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With the advances in skeletal muscle tissue engineering, new platforms have arisen with important applications in biology studies, disease modeling, and drug testing. Current developments highlight the quest for engineering skeletal muscle tissues with higher complexity that can better mimicThe authors review the latest advances inIn recent years, more sophisticated

Recent years have witnessed the emergence of several viruses and other pathogens. Some of these infectious diseases have spread globally, resulting in pandemics. Although biosensors of various types have been utilized for virus detection, their limited sensitivity remains an issue. Therefore, the development of better diagnostic tools that facilitate the more efficient detection of viruses and other pathogens has become important. Nanotechnology has been recognized as a powerful tool for the detection of viruses, and it is expected to change the landscape of virus detection and analysis. Recently, nanomaterials have gained enormous attention for their value in improving biosensor performance owing to their high surface-to-volume ratio and quantum size effects. This article reviews the impact of nanotechnology on the design, development, and performance of sensors for the detection of viruses. Special attention has been paid to nanoscale materials, various types of nanobiosensors, the internet of medical things, and artificial intelligence-based viral diagnostic techniques.

Organ-on-chip (OoC) platforms are a new class of advanced microfluidic devices that have been attracting significant attention for drug development since they can allow a faster and more patient specific evaluation of the effects of drugs due to the mimicking of organ human models. As most microfluidic devices, the great majority of the OoC is made from polydimethylsiloxane (PDMS), due to the easiness to handle and due to its physicochemical properties. Nevertheless, PDMS can leach uncrosslinked oligomer and absorb small hydrophobic molecules, which makes it less suitable for cell culture. In this work, we review different kinds of materials that are used together with, or in replacement, of PDMS to manufacture the OoC platforms. Several studies have replicated differentiated structures and their key physiological functions, such as muscle contraction, skin elasticity and deformability, gas exchange in alveolar sacs, electrical stimulation or even to monitor the microenvironment, inside the chip. Therefore, in this Review, recent advances on OoC platforms, including fabrication and miniaturization of biosensors for an end-use perspective are revised., This work has been supported by the projects NORTE-01-0145-FEDER-030171 (project reference PTDC/EME-SIS/30171/2017), NORTE-01-0145-FEDER-029394 (project reference PTDC/EMD-EMD/ 29394/2017), through the COMPETE2020, the Lisb@2020, the Programa Operacional Regional do Norte–Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and by Fundação para a Ciência e Tecnologia (FCT), and through FEDER and FCT, project references EXPL/EMD-EMD/0650/2021 and PTDC/EEI-EEE/2846/2021. The authors also acknowledge the partial financial support within the R&D Units Project Scope: UIDB/00319/2020, UIDB/04077/2020, UIDB/00690/2020, UIDB/04436/2020, BD/08646/2020. This work was also funded by AMED-CREST Grant Number JP21gm1310001h0002. Raquel O. Rodrigues (R.O.R.) thanks FCT for her contract funding provided through 2020.03975.CEECIND.

A grid micropattern of neuronal cells was formed on a free-standing collagen film (35 μm thickness) by directing migration and extension of neurons along a Matrigel pattern previously prepared on the film by the microcontact printing method. The neurons migrated to reach the nodes on the grid pattern and extended neurites to bridge cell bodies at the nodes. The resulting neuronal micropattern on the collagen film containing culture medium can be handled and deformed with tweezers with maintenance of physiological activity of the neurons, as examined by response of cytosolic Ca2+ concentration to a dose of bradykinin. This portability is the unique advantage of the present system that will open novel possibility of cellular engineering including the on-demand combination with analytical devices. The repetitive lamination of the film on a microelectrode chip was demonstrated for local electrical stimulation of a specific part of the grid micropattern of neurons, showing Ca2+ wave propagation along the neuri...

