Journal of Neural Engineering PAPER First steps for the development of silk fibroin- based 3D biohybrid retina for age-related macular degeneration (AMD) To cite this article: Nahla Jemni-Damer et al 2020 J. Neural Eng. 17 055003   View the article online for updates and enhancements. 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Neural Eng. 17 (2020) 055003 https://doi.org/10.1088/1741-2552/abb9c0 Journal of Neural Engineering RECEIVED 29 February 2020 REVISED 10 September 2020 ACCEPTED FOR PUBLICATION 18 September 2020 PUBLISHED 28 October 2020 PAPER First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD) Nahla Jemni-Damer1,2,3, Atocha Guedan-Duran1,2,3,4, Jasmin Cichy1,5, Paloma Lozano-Picazo6, Daniel Gonzalez-Nieto6,7,8,9, José Perez-Rigueiro6,7,8,10, Francisco Rojo6,7,8,10, Gustavo V. Guinea6,7,8,10, Assunta Virtuoso11, Giovanni Cirillo11, Michele Papa11, Félix Armada-Maresca12, Carlota Largo-Aramburu13, Salvador D Aznar-Cervantes14, José L Cenis14 and Fivos Panetsos1,2,8 1 Neuro-computing & Neuro-robotics Research Group, Complutense University of Madrid, Spain 2 Innovation Research Group, Institute for Health Research San Carlos Clinical Hospital (IdISSC), Madrid, Spain 3 These authors equally contributed to this article 4 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States of America 5 Faculty of Biology, Christian Albrechts University, Kiel, Germany 6 Center for Biomedical Technology, Polytechnic University of Madrid, Pozuelo de Alarcón, Madrid, Spain 7 Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain 8 Silk Biomed SL, Madrid, Spain 9 Department of Photonics Technology and Bioengineering. ETSI Telecomunications, Polytechnic University of Madrid, Spain 10 Department Materials Science, ETSI de Caminos, Canales y Puertos. Polytechnic University of Madrid, Spain 11 Division of Human Anatomy – Neuronal Networks Morphology Lab, Department Mental, Physical Health and Preventive Medicine, University of Campania ‘Luigi Vanvitelli’, Naples, Italy 12 Ophthalmology Service, La Paz University Hospital, Madrid, Spain 13 Experimental Surgery. University Hospital, La Paz (IdiPAZ), Madrid, Spain 14 Department of Biotechnology La Alberca (Murcia), InstitutoMurciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), E-30150, Spain E-mail: fivos@ucm.es Keywords: scaffolds, retinal pigment epithelium, neurons, cultured cells, in vitro, macular degeneration Abstract Age-related macular degeneration is an incurable chronic neurodegenerative disease, causing progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches can only slow down he progression of the disease. Objective. Feasibility study for a multilayered, silk fibroin-based, 3D biohybrid retina. Approach. Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Müller and mesenchymal stem cells ; creation of a layered structure glued with silk fibroin hydrogel.Main results. In vitro evidence for the feasibility of layered 3D biohybrid retinas; primary culture neurons grow and develop neurites on silk fibroin biofilms, either alone or in presence of other cells cultivated on the same biomaterial; cell organization and cellular phenotypes are maintained in vitro for the seven days of the experiment. Significance. 3D biohybrid retina can be built using silk silkworm fibroin films and hydrogels to be used in cell replacement therapy for AMD and similar retinal neurodegenerative diseases. 1. Introduction Age-related macular degeneration (AMD) is an incurable chronic neurodegenerative disease, which causes progressive loss of the central vision and even blindness at its most advanced stage [1– 4]. Triggered by heterogeneous, complex and still poorly understood pathogenetic mechanisms, it is the main cause of irreversible loss of vision among over 65 y.o. subjects, and affects over 196 million people worldwide [1, 2, 4, 5]. AMD acts on Bruch membrane, a 2–5 µm-thick layer that lies between the choriocapillary and the retinal pigment epithelium (RPE) (figure 1). Under normal conditions this membrane (i) reg- ulates the exchange of molecules, oxygen, nutri- ents and metabolic residues between the retina and the choroid (ii) together with the retinal pig- ment epithelium constitutes the external blood- retinal barrier and (iii) gives physical support for the correct adhesion and functioning of epithelium cells [6, 7]. However, aging [8–10], oxidative stress © 2020 IOP Publishing Ltd https://doi.org/10.1088/1741-2552/abb9c0 https://crossmark.crossref.org/dialog/?doi=10.1088/1741-2552/abb9c0&domain=pdf&date_stamp=2020-10-28 https://orcid.org/0000-0002-3518-0262 https://orcid.org/0000-0003-2972-729X https://orcid.org/0000-0001-8298-8398 https://orcid.org/0000-0002-6609-7453 https://orcid.org/0000-0003-0897-411X mailto:fivos@ucm.es J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 1. Left. Schematic representation of the human eye with all its structures. Right. Magnification of the macula, central area of the retina affected by age-related macular degeneration, in which the distribution of neurons in the neural retina and structures such as the retinal pigment epithelium, Bruch’s membrane and the choroid can be observed. [11–15] and several genetic factors [16–18] cause the Bruch membrane to weaken, thicken and become more rigid, which provoke a loss of permeability and other physiological dysfunctions [6]. Dysfunc- tions lead to a slowdown of the molecular exchange, and a consequent accumulation of lipids, cellular and metabolic residues (originated in the retinal pigment epithelium and the surrounding tissue known also as ‘drusen’) which are accumulated between the basal membrane of the retinal pigment epithelium and the Bruch membrane [19–22]. All these changes cause a chain of pathological events: loss of the integrity of the external blood-retinal barrier, death of retinal pig- ment epithelium cells, then, death of photoreceptors and, at the end, permanent loss of the visual function. Furthermore, the most aggressive stage of the disease (neovascular/wet form) is characterize by the gener- ation of sub-choroidal neovessels that penetrate the subretinal space through the epithelium and the dam- aged Bruch membrane, and alter the 3D structure of the retina [22]. All current treatments are aimed at relieving symptoms and at slowing down the progression of the disease [23]. Due to the high prevalence and the wide socio-economic impact of the disease, as well as to the absence of clinical treatments and the growing aging of the world population, the development of effective therapies forAMD is an imperative need forwhich are collaborating biologists, doctors, chemists/biochem- ists and engineers [10, 24, 25]. Several research studies, suggest that the cre- ation and transplantation of artificial membranes with RPE cells slow down the progression of AMD and even improve visual function [26–30]. Photore- ceptor transplantation has also shown good poten- tial as therapy for retinal degenerative pathologies with photoreceptor death, as in AMD advanced stages [28, 31–34]. Furthermore, with the great advances in differentiation of neural retinal cells from human stem cells, an unlimited number of photoreceptors can be obtained for transplantation purposes.However,most of the studies being conduc- ted with photoreceptors focus on injecting photore- ceptor precursors at the site of the injury or gen- erating 3D retinal organoids in vitro [27, 34–38]. These last structures have shown great potential for transplanting damaged cells. However, they have the disadvantage of their spheroid shape, which hinders synaptic integration and functional interaction with the host. Despite all progresses, the great challenge of AMD cell therapies is the development of retinal implants, either biological or biohybrid, capable to establish synaptic connectivity with the host tissue and restore visual function [23, 25]. Furthermore, great effort is being made to develop biomimetic Bruch membranes with good adherence and survival