26 INTRODUCTION Age-related macular degeneration (AMD) is the leading cause of irreparable blindness in the developed world, affecting approximately 170 million people worldwide. Over 11 million people in the United States suffer from AMD, with that number projected to reach over 22 million over the coming decades [1]. People affected by AMD experience a significant de- crease in their quality of life due to impaired visual acuity at or near 20/200 (Figure 1) [2]. While methods such as complement therapy and neuroprotection are currently be- ing researched, there are no efficacious treat- ments for dry AMD. AMD must progress into the later and more severe wet AMD stage for current treatments to be effective [3]. The pre- vailing therapies of photodynamic therapy and anti-vascular endothelial growth factor (VEGF) therapy are inadequate, as their focus is not on curing AMD and restoring sight, but on slow- ing down and preventing further vision loss [4]. Cellular and Biomaterial Approaches for Treating Age-Relat- ed Macular Degeneration Ashrit Challa*, Srikar Sama, Selik Morrishetty © 2022 Challa, Sama, Morishetty. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits the user to copy, distribute, and transmit the work provided that the original authors and source are credited. University of Pennsylvania, Philadelphia PA KEYWORDS: Age-Related Macular Degeneration, Bioplastics, Tissue Engineering ABSTRACT: Tissue Engineering offers a novel, curative approach to treating Age-Re- lated Macular degeneration (AMD), a disease characterized by excessive drusen depo- sition beneath the retinal surface and consequent vision loss. Preclinical studies in rats have shown that transplanted Retinal Pigment Epithelium (RPE) derived from human Embryonic Stem Cells (hESC) have not only slowed AMD but have also restored vision. There are two main methods of delivering RPE cells: direct injection and monolayer surgical insertion, the latter demonstrating long-term integration. Biocompatible scaf- folds allow for better delivery of RPE cells, induced Pluripotent Stem Cells (iPSC), and Retinal Progenitor Cells (RPC). Unlike animal-derived extracellular matrix components, soft modulus biomaterials such as poly(lactic-co-glycolic acid) (PLGA) and poly(l-lactic acid) (PLLA) are ideal for AMD cell transplants because of fast degradation times, high cellular attachment proliferation, and strong adherence to Bruch’s membrane. These biomaterials can also be created at a 10-100μm thickness so that vision is not distorted. Use of biomaterials could be improved by cross-linking them with anti-vascular endothe- lial growth factors (VEGFs) like Brolucizumab and retinal growth factors such as fibro- blast growth factor (FGF). Similarly, hESC and iPSC cells can be genetically modified to secrete anti-VEGF factors. 27 As such, a tissue-engineered approach for treating AMD has impressive implications, potentially allowing for the reversal of a dis- ease previously thought to be irreversible. This article reviews the clinical features of AMD, its current treatment options, stem cell and bioma- terial tissue engineering therapeutic approach- es, and pitfalls of and suggestions for such tissue-engineered approaches. CLINICAL FEATURES OF AMD The clinical trademark of AMD is the accumulation of fat and protein deposits, com- monly known as drusen, in the macula, an area that is dense with photoreceptors responsible for high visual acuity [5]. Drusen accumulates underneath the photoreceptors beneath the ret- inal pigment epithelium (RPE), which functions as a source of nutrients and growth factors, as well as a photoreceptor phagocytosis mecha- nism. The RPE attaches to Bruch’s Membrane (BM), which acts as a barrier between the retina and the choroid while regulating diffusion between the choroid and the RPE [6]. While the disease can be categorized into early, interme- diate, and late stages based on the extent of drusen proliferation and vision loss, the most important distinction remains between dry and wet AMD. Dry, or non-exudative, AMD occurs when excess drusen is deposited between the RPE and BM. This causes gradual RPE and photoreceptor cell death, as well as cen- tral macular atrophy and blind spots. Wet, or neovascular/exudative, AMD usually follows dry AMD and is characterized by choroidal neovas- cularization (CNV), or the invasion of choroidal blood vessels into the RPE. Bleeding and leak- ing from these vessels result in RPE cell death and rapid progression of blurriness and loss of visual acuity [7]. CURRENT TREATMENTS AND MEDICA- TIONS FOR AMD There are currently no effective curative treatments for AMD; however, existing thera- pies aim to manage the disease and stop its progression. Treatments for Wet AMD include laser photocoagulation and anti-VEGF ther- apy, such as Brolucizumab or Ranibizumab. Inhibiting vascularization in the eye prevents further progress of CNV, but it also contributes to chorioretinal