Self-sustainable release of brain-derived neurotrophic factor (BDNF) to the retina using minimally invasive cell-encapsulation devices is a promising approach to treat retinal degenerative diseases (RDD). Herein, we describe such a self-sustainable drug delivery device with human retinal pigment epithelial (ARPE-19) cells (cultured on collagen coated polystyrene (PS) sheets) enclosed inside a 3D printed semi-porous capsule. The capsule was 3D printed with two photo curable polymers: triethylene glycol dimethacrylate (TEGDM) and polyethylene glycol dimethylacrylate (PEGDM). The capsule&rsquo, s semi-porous membrane (PEGDM) could serve three functions: protecting the cells from body&rsquo, s immune system by limiting diffusion (5.97 &plusmn, 0.11%) of large molecules like immunoglobin G (IgG)(150 kDa), helping the cells to survive inside the capsule by allowing diffusion (43.20 &plusmn, 2.16%) of small molecules (40 kDa) like oxygen and necessary nutrients, and helping in the treatment of RDD by allowing diffusion of cell-secreted BDNF to the outside environment. In vitro results showed a continuous BDNF secretion from the device for at least 16 days, demonstrating future potential of the cell-encapsulation device for the treatment of RDD in a minimally invasive and self-sustainable way through a periocular transplant.

The development of cancer models that rectify the simplicity of monolayer or static cell cultures physiologic microenvironment and, at the same time, replicate the human system more accurately than animal models has been a challenge in biomedical research. Organ-on-a-chip (OoC) devices are a solution that has been explored over the last decade. The combination of microfluidics and cell culture allows the design of a dynamic microenvironment suitable for the evaluation of treatments’ efficacy and effects, closer to the response observed in patients. This systematic review sums the studies from the last decade, where OoC with cancer cell cultures were used for drug screening assays. The studies were selected from three databases and analyzed following the research guidelines for systematic reviews proposed by PRISMA. In the selected studies, several types of cancer cells were evaluated, and the majority of treatments tested were standard chemotherapeutic drugs. Some studies reported higher drug resistance of the cultures on the OoC devices than on 2D cultures, which indicates the better resemblance to in vivo conditions of the former. Several studies also included the replication of the microvasculature or the combination of different cell cultures. The presence of vasculature can influence positively or negatively the drug efficacy since it contributes to a greater diffusion of the drug and also oxygen and nutrients. Co-cultures with liver cells contributed to the evaluation of the systemic toxicity of some drugs metabolites. Nevertheless, few studies used patient cells for the drug screening assays.

Rapid clearance and low ocular bioavailability are drawbacks of conventional ophthalmic eye drops. To increase the ocular drug resistance time and improve efficacy, an in situ forming and thermosensitive chitosan-gelatin hydrogel was developed. The feasibility of using this hydrogel as a topical eye drop formulation for sustained release of timolol maleate was evaluated. The flexible hydrogel that was co-crosslinked with β‑glycerophosphate disodium salt hydrate (β-GD) and genipin showed a fast gel formation at 37 °C. The swelling properties and in vitro biodegradation characteristics showed a strong relationship with the initial genipin concentration. In vitro release profiles demonstrated that crosslinking with genipin reduced the release rate of entrapped model drugs and timolol maleate. In vitro cytotoxicity tests showed that the hydrogel was non-toxic to Chinese hamster fibroblast V79 cells. The hydrogel was further applied as eye drop formulations for sustained release of timolol maleate to reduce intraocular pressure (IOP). A fast gel formation was observed after instilling the chitosan-gelatin solution into the lower conjunctival sac of the rabbit eyes, and the in situ formed hydrogels protected the drugs from clearance by tears, and released the drugs in a sustained manner. Furthermore, administration of timolol maleate containing chitosan-gelatin hydrogels showed a long-lasting and effective IOP lowering efficacy for up to 24 h compared with the conventional eye drops. These results suggested that β-GD and genipin co-crosslinked chitosan-gelatin hydrogels could be a useful ocular drug delivery platform with enhanced therapeutic effects and reduced side effects.

