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.

Transepidermal Potential of the Stretched Skin.

The electrical response of the skin to mechanical stretches is reported here. The electrical potential difference across the epidermis, i.e., transepidermal potential (TEP) of porcine skin samples subjected to cyclic stretching, was measured in real time to observe electrochemical change in epidermal tissue. In addition to a conventional method of TEP measurement for the whole of skin sample, a probe-type system with a fine-needle salt bridge was used for direct measurement of TEP at a targeted local point of the skin. TEP decreased with the increased mechanical stretches, and the change of TEP was found to be mostly occurred in the epidermis but not dermis nor hypodermis by comparing the results of conventional and the probe-type methods. The observed change of TEP value was quick, reversible, and strain-dependent. Considering from such characteristic behaviors, one of the possible mechanisms of the modulation of TEP would be influence of the streaming potential caused by the fluid flow during the physical deformation of the epidermis.

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.

An array of porous microneedles for transdermal monitoring of intercellular swelling.

An array of porous microneedles was developed for minimally-invasive transdermal electrolytic connection through the human skin barrier, the stratum corneum. The length of microneedle was designed to be 100 µm so that it penetrates into the epidermis layer without pain. Each microneedle was supported by a thicker cylindrical post protruding from a planar substrate to realize its effective penetration even into elastic human skin. Since this support (post and substrate) was equally porous as the needles, the needle chip was entirely permeable for electrolyte. This ion-conductive porous microneedle array was applied to the transdermal conductometry with small direct current for local monitoring of intercellular swelling, edema. The porous needle-based electrode system could be a platform for various transdermal electrical diagnosis and treatments.

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.

Minimally-invasive transepidermal potentiometry with microneedle salt bridge.

A commercial painless microneedle was filled with physiological saline agar, and this needle-based salt bridge was inserted into the skin (a piece of porcine skin and a flank skin of a live mouse) to make an electrical contact with its subepidermal region. The transepidermal potential (TEP), the potential difference between the skin surface and the subepidermal region, was measured using this inner electrode and a conventional agar electrode on the surface of the skin. Control of penetration depth of the inner electrode with a spacer and hydrophilic pretreatment with ozone plasma were found to be necessary for stable measurement. The TEP was reduced upon damages on the skin surface by tape stripping and acetone defatting, which indicated the fabricated needle electrode is useful for the minimally-invasive measurement of TEP and evaluation of skin barrier functions. Furthermore, we showed that the device integrating two electrodes into a single compact probe was useful to evaluate the local barrier functions and their mapping on a skin. This device could be a personal diagnostic tool in the fields of medicine and cosmetics in future.

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.

Application of clotrimazole via a novel controlled release device provides potent retinal protection.

Age-related macular degeneration is the leading cause of legal blindness among older individuals. Therefore, the development of new therapeutic agents and optimum drug delivery systems for its treatment are crucial. In this study, we investigate whether clotrimazole (CLT) is capable of protecting retinal cells against oxidative-induced injury and the possible inhibitory effect of a sustained CLT-release device against light-induced retinal damage in rats. In vitro results indicated pretreatment of immortalized retinal pigment epithelium cells (RPE-J cells) with 10-50 µM CLT before exposure to oxygen/glucose deprivation conditions for 48 h decreased the extent of cell death, attenuated the percentage of reactive oxygen species-positive cells, and decreased the levels of cleaved caspase-3. The device consists of a separately fabricated reservoir, a CLT formulation, and a controlled release cover, which are made of poly(ethyleneglycol) dimethacrylate (PEGDM) and tri(ethyleneglycol) dimethacrylate (TEGDM). The release rate of CLT was successfully tuned by changing the ratio of PEGDM/TEGDM in the cover. In vivo results showed that use of a CLT-loaded device lessened the reduction of electroretinographic amplitudes after light exposure. These findings indicate that the application of a polymeric CLT-loaded device may be a promising method for the treatment of some retinal disorders.

Surfactant-assisted direct electron transfer between multi-copper oxidases and carbon nanotube-based porous electrodes.

The effects of pre-treatment with surfactants on the electrocatalytic reaction of multi-copper oxidases were quantitatively evaluated using a well-structured carbon nanotube forest electrode. It was found that both the charge polarity of the head group and the aromatics in the tail part of the surfactants affect the efficiency of enzymatic electrocatalysis.

Intrascleral transplantation of a collagen sheet with cultured brain-derived neurotrophic factor expressing cells partially rescues the retina from damage due to acute high intraocular pressure.

We constructed brain-derived neurotrophic factor (BDNF) expressing rat retinal pigment epithelial (RPE) cells by stable transfection of BDNF cDNA, and the RPE cells were cultured on a cross-linked collagen sheet (Coll-RPE-BDNF). BDNF expression of the Coll-RPE-BDNF was confirmed by western blot, and the Coll-RPE-BDNF was transplanted into the rabbit sclera. In vivo BDNF expression was confirmed by His expression that was linked to the expressing BDNF. The effect of the released BDNF was examined in a rabbit acute high intraocular pressure system by electroretinogram and histological examination. Statistically significant preservation of ERG b wave amplitude was observed in the rabbits treated by Coll-RPE-BDNF when compared to that of no treatment. Statistically significant preservation of the thickness of the inner nuclear layer at the transplanted area was observed in the rabbits treated by Coll-RPE-BDNF compared to that of no treatment. Intra-scleral Coll-RPE-BDNF transplantation may partially rescue retinal cells from acute high intraocular pressure.

