The inability to monitor the molecular trajectories of whole organs throughout the clinically relevant ischemic interval is a critical problem underlying the organ shortage crisis. Here, we report a novel technique for fabricating manufacturing conformal microfluidic devices for organ interface. 3D conformal printing was leveraged to engineer and fabricate novel organ-conforming microfluidic devices that endow the interface between microfluidic channels and the organ cortex. Large animal studies reveal microfluidic biopsy samples contain rich diagnostic information, including clinically relevant biomarkers of ischemic pathophysiology. Overall, these results suggest microfluidic biopsy via 3D printed organ-conforming microfluidic devices could shift the paradigm for whole organ preservation and assessment, thereby relieving the organ shortage crisis through increased availability and quality of donor organs. / Master of Science

abstract: Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

The combined use of additive manufacturing (AM), biocompatible and biodegradable materials, cells and biomolecular signals is the most common biomanufacturing strategy applied in scaffold fabrication. AM processes offer a better control and the ability to actively design the porosity and interconnectivity of the scaffolds. When combined with clinical imaging data, these fabrication techniques can be used to produce constructs that are customised to the shape of the defect or injury. However, due to the hydrophobicity of the commonly used synthetic biopolymers, cell-seeding and proliferation efficiency are limited. Moreover, due to the tortuosity of the scaffolds, non-uniform cell distribution with rare cell adhesion in the core region also commonly exists. Additionally, the commercial available machines are not able to create multi-material and material gradient scaffolds that are required to mimic the nature of nature tissues. To overcome the above limitations, this thesis describes the development of a hybrid bio-additive manufacturing system, called plasma-assisted bioextruson system (PABS), to produce smart scaffold by combining multi-head polymer extrusion and the plasma surface modification layer by layer, in the same chamber. PABS allows not only multiple biomaterials printing with the multi-extrusion heads, but also enables in-process plasma surface modification for zonal plasma-treated scaffolds fabrication. The in-house user interface enables a high degree of scaffold design freedom as it allows users to create single or multi-material constructs with uniform pore size or pore size gradient by changing process parameters such as lay-down pattern, filament distance, feed rate and layer thickness. Water contact angle tests and in vitro biological tests confirm that the hydrophilicity of synthetic polymers is improved and cell attachment and proliferation are enhanced after the in-process plasma modification. The effect of plasma treatment is also investigated by using different plasma modification strategies and various plasma modification parameters, including the plasma deposition velocity and the distance between the plasma jet and the printed scaffolds. The biological results also show dependence between the surface modification strategies and cell proliferation. The mechanical compression results show that for a fixed plasma deposition velocity, the effect of changing the distance between the plasma head and the deposited material is not significant. However, for a fixed distance, the compressive modulus increases with the increase in the plasma deposition velocity.

<p>Additive manufacturing (AM) of hydrogels has gained increasing interest across various fields. Drop-on-demand (DOD) printing (also known as inkjet printing) shows the great potential to construct 3D hydrogels with spatially controlled properties and compositions. However, a limited mechanistic understanding of the behavior of printed polymer drops makes it challenging to design and optimize DOD printing protocols for a wide variety of hydrogels. Here, we have demonstrated an extensive and in-depth study from the theoretical and experimental research of drop-wise structure to the development of functional materials by DOD printing of polymer inks. First, computational and experimental studies are performed to establish a mechanism of the water-matrix interaction within printed polymer drops. The results ultimately enabled a dimensionless parameter that characterizes water transport during the dehydration process of printed polymer drops. Next, as particles are suspended in polymer inks to add functionality, this dimensionless parameter was further extended to characterize particle movement and distribution patterns in the printed particle-laden hydrogels. By correlating the intra-drop particle distribution to the similarity parameter, a scaling law is confirmed to guide ink formulation and printing protocol that enables advanced materials with spatially digitized functionality (i.e., digital hydrogels). Finally, cells that serve as active particles are embedded in the hydrogels to mimic the native tissues. A "digital cell printing" method based on DOD printing of "two colors" cell-laden (i.e., cancer cells and CAFs) polymer inks is developed to rapidly (< 1 day) create 3D tumor models with tumor-stroma interface (i.e., tumoroids) and high cell density (~108 cells/cm3) that closely recapitulate the tumor microenvironment in vivo. Overall, DOD printing of particulate-laden polymer inks showed the great potential to construct 3D functional hydrogels with spatially controlled properties and compositions.</p>

No description available.

