{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:06:03Z","timestamp":1760144763117,"version":"build-2065373602"},"reference-count":42,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T00:00:00Z","timestamp":1715731200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Zimin Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Gels"],"abstract":"<jats:p>Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel\u2013RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.<\/jats:p>","DOI":"10.3390\/gels10050336","type":"journal-article","created":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T11:31:52Z","timestamp":1715772712000},"page":"336","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Sequential Fabrication of a Three-Layer Retina-like Structure"],"prefix":"10.3390","volume":"10","author":[{"given":"Yahel","family":"Shechter","sequence":"first","affiliation":[{"name":"Shmunis School of Biomedicine and Cancer Research, Faculty of Life Science, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roni","family":"Cohen","sequence":"additional","affiliation":[{"name":"Shmunis School of Biomedicine and Cancer Research, Faculty of Life Science, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Namestnikov","sequence":"additional","affiliation":[{"name":"Shmunis School of Biomedicine and Cancer Research, Faculty of Life Science, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Assaf","family":"Shapira","sequence":"additional","affiliation":[{"name":"Shmunis School of Biomedicine and Cancer Research, Faculty of Life Science, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adiel","family":"Barak","sequence":"additional","affiliation":[{"name":"Division of Ophthalmology, Tel Aviv Medical Center, Tel Aviv 6423906, Israel"},{"name":"Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aya","family":"Barzelay","sequence":"additional","affiliation":[{"name":"Division of Ophthalmology, Tel Aviv Medical Center, Tel Aviv 6423906, Israel"},{"name":"Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3153-9928","authenticated-orcid":false,"given":"Tal","family":"Dvir","sequence":"additional","affiliation":[{"name":"Shmunis School of Biomedicine and Cancer Research, Faculty of Life Science, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"The Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Sagol Center for Regenerative Biotechnology, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,15]]},"reference":[{"key":"ref_1","first-page":"353","article-title":"Age-Related Macular Degeneration (AMD): Pathogenesis and Therapy","volume":"58","author":"Nowak","year":"2006","journal-title":"Pharmacol. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1167\/iovs.10-5619","article-title":"Optical Coherence Tomography and Autofluorescence Findings in Areas with Geographic Atrophy Due to Age-Related Macular Degeneration","volume":"52","author":"Fleckenstein","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"doi-asserted-by":"crossref","unstructured":"Cruz-Pimentel, M., and Wu, L. (2023). Complement Inhibitors for Advanced Dry Age-Related Macular Degeneration (Geographic Atrophy): Some Light at the End of the Tunnel?. J. Clin. Med., 12.","key":"ref_3","DOI":"10.3390\/jcm12155131"},{"doi-asserted-by":"crossref","unstructured":"Bhutto, I., and Lutty, G. (2012). Understanding Age-Related Macular Degeneration (AMD): Relationships between the Photoreceptor\/Retinal Pigment Epithelium\/Bruch\u2019s Membrane\/Choriocapillaris Complex. Mol. Asp. Med., 33.","key":"ref_4","DOI":"10.1016\/j.mam.2012.04.005"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.preteyeres.2013.11.001","article-title":"Macular Dystrophies Mimicking Age-Related Macular Degeneration","volume":"39","author":"Saksens","year":"2014","journal-title":"Prog. Retin. Eye Res."