atrophy due to less vascu- larization and potential narrowing of choroid capillaries. In fact, followups with anti-VEGF treated eyes indicate an extremely high level (98%) of macular atrophy, particularly in the fovea [8]. Photodynamic therapy (PDT) like Vertreporfin aims to simply stop the progres- sion of destructive vascularization through laser-activated medication. However, there is no clinically significant improvement of visual acuity because of this therapeutic method [9]. As such, monthly anti-VEGF drug injections are currently prescribed and have had minimal to moderate success in restoring some vision to patients (about 30% of treated individuals), only maintaining the eye equivalent to its initial Figure 1. As AMD progresses, greater amounts of drusen disrupt the RPE-BM inter- face which leads to increased photoreceptor death. Columbia Undergraduate Science Journal Vol. 16, 2022 28 state in which treatment first began. Even when PDT and anti-VEGF drugs are used in conjunction, a secondary approach when neither therapy is individually effective, there is a similar rate of visual acuity improvement (~11-13 letters) [10]. Recent advancements in pharmacology have led to the development of better-performing injections, such as Broluci- zumab, which has a higher rate of visual acuity correction than Ranibizumab. Coupled with its small size, this compound allows for better vision improvement with fewer injections, but still holds the risk of injection-related complica- tions and excessive drying [11]. Currently, there are no current treatments for dry AMD besides a surgical transplantation of a homologous donor retina. Transplantation has previously been shown to be ineffective due to a failure in synapse formation between fully differentiated tissue and the host. Surgical approaches of cleaning the debris near the retina and attempt- ing to replace the degenerating retina with bolus injections have only offered temporary respite to the afflicted retina as it regresses back to its damaged form [12]. CELL THERAPY APPROACHES Due to the difficulties of conventional therapies, recent years have witnessed much research and development in the use of stem cells and induced pluripotent cells as treatment or cures for AMD. In 1987, the first significant study on the use of transplanted RPE cells for treatment was conducted by Gouras et al, who placed transplanted rabbit RPE adjacent to the neural retina of a different rabbit [13]. Anoth- er landmark occurred ten years later, when Al-gvere et al. transplanted human fetal RPE patches into the subretinal space of human patients with wet or dry AMD. The results of this grafting indicated that RPE transplants would not be rejected and further degrade vision, even without immunosuppression, and that dry AMD had a lower graft rejection rate [14]. Since these landmark studies of RPE transplants, iris pigment epithelium (IPE) cells and human embryonic stem cells (hESC) have been trans- planted in many animal models, showing vision improvement. Additionally, researchers have transplanted RPE cells, choroid-Bruch’s-RPE explants, IPE cells and hESC in human pa- tients with AMD, aiming to evaluate transplan- tation safety (Figure 2) [15]. Preclinical studies conducted in rats have shown that using transplanted RPE cells derived from hESC have slowed retinal degen- eration [16] and even improved visual acuity [17][18]. Additionally, hESC-RPE has formed a polarized epithelial layer in vitro, secreting growth factors such as pigment epithelium de- scribed factor and VEGF, all while expressing the barrier properties of normal adult human RPE cells [19]. Currently the two preferred methods for delivering hESC-RPE into the subretinal space are either injections of cells suspended in a fluid, which are inexpensive and simple but carry the risk of RPE cell dedif- ferentiation, or creating monolayers of hESC- RPE which can be surgically placed subreti- nally, necessitating a biologically compatible substrate [20]. Monolayers have shown higher rates of cell survival in comparison to injected Figure 2: hESC-RPE cells injected into the subretinal space of human patients exhibit increased pigmentation and result in regener- ation of the macula. Columbia Undergraduate Science Journal Vol. 16, 2022 29 cell suspensions, as well as less clumping of cells [21]. Recent advancements with hESC- RPE therapy include a completed three-year and an ongoing clinical trial conducted in part by the London Project to Cure Blindness, with the former demonstrating the safety of long-term grafts while being the first to record longitudinal effects of hESC-RPE monolayer implantations. The completed study also showed lasting improvement in visual acuity of about 14-15 letters, as well as no adverse proliferative re- actions such as teratoma formation, even after 37 months of observation. Similarly, the phase I results of the ongoing clinical trial, with interven- tions provided