Browsing by Author "Veiseh, Omid"
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Item A 3D printable perfused hydrogel vascular model to assay ultrasound-induced permeability(Royal Society of Chemistry, 2022) Royse, Madison K.; Means, A. Kristen; Calderon, Gisele A.; Kinstlinger, Ian S.; He, Yufang; Durante, Marc R.; Procopio, Adam T.; Veiseh, Omid; Xu, JunThe development of an in vitro model to study vascular permeability is vital for clinical applications such as the targeted delivery of therapeutics. This work demonstrates the use of a perfusion-based 3D printable hydrogel vascular model as an assessment for endothelial permeability and its barrier function. Aside from providing a platform that more closely mimics the dynamic vascular conditions in vivo, this model enables the real-time observation of changes in the endothelial monolayer during the application of ultrasound to investigate the downstream effect of ultrasound-induced permeability. We show an increase in the apparent permeability coefficient of a fluorescently labeled tracer molecule after ultrasound treatment via a custom MATLAB algorithm, which implemented advanced features such as edge detection and a dynamic region of interest, thus supporting the use of ultrasound as a non-invasive method to enhance vascular permeability for targeted drug therapies. Notably, live-cell imaging with VE-cadherin-GFP HUVECs provides some of the first real-time acquisitions of the dynamics of endothelial cell–cell junctions under the application of ultrasound in a 3D perfusable model. This model demonstrates potential as a new scalable platform to investigate ultrasound-assisted delivery of therapeutics across a cellular barrier that more accurately mimics the physiologic matrix and fluid dynamics.Item Embargo A cell-based therapeutics biomaterial patch for accelerated wound healing(2023-04-21) Schreib, Christian Cody; Veiseh, OmidChronic wounds have a major negative impact on human health. Statistics show that ~2% of the United States population, about 7 million people, suffer from chronic wounds. Globally, it is estimated that chronic wound care costs a total of $3.5 billion. Wound healing is a highly dynamic process, with different phases that require different growth factors and cytokines. Despite this dynamic nature, the current format of wound healing treatments is to use static treatments that do not change over time to encourage wound healing, such as wound vacuums and skin grafts. Although these treatments have shown to help in wound healing, they can still be improved. Here I present my work on developing a dynamic wound-healing patch that utilizes cell-based therapeutics to allow for precise temporal control of the delivery of different cytokines and growth factors into the wound bed.Item Activation of Adaptive and Innate Immune Cells via Localized IL2 Cytokine Factories Eradicates Mesothelioma Tumors(AACR, 2022) Nash, Amanda M.; Aghlara-Fotovat, Samira; Castillio, Bertha; Hernandez, Andrea; Pugazenthi, Aarthi; Lee, Hyun-Sung; Jang, Hee-Jin; Nguyen, Annie; Lu, Alexander; Burt, Bryan M.; Ghanta, Ravi K.; Veiseh, OmidIL2 immunotherapy has the potential to elicit immune-mediated tumor lysis via activation of effector immune cells, but clinical utility is limited due to pharmacokinetic challenges as well as vascular leak syndrome and other life-threatening toxicities experienced by patients. We developed a safe and clinically translatable localized IL2 delivery system to boost the potency of therapy while minimizing systemic cytokine exposure.We evaluated the therapeutic efficacy of IL2 cytokine factories in a mouse model of malignant mesothelioma. Changes in immune populations were analyzed using time-of-flight mass cytometry (CyTOF), and the safety and translatability of the platform were evaluated using complete blood counts and serum chemistry analysis.IL2 cytokine factories enabled 150× higher IL2 concentrations in the local compartment with limited leakage into the systemic circulation. AB1 tumor burden was reduced by 80% after 1 week of monotherapy treatment, and 7 of 7 of animals exhibited tumor eradication without recurrence when IL2 cytokine factories were combined with anti–programmed cell death protein 1 (aPD1). Furthermore, CyTOF analysis showed an increase in CD69+CD44+ and CD69−CD44+CD62L− T cells, reduction of CD86−PD-L1− M2-like macrophages, and a corresponding increase in CD86+PD-L1+ M1-like macrophages and MHC-II+ dendritic cells after treatment. Finally, blood chemistry ranges in rodents demonstrated the safety of cytokine factory treatment and reinforced its potential for clinical use.IL2 cytokine factories led to the eradication of aggressive mouse malignant mesothelioma tumors and