Hydrogel-based sealed microchamber arrays for rapid medium exchange and drug testing of cell spheroids.

Culturing cell spheroids in microchamber arrays is a widely used method in regenerative medicine and drug discovery while it requires laborious procedures during medium exchange and drug administration. Here, we report a simple method for the medium exchange and drug testing using a hydrogel-based sealed microchamber arrays. Owing to the high molecular permeability of poly(vinyl alcohol) hydrogel, the sealed microchamber allows nutrients and drugs in outer medium to pass through. Thus, automatic medium exchange and drug testing for all the cell spheroids inside the microchamber arrays are achieved by simply transferring the microchamber from old medium to fresh medium. Cell spheroids of human induced pluripotent stem cell-derived cardiomyocytes were cultured inside the sealed microchambers, and it was confirmed that the spheroids were stably positioned inside the microchamber even after transferring 10 times. The cell spheroids showed high viability after culturing for 7 days in the sealed microchamber with the transfer-based medium exchange, which allowed cardiac maturation by simultaneous electrical stimulation. Isoproterenol, a model cardiac drug, was administrated from outside the sealed microchamber to demonstrate the feasibility of drug testing by the rapid transfer method.

Totally transparent hydrogel-based subdural electrode with patterned salt bridge.

A totally transparent subdural electrode was developed by embedding a conductive poly (vinyl alcohol) (PVA)-filled microchannel made of poly(dimethylsiloxane) (PDMS) into an another PVA hydrogel substrate. Tight bonding between the PVA substrate and the PDMS microchannel (salt bridge) was achieved by mechanical interlocking utilizing the microprotrusions formed on the microchannel. This simple method of bonding without the use of any additives such as silane molecules or nanofibers is very suitable for constructing biomedical devices. The salt bridge electrode (total thickness, ca. 1.5 mm) was sufficiently soft, and showed superior shape conformability that makes it an excellent choice as a subdural electrode used on the brain surface. In vivo measurement proved that the salt bridge electrode makes close contact to the exposed porcine brain and can record brain wave signals of frequencies 1 ~ 15 Hz. In addition, the high transparency of the electrode provided a clear view of the brain surface that would assist the effective surgical operation and optogenetic research.

Fluid-permeable enzymatic lactate sensors for micro-volume specimen.

Sensing of lactate in perspiration provides a way to monitor health and control exercise. The volume of perspiration is miniscule, and the efficient collection of perspiration is desired for its effective sensing. We developed mesh-type enzymatic electrodes fabricated on textile meshes and integrated the meshes into an enzymatic biofuel cell. We tested them as self-powered lactate sensors for a small volume of lactate solution. A fluid-permeable enzymatic anode was fabricated based on an insulating textile mesh that was coated with carbon nanotubes (CNTs) and lactate oxidase. The anode was further coated with polyurethane to increase the linear range by limiting the diffusion of lactate while maintaining the advantages of the original textile mesh, such as flexibility, stretchability, and permeability. Permeability of the mesh-type lactate-oxidizing anode allowed a vertically stacked structure of the anode and a previously developed air-breathing cathode. This resulted in a small overall device size (1 cm2). The mesh-type sensor was tested using a small flow rate of lactate solution, and a moderate linearity of amperometric response for a wide concentration range (5 to ≥20 mM) was confirmed. The fluid-permeable anode and enzymatic biofuel cell show the potential of the sensor for continuous monitoring of lactate in perspiration on skin.

Physicochemical and biological characterization of sustained isopropyl unoprostone-release device made of poly(ethyleneglycol) dimethacrylates.