properties for the cells to be transplanted and therefore capable to replace the damaged retinal tissue [39]. Researchers are working with both, synthetic and natural materi- als. The former include poly-methyl-methacrylate (PMMA) [40], modified polytetrafluoroethylene (PTFE) [41], polyethylene terephthalate (PET) [42], poly-lactic-co-glycolic acid (PLGA) [43], poly-e- caprolactone (PC) [44], etc, while the later include silk fibroin [45], collagen [46], hyaluronic acid [47], gelatin [48], etc. Cell replacement therapies are a promising approach for AMD due to the surgical accessibil- ity of the damaged tissue and the small number of transplanted cells needed in comparison with other organs. The characteristic degeneration of retinal pigment epithelium and, in advanced stages of AMD, photoreceptors’ atrophy, has led to the development of cellular approaches aiming the replacement of these components [49]. RPE and photoreceptors have been transplanted both as a suspension and integ- rated into biomaterials. The first strategy has proven lower efficacy in terms of transplanted cell survival and integration [50] than biomaterial-encapsulated cells [51], which has led researchers to focus on the latter. 2 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Researchers are working with biomater- ials for RPE transplantation such as colla- gen [52–58], gelatin [59, 60], silk fibroin [61, 62] ; polyethylene terephthalate (PET) [63]; poly(lactic acid-co-glycolic acid) (PLGA) [64, 65], etc. Several biomaterials have shown good biocompatibility and allowed the formation of an RPE polarized monolayer in vitro [61, 66–69]. This RPE cells can be extracted from adult human RPE stem cells, RPE derived from embryonic stem cells or from induced pluripotent stem cells (iPSCs), etc. Maintenance of RPE cells´ functionality on the bio- material is crucial in the success of the transplant and only some in vitro studies assessed it [70–72]. The highest RPE survival once transplanted was reported for a gelatin and silk fibroin/polycaprolactone/gelatin scaffold reaching 3 months on porcine [73] and rab- bit eyes [74], respectively. Also noteworthy are the results of RPE implanted on a plasma modified poly- dimethylsiloxane coated with laminin (PDMS-PmL) scaffold, which preserved macular function up to 2 years after implantation with no inflammation in porcine eyes [75]. Clinical studies of RPE transplant- ation on a vitronectin-coated polyester membrane [76], a Parylene membrane [77] and PLGA scaffold [78] are still in early stages. Biomaterials used in the fabrication of scaffold- ing structures for retinal neural implants include alginate, collagen, polycaprolactone, PLGA, polyben- zyl glutamate (PBG), silk fibroin, etc [79–82] . Stud- ies in vitro and in vivo have tested photoreceptor precursor cells (PPCs), photoreceptors, retinal stem cells (RSCs), etc on several substrates for retinal cell replacement with promising results [83–87]. Never- theless, clinical studies testing named cells on AMD are limited [88–90]. As with RPE, transplanted cells have to remain functional to integrate effectively meaning, in this case, creating synapses with bipolar cells, which remains a challenge. As we have mentioned before, both RPE and photoreceptor transplants alone have achieved excel- lent results, but, in these formats, the crucial integ- ration between the different cell types is difficult. To resolve this problem, 3D cell cultures with struc- tural complexity and functionality similar to the nat- ural retina such as retinal organoids began to be researched. Organoids show better tissue survival and interaction between different types of cells. However, their folded structure complicates its implantation and provokes cell death in its internal layers. Besides, different cell types in organoids stay in an embryonal state [91]. To overcome the named obstacles, several studies analyze the use of biomaterials for the devel- opment of a flat retinal organoid [92]. In the present paper we describe the ‘in vitro’ development and test of a 3D multilayered gel- coated biohybrid retina (figure 2). Our biohybrids are based on RPE cells and neurons, cultured separ- ately on semirigid thick silk fibroin films and then joined together and glued by means of silk hydro- gels enriched with supporting cells (Müller cells and mesenchymal stem cells (MSCs)). Photoreceptors do not form a specific layer, but they are merged with the other retinal cells. In this first study, our objective was to prove the suitability of the biomaterials for the growth and adhesion of all types of retinal neurons, to verify that they are adequate for the RPE cells and also allow the survival of the neuronal cells that are more sensitive to the extraction process. Layered biohybrids built with Bruch’s membrane and RPE cells capable to support the survival of the retinal neurons can be extremely useful for the therapy of age-related macu- lar degeneration (AMD) and similar retinopathies, in a relatively near future. 2. Methods The biomaterials used as scaffolds must be biocom- patible, biodegradable, inert, sterilizable, provide an immunoprotective environment for the adhesion, survival and functionality of the cells, avoid the dis- persion of the cells after the implant and at the same time promote and maintain the adequate phenotype of the cells. In addition, theymust be very porous, fine (<10 µm, mimicking Bruch’s membrane) and mech- anically capable of resistingmanipulation during sur- gery. Silk fibroin-made biomaterials do fulfill these criteria. Silk fibroin is the main component of silk- worm silk fibers and one of the most promising bio- materials in nature [93] because of its high biocom- patibility with body tissues [94] and in particular with those of the central nervous system [94, 95]. Silk fibroin hydrogels employed as vehicles for stem cell therapy enhance the engraftment capabilities of the implanted stem cells [96] while silk fibroin films effi- ciently deliver neuroprotective compounds that pen- etrate the blood–brain barrier [97] and silk fibroin- collagen copolymers combined with neural stem cells promote functional recovery from spinal cord injuries [98]. Finally, in the context of retina and macular degeneration, silk fibroin scaffolds and silk nano- particle have been used to promote the growth of retinal pigment epithelium cells, implanted or injec- ted in the subretinal space [99, 100]. It has also been shown that the use of nanofibrous silk fibroin/poly(L- acid lactic-co-ε-caprolactone) (PLCL) membranes promote retinal progenitor cells (RPCs) proliferation, growth and differentiation to specific retinal neurons of interest in vitro [44]. Therefore, we can say that the use of silk fibroin as a scaffold may have poten- tial applications in retinal cell replacement therapy. Silk silkworm fibroin is a biodegradable material susceptible to biological degradation by proteolytic enzymes such as chymotrypsin, actinase, carboxylase or protease XIV [101, 102]. Under in vivo experimental conditions our gels’ degradation reached percentages of 80% after 4 weeks 3 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 2. Schematic representation of the process for the development of the layered biohybrid retinal implant. Once the ocular globes of 1he rat are enucleated, they are subjected to various incubations with different enzymes to achieve separation of the neural retina, from which the neurons are obtained as well as the retinal pigment epithelium cells from which the epithelial cells are extracted. These retinal pigment epithelium cells and neurons are then seeded on silk fibroin biofilms to create the different layers of the biohybrid retina. Once the layers are obtained, they are assembled to create the 3D structure of the artificial retina. implantation in the striatum [94] and similar degrad- ation rates in the brain have been reported by other authors [103]. 