protection from tumor recurrence, and increased the therapeutic efficacy of aPD1 checkpoint therapy. This study provides support for the clinical evaluation of this IL2-based delivery system.See related commentary by Palanki et al., p. 5010Item Applications of 3D printed vascularized tissue constructs for studies of human physiology and disease(2023-01-12) Janson, Kevin D; Veiseh, OmidFor decades, tissue engineering has been largely guided by the paradigm that engineered tissues require appropriate cell types for a specific application, a scaffold to provide three-dimensional (3D) structure, and soluble factors to stimulate growth and proliferation. While this paradigm has successfully guided the creation of homogeneous, small, or thin de novo structures, native tissues often contain regional structural and cellular differences that tissue engineered models struggle to reproduce. Some of this regional variability results from cellular distances to vasculature, which in turn affects available nutrients and tissue composition. Recently, 3D printing has advanced enough for scientists to create vascular structures of different sizes in a variety of shapes. This technology has the ability to precisely incorporate regional variability into scaffolds, which in turn can influence cellular distribution. Here we use a light-based form of 3D printing to incorporate regional variability into constructs that model possible designs for engineered lung replacements and disease models involving skin. We approach this task by fabricating vascular unit cells featuring vasculature and proximal structures of interest for both of these organs. Additionally, we leverage the design freedoms afforded by 3D printing to improve tissue function by exploring designs that do not appear in native physiology. These unit cells are rigorously assessed for their efficiency, and we evaluate both new and existing metrics to quantify their performance. Finally, we explore these vascular unit cells for their utility as both engineered tissue replacements and disease models. We expect that our results will inform the design of engineered organ replacements and disease models and introduce the idea that engineered tissues do not need to perfectly replicate existing native structures.Item Assessing Gq-GPCR–induced human astrocyte reactivity using bioengineered neural organoids(Rockefeller University Press, 2022) Cvetkovic, Caroline; Patel, Rajan; Shetty, Arya; Hogan, Matthew K.; Anderson, Morgan; Basu, Nupur; Aghlara-Fotovat, Samira; Ramesh, Srivathsan; Sardar, Debosmita; Veiseh, Omid; Ward, Michael E.; Deneen, Benjamin; Horner, Philip J.; Krencik, RobertAstrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor–driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.Item Design and Evaluation of Vascularizing Scaffolds Towards Multi-compartment Engineered Tissues(2022-04-22) Parkhideh, Siavash; Veiseh, OmidTissue engineering research and the goal of developing thick replacement tissues such as heart, liver, and lung require the creation of a functional vascular network. Specifically, naturally occurring vasculature is hierarchical and spatially patterned. Prior work has previously shown that patterned vasculature can enhance engineered tissue function and limb perfusion. While various methods can promote the development of patterned vasculature, bioprinting was used in these studies due to its ability to fabricate complex, multivascular structures. To allow for modular and tissue-agnostic design, we designed and fabricated core-shell hydrogel structures with a non-degradable hydrogel core, containing stromal cells (i.e., the stromal compartment), and a degradable hydrogel shell containing perfusable, patterned vascular structures (i.e., the vascular compartment). Initial studies demonstrated that vascular architectures with stromal cells located within the plane of the vasculature resulted in enhanced nutrient delivery between vascular and stromal compartments. Laminar flow was detected within bioprinted channels, beneficial for channel endothelialization and consistent wall shear stress. Then, vascular cells were printed within the hydrogel matrix and seeded into the bioprinted channels and cultured under perfusion over multiple days. Perfusion culture allowed endothelial cell maintenance, and in co-culture hydrogels, lead to cell-cell coordination within the construct in vitro. Notably, the greatest degree of biomaterial vascularization and influence over vascular patterns was seen within hydrogels fabricated with RFP HUVECs and hMSCs encapsulated within the bulk hydrogel, along with GFP HUVECs lining the walls of the patterned channels, maintained in perfusion culture for three days. Finally, for this