Transscleral drug delivery is becoming increasingly popular to manage posterior eye diseases. To evaluate the clinical application of a transscleral, sustained, unoprostone (UNO)-release device (URD) constructed of photopolymerized tri(ethyleneglycol) dimethacrylate and poly(ethyleneglycol) dimethacrylate, we evaluated physicochemical and biological properties of this device. The URD consists of a drug-impermeable reservoir and a semi-permeable cover. The in vitro release rate of UNO from the URD increased with increasing temperatures from 20 to 45 °C. Scanning electron microscopy and atomic-force microscopy showed that the border between the reservoir and drug formulation was sharply defined but that between the cover and drug was poorly determined, indicating that UNO could permeate only through the cover. For stability tests, the URDs were sterilized with ethylene oxide gas and stored at 40 °C/75% for 3 and 6 months and at 25 °C/60% for 3, 6, 9, 12, 18, and 24 months; UNO content and release rate at 37 °C were then evaluated. There was no significant decrease in either UNO content or release rate after the storage conditions. Cytotoxicity was evaluated by examining the colony formation of Chinese hamster fibroblast V79 cells in a media extract of the URD without UNO. This extract did not affect colony formation of V79 cells, indicating the cytocompatibility of the URD. In conclusion, the URD was physically stable for 24 months and is potentially useful for clinical application.

Transscleral Controlled Delivery of Geranylgeranylaceton Using a Polymeric Device Protects Rat Retina Against Light Injury.

We evaluated the effects of a transscleral drug delivery device, consisting of a reservoir and controlled-release cover, which were made of photopolymerized polyethylene glycol dimethacrylate and triethylene glycol dimethacrylate, combined at different ratios. Geranylgeranylacetone (GGA), a heat-shock protein (HSP) inducer, was loaded into the device. The GGA was released from the device under zero-order kinetics. At both 1 week and 4 weeks after device implantation on rat sclera, HSP70 gene and protein expression were up-regulated in the sclera-choroid-retinal pigment epithelium fraction of rat eyes treated with the GGA-loaded device compared with rat eyes treated with saline-loaded devices or eyes of non-treated rats. Flash electroretinograms were recorded 4 days after white light exposure (8000 lx for 18 h). Electroretinographic amplitudes of the a- and b-waves were preserved significantly in rats treated with GGA-loaded devices compared with rats treated with saline-loaded devices. Histological examination showed that the outer nuclear layer thickness was preserved in rats that had the GGA-loaded device. These results may show that transscleral GGA delivery using our device may offer an alternative method to treat retinal diseases.

Self-regulating enzyme-nanotube ensemble films and their application as flexible electrodes for biofuel cells.

Nanostructured carbons have been widely used for fabricating enzyme-modified electrodes due to their large specific surface area. However, because they are random aggregates of particular or tubular nanocarbons, the postmodification of enzymes to their intrananospace is generally hard to control. Here, we describe a free-standing film of carbon nanotube forest (CNTF) that can form a hybrid ensemble with enzymes through liquid-induced shrinkage. This provides in situ regulation of its intrananospace (inter-CNT pitch) to the size of enzymes and eventually serves as a highly active electrode. The CNTF ensemble with fructose dehydrogenase (FDH) showed the oxidation current density of 16 mA cm(-2) in stirred 200 mM fructose solution. The power density of a biofuel cell using the FDH-CNTF anode and the Laccase-CNTF cathode reached 1.8 mW cm(-2) (at 0.45 V) in the stirred oxygenic fructose solution, more than 80% of which could be maintained after continuous operation for 24 h. Application of the free-standing, flexible character of the enzyme-CNTF ensemble electrodes is demonstrated via their use in the patch or wound form.

Conducting polymer electrodes printed on hydrogel.

We report herein the micropatterning of poly(3,4-ethylenedioxythiophene) (PEDOT) on a hydrogel, agarose, to provide a fully organic, moist, and flexible electrode. The PEDOT/agarose electrodes were prepared through two electrochemical processes: electropolymerization of PEDOT into the hydrogel and electrochemical-actuation-assisted peeling. We also present a typical application of the PEDOT/agarose electrode to the cultivation of contractile myotubes.

Electrodes combined with an agarose stamp for addressable micropatterning.

We have combined a topographically patterned agarose microstamp with an electrode substrate to develop a novel printing device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. The 10 wt % agarose gel containing 0.1 M PBS + 25 mM KBr showed suitable elasticity for forming stamps and served as the electrolytic medium for the electrochemical oxidation of Br(-) to generate HBrO. The electrode substrate patched with an agarose stamp having 50-microm-high bumps was used for the spatially confined detachment of heparin/polyethyleneimine precoated on glass substrates, followed by micropatterned adsorption of fibronectin. Using the microelectrode array, the addressable micropatterning of protein by the controlled delivery of HBrO to each bump was demonstrated.