},{"doi-asserted-by":"crossref","unstructured":"Shapira, A., and Dvir, T. (2021). 3D Tissue and Organ Printing\u2014Hope and Reality. Adv. Sci., 8.","key":"ref_6","DOI":"10.1002\/advs.202003751"},{"doi-asserted-by":"crossref","unstructured":"Rahmati, M., Mills, D.K., Urbanska, A.M., Saeb, M.R., Venugopal, J.R., Ramakrishna, S., and Mozafari, M. (2021). Electrospinning for Tissue Engineering Applications. Prog. Mater. Sci., 117.","key":"ref_7","DOI":"10.1016\/j.pmatsci.2020.100721"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1007\/s11465-018-0496-8","article-title":"sheng Fabrication of Scaffolds in Tissue Engineering: A Review","volume":"13","author":"Zhao","year":"2018","journal-title":"Front. Mech. Eng."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.molmed.2022.03.001","article-title":"Strategies of Pluripotent Stem Cell-Based Therapy for Retinal Degeneration: Update and Challenges","volume":"28","author":"Maeda","year":"2022","journal-title":"Trends Mol. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1136\/bjo.2009.176305","article-title":"Human Retinal Pigment Epithelium (RPE) Transplantation: Outcome after Autologous RPE-Choroid Sheet and RPE Cell-Suspension in a Randomised Clinical Study","volume":"95","author":"Krebs","year":"2011","journal-title":"Br. J. Ophthalmol."},{"doi-asserted-by":"crossref","unstructured":"Hinkle, J.W., Mahmoudzadeh, R., and Kuriyan, A.E. (2021). Cell-Based Therapies for Retinal Diseases: A Review of Clinical Trials and Direct to Consumer \u201cCell Therapy\u201d Clinics. Stem Cell Res. Ther., 12.","key":"ref_11","DOI":"10.1186\/s13287-021-02546-9"},{"doi-asserted-by":"crossref","unstructured":"Barzelay, A., Algor, S.W., Niztan, A., Katz, S., Benhamou, M., Nakdimon, I., Azmon, N., Gozlan, S., Mezad-Koursh, D., and Neudorfer, M. (2018). Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress. Stem Cells Int., 2018.","key":"ref_12","DOI":"10.1155\/2018\/9682856"},{"doi-asserted-by":"crossref","unstructured":"Krief, B., Algor, S.W., Nakdimon, I., Elhikis, A., Benhamou, M., Kadmon, A.S., Keren, S., Ohana, O., Feldman, I., and Cnaan, R.B. (2021). Retinal Lineage Therapeutic Specific Effect of Human Orbital and Abdominal Adipose-Derived Mesenchymal Stem Cells. Stem Cells Int., 2021.","key":"ref_13","DOI":"10.1155\/2021\/7022247"},{"doi-asserted-by":"crossref","unstructured":"Sharma, R., Khristov, V., Rising, A., Jha, B.S., Dejene, R., Hotaling, N., Li, Y., Stoddard, J., Stankewicz, C., and Wan, Q. (2019). Clinical-Grade Stem Cell-Derived Retinal Pigment Epithelium Patch Rescues Retinal Degeneration in Rodents and Pigs. Sci. Transl. Med., 11.","key":"ref_14","DOI":"10.1126\/scitranslmed.aat5580"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1146\/annurev-pharmtox-010919-023245","article-title":"Retinal Pigment Epithelium Replacement Therapy for Age-Related Macular Degeneration: Are We There Yet?","volume":"60","author":"Sharma","year":"2020","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"doi-asserted-by":"crossref","unstructured":"Wang, L., Wu, W., Gu, Q., Liu, Z., Li, Q., Li, Z., Fang, J., Liu, W., Wu, J., and Zhang, Y. (2019). The Effect of Clinical-Grade Retinal Pigment Epithelium Derived from Human Embryonic Stem Cells Using Different Transplantation Strategies. Protein Cell, 10.","key":"ref_16","DOI":"10.1007\/s13238-018-0606-8"},{"unstructured":"De Quadros Costa, M.T.M. (2024, May 15). Autologous Transplantation of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for Geographic Atrophy Associated with Age-Related Macular Degeneration. NCT04339764, Available online: https:\/\/classic.clinicaltrials.gov\/ct2\/show\/NCT04339764.","key":"ref_17"},{"doi-asserted-by":"crossref","unstructured":"Stahl, A. (2020). The Diagnosis and Treatment of Age-Related Macular Degeneration. Dtsch. Arztebl. Int., 117.","key":"ref_18","DOI":"10.3238\/arztebl.2020.0513"},{"doi-asserted-by":"crossref","unstructured":"Fontaine, V., Kinkl, N., Sahel, J., Dreyfus, H., and Hicks, D. (1998). Survival of Purified Rat Photoreceptors In Vitro Is Stimulated Directly by Fibroblast Growth Factor-2. J. Neurosci., 18.","key":"ref_19","DOI":"10.1523\/JNEUROSCI.18-23-09662.1998"},{"doi-asserted-by":"crossref","unstructured":"Noor, N., Shapira, A., Edri, R., Gal, I., Wertheim, L., and Dvir, T. (2019). 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts. Adv. Sci., 6.","key":"ref_20","DOI":"10.1002\/advs.201900344"},{"doi-asserted-by":"crossref","unstructured":"Feiner, R., Engel, L., Fleischer, S., Malki, M., Gal, I., Shapira, A., Shacham-Diamand, Y., and Dvir, T. (2016). Engineered Hybrid Cardiac Patches with Multifunctional Electronics for Online and Regulation of Tissue Function. Nat. Mater., 15.","key":"ref_21","DOI":"10.1038\/nmat4590"},{"doi-asserted-by":"crossref","unstructured":"Shevach, M., Zax, R., Abrahamov, A., Fleischer, S., Shapira, A., and Dvir, T. (2015). Omentum ECM-Based Hydrogel as a Platform for Cardiac Cell Delivery. Biomed. Mater., 10.","key":"ref_22","DOI":"10.1088\/1748-6041\/10\/3\/034106"},{"doi-asserted-by":"crossref","unstructured":"Levato, R., Dudaryeva, O., Garciamendez-Mijares, C.E., Kirkpatrick, B.E., Rizzo, R., Schimelman, J., Anseth, K.S., Chen, S., Zenobi-Wong, M., and Zhang, Y.S. (2023). Light-Based Vat-Polymerization Bioprinting. Nat. Rev. Methods Primers, 3.","key":"ref_23","DOI":"10.1038\/s43586-023-00231-0"},{"doi-asserted-by":"crossref","unstructured":"Ng, W.L., and Shkolnikov, V. (2024). Optimizing Cell Deposition for Inkjet-Based Bioprinting. Int. J. Bioprinting, 10.","key":"ref_24","DOI":"10.36922\/ijb.2135"},{"doi-asserted-by":"crossref","unstructured":"Boularaoui, S., Al Hussein, G., Khan, K.A., Christoforou, N., and Stefanini, C. (2020). An Overview of Extrusion-Based Bioprinting with a Focus on Induced Shear Stress and Its Effect on Cell Viability. Bioprinting, 20.","key":"ref_25","DOI":"10.1016\/j.bprint.2020.e00093"},{"doi-asserted-by":"crossref","unstructured":"Shapira, A., Noor, N., Asulin, M., and Dvir, T. (2018). Stabilization Strategies in Extrusion-Based 3D Bioprinting for Tissue Engineering. Appl. Phys. Rev., 5.","key":"ref_26","DOI":"10.1063\/1.5055659"},{"doi-asserted-by":"crossref","unstructured":"Shapira, A., Noor, N., Oved, H., and Dvir, T. (2020). Transparent Support Media for High Resolution 3D Printing of Volumetric Cell-Containing ECM Structures. Biomed. Mater., 15.","key":"ref_27","DOI":"10.1088\/1748-605X\/ab809f"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1330","DOI":"10.1242\/dev.086603","article-title":"Conditional Knockdown of DNA Methyltransferase 1 Reveals a Key Role of Retinal Pigment Epithelium Integrity in Photoreceptor Outer Segment Morphogenesis","volume":"140","author":"Swaroop","year":"2013","journal-title":"Development"},{"doi-asserted-by":"crossref","unstructured":"Naylor, A., Hopkins, A., Hudson, N., and Campbell, M. (2020). Tight Junctions of the Outer Blood Retina Barrier. Int. J. Mol. Sci., 21.","key":"ref_29","DOI":"10.3390\/ijms21010211"},{"doi-asserted-by":"crossref","unstructured":"Reynolds, J.D., and Olitsky, S.E. (2011). Pediatric Retina, Springer.","key":"ref_30","DOI":"10.1007\/978-3-642-12041-1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1016\/j.preteyeres.2008.08.002","article-title":"Epithelial Phenotype and the RPE: Is the Answer Blowing in the Wnt?","volume":"27","author":"Burke","year":"2008","journal-title":"Prog. Retin. Eye Res."