to two human patients, indicate hESC-RPE integration while presenting im- provement of visual acuity and reading speeds [22][23]. While the former study was conducted using bolus cell injections of hESC-RPEs, the latter utilized a polyester sub-strate for monolay- er insertion. Furthermore, there are also other clinical trials using cell sheets, such as a trial using induced pluripotent stem cell (iPSC)-RPE cell sheet transplantation in patients with dry AMD rather than wet AMD, although this trial has only very recently begun (NCT04339764). Though the retinal space in the eye is immuno- logically privileged, various studies have used immunosuppression (such as tacrolimus and mycophenolate mofetil) during their trials of implanted stem cells, while others have relied purely on the immunological status of the eye [24]. Both methods have had been successful in avoiding immunological rejection. iPSCs have been studied for in vivo curative treatments and for the modeling of AMD and other eye-related diseases. This is because there is an information deficit in the exact mech- anisms for the progression of AMD, specifically the non-exudative form. In response, the wide range of iPSC potential differentiation offers nov- el breakthrough methods in terms of replace- ment therapy and disease modeling [25]. To dedifferentiate human fibroblasts for use in iP- SC-RPE procedures and other iPSC techniques, cells can be transfected with vectors such as the Venezuelan Equine Encephalitis RNA vector, inducing exogenous expression of pluripoten- cy markers such as OCT4, SOX2, KLF4, and GLIS1. With a dedifferentiation rate of >95%, newly formed iPSC cells are then suspended to form embryoid bodies, which are then placed in RPE medium. This allows for iPSC-RPE cells from different lineages to express high levels of RPE genes and proteins, such as RPE65 and MERTK [26]. Already, iPSC-RPE cells have been used to better understand the molecular etiology of AMD, with one study identifying a single-nucleotide polymorphism near the VEG- FA gene in AMD patients that decreases gene expression (Figure 3). In conjunction, another study has been able to identify genes that are differently regulated in AMD patients, alongside cell proliferation and localized immune response changes [27][28]. In this vein, iPSC-RPE re- search is exciting in its ability to better elucidate previously unknown mechanisms in AMD devel- opment. Figure 3. Expression levels of VEGFA for six iPSC-RPE samples, with the risk variant/ AMD SNP sample exhibiting significantly lower amounts of the gene Columbia Undergraduate Science Journal Vol. 16, 2022 30 BIOMATERIALS APPROACHES Surgeons currently do not have com- plete control over where the RPE or retinal progenitor cells (RPCs) are placed subretinally, with random clumps of cells not being condu- cive to regeneration. Additionally, without a way to ascertain the polarity and orientation of inject- ed bolus or unoriented sheet cells, adherence to Bruch’s membrane is drastically lowered. The use of biocompatible materials allows the pa- rameters for delivery to be adjusted so that RPE cells, iPSCs, and other RPCs can be delivered subretinally to regenerate the damaged RPE cell layer, attaching to photoreceptors apically and BM basally. Thus, biomaterials must allow for cell attachment, proliferation, and correct orientation/polarization. To allow for RPE cell proliferation and BM attachment, the material must degrade by 2-3 weeks post-implant, and is ideally 10-100μm to allow for precise manipula- tion without retinal distortion and nutrient diffu- sion limitation [29]. Some biomaterials that have been explored as substrates for RPE monolayer insertion are collagen, Matrigel®️, fibronectin, laminin, vitronectin, and oligopeptides. Howev- er, these organic, animal-derived extracellular matrix components were found to discourage cell proliferation and have variable degradation times based on individual enzymatic digestion rates [30]. Nanowires of poly(e-caprolactone) (PCL) may also be cast, but require precise construction in order to ensure porosity levels conducive to RPC polarization and attachment [31]. Poly(dl-lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poly(ethylene gly- col) (PEG), and poly(dl-lactic acid) (PLA), are synthetic, thin, and degradable bio-materials. However, PLGA has an ideal degradation time of 2-3 weeks (whereas the others do not sig- nificantly degrade until about four weeks after initial cell seeding), can be 10-130μm thick, and has a feasible manufacturing process [32] [33]. Once solvated in chloroform or hexafluo- roisopropanol (HFIP), these polymers are left to deposit on an even glass or Scaffdex surface for 8 hours while the solvent evaporates. The thin sheets are then left to dry, potentially stored in nitrogenous atmospheres over desiccators like calcium sulfate. They may then be crosslinked and sterilized using UV light [34][35]. The RPE, RPC, and