A Novel Microbial Source Tracking DNA Microarray Used for Pathogen Detection in Environmental Systems

Pathogen detection and the identification of fecal contamination sources can be challenging in environmental and engineered treatment systems. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. Therefore, a quick method that can detect and identify waterborne pathogens in environmental systems is needed. In this work, a custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, mitochondria DNA (mtDNA) and antibiotic resistance genes was used to detect over 430 selected gene targets in whole genome amplification (WGA) DNA and complementary DNA (cDNA) isolated from sewage and animal (avian, cattle, poultry and swine) feces, freshwater and marine water samples, sewage spiked surface water samples, treated wastewater and sewage contaminated produce.;A combination of perfect match and mismatch probes on the microarray reduced the likelihood of false positive detections, thus increasing the specificity of the microarray for various gene targets. A linear decrease in fluorescence of positive probes over a 1:10 dilution series demonstrated a semi-quantitative relationship between gene concentrations in a sample and microarray fluorescence. Various pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage via the microarray, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21--33%) was lower than specificity (83--90%, percentage of true negatives). Next generation sequencing (NGS) of DNA from the fecal samples revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources.;Waterborne pathogens were also detectable via the microarray in freshwater, marine water and sewage spiked surface water samples as well as treated wastewater. Ultrafiltration was used to concentrate microorganisms (bacteria, viruses, protozoa and parasites) from several liters of environmental and treated water samples. Dead-end ultrafiltration (DEUF) was shown to have a 61.4 +/- 47.8 % recovery efficiency and 46-fold concentration increasing ability. Then WGA was utilized to increase gene copies and lower the microarray detection limit. Viruses, including adenovirus, bocavirus, Hepatitis A virus, and polyomavirus were detected in human associated water samples as well as pathogens like Legionella pneumophila, Shigella flexneri, C. fetus and genes coding for resistance to aminoglycosides, beta-lactams, tetracycline. Microbial source tracking results indicate that sewage spiked freshwater and marine samples clustered separately from other fecal sources including wild and domestic animals via non-metric dimensional scaling. A linear relationship between qPCR and microarray fluorescence was found, indicating the semi-quantitative nature of the MST microarray.;Multiple displacement amplification (MDA), which is an important type of WGA, is a widely used tool to amplify genomic nucleic acids. The strong amplification efficiency of MDA and low initial template requirement make MDA an attractive method for environmental molecular and NGS studies. However, like other nucleic acid amplification techniques, various factors may influence MDA efficiency including template concentration (e.g. rare species swamping out), GC amplification bias and genome length favoring amplification of longer genomes. It was found that MDA increased nucleic acids in mixed environmental samples approximately 4.24 +/- 1.40 (log, average +/- standard deviation) for 16S rRNA gene of Enterococcus faecalis, 1.90 +/- 1.70 for RNA polymerase gene of human norovirus, 8.83 +/- 2.88 for T antigen gene of human polyomavirus, 3.83 +/- 0.93 for uidA gene of Escherichia coli, 4.96 +/- 0.32 for invA gene of S. enterica and 8.77 +/- 2.85 for 16S rRNA gene of human Bacteroidales. The template length, concentration and GC content were found to influence MDA efficiency. The results mainly show that the MDA will be more efficient the longer the template length, the greater the initial concentration of nucleic acids and the lower the GC content of the template.;Overall, the results of this work show that 1) the microarray and sample handling technique is suitable for pathogen detection from feces and sewage; 2) when combined with ultrafiltration techniques, the microarray can also be used as a pathogen detection tool in environmental waters; 3) template length, and initial concentration increase MDA efficiency, but higher GC content template negatively effects MDA efficiency. The proposed microarray can be used for pathogen detection in feces, wastewater treatment plant sewage, treated wastewater and environmental waters. Further the proposed method is potentially applicable to pathogen/microorganism detections on vegetables, seafood, in hospital settings, industrial wastewater, and aquaculture settings

