archives@tulane.edu / Osteoarthritis (OA) is a common joint disorder with significant economic and healthcare impact. The knee joint is composed of cartilage and the adjoining bone, a synovial capsule, the infrapatellar fat pad (IPFP), and other connective tissues such as tendons and ligaments. Adipose tissue has recently been highlighted as a major contributor to OA through strong inflammation mediating effects. Type II diabetes mellitus (T2D) is a weight independent risk factor for OA, suggesting a link between OA and adipose dysfunction that has yet to be elucidated. There is a critical need for development of new methodologies to investigate the interaction between an osteochondral interface and extra-synovial tissue. There is also a need for investigating adipose derived stem cells (ASCs) isolated from the IPFP of T2D patients as a potential cell source to model diabetic complications. In this work, we develop a novel 3D printed bioreactor model for incorporation into a previously established osteoarthritic knee microphysiological system. Using our established model, we investigated xenoprotein free (XPF) media as a potential commercial product for the MPS industry. Additionally, differences in inflammatory and adipokine related mRNA expression of IPFP-ASCs isolated from non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and type II diabetes mellitus (T2D) patient samples were analyzed. After 28 Days of differentiation, 3D printer bioreactors using commercially available AdipoQual media exhibited robust increase in adipokine expression and neutral lipid accumulation. Bioreactors cultured with a novel XPF supplemented AdipoQual had similar adipokine expression but no neutral lipid accumulation. Pre-T2D IPFP-ASCs exhibited a robust decrease in CD90 and CD105 levels with no corresponding increase in markers for potentially contaminating cell types. Cox-2 expression and PGE2 secretion were significantly increased in IL-1β stimulated Pre-T2D IPFP-ASCs compared to Non-T2D and T2D IPFP-ASCs. When the Pre-T2D ASCs were co-cultured with M1-induced macrophages, the macrophages significantly reduced expressions of tumor necrosis factor α (TNFα) and IL-6 compared to M1-induced macrophages co-cultured with Non-T2D and T2D IPFP-ASCs. Taken together, this work has taken significant steps towards establishment of a model for the IPFP and establishment of important phenotypic and genotypic changes of IPFP-ASCs isolated from Pre-T2D patients. / 1 / Benjamen O'Donnell

archives@tulane.edu / 1 / Benjamin Vinson

The research presented in this dissertation supports the overall goal of producing sensor functionalized neural microphysiological systems to enable deeper fundamental understandings of disease pathology and to provide drug screening and discovery platforms for improved clinical translation. Towards this goal, work addressing three broad objectives has been completed. The first objective was expanding the manufacturing process capabilities for hydrogels and tissues through augmentation of the 3D printing systems and developing novel modeling capabilities. The second objective was to expand the palette of available materials which exhibit the rheological properties required for 3D printing and the mechanical and biological properties required for neural tissue culture. The third objective was to develop sensing capabilities for both monitoring and control of the manufacturing process and to provide non-destructive assessment of microphysiological systems in real-time to quantify the dynamics of disease progression or response to treatment. The first objective of process improvement was addressed both through modification of the 3D printing system itself and through modeling of process physics. A new manifold was implemented which enabled on-the-fly mixing of bioprinting inks (bioinks) to produce smooth concentration gradients or discrete changes in concentration. Modeling capabilities to understand the transport occurring during both the processing and post-processing windows were developed to provide insight into the relationship between the programmed concentration distribution and its temporal evolution and stability. Vacuum-based pick-and-place capabilities for integration of prefabricated components for sensing and stimulation into the printed hydrogel constructs were developed. Models of the stress profiles, which relate to cell viability, within the printing nozzle during extrusion were produced using parameters extracted from rheological characterization of bioinks. The second objective was addressed through the development hydrogel bioinks which exhibited yield stresses without the use of rheological modifiers (fillers) to enable 3D printing of free-standing neural tissue constructs. A hybrid bioink was developed using the combination of a synthetic polaxamer with biomacromolecules present in native neural tissue. Functionalization of the biomacromolecules with catechol or methacrylate groups enabled two crosslinking mechanisms: chelation and UV exposure. Crosslinked gels exhibited moduli in the range of native neural tissue and enabled high viability culture of multiple neural cell types. The third objective was addressed through the characterization