Contractile skeletal muscle cells were cultured so as to wrap around an electrode wire to enable their selective stimulation even when they were co-cultured with other electrically-excitable cells. Since the electrode wire was composed of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU), which is soft and highly capacitive (~10 mF cm−2), non-faradaic electrical stimulation with charge/discharge currents could be applied to the surrounding cells without causing significant damage even for longer periods (more than a week). The advantage of this new culture system was demonstrated in the study of chemotactic interaction of monocytes and skeletal muscle cells via myokines.

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.

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.

An Au film electrode supported by a conductive elastic film was tightly bonded on a stretchable double-network hydrogel sheet by means of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) grown from the electrode surface into the hydrogel. This electrode/hydrogel composite showed stable resistance of 35 ± 5 Ω sq −1 even during successive 20% stretching because of the pre-formed, designed cracks in the Au film. The large interfacial electric double layer capacitance (9.5 ± 0.3 mF cm −2 ) of the PEDOT adhesive layer at the interface of the layered composite was found to stabilize the electrode potential against external noises, and decrease the electric impedance at the frequency of 5–500 Hz, which is the typical range of electromyographic signals. The electrical robustness and shape-conformability of the composite electrode were demonstrated by monitoring electromyographic signals of the joint of a human forefinger. In addition, it was also demonstrated that an ionic liquid-containing gel (ionogel) serves as a substrate of the composite for longer-term monitoring over 3 days on air-exposed human skin.

In this study, we developed a subcutaneous insulin-releasing device consisting of a disk-shaped capsule and drug formulation comprised of poly(ethylene glycol) dimethacrylates, then evaluated its efficacy on retinal function in streptozotocin (STZ)-induced diabetic rats. In vitro release studies showed that recombinant human insulin was released with a constant rate for more than 30 days. The device was able to maintain a basal level of blood glucose in diabetic rats for a prolonged period of more than 30 days, simultaneously preventing a decrease in body weight. For assessing the pharmacological effect of the device on retinal function in diabetic rats, electroretinograms were conducted for 12 weeks. The reduction in amplitude and delay in implicit time were attenuated by the device during the initial 4 weeks of application. The increase in gene expression of protein kinase C (PKC)-γ and caspase-3 in the diabetic retina was also attenuated by the device. Immunohistochemistry showed that the increase in glial fibrillary acidic protein expression in the diabetic retina was attenuated by the device. Histological evaluation of subcutaneous tissue around the device showed the biocompatibility of the device. In conclusion, the insulin-releasing device attenuated the reduction of retinal function in STZ-induced diabetic conditions for 4 weeks and the efficacy of the device might be partially related to PKC signaling in the retina. The long-term ability to control the blood glucose level might help to reduce the daily frequency of insulin injections.

Due to the fact that ~ 80% of all sensory input is received via the eyes, suffering from chronic retinal diseases that lead to blindness causes a significant decrease in the quality of life. In addition, retinal diseases are common in the elderly; developing pathological analyses and treatments for retinal diseases have become an urgent issue especially in superaging countries such as Japan. Although evaluation of drug candidates against retinal diseases has been done on animal models, serious concerns arise regarding the ethics and costs in addition to the limitations of translating data from animal models to clinical settings. Pathophysiology of neovascularization in diabetic retinopathy and age-related macular degeneration (AMD) is complicated due to various factors involved, which include but are not limited to aging, oxygen concentration, energy metabolism, pressure, blood flow, and genetics. For more reliable outcomes, it is essential to understand the pathological mechanisms of eye diseases. in vitro cell culture models, an alternative to animal models, enable investigations on specific molecule of interest and simply recapitulate complex and chronic conditions. Recently, studies on organ-on-a-chip for drug discovery have been extensively carried out. This chapter describes organ-on-a-chip approaches that target the retinal tissue particularly focusing on the blood-retinal barriers and related diseases.