Microfluidic organ-on-chip for assessing the transport of therapeutic molecules and polymeric nanoconstructs

openThe selective permeation of molecules and nanomedicines across the diseased vasculature dictates the success of a therapeutic intervention. Yet, in vitro assays cannot recapitulate relevant differences between the physiological and pathological microvasculature. Here, a double-channel microfluidic device was engineered to comprise vascular and extravascular compartments connected through a micropillar membrane with tunable permeability. The vascular compartment was coated by endothelial cells to achieve permeability values ranging from 0.1 um/sec, following a cyclic adenosine monophosphate (cAMP) pre-treatment (25 ug/mL), up to 2 um/sec, upon exposure to Mannitol, Lexiscan or in the absence of cells. Fluorescent microscopy was used to monitor the vascular behavior of 250 kDa Dextran molecules, 200 nm polystyrene nanoparticles (PB), and 1,000-400 nm discoidal polymeric nanoconstructs (DPN), under different permeability and flow conditions. In the proposed on-chip microvasculature, it was confirmed that permeation enhancers could favor the perivascular accumulation of ~ 200 nm, in a dose and time dependent fashion, while have no effect on larger particles. Moreover, the microfluidic device was used to interrogate the role of particle deformability in vascular dynamics. In the presence of a continuous endothelium, soft DPN attached to the vasculature more avidly at sub-physiological flows (100 um/sec) than rigid DPN, whose deposition was larger at higher flow rates (1 mm/sec). The proposed double-channel microfluidic device can be efficiently used to systematically analyze the vascular behavior of drug delivery systems to enhance their tissue specific accumulation.openXXXIII CICLO - BIOINGEGNERIA E ROBOTICA - BIOENGINEERING AND ROBOTICSPAOLO DECUZZIBarbato, MARIA GRAZI

Microfabricated Poly (ethylene glycol) Based Hydrogels for Microvascular Tissue Engineering Applications

Shortages in donor organs and the lack of therapeutic treatment options to address tissue loss and end-organ failure has led to intense research into tissue engineering based therapeutics. Cellular, tissue, and organ level therapeutics hold the potential to shift clinical paradigms and drastically improve healthcare outcomes. However, to date the only successful tissue engineering therapeutics have been limited to thin and avascular tissues such as skin, cartilage and the bladder. This is primarily due to the absence of a perfusable vasculature to transport nutrients and waste during in vitro tissue development and inadequate host-implant vascular integration upon implantation. In this thesis we set out to develop hydrogel microfabrication technologies to (1) improve in vitro mass transport, (2) integrate self-assembling microvascular networks with microfabricated channels and (3) incorporate and support functional parenchymal cellular elements within in vitro constructs. Application of microfabrication technologies to PEG hydrogels requires that fabrication schemes are cell compatible, robust for handling and imaging and most importantly allow for precise micron level control of both fluid perfusion and hydrogel structure fabrication. Herein we report multiple cell compatible 1 microfabrication schemes that employ multilayer replica molding and photolithographic hydrogel fabrication techniques. Systems designed with these techniques resulted in improved in vitro mass transport, integration of selfassembled microvascular networks with fabricated structures and the ability to pattern multilayer heterogeneous hydrogel structures that contain and support multiple cellular elements. The progress reported herein has broad applicability towards the development of biomaterials with highly biomimetic structural-functional characteristics. More specifically these hydrogel microfabrication technologies hold the promise to improve the therapeutic potential of tissue engineered constructs and provide more biologically applicable pre-clinical tissue models.

