RESUMEN
A transscleral drug-delivery device, designed for the administration of protein-type drugs, that consists of a drug reservoir covered with a controlled-release membrane was manufactured and tested. The controlled-release membrane is made of photopolymerized polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticles (COLs), which are the routes for drug permeation. The results showed that the release of 40-kDa FITC-dextran (FD40) was dependent on the COL concentration, which indicated that FD40 travelled through the membrane-embedded COLs. Additionally, the sustained-release drug formulations, FD40-loaded COLs and FD40-loaded COLs pelletized with PEGDM, fine-tuned the release of FD40. Capsules filled with COLs that contained recombinant human brain-derived neurotrophic factor (rhBDNF) released bioactive rhBDNF in a manner dependent on the membrane COL concentration, as was found for FD40 release. When capsules were sutured onto sclerae of rabbit eyes, FD40 was found to spread to the retinal pigment epithelium. Implantation of the device was easy, and it did not damage the eye tissues. In conclusion, our capsule is easily modified to accommodate different release rates for protein-type drugs by altering the membrane COL composition and/or drug formulation and can be implanted and removed with minor surgery. The device thus has great potential as a conduit for continuous, controlled drug release.
Asunto(s)
Dextranos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Retina/metabolismo , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Colágeno/química , Dextranos/administración & dosificación , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Microscopía Electrónica de Rastreo , Conejos , Retina/ultraestructuraRESUMEN
We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.
Asunto(s)
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Microelectrodos , Fibras Musculares Esqueléticas/citología , Mioblastos Esqueléticos/citología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Línea Celular , Estimulación Eléctrica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Dispositivos Laboratorio en un Chip , Ratones , Polímeros/química , Transporte de Proteínas/fisiologíaRESUMEN
A microfluidic device was integrated with a controlled coculture system of HeLa cells and human umbilical vein endothelial cells (HUVECs). This integrated assembly allowed control of the direction of flow of medium (along with signaling factors secreted from cells) across the cultured cells. We grew HeLa cells and HUVECs to confluency on separate substrates and then joined the two substrates. A microfluidic device was then assembled onto the substrates and a cell coculture was initiated with controlled perfusion of the medium. When the medium flow was directed from the HeLa side to the HUVEC side, the HUVECs retreated and the HeLa cells migrated into the newly vacated areas. By contrast, when the medium flow was in the opposite direction, there was essentially no net movement of either cell type. Our results suggest that the migration of HeLa cells and HUVECs in coculture was likely mediated by soluble factors produced by HeLa cells.
Asunto(s)
Técnicas de Cocultivo/instrumentación , Medios de Cultivo , Células Endoteliales/citología , Técnicas Analíticas Microfluídicas , Venas Umbilicales/citología , Difusión , Células Endoteliales/metabolismo , Células HeLa , Humanos , Solubilidad , Integración de SistemasRESUMEN
In this study, we prepared injectable collagen microspheres for the sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) for tissue engineering. Collagen solution was formed into microspheres under a water-in-oil emulsion condition, followed by crosslinking with water-soluble carbodiimide. Various sizes of collagen microspheres in the range of 1-30 mum diameters could be obtained by controlling the surfactant concentration and rotating speed of the emulsified mixture. Particle size proportionally decreased with increasing the rotating speed (1.8 mum per 100 rpm increase in the range of 300-1,200 rpm) and surfactant concentration (3.1 mum per 0.1% increase in the range of 0.1-0.5%). The collagen microspheres showed a slight positive charge of 8.86 and 3.15 mV in phosphate-buffered saline and culture medium, respectively. Release study showed the sustained release of rhVEGF for 4 weeks. Released rhVEGF was able to induce capillary formation of human umbilical vein endothelial cells, indicating the maintenance of rhVEGF bioactivity after release. In conclusion, the results suggest that the collagen microspheres have potential for sustained release of rhVEGF.