Three-dimensional (3D) in vitro models, such as organ-on-a-chip platforms, are an emerging and effective technology that allows the replication of the function of tissues and organs, bridging the gap amid the conventional models based on planar cell cultures or animals and the complex human system. Hence, they have been increasingly used for biomedical research, such as drug discovery and personalized healthcare. A promising strategy for their fabrication is 3D printing, a layer-by-layer fabrication process that allows the construction of complex 3D structures. In contrast, 3D bioprinting, an evolving biofabrication method, focuses on the accurate deposition of hydrogel bioinks loaded with cells to construct tissue-engineered structures. The purpose of the present work is to conduct a systematic review (SR) of the published literature, according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, providing a source of information on the evolution of organ-on-a-chip platforms obtained resorting to 3D printing and bioprinting techniques. In the literature search, PubMed, Scopus, and ScienceDirect databases were used, and two authors independently performed the search, study selection, and data extraction. The goal of this SR is to highlight the importance and advantages of using 3D printing techniques in obtaining organ-on-a-chip platforms, and also to identify potential gaps and future perspectives in this research field. Additionally, challenges in integrating sensors in organs-on-chip platforms are briefly investigated and discussed., The authors are grateful for the funding of FCT through the projects NORTE-01-0145- FEDER-029394, NORTE-01-0145-FEDER-030171 funded by COMPETE2020, NORTE2020, PORTUGAL2020, and FEDER. This work was also supported by Fundação para a Ciência e a Tecnologia (FCT) under the strategic grants UIDB/04077/2020, UIDB/00319/2020, UIDB/04436/2020 and UIDB/00532/2020. This work was also funded by AMED-CREST Grant Number JP20gm1310001h0002., Violeta Carvalho acknowledges the PhD scholarship UI/BD/151028/2021 attributed by FCT. Inês Gonçalves acknowledges the PhD scholarship BD/08646/2020 attributed by FCT.

This study provides design of a low-cost and open source add-on device that enhances the functionality of the popular EVOM® instrument for transepithelial/endothelial electrical resistance (TEER) measurement. The original EVOM® instrument is designed for measuring TEER in transwell samples manually using a pair of Ag/AgCl electrodes. The inconsistency in electrode placement, temperature variation, and a typically large (12–24 h) time interval between measurements result in large data variabilities. Thus, to solve the current limitation of the EVOM® instrument, we built an add-on device using a custom designed electronic board and a 3D printed electrode holder that allowed automated TEER measurements in multiple transwell samples. To demonstrate the functionality of the device prototype, we monitored TEER in 4 transwell samples containing retinal cells (ARPE-19) for 67 h. Furthermore, by monitoring temperature of the cell culture medium, we were able to detect fluctuations in TEER due to temperature change after the medium change process, and were able to correct the data offset. Although we demonstrated the use of our add-on device on EVOM® instrument only, the concept (multiplexing using digitally controlled relays) and hardware (custom data logger) presented here can be applied to more advanced TEER instruments to improve the performance of those devices.