2.1. Silk fibroin supporting structure 2.1.1. Production of freeze-dried silk fibroin and fluorescein isothiocyanate-stained silk fibroin Silk fibroin purification was achieved from bombyx mori cocoons following the protocol described in (Fernández-García et al, 2016). Briefly, cocoons were cut and boiled in 0.2% (w/v) Na2CO3 solution for 30 min at 121 ◦C, 103.4 kPa to remove the seri- cin. After that, silk fibroin fibers were repeatedly rinsed with tap water, followed by rinsing in distilled water and let to dry overnight at room temperat- ure. Then, dried fibers were dissolved in 9.3 M lith- ium bromide (LiBr) solution during 4 h at 60 ◦C under stirring. LiBr was cleaned from solution by dialysis against 50 mM Tris base, pH 8 and 40 ◦C for 72 h using BioDesignDialysis TubingTM (MWCO 3.5 KDa). After dialyzing, the solution was cent- rifuged at 5000 rpm for 20 min and the super- natant was frozen at −800 ◦C and lyophilized. In the case of fluorescein isothiocyanate-stained silk fibroin, 0.5 mg ml−1 of fluorescein isothiocyanate was added to the silk fibroin solution. It was mixed for 1.0 h and a step of 24 h of dialysis against 50mMTris base, pH 8 and 4 ◦Cwas done. After that, the solution was frozen similarly and lyophilized. Finally, both, silk fibroin and fluorescein isothiocyanate-stained silk fibroin were grinded, and powder was sealed and stored at −20 ◦C until being used. 2.1.2. Hydrogels fabrication Silk fibroin and fluorescein isothiocyanate-stained silk fibroin powder at a ratio of 1:1 was used to prepare solutions of 2.0 and 8.0% (w/v) in phos- phate buffer saline (PBS) and stirred at 600 rpm for 1.0 h. Then, solutions were centrifuged at 4,000 rpm for 20 min and supernatant was collected. Each 6.0 ml of solution was sonicated (Branson 450 Soni- fier, Branson Ultrasonics Corporation, Danbury, CT) coupled to a 3.0 mm diameter Tapered Microtip at 15% of amplitude for 15 min. Then, sonicated solu- tion was filtered with 0.22 µm filters and transferred to a sterile multiwell dish of 24 wells (300 µl/well) or to a cylindrical mold for cell culture or mechanical characterization respectively. 2.1.3. Films fabrication Silk fibroin powder was dissolved in hexafluoroiso- propanol at 8% (w/v) stirred at 600 rpm. Solution was filtered with 0.22 µm filters and transferred to ø35 mm sterile Petri dishes (500 µl/dish) or to ø90 mm non-treated Petri dishes for cell culture or characterization respectively. Dishes were covered with parafilm and left 16 h under a hood. Holes were done to the parafilm to help hexafluoroisopropanol evaporate. Afterwards, films produced over the dishes were insolubilized by incubation for 1.0 h with 80% (v/v) solution of ethanol. Then, films were washed with solutions of ethanol at 70% (v/v) for 30 min, 50% 10 min and 20% 10 min. Finally, sterile PBS 1x was used to wash the films for 10 min three times to remove the remaining ethanol. 2.1.4. Mechanical characterization of the materials Hydrogels were cut in 8 mm-height cylinders. Com- pression assays were carried out with an Instron 4411 mechanical tester at 1.0mmmin−1 work speed. Films were cut in 35× 5mmpieces while wetted with water. Traction assays in water were applied with an Instron 55 434 A at 1.0 mmmin−1 traction speed. 2.1.5. Biocompatibility study of the materials Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose supplemented with 10% (v/v) of Fetal Bovine Serum, 1.0% (v/v) penicillin-streptomycin and 1.0% (v/v) L-glutamine was used as cell culture medium for CD1 mice bone marrow MSCs. A total of 15 000 cells/cm2 were seeded over hydrogels and films and the cultures were incubated at 37 ◦C, 5% of CO2 and 95% of relative humidity. Biocompatibility was checked at different times of culture. Tissue culture 4 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al plastic (TCP) was used as positive control of cell growth. 2.2. In vitro building and testing of the biohybrid retinas 2.2.1. Use of animal tissues Experiments were carried out with adults female Lister Hood rats, weighing 200 to 250. All anim- als were treated according to European (Directive 86/609/EEC) and national (RD 53/2013) regulations in force on the protection of animals that are used for experimentation andother scientific purposes and Law 39/2015 of the Procedure Common Administra- tion of Public Administrations for the care of anim- als. The study of the biohybrid retina has been carried out in accordance with the ARVO Declaration (The Association for Research in Vision and Ophthalmo- logy, ARVO) for the use of animals in Vision Research and Ophthalmology. All the experiments have been previously approved by the Ethical Committee of the Research Institution and with the approval of the Committee of Experimentation and Animal Welfare of the Community of Madrid (Spain). 2.2.2. Retina dissection, primary cell cultures and retinal pigment epithelium cells isolation Isolation of retinal pigment epithelium cells was per- formed according to the procedure described in [104] with some modifications. Briefly, 3-month-old Lister Hood rats were sacrificed via CO2 inhalation followed by cervical dislocation. Eyes were extracted using scissors and immediately dipped in a 3.5 cm petri dish containing freshGBSS (Sigma-Aldrich, St. Louis, CA) + 1% penicillin-streptomycin (P/S) (GIBCO, NY) on ice. All tissue around the eyeball was removed, then eyes were washed three times (3 × 5 min each) under a sterile hood, and placed in 2%Hyaluronidase (GIBCO, NY, USA) in GBSS for 45 min at 37 ◦C in a 5% CO2 incubator. Then, enzyme activity was neut- ralized by dipping the eyeballs in DMEM-F12+ 10% FBS+ 1% P/S (M) medium. Eyes were then dissected along the cornea-sclera edge and cornea, iris epithelium, and lens were gently removed. Retina and posterior sclera were put in M and incubated for 25 min in presence of 37 ◦C 5% CO2 to help the separation of the neural ret- ina from the retinal pigment epithelium. After neural retina removal, the retinal pigment epithelium layer attached to the choroid was cut into a four-leaf shape structure. Epithelium sheet was carefully scrapped of from the inside to outside and the fragments on each flap were collected and transferred into a 1.5 ml Eppendorf tube prior to centrifugation at 1,000 rpm for 5 min at room temperature. The supernatant was discharged, and retinal pigment epithelium cells were gently resuspended in a 1.0 ml complete medium (DMEM-F12 + 10% FBS + 1% P/S). Cells from two rats’ eyes were put into one well of the 48-well plate with 500 µl complete medium. Cells were incubated in growth medium containing in presence of 37 ◦C 5% CO2. 2.2.3. Retina dissection, primary cell cultures and neurons isolation Cell isolation performed according to the proced- ure described in Rocco et al, 2015 [105] with some modifications. Briefly, retinas were dissected out from whole eyeball according to the standard retina peel- off (as already explained in the previous section) and treated with 0.25% Trypsin-HBSS solution (Sigma- Aldrich, St. Louis, CA,USA) for 10min for single cells release. Single cells (5,000 cells/well) were seeded on precoated (10 mg poly-L-lysine and 100 mg laminin) silk fibroin films in 48-well plates and cultured in Neurobasal media supplemented with 5% FBS and 1% P/S. Under a microscope cells put in wells with complete medium were confirmed to be evenly dis- tributed before transferring into a 37 ◦C 5% CO2 incubator. Culture dish was kept in undisturbed con- dition for at least 48 h to allow cell attachment to the dish. Culture medium was half-changed every 2 d through removing half of it and then by adding fresh culture medium. 