Islet-on-a-chip for the study of pancreatic β-cell function

Diabetes mellitus is a significant public health problem worldwide. It encompasses a group of chronic disorders characterized by hyperglycemia, resulting from pancreatic islet dysfunction or as a consequence of insulin-producing β-cell death. Organ-on-a-chip platforms have emerged as technological systems combining cell biology, engineering, and biomaterial technological advances with microfluidics to recapitulate a specific organ’s physiological or pathophysiological environment. These devices offer a novel model for the screening of pharmaceutical agents and to study a particular disease. In the field of diabetes, a variety of microfluidic devices have been introduced to recreate native islet microenvironments and to understand pancreatic β-cell kinetics in vitro. This kind of platforms has been shown fundamental for the study of the islet function and to assess the quality of these islets for subsequent in vivo transplantation. However, islet physiological systems are still limited compared to other organs and tissues, evidencing the difficulty to study this “organ” and the need for further technological advances. In this review, we summarize the current state of islet-on-a-chip platforms that have been developed so far. We recapitulate the most relevant studies involving pancreatic islets and microfluidics, focusing on the molecular and cellular-scale activities that underlie pancreatic β-cell function.This review received financial support from the European Research Council program under grants ERC-StG-DAMOC (714317), the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Program for Centres of Excellence in R&D (SEV-2016-2019) and “Retos de investigación: Proyectos I+D+i” (TEC2017-83716-C2-2-R), the CERCA Programme/Generalitat de Catalunya (2017-SGR-1079), and Fundación Bancaria “la Caixa”- Obra Social “la Caixa” (project IBEC-La Caixa Healthy Ageing)

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

The second generation of the CCCM system for in-vitro cardiac tissue engineering.

Cardiovascular disease is the leading cause of death worldwide. When a myocardial infarction occurs, scar tissue compensates the damaged myocardial tissue. This scar tissue increases the stiffness of the heart tissue, reduces the heart’s function, and finally leads to the heart failure (HF) disease. To have the tissue engraftment, in-vitro cardiac tissue should have the same properties as the native mature cardiac tissue. However, current in-vitro cell culture technologies fail to accurately recreate the in-vivo like mechanically physiological environment for in-vitro cardiac tissue culture, and therefore, fail to regenerate the in-vivo like mature cardiac tissue. Hence, a microfluidic cardiac cell culture model (CCCM) system was developed to better recreate the cellular environment and advance cardiac regeneration. CCCM system replicates the hemodynamic loading and unloading conditions occurring inside the left ventricle of a heart. With this system, different pressures of human heart conditions may be replicated for a variety of clinical and physiologic conditions. For proof-of-concept, embryonic chick cardiac cells with normal heart condition were applied. Compared to the tissue cultured in a static condition, tissues stimulated in the CCCM system achieved an in-vivo like cardiac matured phenotype, had higher proliferating rate, showed more maturity, and expressed more contractile proteins. These results demonstrated that the CCCM system can be used to study the behavior of cardiomyocytes in different mechanical heart conditions and to create mature cardiac tissue which will benefit cardiac tissue transplant for HF

Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans.

Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta-cells found in the islets of Langerhans of the pancreas. Severe cases can be treated in a minimally invasive way by islet transplantation; however, islet transplantation has been limited by an inability to measure islet viability and potency prior to transplant. To address this need, we have developed a microfluidic platform to measure both intracellular calcium flux and insulin secretion, two important indicators of beta-cell function, at high temporal resolution during glucose treatment. Combining these measures on islets required methods for measuring fluorescence at two separate locations on a microfluidic system. To accomplish this objective, we used a 2-chip system in which perfusate was collected in fractions while intracellular calcium was measured using fluorescence imaging. The perfusate was subsequently analyzed for insulin by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) using the same fluorescence microscope. We were able to distinguish first and second phase insulin secretion from batches of 8-10 islets with 80 s temporal resolution. Measured basal and peak first phase insulin secretion correlated well with previously reported results. Total analysis time using this system was <90 min. For an alternative approach to islet evaluation, we developed a metabolomic method to identify potential biomarkers of islet health for transplant. Using a miniaturized sample preparation method and HPLC-TOF-MS, we were able to identify 62 metabolites reliably in whole islet samples. To mimic damage that can occur during islet transplant, we induced oxidative stress in islets using hydrogen peroxide and measured their immediate metabolomic response as well as their response 1-4 h following stress removal. Increased concentrations of pentose phosphates, glucose-6-phosphate, and fructose bisphosphate in the immediate response corresponded to glycolysis blockage and possibly increased flux through the pentose phosphate pathway. Post-stress responses included increased levels of free fatty acids, phospholipids, long chain CoAs, and HMG-CoA as well blunted malonyl CoA concentrations, potentially relating to alterations in the glycerolipid/free fatty acid cycle and mevalonate pathway. These metabolites could comprise a metabolic signature of stressed cells for islet evaluation prior to transplantation.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113374/1/ccipolla_1.pd

Neuronal filopodia borne along tips and shafts of dendrites comprise two distinct populations as evidenced by differences in structure and dynamics

Ever since their discovery in 1880 by Ramon y Cajal, dendritic spines have evoked considerable interest in the field of cellular and molecular neuroscience. Subsequent studies into their morphogenesis, and into synaptogenesis, brought into the spotlight their putative precursors – the dendritic filopodia. This set off several lines of investigation into filopodial structure and function, notable among which is the work by Portera-Cailliau et al. who showed in 2003 that growth cone filopodia differ from shaft filopodia in terms of densities and lengths, and in their response to blocking of synaptic transmission, and of ionotropic glutamate receptors. However, they observed these differences only up to postnatal day 5. In 2010, Korobova and Svitkina reported the existence of a different actin organization in shaft filopodia at 10 days in vitro (DIV). This work fills the gap between those two studies, investigating differences between tip and shaft filopodia at 4, 7, 10 and 14 DIV, and examining structure and dynamics, as well as responses to developmental cues, specifically, Semaphorin3A (Sema3A). Using confocal microscopy to visualize filopodial membrane and actin we found that shaft filopodia are shorter than tip filopodia, and show a less dense presentation along the dendrite. We then employed the quantitative phase imaging technology of Spatial Light Interference Microscopy (SLIM) for analysis of mass change dynamics of individual filopodia. We found that tip and shaft filopodia show similar dynamics early on, but further on in development by 7 DIV shaft filopodia slow down considerably while tip filopodia continue to show fast increases and decreases in mass. Further analysis of growth rates showed that both types filopodia exhibit exponential growth during their extension, implying that the bigger the filopodium the faster it grows. Next we sought to examine the functional ramifications of these differences in tip and shaft filopodia. We investigated the differential responses of the two populations to Sema3A. We found that a 24 h exposure to Sema3A at 0-1 DIV leads to accelerated maturation of shaft filopodia as evidenced by (1) an increase in dendritic branching, (2) an acceleration of maturation into spines, and (3) into synapses. An analysis of the underlying dynamics showed that Sema3A treatment results in (1) tip filopodial movement becoming more deterministic, (2) an increase in average growth and shrinkage rates in shaft filopodia, and, (3) an increase in speed of the fastest growth and shrinkage in tip and shaft filopodia at 4 and 7 DIV. Together these findings show that Sema3A is a unique cue that acts on both tip filopodia and shaft filopodia, but with different outcomes – the former to increase dendrite lengths, and the latter to increase branching, spinogenesis and synaptogenesis. Bath application of Sema3A also elicits an axonal response, which might itself affect the cells as a whole, and could confound the filopodial read out. To avoid this, we supplemented bath application studies with investigations using microfluidic devices that enable focal, dendrite specific application of Sema3A, and, also, better replicate the in vivo layered structure of the hippocampus. Our results held true even with this sub-cellular administration of Sema3A. Taken together our findings provide further evidence for differences in the two dendritic filopodial populations – those borne on the tips, and those along the shafts, and help deconstruct the role of Sema3A in dendritic development. A greater comprehension of this diversity in the filopodial population, and its role in shaping the development of neuronal networks will not only further our understanding of the nervous system, but will also help unravel the mechanistic bases of developmental disorders and diseases