Nano-biosupercapacitors enable autarkic sensor operation in blood

Today’s smallest energy storage devices for in-vivo applications are larger than 3 mm3 and lack the ability to continuously drive the complex functions of smart dust electronic and microrobotic systems. Here, we create a tubular biosupercapacitor occupying a mere volume of 1/1000 mm3 (=1 nanoliter), yet delivering up to 1.6 V in blood. The tubular geometry of this nano-biosupercapacitor provides efficient self-protection against external forces from pulsating blood or muscle contraction. Redox enzymes and living cells, naturally present in blood boost the performance of the device by 40% and help to solve the self-discharging problem persistently encountered by miniaturized supercapacitors. At full capacity, the nano-biosupercapacitors drive a complex integrated sensor system to measure the pH-value in blood. This demonstration opens up opportunities for next generation intravascular implants and microrobotic systems operating in hard-to-reach small spaces deep inside the human body

Exploration of large scale manufacturing of polydimethylsiloxane (PDMS) microfluidic devices

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Includes bibliographical references (leaves 54-56).Discussion of the current manufacturing process of polydimethylsiloxane (PDMS) parts and the emergence of PDMS use in biomedical microfluidic devices addresses the need to develop large scale manufacturing processes for the fabrication of said devices. Casting PDMS parts is found to be the best mass production process after evaluating several different production methods. Automation of the manufacturing process is introduced as a solution to the need for mass production. Changing variables within the production process and its effects are also discussed with the recommendation being made for using low viscosity pre-cured PDMS, high temperature curing and high vacuum degassing techniques to produce high quality parts at high production rates. The further development of producing two-sided PDMS parts is recommended by investigating the usage of a non-closed aspect limited casting process.by Philip W. Hum.S.B

Deformability-induced effects of red blood cells in flow

To ensure a proper health state in the human body, a steady transport of blood is necessary. As the main cellular constituent in the blood suspension, red blood cells (RBCs) are governing the physical properties of the entire blood flow. Remarkably, these RBCs can adapt their shape to the prevailing surrounding flow conditions, ultimately allowing them to pass through narrow capillaries smaller than their equilibrium diameter. However, several diseases such as diabetes mellitus or malaria are linked to an alteration of the deformability. In this work, we investigate the shapes of RBCs in microcapillary flow in vitro, culminating in a shape phase diagram of two distinct, hydrodynamically induced shapes, the croissant and the slipper. Due to the simplicity of the RBC structure, the obtained phase diagram leads to further insights into the complex interaction between deformable objects in general, such as vesicles, and the surrounding fluid. Furthermore, the phase diagram is highly correlated to the deformability of the RBCs and represents thus a cornerstone of a potential diagnostic tool to detect pathological blood parameters. To further promote this idea, we train a convolutional