Many drugs and environmental chemicals, such as antibiotics and chemotherapeutic agents, are nephrotoxic (toxic to the kidney) and are a common cause of acute kidney injury and chronic kidney disease. Conventional tissue models for assessment of drug-induced nephrotoxicity rely on animals or simple cell culture models, which lack tissue characteristics of the human kidney required to accurately predict a drug’s effect in clinical trials. Microfluidic kidney-on-chips can generate tissue with improved human relevance compared to traditional models, however, generally lack high-throughput and multiparametric data collection capabilities for evaluation of nephrotoxic drug exposures. Standard data collection techniques remain limited to fluorescent imaging or colorimetric assays that often focus on single endpoints, are invasive due to the addition of labels, and fail to capture dynamic changes in tissue function. Additionally, conventional toxicological readouts rely on bulk measures of injury, such as cell death, which are less sensitive than sub-lethal changes in cell function and morphology that occur prior to cell death. Due to the challenges above, there is a need for new measurement approaches that enable collection of kinetic, multi-parametric, and sub-lethal readouts of injury in kidney-on-chip systems. In this work, we developed and characterized several measurement approaches for evaluation of tissue function in kidney-on-chip systems and assessment of drug-induced nephrotoxicity. In chapter 2, we developed a novel optical-based oxygen sensing technique for measurement of sub-lethal mitochondrial dysfunction in an array of kidney-on-chips. In chapter 3, we investigated an approach for simultaneous transepithelial electrical resistance (TEER) sensing and flow control to enable near-continuous monitoring of tissue barrier function under different flow conditions. In chapter 4, we demonstrated the use of different data collection modalities, including multiple sensors, fluorescent imaging, and colorimetric-based assays, to generate multi-parametric readouts for evaluation of drug-induced nephrotoxicity in kidney-on-chips. / 2024-05-24T00:00:00Z

Inflammation is a crucial physiological defense mechanism of the human body to injury or infection. However, dysregulation of the magnitude or duration of inflammation response underlies multiple disease pathologies and may cause organ damage. Sepsis is a severe inflammatory disease now known as a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis patients often die of organ failure and the endothelium and neutrophil-endothelial cell (EC) interactions play an active role in the regulation of the systemic inflammatory response. Systemic inflammatory disease often results in alterations in vascular endothelium barrier function, increased permeability, excessive leukocyte trafficking, and reactive oxygen species production, leading to organ damage. While neutrophils are critical to host defense, neutrophil dysregulation has a critical role in organ damage through release of proteases, neutrophil extracellular traps (NETs), and reactive oxygen species (ROS), which can damage host tissue leading to organ failure. To date therapeutic approaches are largely supportive and therapeutics targeting endothelium inflammation and immune cell dysregulation are urgently needed. However, strong concerns regarding the level of phenotypic heterogeneity of microvascular ECs between different organs have been expressed. Microvascular EC heterogeneity in different organs and organ-specific variations in EC structure and function are regulated by intrinsic signals that are differentially expressed across organs and species, as a result of which neutrophil recruitment to discrete organs may be regulated differently. In addition, therapeutic development is hindered due to the heterogeneous nature of sepsis and the presence of multiple distinct immune phenotypes that can impact function and response to infection. In fact, clinically sepsis is a heterogeneous syndrome and diagnosis is complicated due to the broad spectrum of non-specific clinical features. Patients with similar clinical symptoms can be associated with distinct immune cell phenotypes ranging from excessive immune activation to immunosuppression, which means different therapeutics are required. In this work, the morphological and functional variations of differently originated microvascular endothelium are discussed and how these variances affect systemic function in response to inflammation. Emerging in vivo and in vitro models and