The research presented in this dissertation supports the overall goal of creating piezoelectric measurement technology for the analysis and characterization of soft materials that serve as feedstocks (inputs) and products (outputs) of emerging biomanufacturing processes, including cell and additive biomanufacturing processes. The first objective was to define measurement challenges associated with real-time monitoring of material compositional profiles using biosensors in practical biomanufacturing and bioprocessing formats, as insight into a material's composition (i.e., concentration of a given biologic within a material or product) provides molecular-scale insight into processes and product quality. The second objective was to design, fabricate, and characterize continuous flow cell separation technology based on 3D printed self-exciting and -sensing millimeter-scale piezoelectric transducers and microfluidic networks for separation and characterization of expanded therapeutic cells. The third objective was to establish a sensor-based characterization approach for viscoelastic properties of hydrogels and gelation processes using high-order modes of piezoelectric-excited millimeter cantilever (PEMC) sensors and understand the influence of cantilever mode number on critical sensor characteristics, including sensitivity, dynamic range, and limit of detection. The first objective was addressed through a comprehensive review of recent progress in electrochemical and hybrid biosensors, which included discussions of measurement formats, sensor performance, and measurement challenges associated with use in practical bioprocessing environments. This critical review revealed that cost, disposability, form factor, complex measurement matrices, multiplexing, and sensor regeneration/reusability are among the most pressing challenges that require solutions through advancement of sensor design and manufacturing approaches before biosensors can facilitate high-confidence long-term continuous bioprocess monitoring. The second objective was addressed by creating a microextrusion-based additive manufacturing approach for fabrication of piezoelectric-based MEMS devices that enabled integration of 3D configurations of piezoelectric transducers and microfluidic networks in a one-pot manufacturing process. The devices contained orthogonal out-of-plane piezoelectric sensors and actuators and generated tunable bulk acoustic waves (BAWs) capable of size-selective manipulation, trapping, and separation of suspended particles in droplets and microchannels. This work suggests that additive manufacturing potentially provides new opportunities for the fabrication of sensor-integrated microfluidic platforms for cell culture analysis. The third objective was addressed through resonant frequency tracking of low- and high-order modes in dynamic-mode cantilevers to enable the real-time characterization of hydrogel viscoelastic properties and continuous monitoring of sol-gel phase transitions over a wide dynamic range using practically relevant hydrogel systems used commonly in additive biomanufacturing. This work suggests that high-order modes of PEMC sensors facilitate characterization of hydrogel viscoelastic properties and gelation processes with improved dynamic range and limit of detection that can complement the performance of low-order modes. Through this research, new approaches for sensor-based characterization of soft material composition and mechanical properties using millimeter-scale piezoelectric devices are presented as solutions for current challenges in biomanufacturing and biosensing to advance capability in real-time sensing of quality attributes among biomanufactured products. / Doctor of Philosophy / The research presented in this dissertation supports the overall goal of creating sensor-based measurement technology for quality assessment of soft materials within practical online biosensing and biomanufacturing processing formats. This technology seeks to enable monitoring and control of product quality in real-time. Soft biomaterials used in these processes, including cells and hydrogels, can be characterized by quality signatures such as concentration of analytes and physical and mechanical properties. Separation and fluid handling technologies aid real-time characterization when integrated with the processing system. By improving sensor-based measurement capability of soft materials, sensing platforms can provide online quality assurance and control, thereby increasing the product quality and process efficiency – or yield– at reduced cost. The first objective was to define measurement challenges and limitations associated with detection of biologics in practical biomanufacturing and bioprocessing formats (with focus on pathogen detection, as the detection of adventitious agents and pathogens remains a critical aspect of bioprocess monitoring). This was addressed through a comprehensive review of recent progress in the field of electrochemical and hybrid biosensors. The second objective was to design and fabricate sensor-integrated microfluidic technology for cell separation applications using a combination of multi-material 3D printing and pick-and-place techniques. The third objective was to improve measurement capability of piezoelectric sensors for characterization of viscoelastic properties of hydrogel formulations commonly used in additive biomanufacturing processes and tissue engineering. Through this research, new approaches for sensor-based characterization of soft materials using millimeter-scale piezoelectric devices are presented as solutions for current challenges in biomanufacturing and biosensing platforms in order to advance quality assessment capability.