optimal formulation, vascularization was detected as early as two weeks, and vessels up to 100 µm in diameter had formed by eight weeks, demonstrating vessel development and maturation over time. The ability for spatially controlled endothelial structures to influence vascular patterning in vivo can inform future studies in developing thick, vascularized tissues and organs. Furthermore, we fabricate retrievable, immunoisolating hydrogels to comprise the stromal compartment and determine that islets maintain viability, functionality, and ability to restore normoglycemia within these matrices.Item Developing Novel and Interdisciplinary Methods for DNA Detection and DNA Structure Profiling(2021-11-30) Li, Jiaming; Zhang, David Yu; Veiseh, OmidUnderstanding the secondary structures of nucleic acid polymers, i.e. DNA and RNA, is fundamentally important for both biochemistry and molecular biology, as structures often influence biological function, such as the affinity of protein binding and accessibility to DNA-binding drugs. Software currently used to predict secondary structures of nucleic acids from sequence exhibits limited accuracy, and furthermore there are limited datasets of DNA sequence and structure to improve the accuracy of biophysical models and secondary structure prediction software. Additionally, secondary structure prediction software is known to have significant qualitative limitations, such as the inability to predict pseudoknots. Recently there arose new chemical probing methods to profile RNA secondary structures such as SHAPE-Seq and DMS-Seq, but no experimental method has been demonstrated for profiling DNA secondary structures. I developed a novel, robust, and high-throughput method to experimentally characterize the DNA secondary structures at the single-molecule resolution by applying low-yield bisulfite conversion and next-generation sequencing (NGS) to a mixture of thousands of DNA species. Bisulfite conversion is a chemical reaction in which cytosines are converted to uracils when the DNA is treated with sodium bisulfite. Importantly, the efficiency of the bisulfite conversion reaction is lowered when the cytosine nucleotide is in a double-stranded state, so the statistical observation of the conversion yield across a large number of molecules suggests the base pairing status of the nucleotide. By lowering the concentration of bisulfite and the reaction time, I was able to modulate the conversion yield to values that optimize determination of base pairing state. By using chip-synthesized oligo pools of over 10,000 strands, I was be able to build a large database that pairs DNA sequences to observed DNA secondary structures and used this database to develop an analytical model to determine the secondary structures of any DNA sequence given its experimental bisulfite conversion data. I found that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.Item Development of a 3D printable in vitro vascular model for therapeutic applications(2022-08-08) Royse, Madison K.; Veiseh, OmidThe process of drug discovery and development is long and costly, often resulting in limited or expensive treatment options for patients. The translation of drug candidates and therapeutic strategies from preclinical screening to clinical studies is a substantial hurdle in the field and has underscored the need for technologies that can accurately predict therapeutic outcomes in vitro before translation to clinical studies. Recent advances in 3D printing have enabled the fabrication of in vitro models that more accurately recapitulate physiologic forces and material composition in comparison to traditional in vitro methods such as 2D cell culture and microfluidic devices. Here, we demonstrate the development of a 3D-printed perfusable gelatin-based hydrogel as an in vitro vascular model for screening therapeutics in an accelerated and cost-effective manner. Biocompatible hydrogels of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) with hollow vascular architectures were fabricated via projection stereolithography, endothelialized with human umbilical vein endothelial cells (HUVECs), and subjected to fluid flow with varying biochemical stimuli to promote endothelial maturation and stability. The ability of these endothelialized channels to respond to external barrier-disrupting stimuli was validated by showing a significant increase in vascular permeability after ultrasound treatment, demonstrating the utility of this model in assessing therapeutic strategies targeting vasculature. Finally, this model was adapted to recreate the highly restrictive vasculature of the central nervous system, the blood-brain barrier (BBB), via incorporation of human brain microvascular endothelial