Transdermal drug delivery using a porous microneedle device driven by a hydrogel electroosmotic pump.

Integrating a hydrogel electroosmotic pump with a parylene C-coated porous microneedle (PMN) is developed for transdermal drug delivery applications. The hydrogel pump is fabricated by combining an anionic and a cationic hydrogel to generate enhanced electroosmosis flow (EOF) to drive the transportation of molecules via PMN.

Bilaterally Aligned Electroosmotic Flow Generated by Porous Microneedle Device for Dual-Mode Delivery.

Here, a novel porous microneedle (PMN) device with bilaterally aligned electroosmotic flow (EOF) enabling controllable dual-mode delivery of molecules is developed. The PMNs placed at anode and cathode compartments are modified with anionic poly-2-acrylamido-2-methyl-1-propanesulfonic acid and cationic poly-(3-acrylamidopropyl) trimethylammonium, respectively. The direction of EOF generated by PMN at the cathode compartment is, therefore, reversed from cathode to anode, countering the unwanted cathodal suctioning of interstitial fluid caused by reverse iontophoresis. With the bilateral alignment of EOF, the versatility of the proposed device is evaluated by delivering molecules with different charges and sizes using Franz cell. In addition, a 3D printed probe device is developed to ease practical handling and minimize electrical stimulation by integrating two PMNs in closed proximity. Finally, the performance of the integrated probe device is demonstrated by dual delivery of a variety of molecules (methylene blue, rhodamine B, and fluorescein isothiocyanate-dextran) using pig skin and vaccination using mice with delivered ovalbumin.

Frustoconical porous microneedle for electroosmotic transdermal drug delivery.

A truncated cone-shaped porous microneedle (PMN) made of poly-glycidyl methacrylate was studied as a minimally invasive tool for transdermal drug delivery. The transdermal electrical resistance of a pig skin was evaluated during the indentation of the PMNs, revealing that the frustoconical PMN (300 µm height) significantly reduced the resistance of the skin by expanding the stratum corneum without penetrating into the skin. A thin film of poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) was grafted onto the inner wall of the microchannels of the frustoconical PMN to generate electroosmotic flow (EOF) upon current application in the direction of injection of the drug into the skin. Owing to the synergy of the expansion of the stratum corneum and the EOF-promotion, the PAMPS-modified frustoconical PMN effectively enhances the penetration of larger (over 500 Da) molecules, such as dextran (∼10 kDa).

Totally Organic Hydrogel-Based Self-Closing Cuff Electrode for Vagus Nerve Stimulation.

An intrinsically soft organic electrode consisting of poly(3,4-ethylenedioxythiophene)-modified polyurethane (PEDOT-PU) is embedded into a bilayer film of polyvinyl alcohol (PVA) hydrogels for developing a self-closing cuff electrode for neuromodulation. The curled form of the PVA hydrogel is prepared by releasing internal stress in the bilayer structure. The inner diameter of the cuff electrode is set to less than 2 mm for immobilization to the vagus nerve (VN) of humans and pigs. The stability of the immobilization is examined, while the pressure applied to a nerve bundle is at a harmless level (≈200 Pa). Since the electrode is totally organic, MRI measurements can be conducted without image artifacts. The large electric capacitance of the PEDOT-PU (≈27 mF cm-2 ) ensures a safe stimulation of living tissues without Faradaic reactions. The practical performance of the cuff electrode for VN stimulation is demonstrated by observation of bradycardia induction in a pig.

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.

Electrical aspects of skin as a pathway to engineering skin devices.

Skin is one of the indispensable organs for life. The epidermis at the outermost surface provides a permeability barrier to infectious agents, chemicals, and excessive loss of water, while the dermis and subcutaneous tissue mechanically support the structure of the skin and appendages, including hairs and secretory glands. The integrity of the integumentary system is a key for general health, and many techniques have been developed to measure and control this protective function. In contrast, the effective skin barrier is the major obstacle for transdermal delivery and detection. Changes in the electrical properties of skin, such as impedance and ionic activity, is a practical indicator that reflects the structures and functions of the skin. For example, the impedance that reflects the hydration of the skin is measured for quantitative assessment in skincare, and the current generated across a wound is used for the evaluation and control of wound healing. Furthermore, the electrically charged structure of the skin enables transdermal drug delivery and chemical extraction. This paper provides an overview of the electrical aspects of the skin and summarizes current advances in the development of devices based on these features.

Transdermal electroosmotic flow generated by a porous microneedle array patch.

A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O2 battery) is also demonstrated as a possible totally organic iontophoresis patch.

Directing the flow of medium in controlled cocultures of HeLa cells and human umbilical vein endothelial cells with a microfluidic device.

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.

Automatic, sequential power generation for prolonging the net lifetime of a miniature biofuel cell stack.

A sequential power generation system for prolonging the net lifetime of a miniature biofuel cell stack has been developed. The system consists of layered chambers of enzyme fuel cells designed to be exposed sequentially to fuel solution by automatically switched fuel flow. The cell chambers were initially separated by magnetized plastic covers sealed with a degradable glue, poly(lactic-co-glycolic acid) (PLGA). The time that the cover was opened by attraction with an external magnet, thereby activating the following cell, was adjustable from a few hours to a few weeks by controlling the weight ratio of Fe(3)O(4) in the covers and the molecular weight of PLGA. By using sequential power generation in this way, the power output of the system was stable for longer periods, and therefore the net lifetime of the stack has been extended as compared with that of a single biofuel cell.