techniques including microphysiological devices, proteomics, and RNA-Sequencing used to study the cellular and molecular heterogeneity of endothelium from different organs will also be discussed. Our group have developed a novel Organ-on-Chip, the biomimetic microfluidic assay (bMFA) that mimics physiological conditions, allowing us to observe real-time neutrophil-endothelial interactions, including rolling, adhesion, and migration, and to study endothelial barrier function under physiologically relevant conditions including the effect of shear forces and vascular geometry. The bMFA enables the quantification of leukocyte-EC interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, EC permeability, adhesion molecule expression and other important variables. Furthermore, by using human related samples, such as human ECs and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability. In this work, a protocol was developed to study endothelium function and neutrophil-endothelial interactions during inflammation in the bMFA. Lastly, to develop targeted therapeutics, immunophenotyping is needed to identify distinct immune cell functional phenotypes. We have developed a methodology to classify ICU sepsis patients into three phenotypes using patient data, Organ-on-Chip-based neutrophil functional analysis and proteomics. The findings of the study will help identify different sepsis patient immune-phenotypes and personalize treatment accordingly. / Mechanical Engineering

Drug metabolism plays an important role in drug absorption and drug elimination. Therefore, it is crucial to understand the mechanism and kinetics of drug metabolism by various drug-metabolizing enzymes (DMEs). Cytochrome P450 enzymes (CYPs) are responsible for the metabolism of more than 60% of the top 200 prescribed drugs. X-ray and NMR data of CYP enzyme suggest that relatively large and flexible active sites are capable of multi-substrate binding. Due to the multiple substrate-binding, CYP reactions tend to show non-Michaelis Menten kinetics (atypical kinetics), multiple metabolite formation and sequential metabolism.To investigate the complexity of cytochrome P450 kinetics, saturation curves and intrinsic clearances (CLint) were simulated for single substrate and multi-substrate models using rate equations and numerical analysis. These models were combined with multiple product formation and sequential metabolism and simulations were performed with random error. All simulation and model fitting was performed using Mathematica. A concentration-dependent metabolite ratio plot can be observed from multi-substrate binding kinetics. Use of single substrate models to characterize multi-substrate data can result in inaccurate kinetic parameters and poor clearance predictions. It has been shown that use of different substrate concentrations may lead to highly variable in vitro CLint estimations when sigmoidal kinetics are observed. Comparing results for use of standard velocity equations with ordinary differential equations (ODEs) clearly shows that ODEs are more versatile and provide better parameter estimates. It would be difficult to derive concentration-velocity relationships for complex models, but these relationships can be easily modeled using numerical methods and ODEs. The model drug diazepam (DZP) was chosen as the probe substrate to demonstrate complex CYP kinetics with specific CYP enzyme sources, including rat liver microsome (RLM), human liver microsome (HLM), purified CYP enzyme isoforms and rat hepatocytes. All saturation curves display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. In addition, the sequential metabolism and disposition would be characterized in hepatocytes incubation under flow conditions. To provide in vivo evidence of the atypical kinetics and investigate CYP-mediated sequential metabolism, preliminary intravascular (IV) dosing PK studies with male rats was performed for DZP. In general, DZP and its metabolites were quantitated by LC/MS/MS. Numerical methods were used to solve ODEs and parameterize micro and macro rate constants for the models. It has been shown that more complex models that include explicit enzyme-product complexes can well characterize the datasets for diazepam sequential metabolism with CYP3A4. Uncommon DZP metabolite PK profiles are observed in rat PK studies. In summary, methods of in vitro data analysis are compared, new assays are developed, and new modeling approaches for complex drug and metabolite pharmacokinetics are being investigated. / Pharmaceutical Sciences

No description available.