cells, human brain microvascular pericytes, and human brain astrocytes. The presence of essential blood-brain barrier junctional complexes and transporters were confirmed, and pilot work demonstrated the capacity of this in vitro BBB model to be used as a tool for evaluating the transport of therapeutics across the blood-brain barrier. This 3D printed perfusable vascular model offers an alternative route to assess vascular-focused therapies, providing a preclinical model that bridges less physiologic in vitro methods and more complex, costly in vivo animal studies.Item Development of a Sterilizable Hydrogel Platform for Biocontained Cell Therapy Delivery to the GI Tract(2022-04-22) Doerfert, Michael David; Veiseh, OmidBacteria are a ubiquitous and diverse class of microorganism that play a wide variety of roles in nature, industry, and human health. Advances in genetic engineering of bacteria has given rise to the rapid development of a wide variety of diagnostic and therapeutic bacteria strains that have the potential to greatly improve healthcare, particularly for diseases of the digestive system. However, the use of genetically modified organisms in medicine has raised multiple concerns of therapeutic delivery, control, and safety, which limits the translation and adoption of these technologies. There exists a gap between current biocontainment technologies and platforms made for bacteria delivery to the GI tract that ensures a safe and effective delivery of the engineered bacteria to the patient. This report details the design and development of an externally sterilizable cell therapy platform that uses a UV-absorbent, reinforced hydrogel formulation to improve biocontainment of the encapsulated bacteria.Item Development of an automated biomaterial platform to study mosquito feeding behavior(Frontiers Media S.A., 2023) Janson, Kevin D.; Carter, Brendan H.; Jameson, Samuel B.; de Verges, Jane E.; Dalliance, Erika S.; Royse, Madison K.; Kim, Paul; Wesson, Dawn M.; Veiseh, OmidMosquitoes carry a number of deadly pathogens that are transmitted while feeding on blood through the skin, and studying mosquito feeding behavior could elucidate countermeasures to mitigate biting. Although this type of research has existed for decades, there has yet to be a compelling example of a controlled environment to test the impact of multiple variables on mosquito feeding behavior. In this study, we leveraged uniformly bioprinted vascularized skin mimics to create a mosquito feeding platform with independently tunable feeding sites. Our platform allows us to observe mosquito feeding behavior and collect video data for 30–45 min. We maximized throughput by developing a highly accurate computer vision model (mean average precision: 92.5%) that automatically processes videos and increases measurement objectivity. This model enables assessment of critical factors such as feeding and activity around feeding sites, and we used it to evaluate the repellent effect of DEET and oil of lemon eucalyptus-based repellents. We validated that both repellents effectively repel mosquitoes in laboratory settings (0% feeding in experimental groups, 13.8% feeding in control group, p < 0.0001), suggesting our platform’s use as a repellent screening assay in the future. The platform is scalable, compact, and reduces dependence on vertebrate hosts in mosquito research.Item Development of highly multiplex nucleic acid-based diagnostic technologies(2021-12-02) Xie, Guanyi; Zhang, David Yu; Veiseh, OmidThe design of highly multiplex nucleic acid primers and probes to enrich and detect many different DNA sequences is increasing in biomedical importance as new mutations and pathogens are identified. One major challenge in the design of highly multiplex PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. During my Ph.D., one of my main focuses is how to design highly multiplex PCR primer sets that minimize primer dimer formation. Here I present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for the design of highly multiplex PCR primer sets that minimize primer dimer formation. I also worked on the design of multiplex probes for variants detection. Many diseases are related to multiple genetic alterations along a single gene. Probing for highly multiple (>10) variants in a single qPCR tube is impossible due to a limited number of fluorescence channels and one variant per channel, so many more tubes are needed. Here, I experimentally validate a novel color-mixing strategy that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously.Item Embargo Development of Implantable Cytokine Factories for Controlled Modulation of the Immune Response(2023-04-21) Nash, Amanda; Veiseh, OmidCytokines are soluble