According to data provided by the U.S. Department of Health and Human Services, the waiting list of organ transplantation as of April 2021 is approximately 107,550 out of which 90,908 patients are waiting for a kidney and 11,871 are waiting for a liver. In 2020, only 39,000 transplants were performed. A promising potential solution to this organ shortage crisis is rapid development of drugs for end-stage kidney and liver failure and the fabrication of organs using additive biomanufacturing (Bio-AM) processes. While progress toward industrial-scale production of 3D-bioprinted tissue models and organs remains hindered by various biological and tissue engineering challenges, such as vascularization and innervation, quality Bio-AM is impeded by lack of integrated process monitoring and control strategies. This dissertation aims to address the compelling need to incorporate sensing and control with Bio-AM processes, which are currently open-loop processes and improve the scalability and reliability of additively biomanufactured products. The specific aim is to develop a closed loop-controlled additive biomanufacturing process capable of fabricating 3D-bioprinted biological constructs (mini-tissues) of controlled mechanical properties. The proposed methodology is based on the use of embedded sensors and real-time material property sensing for feedback control of the bioprinted constructs mechanical property. There are three objectives of this dissertation: (1) experimenting and modeling the processes to understand the causal effect of process-material interactions on Bio-AM defects, (2) use of sensors to detect defects during printing, (3) prevention of the propagation of defects through closed-loop process control. This will help us understand the fundamentals of the bio-physical process interactions that govern the quality of printed biological tissue through empirical investigation of the sensor-based data This will also provide us with a real-time monitoring, closed-loop quality control strategy to prevent the propagation of quality defects by executing corrective actions during the whole duration of the printing process. / Doctor of Philosophy / As of April 2021, there are 107,550 patients on the national transplant list out of which approximately 39,000 patients received a transplant. Simultaneously, drug development remains an expensive and time-consuming endeavor. These burden on the public and healthcare system are expected to further increase compounded by the rapidly aging population in the United States with 80 million people expected to be older than 65 years old by 2040. Additive biomanufacturing processes, commonly referred to as 3D bioprinting processes, are automated biofabrication processes that offer great potential toward manufacturing future therapeutics and models for drug discovery. Despite all the benefits and the versatility that 3D printing provides, it does not come without its own shortcomings. Additive biomanufacturing is traditionally an open-loop process, meaning the process parameters are not adjusted during the biofabrication process making it challenging to detect and correct defects during processing and achieve high reproducibility and product quality. While the dimensional characteristics and material properties are important quality signatures of a cell-based products, there are additional signatures associated with the cell quality. Some of these quality attributes include cell viability, cell proliferation, metabolic activity, morphology, and gene expression profile. Given the clinical importance and invasive nature of bio-products such as scaffolds for tissue regeneration and stem cell therapy, rigorous approaches for characterization, monitoring, and control of quality are critical to future additive bio-manufacturing paradigms. In particular, the elastic modulus of the extracellular matrix has been found to have an influence on the cell morphology, proliferation, and differentiation process. Hence, it is an excellent parameter to monitor as a measure of tissue quality. However, the traditional techniques used to characterize tissue elastic modulus are low-throughput, offline techniques and face challenges with tissue integration. Thus, there is a need for integrated sensors that can measure the modulus of tissues during 3D bioprinting. This dissertation aims to address some of these issues by developing a multi-material 3D printing and pick-and-place approach to develop smart tissue cultureware and designing a tissue integrated closed-loop feedback sensor system for polymerization of hydrogels.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-10T20:31:55Z No. of bitstreams: 1 DissLAVF.pdf: 2396434 bytes, checksum: d18db543d45b99a8efd280285db823b0 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T12:05:36Z (GMT) No. of bitstreams: 1 DissLAVF.pdf: 2396434 bytes, checksum: d18db543d45b99a8efd280285db823b0 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T12:05:43Z (GMT) No. of bitstreams: 1 DissLAVF.pdf: 2396434 bytes, checksum: d18db543d45b99a8efd280285db823b0 (MD5) / Made available in DSpace on 2016-10-21T12:05:51Z (GMT). No. of bitstreams: 1 DissLAVF.pdf: 2396434 bytes, checksum: d18db543d45b99a8efd280285db823b0 (MD5) Previous issue date: 2016-03-24 / Não recebi financiamento / Currently, there are several studies directed to the manufacture of biosystems (biomaterials, living tissues or organs). These studies include several practice areas ranging from virtual representation of an organ or tissue to its biomanufacturing (bioprinting) itself. But for biomanufacturing a complex organ, it is still needed a long walk, because this process requires a very large wealth of information. Experiments to aid in surgical planning have been made based on medical image data and use of 3D printer rapid prototyping through STL specifications (STereoLitography). This work aims the study biomanufacturing processes of biomaterials, living tissues and organs aiming to establish the requirements for building a computer model to assist in the development of a project description framework for bioprinting living tissues and organs via STL specifications. This model was designed through research processes and parameters required for bioprinting of living tissues or organs resulting from the state of the art in this area and forms of representation in a computer model. For the evaluation of the model and the developed framework, an experiment was conducted where the data of a cartilage bioprinting experiment conducted by other authors were expressed through a bioprinting project. / Atualmente, existem vários estudos voltados para a fabricação de biosistemas (biomateriais, tecidos vivos ou órgãos). Esses estudos contemplam várias áreas de atuação que vão desde a representação virtual de um órgão ou tecido até a sua biofabricação (bioimpressão) propriamente dita. Porém, para a biofabricação de um órgão complexo, ainda é necessária uma longa caminhada, pois esse processo exige uma riqueza muito grande de informações. Experimentos para auxilio em planejamento cirúrgico têm sido feitos baseados em dados de imagens médicas e uso de impressoras 3D de prototipagem rápida, através de especificações STL (STereoLitography). Este trabalho, tem como objetivo, o estudo de processos de biofabricação de biomateriais, tecidos vivos e órgãos visando, estabelecer os requisitos necessários para a construção de um modelo computacional que auxilie no desenvolvimento de um framework de descrição de projetos para bioimpressão de tecidos vivos e órgãos por intermédio de especificações STL. Esse modelo foi concebido através da investigação de processos e parâmetros necessários para a bioimpressão de tecidos vivos ou órgãos, decorrentes do estado da arte nessa área e das formas de sua representação em um modelo computacional. Para a avaliação do modelo e do framework desenvolvido, foi realizado um experimento onde os dados de um experimento de bioimpressão de cartilagem realizado por outros autores foram expressados através de um projeto de bioimpressão.

In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007. In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy. As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0). / Ph. D.