Mesh-supported submicron parylene-C membranes for culturing retinal pigment epithelial cells.

In this work, a mesh-supported submicron parylene-C membrane (MSPM) is proposed as an artificial Bruch's membrane for the therapy of age-related macular degeneration (AMD). Any artificial Bruch's membrane must first satisfy two important requirements. First, it should be as permeable as healthy human Bruch's membrane to support nutrients transportation. Secondly, it should be able to support the adherence and proliferation of retinal pigment epithelial (RPE) cells with in vivo-like morphologies and functions. Although parylene-C is widely used as a barrier layer in many biomedical applications, it is found that parylene-C membranes with submicron thickness are semipermeable to macromolecules. We first measure the permeability of submicron parylene-C and find that 0.15-0.30 µm parylene-C has similar permeability to healthy human Bruch's membranes. Blind-well perfusion cell viability experiments further demonstrate that nutrients and macromolecules can diffuse across 0.30 µm parylene-C to nourish the cells. A mesh-supported submicron parylene-C membrane (MSPM) structure is design to enhance the mechanical strength of the substrate. In vitro cells culture on the MSPM (with 0.30 µm ultrathin parylene-C) shows that H9-RPE cells are able to adhere, proliferate, form epithelial monolayer with tight intracellular junctions, and become well-polarized with microvilli, which exhibit similar characteristics to RPE cells in vivo. These studies have demonstrated the potential of the MSPM as an artificial Bruch's membrane for RPE cell transplantation.

A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.

OBJECTIVE: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. METHODS: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. RESULTS: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat's subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. CONCLUSION: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat's subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

Long-Term Transplant Effects of iPSC-RPE Monolayer in Immunodeficient RCS Rats.

Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.

Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

A refillable microfabricated drug delivery device for treatment of ocular diseases.

An implantable manually-actuated microfabricated drug delivery device was demonstrated as a new approach for delivering therapeutic compounds to ocular tissue in acute in vitro, ex vivo, and in vivo studies.

A reversible thermoresponsive sealant for temporary closure of ocular trauma.

Open globe injuries are full-thickness injuries sustained to the eye wall (cornea or sclera), which cause immediate drops in intraocular pressure that may lead to retinal detachment and permanent vision loss if not treated rapidly after injury. The current standard of care for open globe injuries consists of suturing the margins closed, but the technique can be time-consuming, requires specialized training and equipment, and can lead to patient discomfort, abrasion, and infection from eye rubbing. We engineered an injectable, thermoresponsive sealant (TRS) and a custom tool to occlude open globe injuries. The smart hydrogel sealant consists of physically cross-linked N-isopropylacrylamide copolymerized with butylacrylate. At low temperatures, it can be injected as a liquid, and when raised to body temperature, a heat-induced gelation converts the hydrogel into a solidified occlusion. The sealant can be repositioned or removed without causing additional trauma via exposure to cold water. In vitro and ex vivo assessments of mechanical adhesion to eye tissue revealed maintenance of intraocular pressure that is five times greater than the physiological range with reversible seal strength comparable to cyanoacrylate (super glue). In vivo assessment in a rabbit model of ocular trauma demonstrated ease of use for TRS deployment, statistically significant improvement in wound sealing, and no evidence of neurotoxicity, retinal tissue degradation, or significant chronic inflammatory response after 30 days of exposure. Given the advantages of body heat-induced gelation, rapid reversible occlusion, and in vivo safety and efficacy, shape-adaptable TRSs have translational potential as smart wound sealants for temporary occlusion of surgical incisions or traumatic injuries.

In vitro and in vivo evaluation of ultrananocrystalline diamond for coating of implantable retinal microchips.

In this work, ultrananocrystalline diamond (UNCD) thin films were evaluated for use as hermetic and bioinert coatings for a retinal microchip. These films were deposited on highly conductive Si substrates at different temperatures (from 400 to 800 degrees C), using microwave plasma enhanced chemical vapor deposition with argon-rich Ar/CH4 gas mixtures and different relative amounts of hydrogen (0-20%). Scanning electron microscopy studies showed that all the films are dense and continuous. Results of cyclic voltammetry test revealed that when there was <2% of hydrogen in the plasma, the film obtained renders the surface electrochemically inactive, with very low leakage currents ( approximately 4 x 10(-7) A/cm2 at +/-5 V). In addition, in vivo tests of the UNCD-coated Si samples were performed by implanting them in the eyes of rabbits for 4-6 months within the eye physiological environment. According to all these results, it was concluded that UNCD is a promising candidate for use as the encapsulating coatings for implantable retinal microelectronic devices.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

PURPOSE: To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS: Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. RESULTS: Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS: These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

An Innovative Surgical Technique for Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results.