The evolution of the axonal transport toolkit

Neurons are highly polarized cells that critically depend on long‐range, bidirectional transport between the cell body and synapse for their function. This continual and highly coordinated trafficking process, which takes place via the axon, has fascinated researchers since the early 20th century. Ramon y Cajal first proposed the existence of axonal trafficking of biological material after observing that dissociation of the axon from the cell body led to neuronal degeneration. Since these first indirect observations, the field has come a long way in its understanding of this fundamental process. However, these advances in our knowledge have been aided by breakthroughs in other scientific disciplines, as well as the parallel development of novel tools, techniques and model systems. In this review, we summarize the evolution of tools used to study axonal transport and discuss how their deployment has refined our understanding of this process. We also highlight innovative tools currently being developed and how their addition to the available axonal transport toolkit might help to address key outstanding questions

Biophysical properties of blood-stage Plasmodium falciparum malaria: from single-cell host-pathogen interactions to human protective polymorphisms

Malaria is a mosquito-borne infectious disease responsible for half a million deaths every year and long-term economic stagnation in many countries where it is endemic. All symptoms and pathology of malaria are caused by Plasmodium falciparum parasite, and are initiated when parasites invade human red blood cells, then mature and multiply inside them in approximately 48 hours. The invasion process is completed in less than a minute and is one of the most crucial, yet least understood, phases of malaria infection. It also represents a brief window in which the parasites are extracellular and hence exposed to the host immune system, therefore representing a potential target for vaccines and treatments. The work described in this thesis firstly includes the optimisation of a real-time live microscopy platform for recording parasite egress-invasion sequences under controlled conditions, and to investigate their morphology and kinetics. This set-up was employed to address the role of calcium in mediating successful invasion by observing the invasion process simultaneously in bright-field and fluorescence. Elevated calcium signal was found to be absent during the early steps of the process, implying that calcium does not trigger invasion, and an alternative invasion mechanism was suggested. To investigate whether parasite and host cell physical parameters were actively involved in invasion, adhesion forces between parasites and red cells were measured with optical tweezers, while the biophysical properties of the red blood cells such as bending modulus, tension, radius, and viscosity were assessed by analysing their plasma membrane fluctuations. In particular, cells from the Dantu blood group, a rare blood variant found mainly in East Africa that provides up to 70% protection against malaria, and from Beta-thalassaemia individuals, were studied. A general correlation between red blood cell membrane tension, invasion efficiency and dynamics was established, determining a protective tension threshold above which cells are less likely to be invaded. Finally, mature parasites have the ability to bind to the endothelium of peripheral blood vessels, causing impair flow that can lead to a range of fatal conditions. To study malaria cytoadherence to endothelial cells, a microfluidic device was designed to produce an in vitro physiologically relevant model of human circulation. Increasing cytoadhesion was experimentally associated with endothelial glycocalyx disruption as initial factor for malaria pathogenesis. Live imaging methods and techniques adopted in this study highlight mechanisms crucial for malaria infection, and represent an innovative and complementary study of this disease with respect to purely biological approaches. These findings show how changes in red blood cell biophysics can be linked to human evolutionary response against malaria with tangible effects on the population