Preparation and characterization of collagen microspheres for sustained release of VEGF.

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.

Controlled cocultures of HeLa cells and human umbilical vein endothelial cells on detachable substrates.

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.

Localized electrical stimulation to C2C12 myotubes cultured on a porous membrane-based substrate.

We report a porous membrane-based cell culture device that can conduct localized electrical stimulation of a cell monolayer. The device's cell culture substrate is a microporous alumina membrane with an underlying thin poly(dimethylsiloxane) (PDMS) film spotted with holes. When electric current is generated between the device's Pt ring electrodes--one of which is placed above the cells and the other below the PDMS layer--the current density condenses at the holes in the PDMS film, and cells located above the holes can be electrically stimulated. C2C12 cells were confluently cultured on the substrate and were differentiated to myotubes. To control the stimulated area in the substrate, we attempted to seal and reopen the holes of the PDMS film by using an air bubble. Since the current pulse could be effectively blocked at the sealed holes, fluorescent Ca2+ transients, indicative of cellular excitation, were observed from the myotubes located above holes in the open state.

Organic electrochromic timer for enzymatic skin patches.

A totally organic and disposable electrochromic timer integrated with an enzymatic electrode and powered by biofuel cells is developed. The cathode of the self-powered electrochromic timer consists of a composite electrochromic film of poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU), while the anode is made up of a fructose dehydrogenase (FDH) enzymatic electrode. The electrochromic changes over time (up to 100 min) can be displayed in the device, and the speed of color change can be controlled by changing the resistance between the anode and the cathode. Automatic activation of the timer after placement on a skin is achieved by integrating a porous microneedle array. The electrochromic timer would be used along with a skin patch as a time-lapse display of medical and cosmetic treatments.

Hydrogel-Based Organic Subdural Electrode with High Conformability to Brain Surface.

A totally soft organic subdural electrode has been developed by embedding an array of poly(3,4-ethylenedioxythiophene)-modified carbon fabric (PEDOT-CF) into the polyvinyl alcohol (PVA) hydrogel substrate. The mesh structure of the stretchable PEDOT-CF allowed stable structural integration with the PVA substrate. The electrode performance for monitoring electrocorticography (ECoG) was evaluated in saline solution, on ex vivo brains, and in vivo animal experiments using rats and porcines. It was demonstrated that the large double-layer capacitance of the PEDOT-CF brings low impedance at the frequency of brain wave including epileptic seizures, and PVA hydrogel substrate minimized the contact impedance on the brain. The most important unique feature of the hydrogel-based ECoG electrode was its shape conformability to enable tight adhesion even to curved, grooved surface of brains by just being placed. In addition, since the hydrogel-based electrode is totally organic, the simultaneous ECoG-fMRI measurements could be conducted without image artifacts, avoiding problems induced by conventional metallic electrodes.

Transscleral sustained ranibizumab delivery using an episcleral implantable device: Suppression of laser-induced choroidal neovascularization in rats.

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.

Integration of an electrochemical-based biolithography technique into an AFM system.

An ordinary atomic force microscopy (AFM) was functionalized and applied to electrochemically draw micropatterns of biomolecules. To fabricate an electrochemical AFM probe having an electrode at the tip, a metal-coated AFM probe was first insulated with Parylene C, and then the apex of the tip was ground mechanically to expose the electrode. The effective electrode diameter was estimated to be ca. 500 nm. The electrode probe was positioned close to a heparin-coated antibiofouling substrate and used to locally generate hypobromous acid from a dilute Br(-) solution to render the substrate surface protein-adhesive. In situ topographical imaging after the electrochemical treatment suggested the heparin layer became detached to allow the adsorption of proteins, in this case fibronectin. The diameter of the drawn fibronectin pattern was 2 microm, which is one order of magnitude smaller than we achieved previously using a microdisk electrode (tip diameter 10 microm).

Pharmacokinetic and Safety Evaluation of a Transscleral Sustained Unoprostone Release Device in Monkey Eyes.