and implementation of physical and electronic sensors. The resonance of millimeter-scale dynamic-mode piezoelectric cantilevers submerged in polymer solutions was found to persist into the gel phase enabling viscoelastic sensing in hydrogels and monitoring of sol-gel transitions. Resonant frequency and quality factor of the cantilevers were related with the viscoelastic properties of hydrogels through both a first principles approach and empirical correlation. Electrode functionalized hollow fibers were implemented as impedimetric sensors to monitor bioink quality during 3D printing. Impedance spectra were collected during extrusion of cell-laden bioinks and the magnitude and phased angle of the impedance response correlated with quality measures such as cell viability, cell type, and stemness which were validated with traditional off-line assays. / Doctor of Philosophy / The research presented in this dissertation supports the overall goal of producing sensor functionalized neural microphysiological systems to enable deeper fundamental understandings of disease pathology and to provide drug screening and discovery platforms for improved clinical translation. Microphysiological systems are miniaturized tissue constructs which strive to mimic the complex conditions present in-vivo within an in-vitro platform. By producing these microphysiological systems with sensing functionality, new insight into the mechanistic progression of diseases and the response to new treatment options can be realized. Towards this goal, work addressing three broad objectives has been completed. The first objective was expanding the manufacturing process capabilities for hydrogels and tissues through augmentation of the 3D printing systems and developing novel modeling capabilities. The second objective was to expand the palette of available materials which exhibit both the properties required for 3D printing and the mechanical and biological properties required for neural tissue culture. The third objective was to develop sensing capabilities for both monitoring and control of the manufacturing process and to provide non-destructive assessment of microphysiological systems in real-time to quantify the dynamics of disease progression or response to treatment. Through these efforts higher quality microphysiological systems may be produced benefitting future researchers, medical professionals, and patients.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24610号 / 工博第5116号 / 新制||工||1978(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 土屋 智由, 教授 横川 隆司, 教授 安達 泰治, 教授 田畑 修(京都先端科学大学) / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The drug development process is lengthy and expensive, with a 90% failure rate among drugs entering clinical trials due to the inadequacy of predictive models in the initial phases of drug development. To overcome these limitations, there is a paradigm shift towards developing micro physiological systems often referred as Organ-on-a-Chip that have be shown to recapitulate organ level functions in vitro. However, despite their promise, these systems often have limited throughput, restricting their widespread use in the drug development process. The work outlined in this thesis aims to bridge this gap by integrating the physiological relevance offered by micro physiological systems with the high throughput capabilities of traditional 2D multi-well plate cultures. The thesis outlines the development of two novel micro physiological systems, engineered in a high throughput multi-well format called the IFlowPlate and AngioPlate. Both the platforms have an open-top design and unlike tradition microphysiological platforms does not need complex pump systems and have built-in connections to achieve perfusion making it more scalable and user-friendly. The IFlowPlate leverages the self-assembly capability of endothelial cells to create a perfusable vascular network. This platform technology was utilized in this work to achieve intravascular perfusion of colon organoids for the first time and demonstrated immune cell circulation and recruitment in response to injury. AngioPlate, the other platform that was developed as a part of this work, utilizes a pre-patterned scaffold completely embedded in native hydrogel matrix to guide cells in forming organ-specific geometries and tubular structures using a novel subtractive manufacturing technique. This platform allowed for fabricating complex and intricate networks to model vascularized terminal lung alveoli and renal proximal tubules. This work demonstrated for the first time that highly complex perfusable tissues embedded in hydrogel can be integrated with multi-well plates to mimic tissue specific structures and interfaces without the use of synthetic membranes or plastic channels. The built-in perfusion channel and the flexible hydrogel matrix allowed for the terminal lung alveoli model to be mechanically actuated to mimic breathing motions. The renal proximal tubule model was used to mimic glucose reabsorption in kidney and model renal inflammation. The latter part of this work focusses on further improving this platform to increase platform robustness and to allow for incorporating supporting cells such as