Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.

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Improved in vivo imaging method for individual islets across the mouse pancreas reveals a heterogeneous insulin secretion response to glucose

Scientific Reports ◽

10.1038/s41598-020-79727-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Henriette Frikke-Schmidt ◽

Peter Arvan ◽

Randy J. Seeley ◽

Corentin Cras-Méneur

Keyword(s):

Insulin Secretion ◽

In Vivo Imaging ◽

Motion Blur ◽

Isolated Islets ◽

Imaging Method ◽

Glucose Stimulation ◽

Powerful Approach ◽

Glucose Challenge ◽

The Impact

AbstractWhile numerous techniques can be used to measure and analyze insulin secretion in isolated islets in culture, assessments of insulin secretion in vivo are typically indirect and only semiquantitative. The CpepSfGFP reporter mouse line allows the in vivo imaging of insulin secretion from individual islets after a glucose stimulation, in live, anesthetized mice. Imaging the whole pancreas at high resolution in live mice to track the response of each individual islet over time includes numerous technical challenges and previous reports were only limited in scope and non-quantitative. Elaborating on this previous model—through the development of an improved methodology addressing anesthesia, temperature control and motion blur—we were able to track and quantify longitudinally insulin content throughout a glucose challenge in up to two hundred individual islets simultaneously. Through this approach we demonstrate quantitatively for the first time that while isolated islets respond homogeneously to glucose in culture, their profiles differ significantly in vivo. Independent of size or location, some islets respond sharply to a glucose stimulation while others barely secrete at all. This platform therefore provides a powerful approach to study the impact of disease, diet, surgery or pharmacological treatments on insulin secretion in the intact pancreas in vivo.

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In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04324-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2113-2121 ◽

Cited By ~ 9

Author(s):

U. Malik ◽

O. N. Silva ◽

I. C. M. Fensterseifer ◽

L. Y. Chan ◽

R. J. Clark ◽

...

Keyword(s):

Antimicrobial Agents ◽

Cyclic Peptide ◽

Sequence Similarity ◽

Infection Model ◽

Content Type ◽

Wide Range ◽

Inhibitory Activities ◽

The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

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Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle

Biochemical Journal ◽

10.1042/bj20110366 ◽

2011 ◽

Vol 437 (2) ◽

pp. 215-222 ◽

Cited By ~ 93

Author(s):

Christopher G. R. Perry ◽

Daniel A. Kane ◽

Chien-Te Lin ◽

Rachel Kozy ◽

Brook L. Cathey ◽

...

Keyword(s):

Skeletal Muscle ◽

Dynamic Range ◽

Energy Charge ◽

Respiratory Control ◽

Basic Research ◽

Dependent Manner ◽

Atpase Inhibitor ◽

Wide Range ◽

The Impact

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20–300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2–10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

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Differential responsiveness of glabrous and non-glabrous skin to local transmural pressure elevations; the impact of 5 weeks of iterative local pressure loading

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00151.2021 ◽

2021 ◽

Author(s):

Michail E. Keramidas ◽

Roger Kölegård ◽

Patrik Sundblad ◽

Håkan Sköldefors ◽

Ola Eiken

Keyword(s):

Transmural Pressure ◽

Glabrous Skin ◽

Local Pressure ◽

Wide Range ◽

Forearm Skin ◽

Differential Responsiveness ◽

Pressure Resistance ◽

The Impact ◽

Pressure Perturbations

We examined the in vivo pressure-flow relationship in human cutaneous vessels during acute and repeated elevations of local transmural pressure. In 10 healthy men, red blood cell flux was monitored simultaneously on the non-glabrous skin of the forearm and the glabrous skin of a finger during a vascular pressure provocation, wherein the blood vessels of an arm were exposed to a wide range of stepwise increasing distending pressures. Forearm skin blood flux was relatively stable at slight and moderate elevations of distending pressure, whereas it increased ~3-4-fold at the highest levels (P = 0.004). Finger blood flux on the contrary, dropped promptly and consistently throughout the provocation (P < 0.001). Eight of the subjects repeated the provocation trial after a 5-week pressure-training regimen, during which the vasculature in one arm was exposed intermittently (40 min, 3 times･week-1) to increased transmural pressure (from +65 mmHg week-1 to +105 mmHg week-5). The training regimen diminished the pressure-induced increase in forearm blood flux by ~34% (P = 0.02), whereas it inhibited the reduction in finger blood flux (P < 0.001) in response to slight and moderate distending pressure elevations. The present findings demonstrate that, during local pressure perturbations, the cutaneous autoregulatory function is accentuated in glabrous compared to in the non-glabrous skin regions. Prolonged intermittent regional exposures to augmented intravascular pressure blunt the responsiveness of the glabrous skin, but enhance arteriolar pressure resistance in the non-glabrous skin.