Purpose: We evaluate the ocular tissue distribution and retinal toxicity of unoprostone (UNO) during 12 months, after transscleral sustained-UNO administration using a drug delivery device in monkey eyes. Methods: The device consisted of a reservoir, controlled-release cover, and a drug formulation of photopolymerized polyethylene glycol dimethacrylate. Six mg UNO was loaded into the device (length, 17 mm; width, 4.4 mm; height, 1 mm). The concentrations of M1, a primary metabolite of UNO, in the retina, choroid, vitreous, lens, aqueous humor, iris, ciliary body, and plasma were determined by liquid chromatography-tandem mass spectrometry at 3, 6, and 12 months after implantation. Retinal toxicity was evaluated by electroretinography (ERG), optical coherence tomography (OCT), and IOP at preimplantation, and at 6, 9, and 12 months after implantation. Focal ERGs were performed at 9 and 12 months after implantation. Results: M1 was detected in the choroid and retina with maximum peaks of 243.2 and 8.41 ng/g at 6 months, respectively. M1 in the ciliary body and iris was detected with maximum peaks of 7.66 and 10.4 ng/g at 6 and 12 months, respectively. Less than 1 ng/mL or ng/g of M1 was detected in the aqueous humor, vitreous, and lens. No changes were observed in retinal function as assessed by ERG, IOP, or macula thickness and retinal histology by OCT examinations during the 12-month period. No differences in focal ERG amplitudes, especially in the macula, were observed. Conclusions: The device provided intraocular sustained delivery of UNO for 12 months without producing severe retinal toxicity.

A self-deploying drug release device using polymeric films.

Herein, we report a sheet-type device capable of self-deployment and sustained release of protein type drugs. The device consisted of a thin photopolymerized polyethylene glycol dimethacrylate (PEGDM) sheet and collagen microparticles (COLs), which were embedded in the sheet as drug carriers and for increased drug permeation. When the density of the COLs in the sheet was increased to be sufficiently interconnected, the drug permeability was increased. In addition, since protein type drugs electrostatically interacted with the COLs, a prolonged sustained release was possible. The PEGDM/COLs device was flexible enough to be rolled up, and the device maintained its structure due to van der Waals attractive forces between the sheet surfaces. When the device was immersed in water, the attractive forces acting between the sheet surfaces were relieved by water. Subsequently, the device unfolded by bending-stress relaxation. Moreover, the rolled-up device could be injected through a conventional syringe needle into water to recover its original shape. The developed sheet-type device provides the possibility of minimally invasive transplantation into diseased tissues and organs, and could provide better therapeutic outcomes and reduce possible side effects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 780-786, 2018.

Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes.

The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the PI surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8 cm(2) sheet of myocytes was around 0.2 microC, roughly corresponding to a membrane depolarization of 250 mV.

Electrochemical manipulation of cell populations supported by biodegradable polymeric nanosheets for cell transplantation therapy.

We describe an electrochemical method of harvesting cells cultured on a biodegradable polymeric nanosheet (cell/nanosheet construct), which is stabilized on a self-assembled monolayer (SAM) of thiol molecules. A poly(lactic-co-glycolic acid) (PLGA) nanosheet was attached by hydrophobic interactions onto the surface of a SAM of l-cysteine coated onto a gold electrode. Retinal pigment epithelial cell lines (RPE-J cells) were cultured on the nanosheet to form a monolayer. An AA-size dry battery was used to apply a negative electrical potential, causing reductive desorption of the SAM from the gold surface. Within one minute of application of the voltage, the cell/nanosheet of several mm in diameter was successfully detached without the loss of cell viability in a gentle stream of the electrolyte solution. The use of a porous electrode shortened the detachment time due to the more efficient permeation of the electrolyte solution to the electrode surface. Cell transplantation following the harvesting process was demonstrated by the local delivery of RPE-J cell/nanosheet constructs into the subretinal space of rat eyes through a capillary needle. This nanosheet-based approach that allows the on-demand harvesting of cell/nanosheet constructs and their subsequent transplantation in a minimally-invasive manner could play an important role in cell transplantation therapy.

Controlled basic fibroblast growth factor release device made of poly(ethyleneglycol) dimethacrylates for creating a subcutaneous neovascular bed for cell transplantation.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.