Les matériaux à structure tridimensionnelle laissent entrevoir de nombreuses applications prometteuses dans le domaine de l'ingénierie tissulaire et de la médecine régénérative en fournissant un micro-environnement approprié pour l'incorporation de cellules ou de facteurs de croissance afin de régénérer des tissus ou organes endommagés. Dans ce contexte, une membrane a été élaborée à partir d'un mélange de poly (ε-caprolactone) PCL / chitosan CHT à partir d'une technique d'inversion de phase permettant un apport localisé de non solvent. La technique permet d'obtenir une double morphologie poreuse : (i) des macrovides en surface (gros pores) facilement accessibles pour l'invasion et la viabilité des cellules; (ii) un réseau macroporeux interconnecté (petits pores) pour transférer les nutriments, l'oxygène, le facteur de croissance à travers le matériau. Les propriétés physico-chimiques (taille des pores, chimie de surface et biodégradabilité) des matériaux ont été caractérisées. Il est montré comment il est possible d'ajuster les propriétés de la membrane en modifiant le rapport PCL / CHT. Des cultures de cellules souches mésenchymateuses humaines (CSMh) ont été réalisées sur la membrane. La viabilité et la prolifération cellulaires ont été étudiées par des essais de test au MTT et de taux d'absorption d'oxygène. Les expériences démontrent que la membrane est biocompatible et peut être colonisée par les cellules. La microscopie confocale montre que les cellules sont capables de pénétrer à l'intérieur des macrovides de la membrane. La prolifération cellulaire de CSM dans ce matériau pourrait être utile pour augmenter la longévité d'autres cellules primaires en modifiant les CSM pour produire des facteurs de croissance. Pour tester le comportement dynamique des cellules sur la membrane, un dispositif d'organe sur puce a été développé avec des cellules endothéliales ombilicales humaines ensemencées sur la membrane. Les résistances hydrauliques de la barrière cellulaire sur la membrane ont été quantifiées en temps réel pour une pression trans-endothéliale (PTE), 20 cm H2O à 37 ° C et avec des cellules vivantes après 1 jour et 3 jours après l'ensemencement. Les résultats suggèrent que ce type d'échafaudages polymères peut être utile à l'avenir comme patch in vivo pour réparer des vaisseaux endommagés. / Over the last decades, three-dimensional (3D) scaffolds are unfolding many promising applications in tissue engineering and regenerative medicine field by providing suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. The three-dimensional polymeric porous scaffolds with higher porosities having homogeneous interconnected pore network are highly useful for tissue engineering. In this context, a poly (ε- caprolactone) PCL/chitosan CHT blend membrane with a double porous morphology was developed by modified liquid induced phase inversion technique. The membrane shows: (i) surface macrovoids (big pores) which could be easily accessible for cells invasion and viability; (ii) interconnected microporous (small pores) network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. The physico-chemical properties (pore size, surface chemistry and biodegradability) of the materials have been characterized. This study shows how it is possible to tune the membrane properties by changing the PCL/CHT ratio. Human mesenchymal stem cell (hMSCs) culture was performed on the membranes and the cell viability and proliferation was investigated by MTT assay and oxygen uptake rate experiments. The experiments demonstrate that the membranes are biocompatible and can be colonized by the cells at micron scale. Confocal microscopy images show that the cells are able to adhere and penetrate inside the macrovoids of the membranes. Both cell proliferation and oxygen uptake increase with time especially on membranes with lower chitosan concentration. The presence of chitosan in the blend produces an increase of porosity that affect the entrapment of the cells inside the porous bulk of the membranes. Successful cellular proliferation of hMSCs could be useful to enhance longevity of other primary cells by production of corresponding growth factors. To test the dynamic behavior of cells on the membranes, an organ-on-chip (OOC) device has been developed with human umbilical endothelial cells (HUVECs) seeded on the membrane. The hydraulic resistance of the cellular barrier on the membrane has been quantified for real time trans-endothelial pressure (TEP) 20 cmH2O at 37 degree C and with living cells after 1 day and 3 day of post seeding. Results suggests this kind of polymeric scaffolds can be useful in future as an in vivo patch to repair disrupted vessels.

No description available.

