Organoid technology, as an emerging field within biotechnology, has demonstrated transformative potential in advancing precision medicine. This review systematically outlines the translational trajectory of organoids from bench to bedside, emphasizing their construction methodologies, key regulatory factors, and multifaceted applications in personalized healthcare. By recapitulating physiological architectures and disease phenotypes through three-dimensional culture systems, organoids leverage natural and synthetic scaffolds, stem cell sources, and spatiotemporal cytokine regulation to model tissue-specific microenvironments. Diverse organoid types-including skin, intestinal, lung, and tumor organoids-have facilitated breakthroughs in modeling tissue development, drug efficacy and toxicity screening, disease pathogenesis studies, and patient-tailored diagnostics. For instance, patient-derived tumor organoids preserve tumor heterogeneity and genomic profiles, serving as predictive platforms for individualized chemotherapy responses. In precision medicine, organoid-guided multiomics analyses identify actionable biomarkers and resistance mechanisms, while clustered regularly interspaced short palindromic repeats-based functional screens optimize therapeutic targeting. Despite preclinical successes, challenges persist in standardization, vascularization, and ethical considerations. Future integration of artificial intelligence, microfluidics, and spatial transcriptomics will enhance organoid scalability, reproducibility, and clinical relevance. By bridging molecular insights with patient-specific therapies, organoids are poised to revolutionize precision medicine, offering dynamic platforms for drug development, regenerative strategies, and individualized treatment paradigms.

Several clinical subtypes of uveitis exist yet specific immunopathogenic mechanisms involved remain unclear. Ex vivo studies are limited by lack of fresh retinal biopsies and studies have relied on aqueous humour or peripheral blood, which may not directly reflect disease. The aim of this review is to compare the various in vivo models and review their contributions to our understanding of disease processes. These models, although unable to reflect all clinical signs, have provided insight into the contribution of genes and molecules, characterisation of effector T-cells, cell trafficking into retinal tissues, the contribution of tissue-resident myeloid cells and the mechanism(s) of action of several anti-inflammatory compounds. In vivo uveitis models have provided an excellent resource with which to study the molecular and cellular processes involved. Recent refinements in models, improved imaging, and the application of omics have greatly increased the number of readouts and translational opportunities. Future approaches with in vitro models will also be discussed.

The human lacrimal gland (LG), located above the outer orbital region within the frontal bone socket, is essential in maintaining eye surface health and lubrication. It is firmly anchored to the orbital periosteum by the connective tissue, and it is vital for protecting and lubricating the eye by secreting lacrimal fluid. Disruption in the production, composition, or secretion of lacrimal fluid can lead to dry eye syndrome, a condition characterized by ocular discomfort and potential eye surface damage. This review explores the recent advancements in LG organoid generation using tissues and stem cells, highlighting cutting-edge techniques in biomaterial-based and scaffold-free technologies. Additionally, we shed light on the complex pathophysiology of LG dysfunction, providing insights into the LG physiological roles while identifying strategies for generating LG organoids and exploring their potential clinical applications. Alterations in LG morphology or secretory function can affect the tear film stability and quality, leading to various ocular pathological conditions. This comprehensive review underlines the critical crosslink of LG organoid development with disease modeling and drug screening, underscoring their potential for advancing therapeutic applications.

Aniridia is a rare panocular disease caused by gene mutation in the PAX6, which is essential for eye development. Aniridia is inherited in an autosomal dominant manner, but its phenotype can vary significantly among individuals with the same mutation. Animal models, such as drosophila, zebrafish, and rodents, have been used to study aniridia through Pax6 deletions. Recently, patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro, providing new insights into therapeutic strategies.

In this study, corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations, allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived samples.

Untargeted metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts, with amino acid metabolism pathways being consistently enriched in AAK organoids.

This study emphasizes the profound impact of AAK mutations on metabolism, particularly in amino acid biosynthesis and energy metabolism pathways.

Ocular vision can be hampered by corneal damages, sensibly reducing patients' quality of life and having important social and economic consequences. Ocular surface diseases, which often lead to corneal opacities with visual impairment are the most severe forms of the Limbal Stem Cell Deficiency (LSCD). The present review provides an updated perspective on the available treatments for LSCD, focusing on clinical and biological features, as well as critical points to monitor during clinical translation. Recently developed surgical treatments for LSCD are described, along with their benefits and limitations, with the aim of addressing the issue of correct patient selection. Autologous surgical approaches have been attempted, such as conjunctival limbal autograft (CLAU), simple limbal epithelial transplantation (SLET), and others. Allogeneic limbal stem cell transplantation represents an alternative but carries risk of rejection and requires immunosuppression. Other potential treatments are based on induced pluripotent stem cells (iPSCs), but they require further investigation. The development of advanced therapy medicinal products (ATMPs) such as cultivated limbal epithelial transplantation (CLET), or the use of other epithelia as cultivated oral mucosal epithelial cell transplantation (COMET), has opened additional therapeutic possibilities. Some common critical issues in clinical translation are described, such as patient selection, biopsy procurement, or the use of human/animal derived components, which require rigorous validation to ensure safety and efficacy. Personalized medicine is a promising field for ocular surface restoration, where long-term follow-up studies and standardized criteria are crucial to evaluate the efficacy of these treatments and their cost-effectiveness in providing high-value healthcare.