neural network (CNN) to classify the distinct RBC shapes. The benchmark of the CNN is validated by manual classification of the cellular shapes and yields very good performance. In the second part, we investigate an effect that is associated with the deformability of RBCs, the lingering phenomenon. Lingering events may occur at bifurcation apices and are characterized by a straddling of RBCs at an apex, which have been shown in silico to cause a piling up of subsequent RBCs. Here, we provide insight into the dynamics of such lingering events in vivo, which we consequently relate to the partitioning of RBCs at bifurcating vessels in the microvasculature. Specifically, the lingering of RBCs causes an increased intercellular distance to RBCs further downstream, and thus, a reduced hematocrit.Um die biologischen Funktionen im menschlichen Körper aufrechtzuerhalten ist eine stetige Versorgung mit Blut notwendig. Rote Blutzellen bilden den Hauptanteil aller zellulären Komponenten im Blut und beeinflussen somit maßgeblich dessen Fließeigenschaften. Eine bemerkenswerte Eigenschaft dieser roten Blutzellen ist ihre Deformierbarkeit, die es ihnen ermöglicht, ihre Form den vorherrschenden Strömungsbedingungen anzupassen und sogar durch Kapillaren zu strömen, deren Durchmesser kleiner ist als der Gleichgewichtsdurchmesser einer roten Blutzelle. Zahlreiche Erkrankungen wie beispielsweise Diabetes mellitus oder Malaria sind jedoch mit einer Veränderung dieser Deformierbarkeit verbunden. In der vorliegenden Arbeit untersuchen wir die hydrodynamisch induzierten Formen der roten Blutzellen in mikrokapillarer Strömung in vitro systematisch für verschiedene Fließgeschwindigkeiten. Aus diesen Daten erzeugen wir ein Phasendiagramm zweier charakteristischer auftretender Formen: dem Croissant und dem Slipper. Aufgrund der Einfachheit der Struktur der roten Blutzellen führt das erhaltene Phasendiagramm zu weiteren Erkenntnissen über die komplexe Interaktion zwischen deformierbaren Objekten im Allgemeinen, wie z.B. Vesikeln, und des sie umgebenden Fluids. Darüber hinaus ist das Phasendiagramm korreliert mit der Deformierbarkeit der Erythrozyten und stellt somit einen Eckpfeiler eines potentiellen Diagnosewerkzeugs zur Erkennung pathologischer Blutparameter dar. Um diese Idee weiter voranzutreiben, trainieren wir ein künstliches neuronales Netz, um die auftretenden Formen der Erythrozyten zu klassifizieren. Die Ausgabe dieses künstlichen neuronalen Netzes wird durch manuelle Klassifizierung der Zellformen validiert und weist eine sehr hohe Übereinstimmung mit dieser manuellen Klassifikation auf. Im zweiten Teil der Arbeit untersuchen wir einen Effekt, der sich direkt aus der Deformierbarkeit der roten Blutzellen ergibt, das Lingering-Phänomen. Diese Lingering-Ereignisse können an Bifurkationsscheiteln zweier benachbarter Kapillaren auftreten und sind durch ein längeres Verweilen von Erythrozyten an einem Scheitelpunkt gekennzeichnet. In Simulationen hat sich gezeigt, dass diese Dynamik eine Anhäufung von nachfolgenden roten Blutzellen verursacht. Wir analysieren die Dynamik solcher Verweilereignisse in vivo, die wir folglich mit der Aufteilung von Erythrozyten an sich gabelnden Gefäßen in der Mikrovaskulatur in Verbindung bringen. Insbesondere verursacht das Verweilen von Erythrozyten einen erhöhten interzellulären Abstand zu weiter stromabwärts liegenden Erythrozyten und damit einen reduzierten Hämatokrit