molecular messengers that activate and propagate disease-fighting immune cascades in the body in response to stimuli from antigen-presenting cells (APC). Many pro-inflammatory cytokines, including IL-2, IL-7, IL-12, IL-15, and IFN-γ, have been tested for anti-tumor efficacy in clinical trials but cannot eradicate tumors without severe off-target effects. However, advances in cell-based therapeutics have enabled effective approaches for genetically engineering cells to produce and secrete proteins continuously. In this work, I have taken advantage of these engineering techniques to genetically engineer cells that continuously produce cytokines and can, thus, be used as living “cytokine factories”. To protect these cells from being rejected in vivo, a porous alginate sphere can be cast around the cells. The ability to fabricate and characterize these cytokine factories, as well as utilize them to initiate and repress immune system responses in vivo, is demonstrated in this work. Briefly, I developed and optimized encapsulated cytokine factories in vitro to tune potency and viability, quantified their ability to activate the immune system with spatial and temporal regulation in multiple pre-clinical mouse models of cancer, evaluated their safety and biocompatibility in rodents and non-human primates, and translated their use into phase I/II clinical trials for women with ovarian cancer. This platform technology can also be rapidly modified to allow for localized immunomodulation in additional diseases including type 1 diabetes, acute respiratory distress syndrome, myocardial tissue repair, and more.Item Development of New Methods for Easy-to-apply, Multiplexed and Ultrasensitive Nucleic-Acid based Diagnostic Technologies(2021-12-09) Zhang, Carol; Zhang, David Yu; Veiseh, OmidThe Polymerase chain reaction (PCR) has been one of the most widely used and easily accessible methods in bio-laboratories. It could be used in a fluorescence detection device together with intercalating dyes or fluorophore-labeled probes to achieve real-time quantitation of nucleic acid targets, or it could be involved in the workflow of high- throughput sequencing library preparation for target enrichment. Clinically and biologically, somatic mutations are becoming significant and informative biomarkers in many diseases, including cancer. The ability to accurately detect rare DNA variant sequences could be beneficial to early-stage cancer diagnosis, recurrence monitoring and precision treatment. However, there are some limitations for the existing common detection technologies. Some qPCR methods are restricted in the limit of detection and require multiple reactions to identify mutations; some qPCR methods are not compatible with high-fidelity DNA polymerase to achieve ultra-sensitive mutation detection and do not allow for multiplexing; some next-generation sequencing methods may require high computing resource or additional steps to reduce dimerization, improve PCR efficiency and increase sensitivity; or some high-throughput sequencing methods may be economically limited due to the expensive chemical synthesis for higher multiplex targets. During my PhD, I have developed several novel PCR-based methods to provide a solution to the unmet needs discussed above, achieving ultra-sensitive and multiplexing variant-enrichment-based detection in either the accessible quantitative PCR (qPCR) or the next-generation sequencing (NGS). This thesis is a collection of 3 manuscripts summarizing the projects during my PhD research: [1] Zhang K, Rodriguez L, Cheng LY, Zhang DY. Single Tube qPCR detection and quantification of hotspot mutations down to 0.01% VAF. Manuscript has been accepted by Analytical Chemistry. [2] Zhang K, Pinto A, Song P, Dai P, Wang MX, Cheng LY, Rodriguez L, Weller C, Zhang DY. Hairpin structure facilitates high-fidelity DNA amplification reactions in both qPCR and high-throughput sequencing. Manuscript under review. [3] Zhang K, Ping S, Zhang JX, Dai P, Wen R, Rodriguez L, Zhang DY. Non- extensible oligonucleotides in DNA amplification reactions. Manuscript under preparation.Item Electrocatalytic on-site oxygenation for transplanted cell-based-therapies(Springer Nature, 2023) Lee, Inkyu; Surendran, Abhijith; Fleury, Samantha; Gimino, Ian; Curtiss, Alexander; Fell, Cody; Shiwarski, Daniel J.; Refy, Omar; Rothrock, Blaine; Jo, Seonghan; Schwartzkopff, Tim; Mehta, Abijeet Singh; Wang, Yingqiao; Sipe, Adam; John, Sharon; Ji, Xudong; Nikiforidis, Georgios; Feinberg, Adam W.; Hester, Josiah; Weber, Douglas J.; Veiseh, Omid; Rivnay, Jonathan; Cohen-Karni, TzahiImplantable cell therapies and tissue transplants require sufficient oxygen supply to function and are limited by a delay or lack of