iPSCs can thus be seeded. It has been found that there is 99-100% attachment of non-hESC human RPE cells to PLGA after 8 hours, and both PLGA and PGA sheets allow for RPE cell metabolism and protein expression. These polymers are therefore viable for cell attachment and proliferation, allowing for apical microvilli and basilar diffusion and molecule excretion [36][37]. However, these RPE cells do not attach significantly to PLA or PEG. After 3-7 days, cells become confluent, forming a multi- layer or monolayer (depending on the cell type) of polarized RPE or RPCs and are ready for implantation. PLGA sheets have been found to be the smoothest, thinnest, have the highest polarized RPE cell attachment, and most proliferation/ material area repopulation. This is due to a 50:50 high molecular weight PGLA that contains an optimal ratio of lactic acid to glycolic acid. In comparison to previous substrates such as collagen, PLGA sheets are much smoother, thinner, and allow for the formation of an RPE monolayer with both correct orientation and polarity of cells. Additionally, multiple PLGA ratios of lactic acid to glycolic acid, such as high molecular weight (high MW) 50:50 and 75:25 PLGA, allowed for significant levels of cell pro- liferation. However, due to its faster degradation time, a high MW 50:50 PLGA blend is thought to be optimal [38]. The porosity of this blend allows correct adherence to BM so that as the biomaterial is degraded, the RPE cells attach Columbia Undergraduate Science Journal Vol. 16, 2022 31 and are integrated into the eye. Studies also indicate that creating a polymer blend of PLGA and poly(l-lactic acid) (PLLA) may improve the porosity of the substrate, thus allowing for higher rates of cell proliferation. The modulus (hardness) of PLGA and poly(L-lactic acid) PLLA 50:50 is low enough to overcome the otherwise stiff composition of pure PLGA, with the flexibility provided by PLLA mitigating the risk of retinal damage. This 50:50 PLGA:PLLA was implanted in the rat model and the viabil- ity of the cells was monitored for 14 days [39]. Unlike previous attempts to directly inject RPC cells into the retina, the PLGA:PLLA bound RPC and RPE cells were still viable and expressed GFP, unlike transplanted cells, which only had a 10% survival rate 14 days after implantation (Figure 4). A more recent study shows that a 25:75 PLLA:PLGA blend might be more efficient, with a higher level of porosity and a lower elastic modulus (Figure 5) [40]. Soft modulus biomate- rials of PLGA and PLLA are ideal for AMD cell delivery because they degrade fast, have high cell attachment, RPE and RPC cell polarization, cell proliferation, adherence to BM, and high viability after implantation. SHORTCOMINGS Although cell therapies have evolved greatly over the past few decades, regarding both transplantations and surgical monolayer integration, there are still many unanswered questions. Transplanted hESC-RPE cells demonstrated an increase in retinal pigmen- tation and visual acuity by about 14 letters in patients [41]. However, a 2008 study that surgically implanted fetal RPE into ten patients saw a four times improvement in visual acuity in one patient, from 20/800 to 20/200, which remained stable for five years [42]. This mag- nitude in visual improvement has not yet been seen from hESC or iPSC approaches, which are also marred by complications such as cataract formation and vitreous inflammation. And while iPSC-RPE clinical trial results are increasing in number, there is no published research on the longitudinal effects of iPSC-RPE cell transplan- tation in human patients with AMD. It is known that iPSC-RPE cells also have a faster rejection time than hESC-derived cells, triggering macro- phage-mediated phagocytosis such that almost no iPSC cells remain after 13 weeks [43]. Given that donor retina scaffolds are dif- ficult to procure, tissue engineering approaches provide alternative avenues. Regardless, the Figure 4. Attachment levels of RPE cells for different cell substrates, with three different compositions of PLGA, the most proliferative substrate Figure 5: Predicted vs. Actual elastic moduli for various PLLA: PLGA ratios, with the 25:75 exhibiting the lowest modulus Columbia Undergraduate Science Journal Vol. 16, 2022 32 use of cutting-edge stem cell engineering tech- niques and substrates like PLGA and PLLA still pose problems. Some of these problems include inflammation and injury due to the injection of cells, incomplete attachment of RPE cells to BM or a lack of subsequent proliferation, and the inability to replicate the true retinal environment. Cells may also dedifferentiate once attached to BM, which would only lead to the presence of more harmful debris in the retina and subretinal space. Both injected and surgically inserted cells have not yet shown complete restorations of vision and have only demonstrated peripheral and minor macular vision improvement. Final- ly, the