Asunto(s)
Microesferas , Factores de Crecimiento Endotelial Vascular/farmacología , Materiales Biocompatibles , Colágeno , Emulsionantes , Emulsiones , Células Endoteliales/efectos de los fármacos , Humanos , Ingeniería de Tejidos , Venas Umbilicales/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Two-dimensional cell patterns prepared on substrate surfaces by an electrochemical-based biolithography method have been transferred into fibrin gels prepared in situ. Line patterns of human umbilical vein endothelial cells (HUVECs) in the gel that was strained after the transfer formed a linear vessel-like structure within 8 days.
Asunto(s)
Endotelio Vascular/citología , Fibrina , Venas Umbilicales/citología , Células Cultivadas , Electroquímica , Humanos , HidrogelesRESUMEN
Contractile C(2)C(12) myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C(2)C(12) myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish.
Asunto(s)
Fibrina/química , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Técnicas de Cultivo de Tejidos/métodos , Andamios del Tejido/química , Animales , Estimulación Eléctrica , Ratones , Microtecnología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/ultraestructuraRESUMEN
We investigated the interactions between HeLa cells and human umbilical vein endothelial cells (HUVECs) by monitoring their movements in a controllable coculture system. Two complementary, detachable, cell-substrates, one of polystyrene (PS) and the other of poly(dimethylsiloxane) (PDMS), were fabricated by replica molding. Coculturing was started by mechanically assembling two complementary substrates. One substrate was covered with a confluent layer of HeLa cells and its complement covered with confluent HUVECs. Using this coculture system as a tumor/endothelium model, we found that the HeLa cells migrated towards the HUVECs, while, simultaneously, the HUVECs retreated and that both types of cells migrated approximately twice as rapidly (two hundred microns per twenty-four hours) as they did alone. Additionally, when direct contact between the two cell types was prevented, the HUVECs initially migrated towards the HeLa cells and then retreated. The characteristics of the cell movements, i.e. direction and speed, probably are consequences of cell-cell signaling, with such signals possibly important during tumor cell intra- and extravasation.
Asunto(s)
Técnicas de Cocultivo/métodos , Células Endoteliales/fisiología , Endotelio Vascular/citología , Comunicación Celular , Movimiento Celular , Células Cultivadas , Dimetilpolisiloxanos/química , Endotelio Vascular/fisiología , Células HeLa , Humanos , Microscopía Fluorescente , Poliestirenos/química , Transducción de Señal , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.
Asunto(s)
Adhesión Celular/fisiología , Separación Celular/instrumentación , Técnicas de Cocultivo/instrumentación , Electroquímica/instrumentación , Microfluídica/instrumentación , Micromanipulación/instrumentación , Siliconas , Animales , Separación Celular/métodos , Técnicas de Cocultivo/métodos , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Ratones , Microfluídica/métodos , Micromanipulación/métodos , Células 3T3 NIH , Electricidad EstáticaRESUMEN
Here, we describe a method for producing patterned cell adhesion inside silicone tubing. A platinum (Pt) needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with layer-by-layer deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The utility of this approach was demonstrated by patterning co-cultures of hepatocytes or endothelial cells with fibroblasts. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.
Asunto(s)
Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Técnicas de Cultivo de Célula/métodos , Técnicas de Cocultivo/métodos , Siliconas/química , Ingeniería de Tejidos/métodos , Células 3T3 , Animales , Línea Celular , Proliferación Celular , Humanos , Ratones , Propiedades de SuperficieRESUMEN
We describe herein a method for controlling the pattern of permissible cell migration and proliferation on a substrate in time and space. Using this method, a confluent monolayer of cells that is confined within a defined region is released into a neighboring region. Incorporated into the method is an electrochemical technique that uses a scanning microelectrode to draw regions on the surface of the system that thereafter can support cell migration and growth. The supporting glass substrate is patterned with regions of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer that are not affected by the electrochemical treatment and also robustly resist cellular overgrowth as well as regions that can be individually switched when electrochemically treated from cell repellent to cell adhering. It is therefore possible to strictly define the areas into which cells can migrate. We found that HeLa cells migrate more rapidly as the width of cell-adhering lanes increases until a width of ca. 50 microm is reached, at which point the migration rate is roughly constant. We also designed a drug assay using our cell migration technique. The technique allows for cell migration only into defined region(s) and therefore may become an important tool for evaluating the biological activity of potential drugs because drug activity and cell motility often directly correlate.