2.2.4. Müller cell line culture Müller cell line MU-PH1 cells were grown to con- fluence in growth medium containing DMEM- F12 + 10% FBS + 1% P/S in presence of 37 ◦C 5% CO2 incubator. At early passage (P6-P8), cells were trypsinized, centrifuged at 1,000 rpm for 7 min and seeded to the films. Before cell seeding, filmswere pre- conditioned overnight in culture medium at 37 ◦C. Cells were left incubating undisturbed at 37 ◦C and 5% CO2 for 48 h to prevent them from flowing out the film. Culture medium was changed every 2 days. 2.2.5. Mesenchymal cells cultures Isolation and expansion of CD-1 mice bone marrow mesenchymal stem cells was performed as described in (Martin-Martin et al, 2019, Scientific Reports). Cell viability on silk fibroin films was assayed Calcein-AM (eBioscience; Cat# 65–0853) staining for 20–30min at 37 ◦C. Cells were imaged under a fluorescence micro- scope (Leica DMI3000, Nussloch, Germany) coupled with a Leica DFC340FX camera. Spreading area and number of processes inmesenchymal cells growing on treated plastic (TCP) or silk fibroin films were evalu- ated with ImageJ software (NIH). 2.2.6. Attachment of cells to silk fibroin scaffolds Silk fibroin films (figure 3) were sterilized and pre- conditioned for cell culture by two successive rinses with ethanol 70◦ for 1.0 h each in a laminar flow cab- inet, and then rinsed with ethanol 50◦ and 30◦ for 10 min each, followed by a final rinse, thoroughly with sterile, deionized water. Then, they were pre- conditioned overnight in culture medium at 37 ◦C. After that, they were adhered to culture well floors 5 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 3. Top. Appearance of the gelled silk fibroin hydrogel as a yellow mass in a tube (A), and of the silk fibroin biofilm in a culture plate (B). Bottom: Curves of elastic moduli of the two materials (hydrogels (C) and biofilms (D)), maximum deformation and tension test. n= 5 for each test. and their surfaces were modified by overnight coat- ingwith 10mg poly-L-lysine+ 100mg laminin under UV light, to increase cells adhesion. Polymers were then rinsed three times with GBSS. Cultured cells were dissociated into cell suspensions separately and 300 µl were seeded onto each scaffold. After cell seed- ing, mats were incubated at 37 ◦C for 30 min to allow cells attachment, mediumwas added to prevent drying, and cells were incubated for further 30 min. Finally, growth medium was added into the inserts in the wells and cultured in the humidified CO2 incub- ator. Medium was changed every 2 days. 2.2.7. In vitro creation of biohybrid retinas The implant of the biohybrid into the eye is not an easy process because (i) fragility does not allow biohybrids to be picked up and introduced to the eye tissue (ii) inflammation provoked to the neural tis- sue by the surgical intervention must be controlled and (iii) to survive, cells in the biohybrid retina must be surrounded by a neuroprotective environment. For these reasons we decided to wrap the biohybrid up with silk fibroin gel (figure 3) with mesenchymal stem cells and Müller cells encapsulated in it, two types of cells well known for their neuroprotective and neuroregenerative secretoms. The structure of the artificial retina was created by attaching a biofilm with retinal pigment epithe- lium cells to a film with neurons, thus allowing con- tact between the two types of cells. In a cylindric (ø8.0 mm) mold we create a layer of silk fibroin hydrogel with mesenchymal stem cells and Müller cells; then we insert a film with retinal pigment epithelium cells; we add a second film with neur- ons; and we conclude by adding a final layer of silk fibroin hydrogel. The hydrogel/mesenchymal stem cells mixture protects cells and keeps the struc- ture together, provides nutrients and neuroprotective and anti-inflammatory molecules while isolating the implant from the hostile environment, thus favoring the survival of the implanted cells. 2.2.8. Evaluation of cell survival (immunohistochemistry/immunofluorescence) After 7 d culturing, cells on films were washed with PBS (0.1 M NaCl, pH 7.2), fixed in cold 4% buf- fered paraformaldehyde (PFA) (15 min), permeab- ilized in 0.01.0% Triton X-100 (15 min), washed with PBS (0.1 M NaCl, pH 7.2) and then incub- ated with primary antibodies overnight at 37 ◦C: anti-RPE-65 (RPE cells), anti-NeuN (neurons) and glial fibrillary acidic protein (anti-GFAP, Müller glia). Samples were then rinsed three times with PBS and reacted with species-specific immunoglobulin conjugated. The secondary antibodies were Cy3 AffiniPure Goat Anti-Rabbit IgG (H + L) (1:800) (111–165-003, Jackson ImmunoResearch) and Fluor- escein (FITC) AffiniPure Goat Anti-Mouse IgG (H + L) (115–095-003, Jackson ImmunoResearch), incubated for 3 h at room temperature. Sections were rinsed three times with PBS and cell nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; 1.0 µg ml−1, Molecular Probes). 3. Results 3.1. Silk fibroin supporting structure Ten to twenty micrometer-thick silk fibroin films and fluorescein isothiocyanate-stained hydrogels (figure 3) were used to build our biohybrids. Mech- anical tests (n= 5 in both cases) showed very similar elastic moduli (13.44 ± 1.67 and 14.90 ± 1.88 MPa, respectively) suitable to be used for the same implants 6 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al [45]. Maximum deformations were in the range of 10%–20% for both materials but with maximum tension of 1194.64± 191.55 kPa and 1.76± 0.09 kPa for films and hydrogels, respectively. We have previously described the excellent com- patibility of silk fibroin to support the growth of specific cellular lineages (Martin-Martin et al, 2019, Scientific Reports). Figure 4 illustrates the ability of silk fibroin (8%) to favor the expansion and prolif- eration of mesenchymal stem cells, a cell population with strong repercussion in regenerative medicine. In agreement with our previous study (Martin-Martin et al, 2019, Scientific Reports) and independently of the substrate (TCP or silk fibroin) these cells were perfectly able to attach and extend processes showing the typical spindle-shaped fibroblast-like morphology characteristic of this cell population (figure 4). Silk tolerability has also been demonstrated in the context of macula and retina degeneration. In a couple of studies, no inflammatory response, neither cytotoxicity was observed after in vivo trans- plantation of silk fibroin scaffolds or nanoparticles after subretinal or intravitreal injection respectively [106, 107]. In both formats, this material supports the growth and function of RPE cells. Other formu- lations based on silk fibroin-tropoelastin composites, support the growth of RPE cells [108]. In an interest- ing study, an advanced photovoltaics semiconductor- based organic prosthesis formulated with silk fibroin was implanted in the degenerated retina to pro- duce photo-stimulation of residual neural networks promoting visual recovery in an animal model of photoreceptor degeneration [109]. These in vitro and in vivo approaches provide the necessary framework to push the fabrication of like-retina structures based on this material. 3.2. In vitro building and testing of the biohybrid retinas 3.2.1. Creation of a biohybrid retina using cell cultures and artificial substrates We created a 3D retina structure with two silk fibroin biofilms attached each other, thus allowing seeded neurons on one film to contact with the seeded ret- inal pigment epithelium cells in the other. A mes- enchymal stem cells-enriched glued the two films together (figure 5). Best results were obtained with films built with functionalized silk fibroin, which provides binding sites for retinal cells (figure 5). Func- tionalization allows us to adapt the substrate for dif- ferent cell types. 3.2.2. Survival of in vitro created biohybrid retinas Films are not cytotoxic and favor cell viabil- ity and adhesion (figure 4). Cells seeded on PLL/laminin-coated films show a higher adhesion rate than on not treated ones. The adequacy of the fabricated substrates is demonstrated by the survival of the cells obtained by primary culture for up to 7 d in vitro (figures 5–8). Cultured retinal cells need a suitable substrate for their survival, so we tested dif- ferent biofunctionalized silk fibroin films and com- binations of seeded cells and ways to assemble the retina. 