toxicity of the material used as the cell substrate must also be considered, as degra- dation of these scaffolds will inevitably lead to the presence of small subunits in the macular region. With the use of PLGA, a potentially toxic and immunogenic substance in the eye, careful construction of scaffolding must be used in accordance with shape and size restraints. As more data comes out regarding biomaterial interactions with the actual human eye, rather than approximating animal models, it will be important to modify scaffold compositions and morphologies accordingly [44]. IMPROVEMENTS FOR CURRENT TECH- NIQUES Based on current research, there are many hypothesized methods of improving the treatment of AMD. Cell therapies utilizing stem cells can be further improved with genetically modified RPE cells, able to impede or revert the progression of AMD through genome and tran- scriptome modifications to counteract the chang- es of the disease. Neovascular AMD is due in part to an imbalance in growth factors such as VEGF. RPE cells naturally express many an- ti-VEGFs like pigment epithelium-derived factor (PEDF), but damage to these cells allows for ex- cess vascularization. To further treatment, trans- planted cells should be genetically modified to upregulate the production of anti-VEGFS, such as endostatin, PEDF, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), or ciliary neurotrophic factor (CNTF). To perform the necessary genetic modifications for the aforementioned suggestions, possible trans- fection methods include the Sleeping Beauty (SB100X)^ transposon system or the CRISPR/ Cas9 system. During the later stages of AMD when damage has occurred to both photorecep- tors and RPE cells, another improvement would be to transplant autologous ESC or iPSC which differentiate under a wider range of factors. This would allow for the reconstruction of the entire BM-RPE-photoreceptor complex, potentially leading to greater improvements in vision. In addition to therapeutic uses, it would also be helpful to use iPSC-RPE cells to model dry AMD alongside wet AMD in humans, as these cells could uncover more about dry AMD while finding use as high throughput drug screens. Gene therapy efficacies could also be tested in this way. This knowledge could lead to not just curative methods, but perhaps preventative interventions as well. In using biomaterials as a substrate for RPE cells, endogenous factors can perhaps be crosslinked to PLGA or PLLA. As protases break the crosslinks in PLGA scaffolds, an- ti-VEGFs such as Ranibizumab and retinal growth factors like FGF could be freed, allowing for retinal regeneration [45]. Delivery of Ran- ibizumab via nanoparticles has already been demonstrated, so it is now a matter of delivering these types of medications on PLGA scaffolds for AMD patients (Figure 6) [46]. Columbia Undergraduate Science Journal Vol. 16, 2022 33 Because RPE cells must be polarized, incorporating PLGA with this cell type alongside RPCs and iPSCs may allow for differentiation into RPE and other retinal cells. In this manner, the eye can heal any damage done by the deg- radation or delivery of the substrate biomaterial and cells in the first place. Similarly, the use of drugs to modulate calcium systems has shown down-regulating effects on proliferation, which could be used in the case of teratomacreating iPSC-RPE cells. If cross-linked in PLGA scaf- folds, these inhibitors could be used as a form of cell control [47]. In regard to surgical place- ment, current methods require the rolling up of PLGA/PLLA scaffolds. These rolled-up scaffolds are then delivered to the subretinal space. An improvement would be to develop a method by which a higher modulus material can be deliv- ered to the eye directly in between the BM and the photoreceptors, perhaps via a small incision. This higher modulus material could then be removed to leave softer biomaterials, which are less harmful to retinal health and vision. Finally, cleaning up dedifferentiated cell and degraded scaffolding debris with lasers may also allow for better vision and healing. CONCLUSION Age-related macular degeneration is one of the leading causes of blindness in aging adults and is prevalent in today’s population. Tissue engi- neering solutions for AMD have improved upon conventional treatments, thanks to their ability to not just stop the progression of AMD, but also result in visual improvement. The use of hESC- RPE and iPSC-RPE, coupled with a thin PLGA/ PLLA scaffold, may allow for effective and safe integration of RPE cells in the macula. With more research on polymer toxicity, molecular crosslinking, and methods of efficient insertion, these cellular engineering techniques may be- come the best way to reverse AMD damage and fully regenerate the patient’s eye. AUTHOR INFORMATION Corresponding Author *Ashrit Challa, University of Pennsylvania, De- partment of Biology; achalla@sas.upenn.edu, 937-750-1530 Author