A simple method to generate human airway epithelial organoids with externally orientated apical membranes.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 105 cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.

Hollow Micropillar Array Method for High-Capacity Drug Screening on Filter-Grown Epithelial Cells.

New high-throughput assay formats and innovative screening technologies are needed for miniaturized screens using small quantities of near-native, patient-derived cells. Here, we developed a hollow micropillar array method to screen compounds using epithelial cells cultured on a porous support, with the goal of screening thousands of compounds using a single 24 mm diameter transwell filter containing cultured cells. Test compounds (∼1 nL) in an alginate hydrogel were printed by microinjection in hollow cylindrical micropillars (height = 150 µm, inner diameter = 100 µm) spaced 300 µm apart in a square array configuration. Compounds were delivered by positioning the array near the surface of a cell layer, with 5-10 µm of distance between the micropillars and cell surface. Micropillar array geometry, and the viscosity of the hydrogel and overlying solutions, were optimized computationally and experimentally to produce sustained exposure of cells to test compounds with minimal cross-talk from compounds in neighboring micropillar wells. The method was implemented using a 10 × 10 micropillar array (size = 3 × 3 mm) on CFTR-expressing epithelial cells, in which CFTR chloride channel function was measured from fluorescence in response to iodide addition using a genetically encoded cytoplasmic yellow fluorescent protein halide indicator. The hollow micropillar array platform developed here should be generally applicable for high-capacity drug screening using small numbers of cells cultured on solid or porous supports.

Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma.

Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed 'glymphatic' clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.

Microfluidic platform for rapid measurement of transepithelial water transport.

Water transport across epithelial monolayers is of central importance in mammalian fluid homeostasis, and epithelial aquaporin (AQP) water channels are potential drug targets. Current methods to measure transepithelial water permeability based on indicator dilution have limited accuracy and can require hours for a single measurement. We report here a microfluidics platform for rapid and accurate measurement of water transport across a conventionally cultured epithelial monolayer on a porous filter requiring only a single image obtained using a standard laboratory fluorescence microscope. The undersurface of a porous polyester filter containing cultured epithelial cells on top is contacted with a perfused microfluidic channel of 100 µm width, 20 µm height and 10 cm length with folded geometry, with in-plane size of 3.2 × 3.2 mm2 for visualization with a 2× objective lens. Osmotic water permeability is measured from the steady-state concentration profile along the length of the channel of a membrane-impermeant fluorescent dye in the perfusate, in which an osmotic gradient is imposed by an anisosmolar solution overlying the epithelial monolayer; diffusional water permeability is measured using a D2O/H2O-sensing fluorescent dye in the perfusate with a D2O-containing isosmolar solution overlying the cell layer. Permeability values are deduced from single fluorescence images. The method, named fluid transport on a chip (FT-on-Chip), was applied to measure transepithelial osmotic and diffusional water permeability in control and AQP4-expressing epithelial cell monolayers. FT-on-Chip allows for rapid, accurate and repeated measurements of transepithelial water permeability, and is generalizable to transport measurements of ions and solutes using suitable indicator dyes.

Spatial model of convective solute transport in brain extracellular space does not support a "glymphatic" mechanism.

A "glymphatic system," which involves convective fluid transport from para-arterial to paravenous cerebrospinal fluid through brain extracellular space (ECS), has been proposed to account for solute clearance in brain, and aquaporin-4 water channels in astrocyte endfeet may have a role in this process. Here, we investigate the major predictions of the glymphatic mechanism by modeling diffusive and convective transport in brain ECS and by solving the Navier-Stokes and convection-diffusion equations, using realistic ECS geometry for short-range transport between para-arterial and paravenous spaces. Major model parameters include para-arterial and paravenous pressures, ECS volume fraction, solute diffusion coefficient, and astrocyte foot-process water permeability. The model predicts solute accumulation and clearance from the ECS after a step change in solute concentration in para-arterial fluid. The principal and robust conclusions of the model are as follows: (a) significant convective transport requires a sustained pressure difference of several mmHg between the para-arterial and paravenous fluid and is not affected by pulsatile pressure fluctuations; (b) astrocyte endfoot water permeability does not substantially alter the rate of convective transport in ECS as the resistance to flow across endfeet is far greater than in the gaps surrounding them; and (c) diffusion (without convection) in the ECS is adequate to account for experimental transport studies in brain parenchyma. Therefore, our modeling results do not support a physiologically important role for local parenchymal convective flow in solute transport through brain ECS.

Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1.

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Droplet-based microfluidic platform for measurement of rapid erythrocyte water transport.

Cell membrane water permeability is an important determinant of epithelial fluid secretion, tissue swelling, angiogenesis, tumor spread and other biological processes. Cellular water channels, aquaporins, are important drug targets. Water permeability is generally measured from the kinetics of cell volume change in response to an osmotic gradient. Here, we developed a microfluidic platform in which cells expressing a cytoplasmic, volume-sensing fluorescent dye are rapidly subjected to an osmotic gradient by solution mixing inside a ~0.1 nL droplet surrounded by oil. The solution mixing time was <10 ms. Osmotic water permeability was deduced from a single, time-integrated fluorescence image of an observation area in which the time after mixing was determined through spatial position. Water permeability was accurately measured in aquaporin-expressing erythrocytes with half-times for osmotic equilibration down to <50 ms. Compared with conventional water permeability measurements using costly stopped-flow instrumentation, the microfluidic platform here utilizes sub-microliter blood sample volume, does not suffer from mixing artifacts, and replaces challenging kinetic measurements by single image capture using a standard laboratory fluorescence microscope.

Aquaporin-4 regulates the velocity and frequency of cortical spreading depression in mice.

The astrocyte water channel aquaporin-4 (AQP4) regulates extracellular space (ECS) K(+) concentration ([K(+)]e) and volume dynamics following neuronal activation. Here, we investigated how AQP4-mediated changes in [K(+)]e and ECS volume affect the velocity, frequency, and amplitude of cortical spreading depression (CSD) depolarizations produced by surface KCl application in wild-type (AQP4(+/+)) and AQP4-deficient (AQP4(-/-)) mice. In contrast to initial expectations, both the velocity and the frequency of CSD were significantly reduced in AQP4(-/-) mice when compared with AQP4(+/+) mice, by 22% and 32%, respectively. Measurement of [K(+)]e with K(+)-selective microelectrodes demonstrated an increase to ∼35 mM during spreading depolarizations in both AQP4(+/+) and AQP4(-/-) mice, but the rates of [K(+)]e increase (3.5 vs. 1.5 mM/s) and reuptake (t1/2 33 vs. 61 s) were significantly reduced in AQP4(-/-) mice. ECS volume fraction measured by tetramethylammonium iontophoresis was greatly reduced during depolarizations from 0.18 to 0.053 in AQP4(+/+) mice, and 0.23 to 0.063 in AQP4(-/-) mice. Analysis of the experimental data using a mathematical model of CSD propagation suggested that the reduced velocity of CSD depolarizations in AQP4(-/-) mice was primarily a consequence of the slowed increase in [K(+)]e during neuronal depolarization. These results demonstrate that AQP4 effects on [K(+)]e and ECS volume dynamics accelerate CSD propagation.

Microfluidics platform for measurement of volume changes in immobilized intestinal enteroids.

Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 µm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a "pinball machine-like" array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling.

Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes.

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1-AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23-AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

Aquaporin-1 gene deletion reduces breast tumor growth and lung metastasis in tumor-producing MMTV-PyVT mice.

Aquaporin 1 (AQP1) is a plasma membrane water-transporting protein expressed strongly in tumor microvascular endothelia. We previously reported impaired angiogenesis in implanted tumors in AQP1-deficient mice and reduced migration of AQP1-deficient endothelial cells in vitro. Here, we investigated the consequences of AQP1 deficiency in mice that spontaneously develop well-differentiated, luminal-type breast adenomas with lung metastases [mouse mammary tumor virus-driven polyoma virus middle T oncogene (MMTV-PyVT)]. AQP1(+/+) MMTV-PyVT mice developed large breast tumors with total tumor mass 3.5 ± 0.5 g and volume 265 ± 36 mm(3) (SE, 11 mice) at age 98 d. Tumor mass (1.6±0.2 g) and volume (131±15 mm(3), 12 mice) were greatly reduced in AQP1(-/-) MMTV-PyVT mice (P<0.005). CD31 immunofluorescence showed abnormal microvascular anatomy in tumors of AQP1(-/-) MMTV-PyVT mice, with reduced vessel density. HIF-1α expression was increased in tumors in AQP1(-/-) MMTV-PyVT mice. The number of lung metastases (5±1/mouse) was much lower than in AQP1(+/+) MMTV-PyVT mice (31±8/mouse, P<0.005). These results implicate AQP1 as an important determinant of tumor angiogenesis and, hence, as a potential drug target for adjuvant therapy of solid tumors.

Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice.

BACKGROUND: Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. OBJECTIVE: To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. DESIGN: Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. RESULTS: Screening of ∼150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a ∼1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. CONCLUSIONS: Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule.

Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

Convective washout reduces the antidiarrheal efficacy of enterocyte surface-targeted antisecretory drugs.

Secretory diarrheas such as cholera are a major cause of morbidity and mortality in developing countries. We previously introduced the concept of antisecretory therapy for diarrhea using chloride channel inhibitors targeting the cystic fibrosis transmembrane conductance regulator channel pore on the extracellular surface of enterocytes. However, a concern with this strategy is that rapid fluid secretion could cause convective drug washout that would limit the efficacy of extracellularly targeted inhibitors. Here, we developed a convection-diffusion model of washout in an anatomically accurate three-dimensional model of human intestine comprising cylindrical crypts and villi secreting fluid into a central lumen. Input parameters included initial lumen flow and inhibitor concentration, inhibitor dissociation constant (K(d)), crypt/villus secretion, and inhibitor diffusion. We modeled both membrane-impermeant and permeable inhibitors. The model predicted greatly reduced inhibitor efficacy for high crypt fluid secretion as occurs in cholera. We conclude that the antisecretory efficacy of an orally administered membrane-impermeant, surface-targeted inhibitor requires both (a) high inhibitor affinity (low nanomolar K(d)) to obtain sufficiently high luminal inhibitor concentration (>100-fold K(d)), and (b) sustained high luminal inhibitor concentration or slow inhibitor dissociation compared with oral administration frequency. Efficacy of a surface-targeted permeable inhibitor delivered from the blood requires high inhibitor permeability and blood concentration (relative to K(d)).

Aquaporin-4-dependent K(+) and water transport modeled in brain extracellular space following neuroexcitation.

Potassium (K(+)) ions released into brain extracellular space (ECS) during neuroexcitation are efficiently taken up by astrocytes. Deletion of astrocyte water channel aquaporin-4 (AQP4) in mice alters neuroexcitation by reducing ECS [K(+)] accumulation and slowing K(+) reuptake. These effects could involve AQP4-dependent: (a) K(+) permeability, (b) resting ECS volume, (c) ECS contraction during K(+) reuptake, and (d) diffusion-limited water/K(+) transport coupling. To investigate the role of these mechanisms, we compared experimental data to predictions of a model of K(+) and water uptake into astrocytes after neuronal release of K(+) into the ECS. The model computed the kinetics of ECS [K(+)] and volume, with input parameters including initial ECS volume, astrocyte K(+) conductance and water permeability, and diffusion in astrocyte cytoplasm. Numerical methods were developed to compute transport and diffusion for a nonstationary astrocyte-ECS interface. The modeling showed that mechanisms b-d, together, can predict experimentally observed impairment in K(+) reuptake from the ECS in AQP4 deficiency, as well as altered K(+) accumulation in the ECS after neuroexcitation, provided that astrocyte water permeability is sufficiently reduced in AQP4 deficiency and that solute diffusion in astrocyte cytoplasm is sufficiently low. The modeling thus provides a potential explanation for AQP4-dependent K(+)/water coupling in the ECS without requiring AQP4-dependent astrocyte K(+) permeability. Our model links the physical and ion/water transport properties of brain cells with the dynamics of neuroexcitation, and supports the conclusion that reduced AQP4-dependent water transport is responsible for defective neuroexcitation in AQP4 deficiency.

Model of aquaporin-4 supramolecular assembly in orthogonal arrays based on heterotetrameric association of M1-M23 isoforms.

Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.

Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching.

Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC-dextran dissolved in the ASL of unperturbed, well-differentiated primary cultures of human bronchial epithelia grown at an air-liquid interface. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection. Bleaching of an in-plane 6-µm-wide region was done in which diffusion coefficients were computed using solution standards of specified viscosity and finite-element computations of 2-layer dye diffusion in 3 dimensions. We found remarkably elevated viscosity in both ML and PCL of CF vs. non-CF bronchial epithelial cell cultures. Relative viscosities (with saline=1) were in the range 7-10 in the non-CF ML and PCL, and 25-30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland-free lower airways.