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Tissue oxygen tension in rabbits measured with a galvanic electrode

Journal of Applied Physiology ◽

10.1152/jappl.1976.41.2.245 ◽

1976 ◽

Vol 41 (2) ◽

pp. 245-250 ◽

Cited By ~ 10

Author(s):

M. E. Towell ◽

I. Lysak ◽

E. C. Layne ◽

S. P. Bessman

Keyword(s):

Oxygen Tension ◽

Peritoneal Cavity ◽

Tissue Oxygen Tension ◽

Tissue Oxygen ◽

Reliable Measure ◽

Air Breathing ◽

Wide Range ◽

Tissue Po2 ◽

Range Of Values

A galvanic electrode which generates current in response to oxygen was used to measure tissue PO2 in 10 rabbits. Sixteen electrodes were calibratedin vitro at PO2 = 0 mmHg and PO2 = 100 mmHg before implantation into muscleand peritoneal cavity. Electrode calibration was checked after removal 7–20days later; mean +/- SE reading at PO2 = 0 was 1.5 +/- 1.83 mmHg and at PO2 = 100 was 98.1 +/- 6.49 mmHg. Continuous recordings of tissue PO2 showed little fluctuation in unanesthetized rabbits resting quietly. Tissue PO2 was lower than arterial or venous PO2 during air breathing but frequently exceeded venous PO2 during O2 breathing. Highly significant correlations(P less than 0.001) were found between blood and tissue PO2 over a wide range of values obtained during air breathing and during 10%, 50%, and 100% O2breathing. Thus, the galvanic electrode provided a reliable measure of tissue PO2 and maintained its stability in vivo for periods as long as 20 days.

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Sensing through Non-Sensing Ocular Ion Channels

International Journal of Molecular Sciences ◽

10.3390/ijms21186925 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6925

Author(s):

Meha Kabra ◽

Bikash Ranjan Pattnaik

Keyword(s):

Ion Channels ◽

Visual Processing ◽

Signal Transmission ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cone Dystrophy ◽

Wide Range ◽

The Impact

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a “sensing” ion channel to “non-sensing,” leading to ocular channelopathies like Leber’s congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a “non-sensing” channel to “sensing” would be life-changing.

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Influence of oxygen tension on the viscoelastic behavior of red blood cells in sickle cell disease

Blood ◽

10.1182/blood.v67.1.110.110 ◽

1986 ◽

Vol 67 (1) ◽

pp. 110-118 ◽

Cited By ~ 37

Author(s):

GB Nash ◽

CS Johnson ◽

HJ Meiselman

Keyword(s):

Oxygen Tension ◽

Sickle Cell ◽

Viscoelastic Behavior ◽

Membrane Properties ◽

Membrane Elasticity ◽

Membrane Deformation ◽

Deformation Response ◽

Equivalent Time ◽

Wide Range

Abstract Although the rheological behavior of sickle cell suspensions and of hemoglobin S solutions is known to be strongly dependent on oxygen tension (PO2), little data exist concerning the influence of PO2 on the viscoelasticity of individual HbSS RBC. We have used micropipette aspiration techniques to test the deformation response of both HbSS and control HbAA RBC over a wide range of PO2 at 23 degrees C. Sickled, spiculed HbSS cells were present for PO2 approximately less than 35 mm Hg; for a number of these cells, the deformation response was essentially elastic and an effective membrane rigidity (EMR) was calculated. EMR increased with decreasing PO2 and was approximately 5 to 50 times higher than the equivalent rigidity of oxygenated HbSS RBC. In addition, the rate of membrane deformation was very slow for sickled cells; the half-time for the deformation process increased as PO2 was lowered and was about two orders of magnitude longer than the equivalent time for normal RBC. Other sickled cells exhibited plastic deformation when subjected to comparable deforming forces and experienced irreversible membrane deformation and budding. At all PO2 levels tested, some HbSS RBC remained as discocytes; these cells had normal membrane elasticity and membrane viscosity. Furthermore, changes in PO2 did not affect the membrane properties of HbAA RBC. Thus, gross abnormalities in the deformation response of HbSS RBC were only detected after morphological sickling had occurred. These abnormalities most likely arose from changes in the cytoplasmic HbS viscoelasticity and, if present in vivo, would be expected to impair the flow of HbSS cells in the microcirculation.