Age-related macular degeneration (AMD) is a major ophthalmic disease that causes visual impairment and blindness, particularly in elderly people. The developing choroidal vessels invade Bruch’s membrane and disrupt the monolayer of retinal pigment epithelial (RPE) cells. Subsequently, degeneration of photoreceptors occurs. Hence, subretinal transplantation of RPE cells to the site of degeneration would offer an ideal treatment providing the transplanted cells could generate a monolayer, reconstruct the RPE–photoreceptor interface, and inhibit further development of choroidal vessels. Although transplantation of autologous peripheral RPE cells has been tested by injection of cell suspensions using a syringe needle, limited visual improvement result due to the low viability of the injected cells, and their restricted distribution and integration into the subretinal tissue. Recently, we investigated the use of biodegradable polymeric nanosheets as cell scaffolds [1,2]. Because this system enabled the easy manipulation of the cell/nanosheet constructs with a syringe needle, it raises the possibility of transplanting cell sheets into a narrow space, such as the subretinal space, in a minimally-invasive manner. So far, poly(lactic-co-glycolic acid) (PLGA) was used as the material for the nanosheets. However, this biodegradable polymer is relatively inflammatory because it produces lactic and glycolic acids as degradation intermediates and these acid dissociation constants are about 3.8. In this study, we fabricated biodegradable nanosheets using polycaprolactone (PCL) and collagen as low inflammatory materials and investigated a minimally invasive and effective method for cell transplantation into the subretinal space (Fig.1(a)). Micropatterned nanosheets consisting of PCL, collagen and Nile red were prepared by a combination of spin-coating and micro-contact printing technique (Fig.1(b)). The diameter and thickness of the nanosheets used for the following experiments were 200 μm and 200 nm, respectively. The cell/nanosheet constructs were prepared by culturing rat retinal pigment epithelial (RPE-J) cells on the micropatterned nanosheets in a monolayer. A hand-made glass capillary needle was as thick as a 27G medical needle, and the tip angle was 30 degrees. Also, it was coated with 2-methayloxyethyl phosphorylcholine (MPC) to prevent cells from attaching the inner surface of the needle. The cell/nanosheet constructs and a sodium hyaluronate solution with the medium were loaded into the capillary needle. Subsequently, the cell/nanosheet was injected into the subretinal space through the sclera of rat. Several days after implantation, the rats were euthanized, and the eyeballs were enucleated. The micropatterned nanosheet was shown in Fig.1(c). The nanosheets had sufficient strength and flexibility to be handled in water. The shape or diameter of the nanosheets could be varied by adjusting the microstructure of the PDMS stamp. Also, the thickness of the nanosheets could be controlled by changing the concentration of the solution. As shown in Fig.1b, the cells formed a monolayer on the nanosheet of PCL/collagen. Finally, we attempted to deliver the RPE-J cell/nanosheet constructs to the subretinal space of a rat eye in vivo. The hand-made glass capillary needle shape allowed the needle insertion along the retina through the sclera. The fluorescence of PCL/collagen nanosheet and cells could be observed in the eyeballs under excitation light. Also, as shown in Fig.1(e), it was confirmed that the cell/nanosheet construct was successively delivered into the subretinal space. We fabricated biodegradable nanosheets using PCL and collagen as low inflammatory materials. Also, we developed a minimally invasive and effective method for cell transplantation into the subretinal space using capillary needles and confirmed that cell/nanosheet was successfully delivered. This method holds great promise for transplantation of monolayer cells and for the development of local cell delivery systems. In order to further improve this method, we plan to evaluate the inflammatory properties of the nanosheets and the efficacy of the delivered cells. References [1] J Suzuki, et al, “Electrochemical manipulation of cell populations supported by biodegradable polymeric nanosheets for cell transplantation therapy”, Biomaterials Science, 5(2), 216-222, (2017) [2] T Fujie, et al, “Micropatterned Polymeric Nanosheets for Local Delivery of an Engineered Epithelial Monolayer”, Advanced Materials, 26(11), 1699-1705, (2014) Figure 1