fibroblasts into the hydrogel matrix. This allowed us to model tubular injuries in kidney such as cisplatin induced -nephrotoxicity and TGF- β1 induced- tubulointerstitial fibrosis. Furthermore this work also describes the development of a high-throughput TEER meter that can be integrated with the AngioPlate platform allowing for rapid, non-invasive measurement of renal epithelial barrier integrity. Given that both platforms are designed in a 384-well plate format, they are high throughput and compatible with existing technologies like high-content imaging systems, robotic liquid handling systems, and microplate readers allowing for widespread adoption across diverse research settings. It is anticipated that the contributions described in this work will significantly advance our understanding of disease propagation and accelerate drug development process. / Thesis / Candidate in Philosophy / Drug development is a complex and expensive process, often hindered by a high failure rate in clinical trials. This failure is partly due to the inadequacy of current predictive models in the early stages of development. To address this, researchers are turning to innovative microphysiological systems known as Organ-on-a-Chip, which mimic organ structure and functions in the lab. However, these systems have been limited in their use due to low throughput. To overcome this limitation, microphysiological systems in multi-well formats called the IFlowPlate and AngioPlate were developed through the works outlined in this thesis. These platforms are designed to be high-throughput, scalable, user-friendly, and are compatible with existing technologies, such as microplate readers, high-content imaging systems and robotic liquid handlers, making them accessible to a wide range of researchers. By combining the physiological relevance of microphysiological systems with the high-throughput capabilities, these platforms aim to transform the way we study diseases and test drugs.

In this thesis we identify device (3D Microelectrode Arrays – 3D MEAs)/system (commercial electronics interface) issues preventing the scaling up of the world’s first in vitro model of afferent synaptic signaling in the spinal cord and develop a potential solution involving spin cast insulation and micromilled/microdrilled/microsoldered/flex circuit integrated 6-well interface board with connectors for analyzing up to six 3D MEAs simultaneously at one time. These novel advances can scale experimentation in the development of new treatments, pharmacological responses, and other electrophysiological discoveries for neurological disorders. In addition, we report on a multi-material palette towards the microfabrication of the aforementioned 3D Microelectrodes Arrays for integration with a variety of 3D electrogenic Microphysiological Systems (MPS) beyond the afferent synaptic model. The goal of this part of the thesis was to fabricate 3D MEAs with six microelectrodes by utilizing materials such as polycarbonate (PC), polymethyl methacrylate (PMMA), and polysulfone (PS). We created a reliable microfabrication process by combining laser micromachining, laser-induced breakdown spectroscopy (LIBS), 3D needle assembly, SU-8 coatings and micromilling/ microdrilling techniques. The 3D MEAs demonstrate impedance characteristics similar to commercial MEAs. Additionally, all material combinations showed outstanding transparency and biocompatibility for applicability in 3D neuronal and cardiac studies.

This research developed methods for culturing self-assembling capillaries in an injectable gel as a potential method for vascularizing tissue-on-a-chip models to mimic physiological drug delivery. Additionally, a mathematical model was developed as a tool for understanding nutrient delivery and comparison of potential delivery systems. Organs-on-a-chip provide novel platforms for studying biology and physiology in 3D, allow exploration of tissue engineering on a manageable scale, and serve as models for drug screening and drug-delivery testing. Methods were first developed for co-culture of endothelial cells and fibroblasts (3T3s or HDFs) in 2D, evaluating culture time, seeding density and ratio of HUVECs and fibroblasts, and immunostaining with a HUVEC-specific marker. Cells formed large sheets with no signs of vessel formation in 2D; therefore, the setup was translated to 3D culture to further induce stress and release of angiogenetic factors, using fibrin gel to suspend cells in 3D. After 9 days of culture, HUVECs had extensive network formation with a high degree of complexity in the experimental cell ratios (especially with 5:1 HUVECs:HDFs). Therefore, these parameters can be used as a starting point for further development of vascularized tissue constructs. A mathematical model was also successfully developed to assess the impact of cell concentration, consumption, and mode of nutrient delivery on 3D cellular constructs which can be used to predict the spatial distribution of glucose over time. Although the model shows flow introduced through a device is sufficient to maintain nutrient levels for cell growth, developing perfusable capillaries is still a critical part of creating physiologically representative tissues.