},{"doi-asserted-by":"crossref","unstructured":"Caceres, P.S., and Rodriguez-Boulan, E. (2020). Retinal Pigment Epithelium Polarity in Health and Blinding Diseases. Curr. Opin. Cell Biol., 62.","key":"ref_32","DOI":"10.1016\/j.ceb.2019.08.001"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1038\/eye.2001.141","article-title":"The Role of the Retinal Pigment Epithelium: Topographical Variation and Ageing Changes","volume":"15","author":"Boulton","year":"2001","journal-title":"Eye"},{"doi-asserted-by":"crossref","unstructured":"Karema-Jokinen, V., Koskela, A., Hytti, M., Hongisto, H., Viheri\u00e4l\u00e4, T., Liukkonen, M., Torsti, T., Skottman, H., Kauppinen, A., and Nymark, S. (2023). Crosstalk of Protein Clearance, Inflammasome, and Ca2+ Channels in Retinal Pigment Epithelium Derived from Age-Related Macular Degeneration Patients. J. Biol. Chem., 299.","key":"ref_34","DOI":"10.1016\/j.jbc.2023.104770"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/j.preteyeres.2006.12.002","article-title":"Ion Channels in the RPE","volume":"26","author":"Wimmers","year":"2007","journal-title":"Prog. Retin. Eye Res."},{"doi-asserted-by":"crossref","unstructured":"Kim, J., Park, J.Y., Kong, J.S., Lee, H., Won, J.Y., and Cho, D.W. (2021). Development of 3D Printed Bruch\u2019s Membrane-Mimetic Substance for the Maturation of Retinal Pigment Epithelial Cells. Int. J. Mol. Sci., 22.","key":"ref_36","DOI":"10.3390\/ijms22031095"},{"doi-asserted-by":"crossref","unstructured":"Tarau, I.S., Berlin, A., Curcio, C.A., and Ach, T. (2019). The Cytoskeleton of the Retinal Pigment Epithelium: From Normal Aging to Age-Related Macular Degeneration. Int. J. Mol. Sci., 20.","key":"ref_37","DOI":"10.3390\/ijms20143578"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1007\/s12015-017-9754-0","article-title":"Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium","volume":"13","author":"Bennis","year":"2017","journal-title":"Stem Cell Rev. Rep."},{"unstructured":"Bonilha, V.L., Rayborn, M.E., Bhattacharya, S.K., Gu, X., Crabb, J.S., Crabb, J.W., and Hollyfield, J.G. (2006). The Retinl Pigment Epirhelium Apical Microvilli and Retinal Function. Adv. Exp. Med. Biol., 572.","key":"ref_39"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.preteyeres.2009.08.003","article-title":"The Dynamic Nature of Bruch\u2019s Membrane","volume":"29","author":"Booij","year":"2010","journal-title":"Prog. Retin. Eye Res."},{"doi-asserted-by":"crossref","unstructured":"Kular, J.K., Basu, S., and Sharma, R.I. (2014). The Extracellular Matrix: Structure, Composition, Age-Related Differences, Tools for Analysis and Applications for Tissue Engineering. J. Tissue Eng., 5.","key":"ref_41","DOI":"10.1177\/2041731414557112"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3089","DOI":"10.1038\/s41596-021-00533-1","article-title":"Perfusion and Endothelialization of Engineered Tissues with Patterned Vascular Networks","volume":"16","author":"Kinstlinger","year":"2021","journal-title":"Nat. Protoc."}],"container-title":["Gels"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2310-2861\/10\/5\/336\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:42:59Z","timestamp":1760107379000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2310-2861\/10\/5\/336"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,15]]},"references-count":42,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5]]}},"alternative-id":["gels10050336"],"URL":"https:\/\/doi.org\/10.3390\/gels10050336","relation":{},"ISSN":["2310-2861"],"issn-type":[{"type":"electronic","value":"2310-2861"}],"subject":[],"published":{"date-parts":[[2024,5,15]]}}}