3.2.3. Creation of a favorable environment for the survival of biohybrid retinas Artificial 3D retinas’ structure is achieved by means of the two attached biofilms which allowed contact establishment between the neurons and the seeded retinal pigment epithelium cells. The combination of support cells, molecules and artificial substrates provided an evenmore favorable environment for the survival of the neurons and the retinal epithelium cells. We used Ø1 mm cylindrical molds of which we are first inserting a layer of silk fibroin hydro- gel with mesenchymal cells included inside, followed with a filmwith the retinal pigment epithelium cells, a second film with the neurons/Müller cells and finally a new layer of silk fibroin hydrogel withmesenchymal cells. We use the hydrogel as a method to protect and keep the structure together, providing nutrients and creating an environment that isolates the implant from the host that favors the survival of the implanted cells. As can be seen in figures 6–9, cells adhere to the films. However, once the entire sandwich is mounted with the silk fibroin hydrogel, it absorbs a large part of the cells and keeps them inside (figure 8). There- fore, the combination of support cells, artificial cells and substrates provide a favorable environment for the survival of implanted cells. 4. Discussion Retinal degeneration is a leading cause of blindness in the world. Its etiology is complex and involves genetic defects and aging associated with stress. In addition to gene therapies for retinal degeneration, cellular therapies have been extensively explored to restore vision in both preclinical animal models and clinical trials. Stem cells from different tissue sources and their derived lineages have been tested to treat ret- inal degeneration. Currently, it is not known whether visual improvement by cell therapy is due to the fact that the grafted cells are capable of replacing lost retinal neurons; or due to the secretion, by these cellular implants, of neuroprotective and neur- otrophic factors that protect the host retina; or both causes [110]. In replacement approaches for cells lost in the posterior retina, both photoreceptors and retinal pro- genitor cells injected subretinally manage to migrate to the correct retinal lamina, form local synapses, and therefore restore some functionality in animalmodels [111, 112]. 7 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 4. Cultures of supporting cells. (A) Representative images of MSCs seeded on plastic (TCP), 8% silk fibroin hydrogels and 8% silk fibroin films. Scale bar: 150 µm. (B) Quantification of MSCs culture on plastic and 8% silk fibroin hydrogels after 24 h and 72 h in culture. (C) Quantification of MSCs culture on plastic and 8% silk fibroin film after 24 h and 72 h in culture. Figure 5. in vitro creation of a 3D multilayer biohybrid retina. To provide shape and resistance to the layered structure, as well as to surround the embedded cells with a favorable environment, the bilayer was covered with a silk fibroin hydrogel biofunctionalized with MSCs, extracted from mice bone marrow (Step 1). These cells secrete neurotrophic factors that improve the survival and viability of both, RPE and neural retinal cells. MSCs are integrated into the porous structure of the silk gel (Central column (Step 2) green cells, fluorescein-stained confocal microscopy image). Once the layers that are going to form the retina are obtained (Step 3, left), they are assembled to get the 3D structure (Step 3, right). After attachment of RPE cells and neurons to the silk fibroin biofilms, the artificial retina is coated with the gel+MSCs, to get the final multilayered biohybrid (Step 4, top). Right column (Step 4), bottom: optical microscopy image of a cross-section of the multilayered biohybrid retina, the film and the embedded cells within the silk fibroin gel are clearly shown. Recently Liu et al (2020) carried out implant experiments of different cell types in the subret- inal space of transgenic mice that develop ret- inal degeneration. They observed that implanting immortalized sphere stem cells (SDSC) into the retina gave rise to cells that were able to differentiate by expressing neural, photoreceptor markers, or syn- thesizing pigment in vivo in the host retina. In addition, a small part of these cells migrated to the layer corresponding to their differentiation, but the vast majority remained in the injection sites. So, it appears that, although there is potential for 8 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 6. RPE cells from primary cells culture seeded over a PLL/LAM-coated silk fibroin film. Cells are double stained with anti-RPE-65 specific protein marker (green), and DAPI cell nuclei counterstaining (cyan). RPE-65-positive cells project to the X and DAPI-positive to the Y axis. The white cross allows the selection and check of each cell. integration, the retinal protection and the improve- ment they observed was mainly due to a parac- rine function of these cells, secreting protective factors. To this paracrine effect, it would be added that the transplanted cells do not survive very long and that some tissues (such as the mammalian central nervous system) have very limited in situ migration [113]. This approach to cell therapy for neurons such as retinal ganglion cells (RGCs), which require even greater development, axonal growth, and synapto- genesis than other retinal cells (greater complexity of local and distal circuits), is even more complex [114]. Transplanted RGCs have been observed to pro- ject dendrites into the inner plexiform layer [115], and stem cells transplanted directly onto the optic nerve head project a process through it [116]. How- ever, transplanted cells located far from the nerve head are unable to extend their axons [117]. The lack of growth and guide factors in adult retinas, added to the reactive environment present due to the injury or disease suffered, may be the cause of not achieving complete integration and functional- ity of cell transplants. To alter their fate, retinal neur- ons must be encapsulated in a permissive microen- vironment for growth, protect themselves from the diseased or harmful environment, present with cell- binding molecules, and be exposed to appropriate mechanical properties to induce and initiate growth [118]. In this way, a functional integration of the implanted cells could be achieved. In the present work we show the promising future of silk fibroin as the base of biohybrid retinas. Our principal achievements are (i) fabrication of a silk fibroin-based layered biohybrid retina (ii) primary culture neurons growth and develop neurites on silk fibroin biofilms alone, or (iii) in presence of other cells cultivated on the same biomaterial, (iv) biocom- patibility of the biomaterials, (v) maintenance of the cellular phenotypes and (vi) creation of a neuropro- tective environment through the use of a silk fibroin- based hydrogel. The fact of getting these different lay- ers with different cell types present in the retina, gives us the opportunity to create an artificial retina based on cell layers that will later allow the creation of con- nections between the different cultured cells and thus develop a stratified biohybrid 3D retina with a struc- ture similar to the real one (through the pores we would get a co-culture between the cells of the dif- ferent layers). The silk fibroin hydrogel gives consist- ency, support and protection to the layers with cul- tured cells. In addition, adding mesenchymal stem cells as suppliers of growth factors to improve the viability and increase the cell longevity (long-term cell survival) of said cultured cells on the biofilms. We used primary culture cells, a cellular source closely resembling natural retinas and allowing us to study the interaction between different cell types with relatively simple experimental protocols. These cells need a suitable substrate for their survival and that facilitates their adhesion. The substrates created are obtained through the functionalization of silk fibroin, which provides binding sites for retinal cells and allows us to adapt the substrate for each cell type (figure 2). We were able to create the structure of the 3D retina with two biofilms attached, thus allowing contact between the neurons and the seeded retinal pigment epithelium cells. 