vascularization from the transplant host. Previous exogenous oxygenation strategies have been bulky and had limited oxygen production or regulation. Here, we show an electrocatalytic approach that enables bioelectronic control of oxygen generation in complex cellular environments to sustain engineered cell viability and therapy under hypoxic stress and at high cell densities. We find that nanostructured sputtered iridium oxide serves as an ideal catalyst for oxygen evolution reaction at neutral pH. We demonstrate that this approach exhibits a lower oxygenation onset and selective oxygen production without evolution of toxic byproducts. We show that this electrocatalytic on site oxygenator can sustain high cell loadings (>60k cells/mm3) in hypoxic conditions in vitro and in vivo. Our results showcase that exogenous oxygen production devices can be readily integrated into bioelectronic platforms, enabling high cell loadings in smaller devices with broad applicability.Item Embargo Encapsulated Cell Systems for Treating Inflammatory Diseases(2024-04-19) Aghlara-Fotovat, Samira; Veiseh, Omid; Ghanta, Ravi KIn response to pathogens and trauma, host immune cells interact bi-directionally with their local environment to receive and deposit molecular signals, which orchestrate cellular activation, proliferation, differentiation, and function to maintain healthy tissue homeostasis. While our immune system functions as a vigilant safeguard against environmental threats, instances of immune dysregulation may occur, leading to uncontrolled responses. In these conditions, it is essential to restore balance to the body through modulation of the immune system and the ECM. Cell-based therapeutics have significant potential in locally monitoring and treating inflammatory diseases, however, their widespread use is hindered by recognition and elimination by the host. Thus, novel technologies that can improve the viability and function of cell-based therapies have significant potential in improving translation. Here, we aim to utilize biomaterial encapsulation as a tool for improving the delivery of cell-based therapeutics for local immunomodulation in various inflammatory diseases including myocardial infarct, acute respiratory distress syndrome, neural inflammation, inflammatory bowel disease.Item Engineering leukocyte mimicking nanoparticles for targeted delivery in triple-negative breast cancer: biological versus cargo-based approach(2021-04-27) Sushnitha, Manuela; Veiseh, Omid; Taraballi, FrancescaNanoparticles offer the ability to achieve targeted drug delivery across many disease contexts, especially difficult to treat cancers like triple-negative breast cancer (TNBC). The lack of targeted of therapies for TNBC patients has warranted the development of novel strategies for targeting the tumor while delivering therapeutics aimed at targeting the underlying drivers of disease. Recent work has demonstrated the promising potential of nanoparticles to achieve both of these desired functions. However, translation of nanoparticles to the clinic has been hampered by the limited ability of synthetic nanoparticles to overcome the biological barriers posed by the complex in vivo milieu. In order to overcome these limitations, nanoparticles designed to mimic native cells through the integration of cell membrane components have been developed. In particular, leukocyte mimicking lipid nanoparticles have demonstrated the ability to target sites of inflammation, evade immune clearance and deliver therapeutic molecules. Leveraging the advantages of this technology, this study aims to demonstrate the utility of these biomimetic nanoparticles (i.e. leukosomes) for the treatment of TNBC. In particular, engineering of the leukosomes was explored from two perspectives: 1) a biological approach that aimed to improve the tumor targeting abilities of the nanoparticles and 2) a cargo-based approach for the development of a leukosome formulation capable of delivering therapeutic RNA molecules. By combining these two approaches for the engineering of leukocyte mimicking nanoparticles, a novel strategy for targeting and treating TNBC was developed while gaining important insights for the future development of these cell mimicking nanoparticle platforms.Item Embargo Immunomodulatory biomaterials to enable long-term delivery of cell-based therapeutics(2023-04-05) Kim, Boram; Veiseh, OmidType 1 diabetes (T1D) is a chronic autoimmune disease that involves the destruction of pancreatic beta-cells. Islet transplantation, a promising treatment for T1D, is restricted by the requirement for lifelong systemic immunosuppression to prevent graft rejection. Encapsulation of transplanted therapeutic cells using selectively permeable