RESUMO
A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, ß-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and ß-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2-4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.
Assuntos
Aquaporina 2/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Capacidade de Concentração Renal/efeitos dos fármacos , Rim/efeitos dos fármacos , Tirfostinas/farmacologia , Agentes Urológicos/farmacologia , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Cães , Exocitose/efeitos dos fármacos , Técnicas In Vitro , Rim/metabolismo , Células LLC-PK1 , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Concentração Osmolar , Transporte Proteico , Ratos Brattleboro , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Suínos , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
A new and simple method for creating superhydrophilic micropatterns on a superhydrophobic surface is demonstrated. The method is based on printing an "ink", an ethanol solution of a phospholipid, onto a porous superhydrophobic surface and, thus, is compatible with a variety of commonly available printing techniques.
Assuntos
Etanol/química , Fosfolipídeos/química , Impressão/métodos , Ensaios de Triagem em Larga Escala , Interações Hidrofóbicas e Hidrofílicas , Tinta , Metacrilatos/química , Microscopia Eletrônica de Varredura , Polímeros/química , Porosidade , Propriedades de SuperfícieRESUMO
High-throughput screening of live cells and chemical reactions in isolated droplets is an important and growing method in areas ranging from studies of gene functions and the search for new drug candidates, to performing combinatorial chemical reactions. Compared with microfluidics and well plates, the facile fabrication, high density, and open structure endow droplet microarrays on planar surfaces with great potential in the development of next-generation miniaturized platforms for high-throughput applications. Surfaces with special wettability have served as substrates to generate and/or address droplets microarrays. Here, the formation of droplet microarrays with designed geometry on chemically prepatterned surfaces is briefly described and some of the newer and emerging applications of these microarrays that are currently being explored are highlighted. Next, some of the available technologies used to add (bio-)chemical libraries to each droplet in parallel are introduced. Current challenges and future prospects that would benefit from using such droplet microarrays are also discussed.
RESUMO
High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings.
Assuntos
Análise Serial de Tecidos/métodos , Transfecção/métodos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibronectinas/química , Gelatina/química , Células HEK293 , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Polímeros/química , Propriedades de SuperfícieRESUMO
A droplet-array (DA) sandwich chip is a miniaturized platform for cell-based high-throughput screening. It is based on sandwiching of a glass slide with a preprinted library and a superhydrophobic-superhydrophilic pattern, which consists of thousands of simultaneously formed microdroplets containing cells. The DA sandwich chip allows for one-step cell seeding, simultaneous initiation of screening, and 1000 times less reagent consumption than a regular 96-well plate.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Técnicas Analíticas Microfluídicas/instrumentação , Análise Serial de Tecidos/instrumentação , Sobrevivência Celular , Células HEK293 , Células HeLa , HumanosRESUMO
Water on superhydrophilic surfaces spreads or is absorbed very quickly, and exhibits water contact angles close to zero. We encounter superhydrophilic materials in our daily life (e.g., paper, sponges, textiles) and they are also ubiquitous in nature (e.g., plant and tree leaves, Nepenthes pitcher plant). On the other hand, water on completely non-wettable, superhydrophobic surfaces forms spherical droplets and rolls off the surface easily. One of the most well-known examples of a superhydrophobic surface is the lotus leaf. Creating novel superhydrophobic surfaces has led to exciting new properties such as complete water repellency, self-cleaning, separation of oil and water, and antibiofouling. However, combining these two extreme states of superhydrophilicity and superhydrophobicity on the same surface in precise two-dimensional micropatterns opens exciting new functionalities and possibilities in a wide variety of applications from cell, droplet, and hydrogel microarrays for screening to surface tension confined microchannels for separation and diagnostic devices. In this Progress Report, we briefly describe the methods for fabricating superhydrophilic-superhydrophobic patterns and highlight some of the newer and emerging applications of these patterned substrates that are currently being explored. We also give an outlook on current and future applications that would benefit from using such superhydrophilic-superhydrophobic micropatterns.
RESUMO
A simple method to form precise micropatterns of hydrophobic liquids using porous hydrophilic-hydrophobic substrates is presented. The micropatterns of hydrophobic liquid exhibit long-term stability, excellent cell-repellency, no cytotoxicity, and are more efficient than conventional PEG or superhydrophobic surfaces in controlling eukaryotic cell adhesion.
Assuntos
Células/citologia , Interações Hidrofóbicas e Hidrofílicas , Microtecnologia/métodos , Polímeros/química , Células/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Porosidade , Propriedades de Superfície , Fatores de TempoRESUMO
Surfaces patterned with both hydrophilic and hydrophobic regions are useful in a variety of applications. For example, they can be used as surface tension-confined microchannels, in paper-based microfluidics, or for patterning cells. To create a new patterned substrate, usually the entire experimental procedure must be repeated, which can be time-consuming and laborious. In this paper, we present a simple and fast method that allows the transfer of superhydrophilic-superhydrophobic micropatterns in porous polymer films onto adhesive tape. Replicating patterns using adhesive tape is economical, as the fabrication of one patterned substrate can be used to create multiple copies of the micropatterns, which can then be used for several different experiments. We demonstrate that at least twelve consecutive copies can be made from 125 µm-thick patterned polymer films. Since the polymer film is transferred to adhesive tape, which is flexible, the copies can be used on curved surfaces and they can also be cut into different shapes and sizes. We also demonstrate an application of the replicated patterned polymer surfaces as a substrate for reverse cell transfection experiments.
Assuntos
Adesivos/química , Interações Hidrofóbicas e Hidrofílicas , Polímeros/química , Microfluídica , Microscopia Eletrônica de Varredura , Porosidade , Propriedades de SuperfícieRESUMO
We describe a one-step method for creating thousands of isolated pico- to microliter-sized droplets with defined geometry and volume. Arrays of droplets are instantly formed as liquid moves along a superhydrophilic-superhydrophobic patterned surface. Bioactive molecules, nonadherent cells, or microorganisms can be trapped in the fully isolated microdroplets for high-throughput screening, or in hydrogel micropads for screening in 3D microenvironments.