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Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure

Gut ◽

10.1136/gutjnl-2019-319960 ◽

2020 ◽

pp. gutjnl-2019-319960 ◽

Cited By ~ 2

Author(s):

Huayu Yang ◽

Lejia Sun ◽

Yuan Pang ◽

Dandan Hu ◽

Haifeng Xu ◽

...

Keyword(s):

Drug Metabolism ◽

Liver Failure ◽

Human Liver ◽

Three Dimensional ◽

3D Bioprinting ◽

Heparg Cells ◽

Liver Functions ◽

Human Specific

ObjectiveShortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo.Design3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production.Results3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of Fah-/-Rag2-/- mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice.ConclusionsOur results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.

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Influence of Supraphysiologic Biomaterial Stiffness on Ventricular Mechanics and Myocardial Infarct Reinforcement

10.1101/2020.05.20.107276 ◽

2020 ◽

Author(s):

Ravi K. Ghanta ◽

Yunge Zhao ◽

Aarthi Pugazenthi ◽

Mathew J. Wall ◽

Lauren N. Russell ◽

...

Keyword(s):

Polyethylene Glycol ◽

Ventricular Remodeling ◽

Myocardial Infarct ◽

Design Criteria ◽

Peg Hydrogels ◽

Ventricular Mechanics ◽

Wide Range ◽

Adverse Remodeling ◽

The Impact

ABSTRACTInjectable intramyocardial biomaterials have promise to limit adverse ventricular remodeling through mechanical and biologic mechanisms. While some success has been observed by injecting materials to regenerate new tissue, optimal biomaterial stiffness to thicken and stiffen infarcted myocardium to limit adverse remodeling has not been determined. In this work, we present an in-vivo study of the impact of biomaterial stiffness over a wide range of stiffness moduli on ventricular mechanics. We utilized injectable methacrylated polyethylene glycol (PEG) hydrogels fabricated at 3 different mechanical moduli: 5 kPa (low), 25 kPa (medium/myocardium), and 250 kPa (high/supraphysiologic). We demonstrate that the supraphysiological high stiffness favorably alters post-infarct ventricular mechanics and prevents negative tissue remodeling. Lower stiffness materials do not alter mechanics and thus to be effective, must instead target biological reparative mechanisms. These results may influence rationale design criteria for biomaterials developed for infarct reinforcement therapy.

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Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models

10.26434/chemrxiv.9759605.v1 ◽

2019 ◽

Author(s):

Sam Hinman ◽

Yuli Wang ◽

Nancy Allbritton

Keyword(s):

Human Colon ◽

Cell Types ◽

Self Renewal ◽

Chemical Gradients ◽

Plate Spacing ◽

Wide Range ◽

Cell Compartmentalization ◽

The Impact

Biochemical gradients across the intestinal epithelium play a major role in governing intestinal stem cell compartmentalization, differentiation dynamics, and organ-level self-renewal. Advances in primary cell-derived in vitro models, in which a full suite of stem and differentiated cell types are present, have vastly accelerated our understanding of intestinal homeostasis and disease. However, scalable platforms that recapitulate the architecture and gradients present in vivo are absent. We present a platform in which individually addressable arrays of chemical gradients along the crypt long axis can be generated, enabling scalable culture of in vitro colonic epithelial replicas. The platform utilizes standardized well plate spacing, maintains access to basal and luminal compartments, and relies on a photopatterned porous membrane to act as diffusion windows while supporting the in vitro crypts. Simultaneous fabrication of 3,875 crypts over a single membrane was developed. Growth factor gradients were modelled and then experimentally optimized to promote long-term health and self-renewal of the crypts which were assayed in situ by confocal fluorescence microscopy. The cultured in vitro crypt arrays successfully recapitulated the architecture, stem/proliferative and differentiated cell compartmentalization, and luminal-to-basal polarity observed in vivo. Furthermore, known signaling regulators produced measurable and predictable effects on the proliferative and differentiated cell compartments. This platform is readily adaptable to the screening of tissue from individual patients to assay the impact of food and bacterial metabolites and/or drugs on colonic crypt dynamics. Importantly, the cassette is compatible with a wide range of sensing/detection modalities, and the developed fabrication methods should find applications for other cell and tissue types.

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