physical barriers offers a possible solution, but preventing fibrosis triggered by inflammatory responses upon biomaterial implantation remains a crucial unmet need. The aim of this thesis is to devise innovative immuno-engineering approaches to modulate immune responses against encapsulated cells within alginate hydrogels. The first strategy involves the chemical modification of encapsulation materials. Novel alginates with triazole modification were synthesized based on a prior library of small molecule-conjugated alginate analogs. Cellular barcoding was used to screen these new biomaterials by encoding the identity of the material with a unique single-nucleotide polymorphism (SNP) profile designed from 20 different human umbilical vein endothelial cell (HUVEC) donors. The lead alginate displayed mitigated anti-fibrotic responses after xeno-transplantation. Human islets implanted in STZ-induced immunocompetent T1D mice were protected by encapsulating with the lead alginate and inhibiting immune cell recognitions, successfully reversing T1D levels to normal glycemia, maintained up to 80 days post-implantation. The second strategy involves the use of engineered cell lines that can serve as sentinels. These sentinel cells detect immune cells and regulate their responses, leading to efficient immunomodulation. Therapeutic efficacy and long-term survival were assessed by performing diabetic reversal studies with T1D mice. Local delivery of immunomodulating cytokine-releasing capsules combined with encapsulated human islets allowed efficient immunomodulation, prevented fibrosis around islets-capsules, and showed long-term glycemic control in the diabetic mouse model. Lastly, we developed a reliable in vivo screening method using live fluorescence imaging to assess the vascularization within biomaterials, which is critical for the long-term survival and function of transplanted cells. By leveraging the IVIS fluorescence probe, we could test various materials in parallel with longitudinal resolutions and cost-effective ways. Taken together, these novel approaches to modulating immune responses have substantial potential for clinical translation in improving the performance of encapsulated cell-based therapeutics, particularly for the treatment of T1D patients. The proposed strategies may overcome the limitations of islet transplantation by providing a long-term solution for glycemic control without the need for systemic immunosuppression.Item mplantable Cell Based Therapy for Treatment of Inflammatory Heart and Lung Conditions(2022-04-22) Lu, Alexander; Veiseh, OmidIn this work, we explore utilizing encapsulated engineered cells for targeted release of anti-inflammatory cytokines. A plasmid encoding the cytokine hIL-10 and suicide system iCasp9 was created and inserted into ARPE-19 cells. After integration, cells were assayed for hIL-10 production and response to an iCasp9 activating molecule. To better understand clinical translatability of a capsule based delivery system, two surgical approaches to delivery of capsules were tested on cadaveric porcine hearts and cadaveric mice. hIL-10 cells showed variable hIL-10 production over 24 hours, ranging from 0.3 - 5.2 pg/cell/24hr. Cells exposed to 1nM of AP1903, 10% of previously reported doses, demonstrated significant cell death over 24 hours. Existing surgical methods involving placement of capsules ultimately proved unsuitable for clinical use. This work serves as an initial proof of concept for treating inflammatory conditions using biologically and physically targeted therapies.Item Perfusable cell-laden matrices to guide patterning of vascularization in vivo(Royal Society of Chemistry, 2023) Parkhideh, Siavash; Calderon, Gisele A.; Janson, Kevin D.; Mukherjee, Sudip; Mai, A. Kristen; Doerfert, Michael D.; Yao, Zhuoran; Sazer, Daniel W.; Veiseh, OmidThe survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 μm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell–cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 μm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.Item Persistent tailoring of MSC activation through genetic priming(Elsevier, 2024) Beauregard, Michael A.; Bedford, Guy C.; Brenner, Daniel A.; Sanchez Solis, Leonardo D.; Nishiguchi, Tomoki; Abhimanyu; Longlax, Santiago Carrero; Mahata, Barun; Veiseh, Omid; Wenzel, Pamela L.; DiNardo, Andrew R.; Hilton, Isaac B.; Diehl, Michael R.Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce the expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor interferon response factor 1 (IRF1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFN-γ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN-γ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.