The placenta is a multifunctional organ essential for fetal development and survival and plays an important role in hormone production and the exchange of substances such as nutrients, gases, and waste products between the mother and fetus. Research on the mechanism of human placental development is important for elucidating the cause of infertility and intrauterine growth restriction and developing new medicines for them, but little is known about the mechanism. Studying the human placenta has been limited because of large species differences, the limited resources of the human placenta, and also the lack of an in vitro cell culture system that can be a good model of the human placenta. To accelerate the study of the human placenta, we recently established a human trophoblast stem (TS) cell lime from the human placenta [1]. This TS cell line could differentiate into the major placental trophoblast subpopulations, i.e. extravillous cytotrophoblast (EVT), and syncytiotrophoblast (ST). In vivo, EVT cells can invade the endometrium and remodel the maternal blood vessels, and ST cells can produce large quantities of placental hormones and mediate the maternal-fetal gas, nutrient, and waste exchange. The purpose of this study is to develop an in vitro human placental model that resembles the human placenta in vivo, using the TS cell line and microfabrication technology. In particular, we have attempted to fabricate a microfluidic device that recapitulates the function of fetal EVT cells that invade the endometrium and remodel the maternal blood vessels. This device could be an excellent model for studying the development and the function of the human placenta (Fig.1a). A two-layer microfluidic device was designed to imitate the fetal and maternal compartments in the placenta and to recapitulate the function of in vivo EVT cells regarding the invasion and remodeling. The upper layer has a small well(Φ4mm) for culturing EVT cells. The lower layer has a microchannel for the formation of perfusable vascular networks of human umbilical vein endothelial cells (HUVEC), as a cell culture space mimicking the maternal environment. A porous membrane was set between the upper layer and the lower layer, and this co-culture system was fabricated to aim to evaluate the migration and interaction of each cell in both layers. This two-layer microfluidic device was made using Polydimethylsiloxane (PDMS). To make each layer, PDMS was poured into silicon substrate molds that were made by photolithography and then cured. The polyester (PET) porous membrane underwent the silane-coupling-treatment and was inserted between the upper and lower layers. To bond all three components thoroughly, they were treated with oxygen plasma before assembly (Fig.1b). In the upper layer, for culturing EVT cells, TS cells were seeded to the small well and were differentiated into EVT cells by culturing cells in a differentiation medium containing Matrigel. In the lower layer, to form vascular networks of HUVEC, HUVEC suspension and a mixed solution of fibrin gel and thrombin were introduced into a channel. Two channels with multi micro-posts were also made in the lower layer to perfuse the fresh medium into the vascular system. The cells in the device were subjected to immunostaining and analyzed by fluorescence microscopy. Results of fluorescence microscopy showed that most of the TS cells in the upper layer were differentiated into EVT cells, which expressed an EVT cell marker HLA-G (Fig1.c). Furthermore, in the lower layer, HUVEC formed vascular networks with branched luminal structures. The fresh medium was successfully introduced into the networks through channels with micro-posts, and the vascular networks of HUVEC were able to be maintained for a long period of the time in this novel EVT-HUVEC co-cultured system (Fig.1c). In this device, these two types of cells are separated by a porous membrane and we would like to analyze in detail how these cells interact through the membrane in the future. Through the analysis, we hope to reveal the mechanism by which fetal EVT cells remodel the uterine vessels. We believe that this device would be an excellent model to examine the role of EVT cells in the human placenta, and we hope it will be a critical step in constructing a human placenta model. [1] Okae. H, Toh. H, Sato. T, Hiura. H, Takahashi. S, Shirane. K, Kabayama. Y, Suyama. M, Sasaki. H, and Arima. T, "Derivation of Human Trophoblast Stem Cells.", Cell Stem Cell 22, 50, 2018. Figure 1

Epithelial and endothelial cells line the surface of the body. Adjacent cells of both epithelial and endothelial cells are connected to each other via intercellular junction called tight junction that regulates the diffusion of molecules and ions between the apical and basolateral side of the body. Quantification of this barrier is important to evaluate the suitability of in vitro cellular barrier models for drug toxicity or transport studies. Transepithelial/endothelial electrical resistance (TEER), a quantitative technique to access electrical resistance of the cellular monolayer during culture has become one of the most popular quantitative techniques to evaluate the integrity of tight junction. The major advantage of the TEER technique over traditional permeability measurements is that it is quick, label-free, and non-invasive. As such, the TEER technique has been widely used to evaluate in vitro models such as the blood-brain barrier, the intestine epithelial barrier, the alveolar epithelial barrier, and the blood-retina barrier. However, the reported normalized TEER values of a specific cell type in transwell inserts are inconsistent between different research groups. A major cause of data variation is the manual technique that is used for measuring TEER. Typically, a “chopstick” like electrode pair is immersed inside a cell cultured semi-porous transwell insert membrane to measure TEER. Due to the variation in the alignment of the electrodes every time a TEER value is measured, the actual values vary significantly between measurements. A second problem with the conventional manual measurement process is time gap between each measurement (typically 12h or 24h) since a more frequent measurement requires frequent opening and closing of cell culture incubator door that can disturb the cells and introduce potential contamination. Thus, to enable consistency in TEER measurement, and allow automated real-time data acquisition in multiple samples, we built an add-on device (used atop EVOM®, a commercially available TEER instrument) consisting of a 3D printed electrode holder, a temperature sensor, and a custom designed data logger. The 3D printed electrode holder allowed data variation to be minimized by allowing perfect alignment of electrodes during measurement. The real-time data acquisition and in-situ temperature measurement allowed detection and correction of data offset during medium replacement process. The concept and device demonstration presented here should enable research labs to minimize data variations allowing them to produce consistent TEER results without having to spend extra time manually measuring multiple samples.