Methods for the Investigation of Microvascular Control of Oxygen Distribution

The purpose of this thesis was to develop tools for studying oxygen-dependent regulation of red blood cell (RBC) flow distribution in the microcirculation. At the microvascular level, arterioles dictate the distribution of oxygen (O2) carrying RBCs to downstream capillaries, a process which needs to be tightly regulated and coupled to O2 off loading from capillaries to the tissue. To investigate potential regulatory mechanisms, an O2 exchange platform was developed to manipulate the RBC hemoglobin O2 saturation (SO2) at the muscle surface while limiting the changes in SO2 to only a single capillary network. Decreasing SO2 in a single capillary network resulted in an increase in supply rate, while increasing SO2 caused a decrease in supply rate. This finding is consistent with our hypothesis that ATP released in capillaries in response to low SO2 is responsible for vasodilation of upstream arterioles to regulate blood flow. To determine whether the dynamics of ATP was fast enough to enable RBC signalling in capillaries, an in vitro microfluidic system was developed to generate a rapid decrease in RBC SO2. The feasibility of this experimental design was first tested computationally using a mathematical model that consisted of blood flow, oxygen and ATP transport as well as a model for hemoglobin binding, ATP release, ATP/luciferin/luciferase reaction and digital camera light detection. The model demonstrated that the concept was theoretically feasible and yielded important insights such as the signal sensitivity to flow rate. The model further revealed that measured light intensity levels would not be directly related to ATP concentrations, thus, care must be taken when interpreting the data. It was determined that the microfluidic device would be fabricated using soft lithography techniques that resulted in a device that differed significantly from our original theoretical design since all of the layers would be oxygen permeable except for a glass coverslip with a small opening for gas exchange between the liquid and gas channel. To optimize the geometric design of this microfluidic device, to maximize the desaturation the RBCs, a finite element model was developed. Based on this design a device was constructed. To test whether the design generated a rapid decrease in RBC SO2, a low hematocrit high SO2 RBC suspension was perfused through the device exposed to 95% N2 and 5% CO2 in the gas channel. Finally, to overcome challenges with existing approaches for measuring SO2 in the device, a novel image analysis technique using digital inpainting was developed. The inpainting approach demonstrated a rapid change in RBC SO2 at the entrance to the window, thus the microfluidic device is ready to be used to measure the dynamics of O2-dependent ATP release from RBCs. The new inpainting algorithm was also applied to in vivo video sequences where it was shown to provide more accurate SO2 measurements and to work under conditions where existing approaches fail. In summary, this thesis provides a set of in vivo, in vitro and computational tools that can be used to study the mechanisms of SO2-dependent regulation of the microvascular blood flow

A Microfluidic Approach for Investigating the Role of Intrathrombus Transport in Thrombosis and Hemostasis

Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Wall shear stress (τw) and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis. This thesis describes the design of a microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) while independently controlling ΔP and τw. Using this device we were able to investigate the impact of physiologic hemodynamics on the growth and architecture of human blood clots. Our results revealed that both wall shear rate and the transthrombus pressure gradient govern clot development leading to the formation of two distinct intrathrombus regions; a core of highly-activated platelets and fibrin covered by a shell of less-activated platelets. These regions mimic the activation gradients of clots formed in vivo. We demonstrated that core development was dependent on the transthrombus pressure gradient restricting thrombin localization while shell development was dependent on wall shear rates. We also found that fibrin polymerization inhibited thrombin activity at both arterial and venous shear rates. However, the mechanism of this inhibition is shear dependent. At venous shears thrombin activity is inhibited by γ\u27-fibrin(ogen) binding. While at arterial and pathological shear rates the clot forms a more dense structure leading to physical trapping of thrombin independent of γ\u27-fibrin(ogen) binding. Taken together our data supports a model where clot architecture is maintained under various conditions by shear-specific thrombin inhibition mechanisms. Lastly, we demonstrated that the prevailing hemodynamics dilute ADP and thromboxane to regulate platelet contractility, a newly defined flow sensing mechanism to regulate clot function. The field of in vitro hemostasis and thrombosis research has lacked an assay capable of independently studying the effects of ΔP and local τw on clot development and function. Our microfluidic device bridges this gap, while providing new insights into the mechanisms of hemostasis and thrombosis, where hemostatic clot development must balance both thrombotic and hemorrhagic risks in order to rapidly and controllably cease bleeding