In vivo label-free tissue histology through a microstructured imaging window

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies

Organ-on-a-Disc: A Scalable Platform Technology for the Generation and Cultivation of Microphysiological Tissues

Organ-on-Chip (OoC) systems culture human tissues in a controllable environment under microfluidic perfusion and enable a precise recapitulation of human physiology. Although recent studies demonstrate the potential of OoCs as alternative to traditional cell assays and animal models in drug development as well as personalized medicine, unmet challenges in device fabrication, parallelization and operation hinder their widespread application. In order to overcome these obstacles, this thesis focuses on the development of the Organ-on-a-Disc technology for the scalable generation and cultivation of microphysiological tissues. Organ-Discs are fabricated using precise, rapid and scalable microfabrication techniques. They enable the pump- and tubing-free perfusion as well as the parallelized generation and culture of tailorable and functional microtissues using rotation-based operations. The Organ-Disc setup is suitable for versatile tissue readouts, treatments and even whole blood perfusion with minimal handling and equipment requirements. Overall, the Organ-Disc creates a scalable and userfriendly platform technology for microphysiological tissue models and paves the way for their transition towards high-throughput systems.:Abbreviations Symbols 1 Introduction 2 Background 2.1 Fluid Dynamics 2.1.1 Flow Equations 2.1.2 Hydraulic Resistance 2.1.3 Wall Shear Stress 2.1.4 Centrifugal Microfluidics 2.2 Microfluidic Chip Fabrication 2.2.1 Chip Materials 2.2.2 Microstructuring 2.2.3 Bonding 3 State of the Art 3.1 Cell Culture Systems 3.2 3D Tissue Generation in Microfluidic Systems 3.3 Organ-on-Chip 3.4 Scale-up of Organ-on-Chip Systems 3.4.1 Scalable Fabrication Technologies 3.4.2 Parallelization Approaches 3.4.3 Integrated Fluid Actuation 3.5 Centrifugal Microfluidics 4 Objectives 5 Materials and Methods 5.1 Organ-Disc Fabrication 5.1.1 Materials 5.1.2 2D Structuring 5.1.3 Hot Embossing Stamp Fabrication TPE Hot Embossing 5.1.4 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 5.1.5 Characterization Methods Structure Sizes Bonding Strength Optical Properties 5.2 Organ-Disc Spinner 5.2.1 Centrifugal Loading Setup 5.2.2 Centrifugal Perfusion Setup 5.2.3 Peristaltic Pumping Setup 5.3 Organ-Disc Perfusion 5.3.1 Centrifugal Perfusion 5.3.2 Peristaltic Perfusion 5.4 Preparatory Cell Culture 5.5 Organ-Disc Cell Loading 5.5.1 Centrifugal Cell Loading 5.5.2 Endothelial-lining 5.6 Organ-Disc Cell Culture 5.6.1 Staining and Imaging Live Cell Labeling Live/Dead Staining CD106 Staining CD41 Staining Fixation, Permeabilization and Blocking Actin/Nuclei Staining CD31/Nuclei Staining 5.6.2 Media Analysis 5.6.3 Endothelial Cell Activation 5.6.4 Whole Blood Perfusion 5.7 Data Presentation and Statistics 6 Concept and Design 6.1 Organ-Disc Technology 6.2 Organ-Disc Design 6.3 Centrifugal Cell Loading 6.4 Endothelial Cell Lining 6.5 Centrifugal Perfusion 6.6 Peristaltic Perfusion 7 Building Blocks 7.1 Microfabrication Technology 7.1.1 Structuring 2D Structuring Hot Embossing 7.1.2 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 7.2 Organ-Disc Spinner 8 Perfusion 8.1 Centrifugal Pumping 8.2 Peristaltic Pumping 9 Tissue Generation and Culture 9.1 3D Tissue Generation 9.2 Stratified Tissue Construction 9.3 Generation of Endothelial-lined Channels 9.4 Perfusion of Endothelial-lined Channels 9.4.1 Media Monitoring Evaporation Cell Metabolism 9.4.2 Inflammatory Cell Stimulation 9.4.3 Whole Blood Perfusion 10 Discussion 10.1 Organ-Disc Technology 10.2 Scalable, Precise and Robust Organ-Disc Fabrication 10.2.1 Fabrication of Thermoplastic Organ-Discs 10.2.2 Fabrication of TPE Modules 10.2.3 Integration of TPE Modules to Organ-Discs 10.3 Tunable, Pump- and Tubing-free Perfusion 10.4 On-Disc Tissue Culture 10.4.1 3D Tissues 10.4.2 Blood Vessel-like Structures 10.4.3 Tissue Characterization and Treatment 10.5 On-Disc Blood