Microfluidic devices are gaining increasing popularity due to their wide applications in various research areas. Herein, we propose a two-layer multi-channel microfluidic device allowing for direct-contact cell-vessel co-culture. Using the device, we built a co-culture model of the outer blood-retina barrier (oBRB), mimicking the in vivo retinal pigment epithelial cells-Bruch membrane-fenestrated choroids. To demonstrate the versatility of the design, we further modified the device by inserting platinum electrodes for trans-epithelial electrical resistance (TEER) measurement, demonstrating the feasibility of on-chip assessment of the epithelial barrier integrity. Our proposed design allows for direct-contact co-culture of cell–cell or cell–vessel, modifiable for real-time evaluation of the state of the epithelial monolayers.

Continuous drug administration with better adherence to treatment and less invasive procedures is important in treating retinal diseases such as age-related macular disease. In this study, we report a drug-refillable device consisting of a silicone reservoir and an injectable gelatin/chitosan gel (iGel). The silicone reservoir was fabricated with polydimethylsiloxane (PDMS) using a computer-aided design and manufacturing to have micropores at a releasing side for uniaxial release to the sclera. A stainless steel wire and sheet were combined in the side and bottom of the reservoir to ensure flexibility and to fit on the curvature of the eyeball and prevent irritation to the sclera through the bottom of the reservoir. The drug was injected and formulated in the reservoir by in situ crosslinking of gelatin/chitosan gel with the crosslinker; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The in vitro release study using fluorescein molecules showed that the release rate from encapsulated iGel in the reservoir was slower than that from the original iGel. After reinjecting the iGel into the reservoir, the same release profile as the first injection was observed. The reservoir containing iGel was placed on the sclera of a rabbit and the distribution of 150 kDa fluorescein isothiocyanate-dextran (FD150) in the retina and choroid/retinal pigment epithelium (choroid/RPE) was studied. The cryosections showed that FD150 was observed in the choroid/RPE. Homogenates of the retina and choroid/RPE showed fluorescence during 12 weeks implantation, indicating the drug could be delivered to the retina by using the device. The drug filling was successful into the reservoir implanted on the sclera through the conjunctiva by using a needle. In conclusion, the refillable drug delivery device is a promising tool to administer drugs long-term by reinjection with less invasiveness to intraocular tissues.

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.

It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 μg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1730-1737, 2016.

A free-standing cell-cultured hydrogel sheet with stretchability was prepared for an in vitro cellular assay with mechanical stimulation. The anti-fouling surface of the stretchable double network (DN) hydrogel composed of poly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt) (PNaAMPS) and poly(N,N-dimethylacrylamide) (PDMAAm) was modified as the cell adhesive by additional polymerization of PNaAMPS at the surface region of the DN hydrogel in the depth of around 170 μm, while preserving the original stretchability and molecular permeability. The mechanically stable and flexible hydrogel sheet holding the culture medium has the advantage that it can be handled with tweezers in air, and cells adhered on the sheet maintain physiological activity during transportation of the film for in vitro assays. The stretchability of the free-standing culture sheet allows for the observation of the Ca2+ response of epidermal keratinocytes upon mechanical stimulation.