The field of organ-on-chip is a relatively new area of research and builds upon the principle of engineering microfluidic systems to mimic the body’s internal environment as precisely as possible. Eventually these models could hopefully simulate whole organ-systems and enable the examination of the cell’s or organ’s reaction to foreign substances like new pharmaceuticals in a better way than current models. Previously this has been done with in vitro models such as petri dishes that only offer static culturing conditions. These are not very realistic environments compared to the human body where the cells are exposed to both variations in pressure and flows among other things. The purpose of this bachelor’s thesis project has been to evaluate and improve the design of an organ-on-chip system developed by the EMBLA-group at Ångströmslaboratoriet, Uppsala university. This has been done by evaluating the manufacturing process to find areas of improvements of the current chip design, as well as conducting a literature study to understand key components of similar organ-on-chip systems and see if it is possible to implement relevant parts to the organ-on-chip of this project. One of these important parts is a so-called bubble trap. A bubble trap is a construction that enables the capturing and elimination of bubbles in the system since the bubbles can harm the chips components, kill the cells, and compromise measurements.  A first prototype of the bubble trap was developed in Polydimethylsioxane (PDMS) and integrated on the EMBLA-group’s chip design. The principle behind the bubble trap was to use the natural buoyancy of the bubbles to trap them. This was done by introducing an upwards going slope before the inlets to the chip. In this manner the bubbles would float up to the top of the slope and accumulate at the roof as the liquid moved on into the chip without bubbles. To make the bubbles leave the chip a low-pressure chamber was added on top of the bubble trap to help the process of the bubble’s diffusion through the roof and out of the chip. The development of an improved chip design turned out to be a time-consuming endeavor and the time left for evaluation the functionality of the chip became too short. One test was performed which showed that the bubbles did accumulate at the top of the slope as expected, but it rapidly became full and thus started to let bubbles through to the microfluidic chip. The bubbles did not diffuse as efficiently as required and the removal of the bubbles became inefficient. To understand and correct the problem areas of this bubble trap design further tests and experiments will have to be conducted. / Organ-på-chip (Organ-on-chip eller OoC) är ett relativt nytt forskningsområde som bygger på att mikrofluidiksystem utvecklas till att efterlikna människokroppen i så stor utsträckning som möjligt. Detta då det är attraktivt att kunna undersöka cellers/organs beteende vid tillförsel av vissa substanser, till exempel nya läkemedel. I tidigare in vitro modeller har det endast observerats och utförts tester på celler odlade i statiska förhållanden vilket inte är likt den omgivning cellerna har i människokroppen där de tex utsätts för olika vätskeflöden och tryckförändringar.    Syftet med detta examensarbete har varit att utvärdera och förbättra designen på ett OoC system utvecklat av EMBLA-gruppen på Ångströmlaboratoriet vid Uppsala universitet. Detta har gjorts genom att studera den nuvarande tillverkningsprocessen för att hitta relevanta förbättringsområden samt att genom en litteraturstudie undersöka viktiga delar som bör ingå i dessa typer av system. En av dessa delar är en bubbelfälla (bubble trap eller BT) vilket innebär att det i chippet bör finnas ett sätt att eliminera/fånga upp bubblor. Detta eftersom bubblorna kan orsaka stor skada på både chipet, cellerna och mätningarna som skall utföras. En första prototyp av en BT design i Polydimetylsiloxan (PDMS) utvecklades och integrerades på EMBLA-gruppens OoC design. Principen bakom BT-designen var att utnyttja bubblornas flytkraft vilket gjordes genom att introducera en uppåtgående backe innan ingångskanalen. Bubblorna kan därmed flyta upp till toppen av lutningen och vätskan kan fortsätta in i mikrochipset utan bubblor. För att bubblorna ska ta sig ut ur chippet integrerades en tryckkammare ovanpå BT-designen för att få bubblorna att diffundera ut genom taket i den uppåtgående kammaren och ut ur chippet. Utvecklingen av den förbättrade chip-designen visade sig var tidskrävande och tiden för att utvärdera designens funktionalitet blev för kort. Ett test gjordes på den nya chip-designen vilket visade att den utvecklade BT som väntat fångade upp bubblor men att den snabbt blev full i och med att bubblorna inte diffunderade ut genom taket i den takt som behövdes. Vidare undersökningar och experiment behövs för att evaluera vad som orsakade detta och rätta till eventuella felkällor i design och experimentuppställning.