Contributions Ashrit Challa – Text Srikar Sama – Figures and Proofreading Selik Morishetty – Figures and Proofreading ABBREVIATIONS AMD – Age-Related Macular Degeneration VEGF – Vascular Endothelial Growth Factor RPE – Retinal Pigment Epithelium BM – Bruch’s Membrane CNV – Choroidal Neovascularization PDT – Photodynamic Therapy IPE – Iris Pigment Epithelium hESC – Human Embryonic Stem Cells iPSC – Induced Pluripotent Stem Cells RPC – Retinal Progenitor Cell PLGA – Poly(dl-lactic-co-glycolic acid) PGA – Poly(glycolic acid) Figure 6: Cumulative Release percentage of Ranibizumab by PLGA nanoparticle system; if modified into PLGA scaffolding, could be used to deliver anti-VEGFs to integrating RPE cells Columbia Undergraduate Science Journal Vol. 16, 2022 34 PEG – Poly(ethylene glycol) PLA – Poly(dl-lactic acid) HFIP - Hexafluoroisopropanol PLLA – Poly(l-lactic acid) PEDF – Pigment epithelium-derived factor bFGF – Basic fibroblast growth factor BDNF – Brain-derived neurotrophic factor CNTF – Ciliary neurotrophic factor REFERENCES [1] Pennington, K. L., & DeAngelis, M. M. (2016). Epidemiology of age-related macular de- generation (AMD): associations with cardio-vas- cular disease phenotypes and lipid factors. Eye and vision (Lon-don, England), 3, 34. https://doi. org/10.1186/s40662-016-0063-5 [2] Fernández-Robredo, P., Sancho, A., Johnen, S., Recalde, S., Gama, N., Thumann, G., Groll, J., & García-Layana, A. (2014). Current treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of ophthalmology, 2014, 510285. https://doi. org/10.1155/2014/510285 [3] Ammar, M. J., Hsu, J., Chiang, A., Ho, A. C., & Regillo, C. D. (2020). Age-related macular degeneration therapy: a review. Current opinion in ophthalmology, 31(3), 215–221. https://doi. org/10.1097/ICU.0000000000000657 [4] Fernández-Robredo, P., Sancho, A., Johnen, S., Recalde, S., Gama, N., Thumann, G., Groll, J., & García-Layana, A. (2014). Current treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of ophthalmology, 2014, 510285. https://doi. org/10.1155/2014/510285 [5] Jager, R. D., Mieler, W. F., & Miller, J. W. (2008). Age-related macular degeneration. The New England journal of medicine, 358(24), 2606–2617. https://doi.org/10.1056/NEJM- ra0801537 [6] Bhutto, I., & Lutty, G. (2012). Understand- ing age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch's membrane/chorio- capillaris complex. Molecular aspects of medi- cine, 33(4), 295–317. https://doi.org/10.1016/j. mam.2012.04.005 [7] Mathenge W. (2014). Age-related macular degeneration. Communi-ty eye health, 27(87), 49–50. [8] Fernández-Robredo, P., Sancho, A., Johnen, S., Recalde, S., Gama, N., Thumann, G., Groll, J., & García-Layana, A. (2014). Current treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of ophthalmology, 2014, 510285. https://doi. org/10.1155/2014/510285 [9] Brown, D. M., Kaiser, P. K., Michels, M., Soubrane, G., Heier, J. S., Kim, R. Y., Sy, J. P., Schneider, S., & ANCHOR Study Group (2006). Ranibizumab versus verteporfin for neovascular age-related macular degeneration. The New En- gland journal of medicine, 355(14), 1432–1444. https://doi.org/10.1056/NEJMoa062655 [10] Fernández-Robredo, P., Sancho, A., Johnen, S., Recalde, S., Ga-ma, N., Thumann, G., Groll, J., & García-Layana, A. (2014). Cur- rent treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of ophthalmology, 2014, 510285. https://doi. org/10.1155/2014/510285 Columbia Undergraduate Science Journal Vol. 16, 2022 35 [11] Yannuzzi, N. A., & Freund, K. B. (2019). Brolucizumab: evidence to date in the treatment of neovascular age-related macular degen- era-tion. Clinical ophthalmology (Auckland, N.Z.), 13, 1323–1329. https://doi.org/10.2147/ OPTH.S184706 [12] MacLaren, R. E., Bird, A. C., Sathia, P. J., & Aylward, G. W. (2005). Long-term results of submacular surgery combined with macular translocation of the retinal pigment epithelium in neovascu-lar age-related macular degeneration. Ophthalmology, 112(12), 2081–2087. https://doi. org/10.1016/j.ophtha.2005.06.029 [13] Lopez, R., Gouras, P., Brittis, M., & Kjeld- bye, H. (1987). Trans-plantation of cultured rabbit retinal epithelium to rabbit retina using a closed-eye method. Investigative ophthalmology & visual science, 28(7), 1131–1137. [14] Algvere, P. V., Berglin, L., Gouras, P., Sheng, Y., & Kopp, E. D. (1997). Transplanta- tion of RPE in age-related macular degenera- tion: observations in disciform lesions and dry RPE atrophy. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 235(3), 149–158. https://doi. org/10.1007/BF00941722 [15] Fernández-Robredo, P., Sancho, A., Johnen, S., Recalde, S., Ga-ma, N., Thumann, G., Groll, J., & García-Layana, A. (2014). Cur- rent treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of ophthalmology, 2014, 510285. https://doi. org/10.1155/2014/510285 [16] Idelson, M., Alper, R., Obolensky, A., Ben- Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., Cohen, M. A., Even-Ram, S., Berman-Zaken, Y., Matzrafi, L., Rechavi, G., Banin, E., & Reubinoff, B. (2009). Di-rected differentiation of human em- bryonic stem cells into functional retinal pigment epithelium cells. Cell stem cell, 5(4), 396–408. https://doi.org/10.1016/j.stem.2009.07.002 [17] Lu, B., Malcuit, C., Wang, S., Girman, S., Francis, P., Lemieux, L., Lanza, R., & Lund, R. (2009). Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem cells (Dayton, Ohio), 27(9), 2126–2135. https://doi. org/10.1002/stem.149 [18] Lund, R. D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B., Girman, S., Bischoff, N., Sauvé, Y., & Lanza, R. (2006). Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning and stem cells, 8(3), 189–199. https:// doi.org/10.1089/clo.2006.8.189 [19] Carr, A. J., Smart, M. J., Ramsden, C. M., Powner, M. B., da Cruz, L., & Coffey, P. J. (2013). Development of human embryonic stem cell therapies for age-related macular degenera- tion. Trends in neuro-sciences, 36(7), 385–395. https://doi.org/10.1016/j.tins.2013.03.006 [20] Carr, A. J., Smart, M. J., Ramsden, C. M., Powner, M. B., da Cruz, L., & Coffey, P. J. (2013). Development of human embryonic stem cell therapies for age-related macular degener- ation. Trends in neuro-sciences, 36(7), 385– 395. https://doi.org/10.1016/j.tins.2013.03.006 [21] Diniz, B., Thomas, P., Thomas, B., Ribeiro, R., Hu, Y., Brant, R., Ahuja, A., Zhu, D., Liu, L., Koss, M., Maia, M., Chader, G., Hinton, D. R., & Humayun, M. S. (2013). Subretinal implantation Columbia Undergraduate Science Journal Vol. 16, 2022 36 of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer. Investigative ophthalmology & visual science, 54(7), 5087– 5096. https://doi.org/10.1167/iovs.12-11239 [22] Schwartz, S. D., Regillo, C. D., Lam, B. L., Eliott, D., Rosenfeld, P. J., Gregori, N. Z., Hub- schman, J. P., Davis, J. L., Heilwell, G., Spirn, M., Maguire, J., Gay, R., Bateman, J., Ostrick, R. M., Morris, D., Vincent, M., Anglade, E., Del Priore, L. V., & Lanza, R. (2015). Human embry- onic stem cell-derived retinal pigment epithelium in patients with age-related macular degenera- tion and Stargardt's macu-lar dystrophy: fol- low-up of two open-label phase 1/2 studies. Lan- cet (London, England), 385(9967), 509–516. https://doi.org/10.1016/S0140-6736(14)61376-3 [23] da Cruz, L., Fynes, K., Georgiadis, O., Kerby, J., Luo, Y. H., Ahmado, A., Vernon, A., Daniels, J. T., Nommiste, B., Hasan, S. M., Gooljar, S. B., Carr, A. F., Vugler, A., Ramsden, C. M., Bictash, M., Fenster, M., Steer, J., Har- binson, T., Wilbrey, A., Tufail, A., … Coffey, P. J. (2018). Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. Na- ture biotechnology, 36(4), 328–337. https://doi. org/10.1038/nbt.4114 [24] Lu, B., Malcuit, C., Wang, S., Girman, S., Francis, P., Lemieux, L., Lanza, R., & Lund, R. (2009). Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem cells (Dayton, Ohio), 27(9), 2126–2135. https://doi. org/10.1002/stem.149 [25] Melville, H., Carpiniello, M., Hollis, K., Staffaroni, A., & Goles-taneh, N. (2013). Stem cells: a new paradigm for disease modeling and developing therapies for age-related macular degeneration. Journal of translational medicine, 11, 53. https://doi.org/10.1186/1479-5876-11-53 [26] Hazim, R. A., Karumbayaram, S., Jiang, M., Dimashkie, A., Lopes, V. S., Li, D., Burgess, B. L., Vijayaraj, P., Alva-Ornelas, J. A., Zack, J. A., Kohn, D. B., Gomperts, B. N., Pyle, A. D., Lowry, W. E., & Williams, D. S. (2017). Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization. Stem cell research & therapy, 8(1), 217. https://doi. org/10.1186/s13287-017-0652-9 [27] Smith, E. N., D'Antonio-Chronowska, A., Greenwald, W. W., Borja, V., Aguiar, L. R., Pogue, R., Matsui, H., Benaglio, P., Bo-rooah, S., D'Antonio, M., Ayyagari, R., & Frazer, K. A. (2019). Human iPSC-Derived Retinal Pigment Epithelium: A Model System for Prioritizing and Functionally Characterizing Causal Variants at AMD Risk Loci. Stem cell reports, 12(6), 1342–1353. https://doi.org/10.1016/j.stem- cr.2019.04.012 [28] Gong, J., Cai, H., NYSCF Global Stem Cell Array Team, Noggle, S., Paull, D., Rizzolo, L. J., Del Priore, L. V., & Fields, M. A. (2020). Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interac- tions. Stem cells translational medicine, 9(3), 364–376. https://doi.org/10.1002/sctm.19-0321 [29] Lu, L., Yaszemski, M. J., & Mikos, A. G. (2001). Retinal pigment epithelium engineer- ing using synthetic biodegradable polymers. Biomaterials, 22(24), 3345–3355. https://doi. org/10.1016/s0142-9612(01)00172-7 [30] Lu, L., Yaszemski, M. J., & Mikos, A. G. (2001). Retinal pigment epithelium engineer- Columbia Undergraduate Science Journal Vol. 16, 2022 37 ing using synthetic biodegradable polymers. Biomaterials, 22(24), 3345–3355. https://doi. org/10.1016/s0142-9612(01)00172-7 [31] McHugh, K. J., Tao, S. L., & Saint-Geniez, M. (2014). Porous poly(ε-caprolactone) scaf- folds for retinal pigment epithelium trans-plan- tation. Investigative ophthalmology & visual sci- ence, 55(3), 1754–1762. https://doi.org/10.1167/ iovs.13-12833 [32] Reed, A. M., Gilding, D. K. (1981). Biode- gradable polymers for use in surgery-poly(gly- colic)/poly(lactic acid) homo and copoly-mers: 2. In vitro degradation. Polymer, 22(4), 494-498. https://doi.org/10.1016/0032-3861(81)90168-3 [33] McCormick, R., Pearce, I., Kaye, S., & Haneef, A. (2020). Opti-misation of a Nov- el Bio-Substrate as a Treatment for Atrophic Age-Related Macular Degeneration. Frontiers in bioengineering and bio-technology, 8, 456. https://doi.org/10.3389/fbioe.2020.00456 [34] Lu, L., Nyalakonda, K., Kam, L., Bizios, R., Göpferich, A., & Mikos, A. G. (2001). Ret- inal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from syn- thetic biode-gradable polymers. Biomaterials, 22(3), 291–297. https://doi.org/10.1016/s0142- 9612(00)00179-4 [35] McCormick, R., Pearce, I., Kaye, S., & Haneef, A. (2020). Opti-misation of a Nov- el Bio-Substrate as a Treatment for Atrophic Age-Related Macular Degeneration. Frontiers in bioengineering and bio-technology, 8, 456. https://doi.org/10.3389/fbioe.2020.00456 [36] Lu, L., Garcia, C. A., & Mikos, A. G. (1998). Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films. Journal of biomaterials science. Poly- mer edi-tion, 9(11), 1187–1205. https://doi. org/10.1163/156856298x00721 [37] McCormick, R., Pearce, I., Kaye, S., & Haneef, A. (2020). Opti-misation of a Nov- el Bio-Substrate as a Treatment for Atrophic Age-Related Macular Degeneration. Frontiers in bioengineering and bio-technology, 8, 456. https://doi.org/10.3389/fbioe.2020.00456 [38] Giordano, G. G., Thomson, R. C., Ishaug, S. L., Mikos, A. G., Cumber, S., Garcia, C. A., & Lahiri-Munir, D. (1997). Retinal pig-ment epi- thelium cells cultured on synthetic biodegrad- able polymers. Journal of biomedical materials research, 34(1), 87–93. https://doi.org/10.1002/ (sici)1097-4636(199701)34:1<87::aid-jb- m12>3.0.co;2-m [39] Lavik, E. B., Klassen, H., Warfvinge, K., Langer, R., & Young, M. J. (2005). Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progen- itors. Biomaterials, 26(16), 3187–3196. https:// doi.org/10.1016/j.biomaterials.2004.08.022 [40] Thomson, H. A., Treharne, A. J., Walker, P., Grossel, M. C., & Lotery, A. J. (2011). Optimi- sation of polymer scaffolds for retinal pigment epithelium (RPE) cell transplantation. The Brit- ish journal of ophthalmology, 95(4), 563–568. https://doi.org/10.1136/bjo.2009.166728 [41] Schwartz, S. D., Regillo, C. D., Lam, B. L., Eliott, D., Rosenfeld, P. J., Gregori, N. Z., Hubschman, J. P., Davis, J. L., Heilwell, G., Spirn, M., Maguire, J., Gay, R., Bateman, J., Ostrick, R. M., Morris, D., Vincent, M., Angla- de, E., Del Priore, L. V., & Lanza, R. (2015). Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related Columbia Undergraduate Science Journal Vol. 16, 2022 38 macular degeneration and Stargardt's ma- cu-lar dystrophy: follow-up of two open-label phase 1/2 studies. Lan-cet (London, England), 385(9967), 509–516. https://doi.org/10.1016/ S0140-6736(14)61376-3 [42] Radtke, N. D., Aramant, R. B., Petry, H. M., Green, P. T., Pidwell, D. J., & Seiler, M. J. (2008). Vision improvement in retinal de- gener-ation patients by implantation of retina together with retinal pigment epithelium. Ameri- can journal of ophthalmology, 146(2), 172–182. https://doi.org/10.1016/j.ajo.2008.04.009 [43] Carr, A. J., Smart, M. J., Ramsden, C. M., Powner, M. B., da Cruz, L., & Coffey, P. J. (2013). Development of human embryonic stem cell therapies for age-related macular degenera- tion. Trends in neuro-sciences, 36(7), 385–395. https://doi.org/10.1016/j.tins.2013.03.006 [44] Thackaberry, E. A., Farman, C., Zhong, F., Lorget, F., Staflin, K., Cercillieux, A., Miller, P. E., Schuetz, C., Chang, D., Famili, A., Daugherty, A. L., Rajagopal, K., & Bantseev, V. (2017). Eval- uation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflamma-to- ry Response. Investigative ophthalmology & visual science, 58(10), 4274–4285. https://doi. org/10.1167/iovs.16-21334 [45] Sievers, J., Hausmann, B., Unsicker, K., Berry, M. (1987). Fibro-blast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neu- roscience Letters, 76(2), 157-162. https://doi. org/10.1016/0304-3940(87)90708-7 [46] Tanetsugu, Y., Tagami, T., Terukina, T., Ogawa, T., Ohta, M., & Ozeki, T. (2017). Devel- opment of a Sustainable Release System for a Ranibizumab Biosimilar Using Poly(lactic-co-gly- colic acid) Biode-gradable Polymer-Based Microparticles as a Platform. Biological & phar- maceutical bulletin, 40(2), 145–150. https://doi. org/10.1248/bpb.b16-00437 [47] Wagner, M., Benson, M. T., Rennie, I. G., & MacNeil, S. (1995). Effects of pharmaco- logical modulation of intracellular signalling systems on retinal pigment epithelial cell at- tachment to extracellular matrix proteins. Cur- rent eye research, 14(5), 373–384. https://doi. org/10.3109/02713689508999935 Columbia Undergraduate Science Journal Vol. 16, 2022