BACKGROUND AND OBJECTIVE: To develop a safe and efficient surgical procedure for subretinal implantation into porcine eyes of a human embryonic stem cell-derived retinal pigmented epithelium (hESC-RPE) monolayer seeded onto a Parylene-C scaffold. This implant is referred to as CPCB-RPE1. MATERIALS AND METHODS: Ultrathin Parylene-C scaffolds were seeded with hESC-RPE and surgically implanted into the subretinal space of Yucatan mini pigs (n = 8). The surgery consisted of pars plana vitrectomy, induction of a limited retinal detachment, and peripheral retinotomy for insertion of the monolayer using a novel tissue injector, followed by silicone oil tamponade injection, laser photocoagulation around the retinotomy site, and inferior iridectomy. Oral cyclosporine was administered from day 1 and during the entire follow-up period. Three months later, the animals were euthanized and the eyes and major organs were submitted for histological analysis. Adjacent sections underwent immunohistochemical analysis to detect human cells using anti-TRA-1-85 (human blood group antigen) antibody and DAPI antibodies. RESULTS: The cell monolayer was immunopositive for TRA-1-85 3 months after implantation and migration from the Parylene-C scaffold was not detected. One eye had a mild inflammatory reaction around the implant that was negative for human biomarkers. No intraocular or systemic tumors were detected. CONCLUSION: The hESC-RPE cells survived for 3 months in this animal model. The surgical procedure for subretinal implantation of CPCB-RPE1 is feasible and safe, without cell migration off the scaffold or development of ocular or systemic tumors.

Use of rotational sutures for limited retinal translocation: a new technique for superior limited macular translocation.

PURPOSE: To report a modified surgical technique for retinal translocation in eyes with subfoveal choroidal neovascularization. DESIGN: Experimental animal study. METHODS: Nine pigmented rabbits were used consecutively to apply this technique. Placement of inferotemporal scleral imbrication sutures was followed by vitrectomy with posterior hyaloid separation. Balanced saline solution (BSS) was injected subretinally with a 30G needle or with a 39G hydrodissection cannula and viscous fluid injector to detach one retinal quadrant. Under low intraocular pressure, the imbrication sutures were tied, the sclerotomy sites were closed, and intravitreal air tamponade was injected. Rotation sutures were passed and the eye globe was rotated approximately 90 degrees counterclockwise. The rotation sutures were removed after 24 hours. Retinal photographs were taken and fundus examination was performed on postoperative days 1, 2 and 7. The animals were sacrificed after 7 to 10 days for postmortem macroscopic examination. RESULTS: The entire procedure was performed in nine eyes of nine rabbits. In eight eyes, translocation could be seen on the first postoperative day after removal of the rotation sutures. The average amount of translocation was 667 microm (range: 500-800 microm) in a nasal to inferonasal direction. Vitreous hemorrhage occurred at the end of surgery in one eye due to hypotony. Iatrogenic small retinal breaks occurred in 2 eyes but did not prevent completion of the procedure. There was only a temporary hyperemia of the eyelids and conjunctiva. CONCLUSION: Limited retinal translocation using rotational sutures provided a predictable amount of translocation in the planned direction. This technique is expected to be useful for superior macular translocation in humans.

In vitro electrical properties for iridium oxide versus titanium nitride stimulating electrodes.

Stimulating electrode materials must be capable of supplying high-density electrical charge to effectively activate neural tissue. Platinum is the most commonly used material for neural stimulation. Two other materials have been considered: iridium oxide and titanium nitride. This study directly compared the electrical characteristics of iridium oxide and titanium nitride by fabricating silicon substrate probes that differed only in the material used to form the electrode. Electrochemical measurements indicated that iridium oxide had lower impedance and a higher charge storage capacity than titanium nitride, suggesting better performance as a stimulating electrode. Direct measurement of the electrode potential in response to a biphasic current pulse confirmed that iridium oxide uses less voltage to transfer the same amount of charge, therefore using less power. The charge injection limit for titanium nitride was 0.87 mC/cm2, contradicting other reports estimating that titanium nitride was capable of injecting 22 mC/cm2. Iridium oxide charge storage was 4 mC/cm2, which is comparable to other published values for iridium oxide. Electrode efficiency will lead to an overall more efficient and effective device.

Evaluation of ultrasound-assisted thrombolysis using custom liposomes in a model of retinal vein occlusion.