9 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 7. RPE and retinal neural cells on silk fibroin biofilms. Rows 1 and 2: RPE cells monolayer from primary cells culture. Row 1, cells seeded over a PLL/LAM-coated silk fibroin. Row 2, cells seeded over a non-coated silk fibroin film. (A) Immunostaining of RPE cells specific protein markers (anti-RPE65, green); (B) cell nuclei counterstained with DAPI (blue) and (C) merge of the two images. As can be seen in these two rows, there is a higher number of RPE cells in the treated film compared to the untreated one, indicating that coating the silk fibroin film with PLL/LAM improves cell adhesion and survival. Rows 3 and 4: neurons monolayer from neuron primary cells culture. Row 3, cells seeded over a PLL/LAM-coated silk fibroin. Row 4, cells seeded over a non-coated silk fibroin film. (A) Immunostaining of neurons specific protein markers (anti-NeuN, red); (B) cell nuclei counterstained with DAPI (blue) and (C) merge of the two images. As can be seen in these two rows, there is a higher number of neurons on the treated film compared to the untreated one, in addition, on the former, the neurons have a better shape, and it can also be seen that some have grown even neurites (see arrows). 4.1. How neurons will integrate with each other? The materials described in this work not only allow us to create a support, but also to be functional- ized with molecules present in the extracellular mat- rix, becoming the perfect substrate for cell adher- ence and growth. The presence of laminin in the films will provide us with the creation of a surface where epithelium cells can grow and can also polar- ized, an essential characteristic for co-culture and for the epithelium cells to be able to create connections with other cell types. These types of materials are also compatible with the functionalization with other types of molecules, such as poly-L-lysine, a necessary component for seeding and survival of neuronal cells, muchmore sensitive to the substrate inwhich they are seeded, and where it is essential not only the presence of a good substrate but also the creation of a favorable environment for its survival. Previous research has shown that transplant- ing retinal stem cells into a biodegradable poly- mer scaffold increases the rate of cell survival [51]. On the other hand, the dimensional conformation 10 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 8. (A) Reconstruction of vertical sections of a 3D multilayer biohybrid retina showing cell survival after 7 d in vitro. Once the biohybrid retina is assembled within the silk fibroin gel, cells are maintained alive. Immunostaining of RPE cells with specific anti-RPE65 protein marker, anti- (B, green), of neurons with anti-NeuN specific protein marker (D, red), and cell nuclei DAPI in specific marker (C, blue). The hydrogel emits fluorescence with the same excitation wavelength as anti-RPE-65 (also in green). or porosity of the scaffold seems to promote the union and subsequent differentiation and orientation of the retinal progenitor cells [119]. Other studies also defend the idea that scaffold topography influ- ences orientation, as it affects cell adhesion, mor- phology, proliferation, differentiation, and migra- tion [32, 120, 121]. Both synthetic polymers and bio- logical materials can be used to generate scaffold- ing. Natural materials (such as alginate, collagen, etc) are suitable to mimic the native tissue archi- tecture [120]. Various synthetic polymers, such as poly-ε-caprolactone and poly-d-co-glycolic l-lactic acid, offer increasedmechanical strength and control- lable degradation rates [122, 123]. However, synthetic polymers tend to cause less biological activity [121]. Silks have been extensively studied for tissue engineering applications. The advantages of this material is that silk fibroin is composed, among other amino acids, of glycine, serine and alanine. This com- position allows it to be easily functionalized with spe- cific bioactive molecules to aid cell binding. Once the sericin that coats fibroin is removed, it leaves a fibrous material that can dissolve and process in many ways, including transparent films with mechanical proper- ties and degradation behavior that can be tailored to a specific application [124]. Different studies show how silk fibroin materials can meet the needs of specific cellular repairs in the engineering of bone, cartilage, adipose tissue, blood vessels and ligaments [125–133]. Another application is the generation of artificial nerve guides, as demon- strated (Tang et al 2009). In this study, they observe the good neuro-compatibility of silk fibroin with hip- pocampal neurons, arguing that fibroin is a suitable material to treat injuries or diseases of the central nervous system. Silk fibroin has also been tested in grafts for peripheral nerve regeneration, with results very close to those of autografts [134, 135]. There is not much research using silk fibroin for the growth and implantation of retinal neurons. However, Wittmer et al (2011) [136], used aligned and multifunctionalized electrospun silk nanofibers with BDNF and CTNF. This scaffolding together with neurotrophic factors significantly increased the sur- vival of the RGC and stimulated the growth of its neurites, which followed the pattern of parallel fibers. Their results suggest that biomaterials based on bio- logically active (biofunctionalized) silk protein may be a good guide support for RGC. However, in this study, they did not manage to perform implants in retinal explants or in vivo, so they did not analyze the migration capacity. Another example is Zhang et al (2015) [44], in which they use scaffolds of silk fibroin and PLCL in a 1:1 ratio. They achieve rapid proliferation of retinal stem cells, high cytocompatib- ility, and improved differentiation to specific retinal 11 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 9. Four and seven days cell viability test over silk fibroin gels: live (green)–dead (red) assay by calcein (green)–propidium iodide (red) staining of the cells. MU-PH1, neural and RPE cells. neurons in vitro; compared to other scaffolding and control. Several studies have shown that scaffolds fromdif- ferent biomaterials allow the development and axonal extension of RGCs [32, 114, 117, 118, 121, 136–138]; but this usually occurs in different directions, and even in a multidirectional way. Tissue engineering of the nervous system has incorporated soluble and immobilized factors, as well as protein gradients, through a variety of methods [139–142], promoting cell survival, axonal cone growth, proper orientation, etc [117]. One of the important factors for the success of artificial constructs is the contribution of an appro- priate cellular environment. Thanks to the facility of obtaining and high performance, mesenchymal stem cells (MSCs) of the bone marrow or adipose tissue, are optimal candidates for a wide variety of applic- ations in regenerative medicine such as cell support in the regeneration of the peripheral nervous sys- tem. The neurons that make up the retina main- tain their homeostasis thanks to the presence of Müller cells, which participate in the recycling of K+, GABA and glutamate ions produced by the intense metabolic activity of the retina. In addition, it has been seen that after damage occurs at the level of the retina, these Müller cells are capable of pro- ducing neurotrophic factors and even proliferate to promote repair. The hydrogel/supporting cells mix- ture protects cells and keeps the structure together, provides nutrients and neuroprotective and anti- inflammatory molecules while isolating the implant 12 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al from the hostile environment, thus favoring the sur- vival of the implanted cells. 