Perfusion 11 Summary and Conclusion 12 References 13 AppendixIn Organ-on-Chip (OoC)-Systemen werden menschliche Gewebe mittels mikrofluidischer Versorgung in einer kontrollierten Umgebung kultiviert und so die Physiologie des Menschen nachgebildet. Obwohl aktuelle Studien zeigen, dass dieser Ansatz Alternativen zu herkömmlichen Zellbasierten Tests und Tiermodellen in der Arzneimittelentwicklung und der personalisierten Medizin bietet, stehen einer breiteren Anwendung Hürden im Bereich der Herstellung, Parallelisierung und Handhabung im Weg. Deshalb ist das Ziel dieser Arbeit die Entwicklung der Organ-on-a-Disc-Technologie, die eine skalierbare Erzeugung und Kultur von mikrophysiologischen Geweben ermöglicht. Für die Herstellung von der Organ-Disc kommen präzise, schnelle und skalierbare Mikrofabrikationsmethoden zum Einsatz. Die Organ-Disc schafft die Basis für die parallelisierte Erzeugung und Kultur von maßgeschneiderten und funktionellen Mikrogeweben, sowie deren Versorgung durch rotationsbasierte Prozesse und ohne zur Hilfenahme von Pumpen oder Schläuchen. Die Organ-Disc eignet sich für unterschiedliche Charakterisierungsmethoden sowie der Gewebestimulation und sogar der Vollblutperfusion mit minimalem Aufwand und Equipment. Insgesamt stellt die Organ-Disc eine skalierbare und benutzerfreundliche Plattformtechnologie für mikrophysiologische Modelle dar und bereitet den Weg für Hochdurchsatzanwendungen.:Abbreviations Symbols 1 Introduction 2 Background 2.1 Fluid Dynamics 2.1.1 Flow Equations 2.1.2 Hydraulic Resistance 2.1.3 Wall Shear Stress 2.1.4 Centrifugal Microfluidics 2.2 Microfluidic Chip Fabrication 2.2.1 Chip Materials 2.2.2 Microstructuring 2.2.3 Bonding 3 State of the Art 3.1 Cell Culture Systems 3.2 3D Tissue Generation in Microfluidic Systems 3.3 Organ-on-Chip 3.4 Scale-up of Organ-on-Chip Systems 3.4.1 Scalable Fabrication Technologies 3.4.2 Parallelization Approaches 3.4.3 Integrated Fluid Actuation 3.5 Centrifugal Microfluidics 4 Objectives 5 Materials and Methods 5.1 Organ-Disc Fabrication 5.1.1 Materials 5.1.2 2D Structuring 5.1.3 Hot Embossing Stamp Fabrication TPE Hot Embossing 5.1.4 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 5.1.5 Characterization Methods Structure Sizes Bonding Strength Optical Properties 5.2 Organ-Disc Spinner 5.2.1 Centrifugal Loading Setup 5.2.2 Centrifugal Perfusion Setup 5.2.3 Peristaltic Pumping Setup 5.3 Organ-Disc Perfusion 5.3.1 Centrifugal Perfusion 5.3.2 Peristaltic Perfusion 5.4 Preparatory Cell Culture 5.5 Organ-Disc Cell Loading 5.5.1 Centrifugal Cell Loading 5.5.2 Endothelial-lining 5.6 Organ-Disc Cell Culture 5.6.1 Staining and Imaging Live Cell Labeling Live/Dead Staining CD106 Staining CD41 Staining Fixation, Permeabilization and Blocking Actin/Nuclei Staining CD31/Nuclei Staining 5.6.2 Media Analysis 5.6.3 Endothelial Cell Activation 5.6.4 Whole Blood Perfusion 5.7 Data Presentation and Statistics 6 Concept and Design 6.1 Organ-Disc Technology 6.2 Organ-Disc Design 6.3 Centrifugal Cell Loading 6.4 Endothelial Cell Lining 6.5 Centrifugal Perfusion 6.6 Peristaltic Perfusion 7 Building Blocks 7.1 Microfabrication Technology 7.1.1 Structuring 2D Structuring Hot Embossing 7.1.2 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 7.2 Organ-Disc Spinner 8 Perfusion 8.1 Centrifugal Pumping 8.2 Peristaltic Pumping 9 Tissue Generation and Culture 9.1 3D Tissue Generation 9.2 Stratified Tissue Construction 9.3 Generation of Endothelial-lined Channels 9.4 Perfusion of Endothelial-lined Channels 9.4.1 Media Monitoring Evaporation Cell Metabolism 9.4.2 Inflammatory Cell Stimulation 9.4.3 Whole Blood Perfusion 10 Discussion 10.1 Organ-Disc Technology 10.2 Scalable, Precise and Robust Organ-Disc Fabrication 10.2.1 Fabrication of Thermoplastic Organ-Discs 10.2.2 Fabrication of TPE Modules 10.2.3 Integration of TPE Modules to Organ-Discs 10.3 Tunable, Pump- and Tubing-free Perfusion 10.4 On-Disc Tissue Culture 10.4.1 3D Tissues 10.4.2 Blood Vessel-like Structures 10.4.3 Tissue Characterization and Treatment 10.5 On-Disc Blood Perfusion 11 Summary and Conclusion 12 References 13 Appendi