PURPOSE: To study the potential efficacy of ultrasound (US) assisted by custom liposome (CLP) destruction as an innovative thrombolytic tool for the treatment of retinal vein occlusion (RVO). METHODS: Experimental RVO was induced in the right eyes of 40 rabbits using laser photothrombosis; the US experiment took place 48 hours later. Rabbits were randomly divided into four equal groups: US+CLP group, US+saline group, CLP+sham US group, and no treatment group. The latter three groups acted as controls. Fundus fluorescein angiography and Doppler US were used to evaluate retinal blood flow. RESULTS: CLP-assisted US thrombolysis resulted in restoration of flow in seven rabbits (70%). None of the control groups showed significant restoration of retinal venous blood flow. CONCLUSIONS: US-assisted thrombolysis using liposomes resulted in a statistically significant reperfusion of retinal vessels in the rabbit experimental model of RVO. This approach might be promising in the treatment of RVO in humans. Further studies are needed to evaluate this approach in patients with RVO. Ultrasound assisted thrombolysis can be an innovative tool in management of retinal vein occlusion.

Mini drug pump for ophthalmic use.

PURPOSE: To evaluate the feasibility of developing a novel mini drug pump for ophthalmic use. METHODS: Using principles of microelectromechanical systems engineering, a mini drug pump was fabricated. The pumping mechanism is based on electrolysis and the pump includes a drug refill port as well as a check valve to control drug delivery. Drug pumps were tested first on the bench-top and then after implantation in rabbits. For the latter, we implanted 4 elliptical (9.9 x 7.7 x 1.8 mm) non-electrically active pumps into 4 rabbits. The procedure is similar to implantation of a glaucoma aqueous drainage device. To determine the ability to refill and also the patency of the cannula, at intervals of 4-6 weeks after implantation, we accessed the drug reservoir with a transconjunctival needle and delivered approximately as low as 1 microL of trypan blue solution (0.06%) into the anterior chamber. Animals were followed by slit lamp examination, photography, and fluorescein angiography. RESULTS: Bench-top testing showed 2.0 microL/min delivery when using 0.4 mW of power for electrolysis. One-way valves showed reliable opening pressures of 470 mmHg. All implanted devices refilled at 4-6 weeks intervals for 4-6 months. No infection was seen. No devices extruded. No filtering bleb formed over the implant. CONCLUSIONS: A prototype ocular mini drug pump was built, implanted, and refilled. Such a platform needs more testing to determine the long term biocompatibility of an electrically-controlled implanted pump. Testing with various pharmacological agents is needed to determine its ultimate potential for ophthalmic use.

A comparison of retinal prosthesis electrode array substrate materials.

Simulations of artificial vision suggest that 1000 electrodes may be required to restore vision to individuals with diseases of the outer retina. In order to achieve such an implant, new technology is needed, since the state-of-the-art implantable neural stimulator has at most 22 contacts with neural tissue. A critical component of this system is the multi-channel, stimulating electrode array. This array must meet very challenging, competing requirements for manufacturing, integration, surgical handling, and biocompatibility. Our lab has evaluated 3 polymers as retinal prosthesis substrates: polyimide, parylene, and silicone.

Implantation of an inactive epiretinal poly(dimethyl siloxane) electrode array in dogs.

The aim of this study is to investigate the long-term, mechanical biocompatibility of a polymer microtechnology that can be used to position electrodes in close proximity to the retina. Poly(dimethylsiloxane) (PDMS) arrays were manufactured by soft-lithography at Lawrence Livermore National Laboratory. The PDMS implant measured 4 mm x 40 mm x 55-60 microm and included 4-8 electrodes. Micromolded ribs were placed at the perimetry for strength and ease of manipulation. The PDMS arrays were implanted epiretinally in four normal dogs, with a single retinal tack used in each case to hold the device on the retina. The mechanical effects of the implant were followed up after surgical implantation by photography, fluorescein angiography, optical coherence tomography (OCT), and electrophysiologic tests. An intraoperative retinal tear occurred in the first implanted dog, causing retinal detachment and necessitating termination. The remaining dogs experienced no gross complications secondary to the array implantation procedure. During the follow-up period of 2 months in one eye and 6 months in three eyes, OCT demonstrated that the arrays were in close contact with the retina. Fluorescein angiography showed good perfusion of the retina under the array. At the end of 6 months, there was no statistical difference from baseline in mean retinal thickness under the array (P=0.43) or peripapillary retinal nerve fibre layer thickness corresponding to the implanted area (P=0.34). The mean distance between the array and the retinal surface varied from 32 to 68 microm throughout the follow-up. Histopathologic evaluation of the retinal implantation site in eyes followed for 6 months showed a general preservation of the normal, layered retinal structure, except for some localized retinal thinning in two eyes, where the array frame had been in direct retinal contact. The PDMS substrate micro array is a new and promising technology that can be scaled to support a high-density retinal stimulating array. Its implantation and handling is surgically manageable, and it forms a mechanically stable, acceptable interface with the inner retinal surface.