4.2. Will the existing dysfunctional Bruch’s membrane impede the normal movement of biological materials and thus impede the function of the implant? An important question is to which extent the existing dysfunctional Bruch’s membrane would impede the normal movement of biological materials and thus impede the function of the implant. Normally, there would be no impediment to the normal exchange of biological materials nor to the proper functioning of our biohybrid retina. Our biohybrid aims at repla- cing the damaged retinal tissue. In particular, the silk fibroin biofilm on which we seed pigment epithelial cells will replace the dysfunctional Bruch membrane. Thanks to its thickness and porosity there will be no problems for a correct exchange of biological mater- ials between the host choroid (damaged natural ret- ina) and the implanted biohybrid one. Pores diamet- ers were in the range of 1–9 µm (figure 10), very similar to [143]. Pores are small enough to keep the supporting cells (e.g. MSCs or Müller cells) into the silk fibroin hydrogel, impeding their diffusion through the host tissue and protecting them from the attacks of the immune system. However, they are big enough to allow both, the arrival of metabol- ites to these cells and the diffusion of MSCs/Müller’s neurotrophic, neuroprotective and neuroregenerat- ive secretome towards the implanted cells and the surrounding host tissue [96, 144]. The silk fibroin hydrogel will provide protection against the immune response and structural consistency at the implanta- tion time [145]. The shape, adhesion, surface conformation, migration functions, and ultimately the fate of cells are known to be governed by the properties of cell- bearing substrates. The latter are typically limited to the topography of the substrate surface, its mechan- ical rigidity, and the bioactive properties of the sub- strate (i.e. the ability of the substrate to target peptides and proteins in a cell). The use of 2D or 3D scaffold- ing has been documented to be an efficient strategy to overcome the limitations of cell transplantation: low cell survival and integration at the transplant site and difficulties in keeping cells injected into target cells [50]. Scaffolds for retinal stem cell (RSC) transplanta- tion can be freely classified into three different types: cylindrical scaffolds designed to mimic the vertical 3D organization of cells in the retina; fibrous scaf- folds designed to mimic the microstructure of the extracellular matrix and hydrogel scaffolds designed to replicate the mechanical properties of the retina. Each type has its advantages and limitations [146]. Cylindrical scaffolds offer orientation of cell growth within the scaffold and cell protection against implantation forces, as well as controlling cell differentiation within them, either by promoting or inhibiting it. However, the rate of cell migration from the implant to the different layers of the host ret- ina, in the few studies in which tests are carried out in vivo, is not very high; having registered amaximumof less than 50% of cells that migrate from the scaffold. In addition, this type of constructions, when made with synthetic polymers, it is necessary to function- alize them with adhesion molecules such as laminin [146]. Fibrous scaffoldsmimic the structure of the extra- cellular matrix. The fibers manage to create a large surface area for cell attachment while creating inter- connected pores for nutrient transport. These struc- tures can be chemically and biologically modified during their construction process; conferring spe- cific advantages to these scaffolds. This allows, for example, to increase the integration rate of the cells implanted in the retina. In general, fibrous scaffolds promote better migration of transplanted cells in nat- ive and degenerate retinas. However, the cell differen- tiation capacity within this type of scaffolding is less compared to the previous ones [146]. In the case of hydrogels, their stiffness can be adapted to match that of the CNS tissue. In addi- tion, there are currently in situ gelation gels, in which cells are implanted as isolated neurospheres or as individual cell suspension; but when they come into contact with the retinal environment, they gel. This property greatly facilitates the implant. However, they have a very limited capacity to promote themigration of the transplanted cells to the retina, and the cells mainly migrate to the RPE [146]. Based on the reviewed literature, a fibroin nan- ofiber scaffold would allow directional adhesion, pro- liferation, and development of retinal neurons. On the other hand, the characteristics of this biomater- ial allow its functionalization with different biological factors of growth, protection, differentiation, signal- ing, etc. This will allow a scaffold to be generated in which cells can differentiate, migrate within it, gen- erate neurites, extend and polarize; obtaining func- tional neurons similar to those found in vivo. On the other hand, as mentioned above, fibrous scaffolds together with their modifications allow better migra- tion of cells to the retina. This characteristic can be exploited in silk fibers. This, together with the fact that silk fibroin allows designs of different degrees of degradability, makes it possible to design a scaffold that will further favor the migration of cells for integ- ration into the retina in vivo. 4.3. In vivo injection and the potential problems and solution that will rise due to this layered hybrid retina The implant of the biohybrid into the eye is not an easy process due to: (i) fragility does not allow biohybrids to bemanipulated and introduced into the eye tissue (ii) inflammation provoked to the neural 13 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al Figure 10. Optic microscopy microphotographs showing the surface of the porous silk fibroin films. Magnification 40X and 1OOX. Bars 1OOµm (left) and 20 µm (right). tissue by the surgical intervention must be controlled and (iii) in order to survive, cells in the biohybrid ret- ina must be surrounded by a neuroprotective envir- onment. For these reasons we decided to wrap the biohybrid up with silk fibroin gel (figure 3) with mes- enchymal stem cells and Müller cells encapsulated in it, two types of cells well known for their neuropro- tective and neuroregenerative secretome. First of all, the material with which the scaf- fold is built must comply with the biocompatibil- ity property, to avoid any toxic response or immune reaction [147]. Scaffolds must also be ultrathin (few micrometers thick), implantable, and flexible to avoid tissue damage; but mechanically strong to resist. Furthermore, in order to promote adequate cell adhe- sion, scaffolds must show sufficient binding signals [148, 149]. On the other hand, these cell transplant constructs must (1) have an enhanced ability to pro- mote cell migration; (2) allow the development of dendrites and axons; (3) lengthening of the RGC axons towards the optic nerve head, and (4) regen- erating the axons over long distances in the injured nerve. In addition, possible biochemical and topo- graphic modifications of the material surface must be added to improve cell differentiation of implanted cells [50]. Thanks to the use of silk fibroin, the structures created are completely biocompatible, being entirely inert and not producing an excessive immunological rejection. In addition, the different types of formats that can be obtained using silk fibroin-based bioma- terials allows us to use: (i) a substrate easy to manipu- late and to be implanted in the form of a biofilm, and (ii) a hydrogel that presents a stiffness similar to the one present in the host tissue, that avoids the rejection of the implant due to the flexibility of the construct. The cells we incorporated in the hydrogel sur- rounding the implant enhance the survival of the construct and favor its integration with the host tissue. They serve as a cellular interface between the implanted cells and the host tissue, and their secretome protect against immune and inflammatory reactions and promote the regeneration of the dam- aged tissues. Finally, the preliminary results that we obtained, cellular survival during the 7 d of our study and the maintenance of a stable configuration in their layered structure, suggest that this approach could be use- ful in long-term implantology for AMD treatments. Showing the ability to produce a stable structure that can be subsequently implanted to study its integration with the host retina. 