Design and fabrication of microgel shapes as red blood cell substitutes (RBCs) by UV-assisted punching

There is a constant high demand for blood transfusions worldwide. Donor-derived blood employed as the current clinical standard has several limitations including insufficient availability, short shelf-life, and risk of pathogenic contamination. Thus, the development of artificial red blood cells (RBCs) has become a crucial field of study. Such artificial RBCs encapsulate hemoglobin (Hb), the oxygen-carrying component of RBCs, and aim to mimic the features of the endogenous cells. Currently, mainly microparticles and nanoparticles are used as encapsulated systems in drug delivery research. Polyethylene glycol (PEG)-based hydrogels are the primary choice for material, due to their biocom-patibility. So far, most carriers developed have spherical shapes and are produced from bottom-up approaches that cannot offer the best geometric control. It has been proven that geometry plays a critical role in the vascular behavior of microparticles, thus the need to obtain non-spherical carriers. This project proposed to employ top-down fabrication methods to better overcome this challenge. By aiming to design and fabricate three different PEG-based microgel shapes (rods, stars, and hexagons) designed within the same size range as RBCs. Firstly, Silicon masters with the designed patterns were produced through photolithography and dry etching. Then they were used to produce cyclo olefin polymer (COP) stamps through hot embossing. These stamps were coated with different PEG formulations (with and without Hb) by slot die coating. Next, they were used in UV-assisted punching to form the microgel shapes and mechanically punch through the hydrogel flash layer onto a poly-vinyl alcohol (PVA) substrate. The microgels were harvested by dissolving the PVA. These microgels shapes obtained had the desired aspect ratio of 3:1 retaining good shape features. However, challenges remained in the achievement of individual microgel shapes after harvest. Due to coating thickness limitations that consequently affected the mechanically punching process afterwards.Existe uma constante procura de transfusões sanguíneas. Isto devido ao sangue proveniente de dadores ter inúmeras limitações incluindo disponibilidade insuficiente, baixa longevidade e risco de contaminação pa-togénica. Assim sendo o desenvolvimento de hemácias artificiais tornou-se um crucial ramo de investigação. Estas hemácias artificiais encapsulam a hemoglobina, componente transportador de oxigénio, visando imitar as características das células endógenas. Atualmente maioritariamente micropartículas e nanopartículas são usadas como sistemas de encapsu-lamento em estudos de entrega de fármacos. Sendo os hidrogéis à base de polietilenoglicol (PEG) a maior escolha como material, devido à sua biocompatibilidade. Até agora, a maioria dos portadores desenvolvidos tem formas esféricas e são produzidos a partir de métodos de bottom-up que não conseguem oferecer o melhor controle geométrico. Foi comprovado que a geometria desempenha um papel crítico no comportamento vas-cular das micropartículas, daí a necessidade de obter portadores não esféricos. Este projeto propôs utilizar métodos de fabricação top-down de forma a superar essa limitação. Com o objetivo principal de desenhar e fabricar três formas diferentes de microgéis à base de PEG (hastes, estrelas e hexágonos) projetadas para dimensões semelhantes às hemácias. Primeiramente, foram produzidos templates de silício com os padrões fabricados por fotolitografia e erosão seca. Em seguida, estes foram usados para imprimir moldes de polímero de ciclo olefina (COP) por hot embossing. A estes moldes foram depositadas diferentes formulações de PEG (com e sem Hb) por slot die coating. Consecutivamente, estes foram usados em UV-assisted punching para obter as desejadas formas microgéis com o molde cortando mecanicamente as formas pelo hidrogel para o substrato de álcool polivinílico (PVA). Posteriormente os microgéis foram obtidos através da dissolução do PVA em água. Sendo que estes mantiveram os tamanhos desejados com uma razão de aspeto de 3:1, conservando também as formas. No entanto, os desafios permaneceram na obtenção de mi-crogéis individuais após a dissolução. Devido a limitações de espessura durante a deposição que consequente-mente afetaram o processo de cortamento mecânico posterior