cell panning method followed by fluorescence-activated cell sorting (FACS). After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These mutant clones were highly specific to the CSPG4 antigen, and displayed nanomolar to picomolar binding affinities. While each of them harbored only two to six amino acid substitutions, they represented approximately 270-3000-fold improvement in affinity compared to the parental clone. These affinity-matured scFvs can be potentially developed into diagnostic or therapeutic agents for evaluation and treatment of CSPG4-expressing tumors.

To facilitate the screening of scFv libraries targeting CSPG4, my second aim is to develop a cell-based fluorescent assay for high-throughput analysis of antibody affinity (KD) in the nanomolar range. In this method, fluorescently labelled antibodies were added to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The results confirmed that the KD values obtained using this method were comparable with values obtained by the conventional flow cytometry and radioactive (I125) scatchard assays. This demonstrates that the cell-based fluorescent method allows for accurate and efficient identification of therapeutically relevant leads.

Finally, to improve the efficacy of ITs targeting CSPG4, especially in the IT-resistant tumor cells, my third aim is to evaluate a multi-pathway therapy that combines anti-CSPG4 ITs and small molecule Bcl-2 inhibitors. To enhance sensitivity of cancer cells to ITs, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting CSPG4 with a Bcl-2 inhibitor (ABT-737, ABT-263, or ABT-199) against patient-derived glioblastoma xenografts, melanoma cell lines, and breast cancer cell lines. Results from the in vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized all three tumors to the IT treatment, and in some cases improved the IC50 values of 9.2.27-PE38KDEL by over 1000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that the rate of IT internalization and the efficiency of cleaved exotoxin accumulation in the cytosol correlated with the enhanced sensitivity of the tumor cells to the combination treatment. Furthermore, the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy was confirmed in an orthotopic GBM xenograft model and a model of melanoma metastasized to the brain. For the first time, our study compares the efficacy of ABT-737 and 9.2.27-PE38KDEL combination therapy in GBM and a different brain metastases model, providing insights into overcoming IT resistance in brain tumors.

In conclusion, I discovered novel human scFvs with high binding affinities to CSPG4, developed a cell-based fluorescent method for accurate and efficient affinity analysis of antibodies, and investigated combination immunotherapies that utilized Bcl-2 inhibitors to sensitize tumor cells to treatment by CSPG4-targeting ITs. The results from these studies helped to facilitate the development of novel antibody-based immunotherapies and combination immunotherapies for CSPG4-expressing tumors.

Chronic respiratory diseases are the leading cause of death and disability. Estimates suggest a ~40% increase in population with chronic respiratory diseases and ~18% increase in respiratory diseases-related deaths between 1990 and 2017 with ~545 million affected and ~3.9 million deaths in 2017. The leading risk factor for men was found to be smoking irrespective of the region while the leading cause of respiratory illness for women varied depending on the region – household air pollution from cooking fuels in part of Asia and Africa, inhalation of particulate matters in parts of Asia and Oceania, and smoking in other regions. With such a large population affected, it is imperative to understand the disease etiologies and find therapies to prevent and manage respiratory illnesses. Animal models of pulmonary diseases serve as invaluable tools towards this; these models have immensely contributed to our understanding especially at a whole-organism level. However, species differences between the models and humans often result in failed translation of therapies and drugs. Additionally, animal models do not allow decoupling of factors contributing to diseases thus making the findings confounding. These bottlenecks can be overcome by utilizing in vitro models wherein human cells can be used and the effect of factors contributing to disorders can be studied systematically. Conventional in vitro models suffer from their own limitations such as being too simplistic and unrepresentative of in vivo conditions. Emergence of organ-on-a-chip and organoid technologies have tried to bridge this gap by recreating key organ-specific features and tissue microenvironment. However, challenges remain with incorporating complex and heterogeneous mechanical cues, long-term culture of primary cells in vitro and creation of multicellular models that accurately capture the architecture and functionalities of the organ.To address several of these challenges, we have developed platforms to capture the complexity of the lung within in vitro devices. Specifically, we developed a breathing alveolus-on-a-platform that utilizes a microfluidic-pneumatic mechanism to subject the cells to heterogeneous strain arising from out-of-plane stretching akin to expansion and contraction of an alveolus during breathing. Using the device, we showed that breathing induced changes in cells such as their alignment, surfactant production, and wound healing response. We used the device to model lung conditions associated with altered pulmonary biomechanics such as changes in lung compliance and ventilator-induced lung injury. We also demonstrated the ability of the device to be used a tool for screening toxicological effects of chemicals such as compounds used as flavors in electronic cigarettes. Finally, we demonstrated the ability of the device to support the culture of primary human alveolar type 2 (AT2) cells and iPSC-derived AT2 cells. One of the limitations of in vitro systems is the inability to culture primary cells, including AT2 cells, for a long time without loss in their phenotype and identity. Until recently, a feeder layer of supporting stromal cells was required to culture AT2 cells in vitro. While medium conditions have now been optimized and defined to avoid the use of feeder cells, Matrigel is still the current state-of-art platform to culture these cells. However, its undefined nature and limited tunability present a barrier to expanding the use and application of such in vitro tools. Through a screening of ECM hydrogels, we identified laminin-111 as a key ECM molecule that supports the culture of AT2 cells and formation of organoids known as alveolospheres. We further showed that laminin-111 can be used by itself or in combination with other ECM proteins such as fibrin or synthetic polymers to grow alveolospheres on par or better than Matrigel. Further, we optimized the laminin and fibrinogen concentrations that can support the formation of alveolospheres and demonstrated cell-mediated degradation of matrix as being critical for alveolosphere formation. We showed the versatility of our system by culturing alveolospheres grown from AT2 cells from various sources – murine, human, and iPSCs-derived. Identification of matrix properties that regulate cellular function such as self-renewal and lineage-specific differentiation will significantly advance the efforts towards organ-specific microphysiological systems. Lung is a common site for metastasis and lung cancer is the most lethal cancer. So, we next examined the applicability of using lung-on-chip platform to study metastasis of distant cancers into the lung. With culture conditions for alveolosphere optimized, we worked towards building more complex models of the lung by including other components such as lung fibroblasts and functional vasculature for their potential use as platforms to model lung metastasis of cancers such as undifferentiated pleomorphic sarcoma. Towards this, we first adapted a vasculature-on-a-chip model and showed that heterogeneous cells populations from undifferentiated pleomorphic sarcoma had different extravasation efficiencies in the vasculature-on-a-chip. We then integrated the vasculature with iAT2 alveolospheres and normal human lung fibroblasts to create multi-cellular models of the lung. To better support the phenotype of individual cells, we also developed novel compartmentalized devices that can house multiple cell types in their corresponding medium while allowing cross-talk with other cell types by means of soluble factors. Such multicellular platforms recapitulating the complexity of the lung using human cells can serve as important tools for gaining mechanistic insights into pulmonary functions as well as models for studying lung disorders, identifying therapeutic targets, and screening candidate drugs.