4.4. Further questions It must be acknowledged that the implementation of a working silk fibroin-based biohybrid retina entails many additional challenges beyond the scope of this study as for example, control of cross-linking density and polymer concentration of the film/gel influence the mechanical properties of the film, including pore size and permeability. Mechanical and elastic proper- ties should be in the range of native retina and provide stability for cells seeded on it in terms of specifica- tion (when using undifferentiated cells) and function [150]. Acuity will depend on the maximum density and correct organization of neural cells that will be achieved to growth on the biofilm and in particular of photoreceptors, bipolar and retinal ganglion cells. Pores diameters, in addition to provide mech- anical/elastic stability, they should allow axons to growth through them. Although intuitively this large pore size should allow the exchanging of soluble molecules/factors responsible of signaling process, cross-linking density and polymer concentration might differently influence the ability of this bioma- terial to permeate soluble factors that are required to maintain cell survival, axonal guiding and functional rewiring. Currently we are working on the temporal dynamics of permeoselectivity of silk fibroin gels to deliver molecules in function of its structure (charge, molecular weight, hydrophobicity). It is clear that a compromise should be reached between mechanic- al/elastic properties for cell fitness and pore size for axonal elongation and delivery of neuroprotective/re- generative factors that anticipates complexity. Ad hoc, controlled silk fibroin degradation is another of the important challenges. In the injured central nervous system, peripheral neutrophils 14 J. Neural Eng. 17 (2020) 055003 N Jemni-Damer et al and monocytes/macrophages as well as resident microglia, are probably responsible for these degrad- ative effects. Degradation depends of inflammatory signals, since a hostile microenvironment exacer- bates the degradation of silk (AMD, brain stroke, etc, manuscript under preparation). At molecular level is largely unknown which proteases might degrade silk fibroin; although in vitro essays identified several proteases (not related to the central nervous sys- tem) that could degrade silk fibroin in a few weeks [151]. The identification of precise factor/s respons- ible for silk fibroin degradation in the central nervous system (and by extension in the retina) will allow us to design functionalized silk-based biofilms (for example carrying proteases inhibitors) to control silk fibroin degradation during pre-defined thera- peutic time-windows. It is worthy to mention that silk biodegradation could be useful also for thera- peutic purposes, since some silk fibroin byproducts are neuroprotective for neurodegenerative diseases [152]. 5. Conclusion In the present paper we provided in vitro evidence for the feasibility of layered biohybrid retinas built with silk silkworm fibroin and cultures of different types of cells: retinal epithelium, retinal neural, Müller and mesenchymal stem cells. Silk fibroin-made biofilms have adequate resist- ance, are easily biofunctionalized, act as a perfect sup- port for cell growth and allow us to create different layers, resulting in increasingly complex structures, while maintaining the ability of the seeded cells to survive and favoring their possible interaction. Our biohybrids are also resistant for physical manipula- tions, that means they are suitable to be implanted for both, animal experiments and clinical studies. Next steps are the in vivo testing of the present work and, in parallel, the engineering of silk-based biohybrid retinas with human iPSCs. Also testing 3D bioprinting technologies to speed up the develop- ment of these retinal prostheses and get closer to the clinical application. Acknowledgments The authors gratefully acknowledge financial support received from the Spanish Ministerio de Economía y Competitividad Spain through grants MAT2016- 76847-R, MAT2016-79832-R and MAT2015-66666- C3-3-R and predoctoral FPI grant (AGD) as well as research contract (ADAC) from theNational Institute for Agricultural and Food Research and Technology (INIA) and the Spanish State Research Agency (AEI) program INIA-CCAA, from the Comunidad deMad- rid through grants Neurocentro-B2017/BMD-3760 and IND2018/BMD-9804, ERDF/FEDER Operative Program of the Region of Murcia (Project No. 14- 20/20). Authors also acknowledge preliminary work of Rocío Fernández Sierra (Silk Biomed, CTB-UPM) and Rebeca Gallego Ruiz (CTB-UPM, UCM), help in experimental work of Nuria Alfageme López, Nora Serrano Bengoechea, Cristina Castro Dominguez, Paula Álvarez Montoya y Miguel Terriza Roberto (UCM, IdISSC) and the invaluable technical support of Soledad Martínez (CTB-UPM). ORCID iDs Nahla Jemni-Damer  https://orcid.org/0000-0002-3518-0262 Daniel Gonzalez-Nieto  https://orcid.org/0000-0003-2972-729X José Perez-Rigueiro  https://orcid.org/0000-0001-8298-8398 Michele Papa  https://orcid.org/0000-0002-6609-7453 Fivos Panetsos  https://orcid.org/0000-0003-0897-411X References [1] Michalska-Małecka K et al 2015 Age related macular degeneration – challenge for future: pathogenesis and new perspectives for the treatment European Geriatric Medicine 6 69–75 [2] Ambati J and Fowler B J 2012 Mechanisms of age-related macular degeneration Neuron 75 26–39 [3] Lacour M, Kiilgaard J F and Nissen M H 2002 Age-related macular degeneration Drugs & Aging 19 101–33 [4] Jager R D, Mieler W F and Miller J W 2008 Age-related macular degeneration New Engl. J. 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Introduction 2. Methods 2.1. Silk fibroin supporting structure 2.1.1. Production of freeze-dried silk fibroin and fluorescein isothiocyanate-stained silk fibroin 2.1.2. Hydrogels fabrication 2.1.3. Films fabrication 2.1.4. Mechanical characterization of the materials 2.1.5. Biocompatibility study of the materials 2.2. In vitro building and testing of the biohybrid retinas 2.2.1. Use of animal tissues 2.2.2. Retina dissection, primary cell cultures and retinal pigment epithelium cells isolation 2.2.3. Retina dissection, primary cell cultures and neurons isolation 2.2.4. Müller cell line culture 2.2.5. Mesenchymal cells cultures 2.2.6. Attachment of cells to silk fibroin scaffolds 2.2.7. In vitro creation of biohybrid retinas 2.2.8. Evaluation of cell survival (immunohistochemistry/immunofluorescence) 3. Results 3.1. Silk fibroin supporting structure 3.2. In vitro building and testing of the biohybrid retinas 3.2.1. Creation of a biohybrid retina using cell cultures and artificial substrates 3.2.2. Survival of in vitro created biohybrid retinas 3.2.3. Creation of a favorable environment for the survival of biohybrid retinas 4. Discussion 4.1. How neurons will integrate with each other? 4.2. Will the existing dysfunctional Bruch's membrane impede the normal movement of biological materials and thus impede the function of the implant? 4.3. In vivo injection and the potential problems and solution that will rise due to this layered hybrid retina 4.4. Further questions 5. Conclusion Acknowledgments References