We developed human cornea organoids (HCOs) from induced pluripotent stem cells (iPSCs) where single-cell RNA-sequence (scRNA-seq) analysis suggested similarity with developing rather than mature human corneas. We performed immunohistology to determine the presence of corneal glycosaminoglycans as an assessment of maturity. We undertook a detailed comparison of the HCO scRNA-seq data with a recent scRNA-seq study of human fetal corneas at different stages to gauge the HCO's maturity.

We generated HCOs from a second iPSC line, NCRM-1, to assess the reproducibility of HCO development. We stained sections from both HCO lines with Alcian blue and picrosirius red to determine deposition of sulfated glycosaminoglycans and fibrillar collagens. We immunolocalized glycosaminoglycan biosynthetic enzymes and proteoglycan core proteins. The scRNA-seq data from IMR90.4 HCOs were compared to that of fetal corneas using MetaNeighbor analysis to assess the similarity of HCOs to different stages of human corneal development.

The MetaNeighbor analysis suggests closer alignment of the IMR90.4 HCOs with 17-18 post-conception week fetal human corneas. HCOs from both iPSC lines deposit sulfated glycosaminoglycans and fibrillar collagens. Immunohistology showed chondroitin/dermatan sulfate (CS/DS) and keratan sulfate in the presumptive stromal and some epithelial layers. The NCRM-1-derived HCOs show increased CS/DS staining compared to the IMR90.4 derived HCOs.

Both HCO lines show similar developmental patterns and timeline. The NCRM-1 HCO line may have more glycosaminoglycan deposition. Overall, the glycosaminoglycan deposition pattern is consistent with an immature tissue. Optimizations based on our current findings may yield more mature stromal cells and cornea-typical proteoglycans.

Ocular diseases such as cataract, refractive error, age-related macular degeneration, glaucoma, and diabetic retinopathy significantly impact vision and quality of life worldwide. Despite advances in conventional treatments, challenges like limited bioavailability, poor patient compliance, and invasive administration methods hinder their effectiveness. Nanomedicine offers a promising solution by enhancing drug delivery to targeted ocular tissues, enabling sustained release, and improving therapeutic outcomes. This review explores the journey of nanomedicine from bench to bedside, focusing on key nanotechnology platforms, preclinical models, and case studies of successful clinical translation. It addresses critical challenges, including pharmacokinetics, regulatory hurdles, and manufacturing scalability, which must be overcome for successful market entry. Additionally, this review highlights safety considerations, current marketed and FDA-approved nanomedicine products, and emerging trends such as gene therapy and personalized approaches. By providing a comprehensive overview of the current landscape and future directions, this article aims to guide researchers, clinicians, and industry stakeholders in advancing the clinical application of nanomedicine in ophthalmology.

In the intricately defined spatial microenvironment, a single fertilized egg remarkably develops into a conserved and well-organized multicellular organism. This observation leads us to hypothesize that stem cells or other seed cell types have the potential to construct fully structured and functional tissues or organs, provided the spatial cues are appropriately configured. Current organoid technology, however, largely depends on spontaneous growth and self-organization, lacking systematic guided intervention. As a result, the structures replicated in vitro often emerge in a disordered and sparse manner during growth phases. Although existing organoids have made significant contributions in many aspects, such as advancing our understanding of development and pathogenesis, aiding personalized drug selection, as well as expediting drug development, their potential in creating large-scale implantable tissue or organ constructs, and constructing multicomponent microphysiological systems, together with functioning at metabolic levels remains underutilized. Recent discoveries have demonstrated that the spatial definition of growth factors not only induces directional growth and migration of organoids but also leads to the formation of assembloids with multiple regional identities. This opens new avenues for the innovative engineering of higher-order organoids. Concurrently, the spatial organization of other microenvironmental cues, such as physical stresses, mechanical loads, and material composition, has been minimally explored. This review delves into the burgeoning field of organoid engineering with a focus on potential spatial microenvironmental control. It offers insight into the molecular principles, expected outcomes, and potential applications, envisioning a future perspective in this domain.

The transparency of the cornea is determined by the extracellular matrix, which is secreted by corneal stromal keratocytes (CSKs). Human-induced pluripotent stem cell (hiPSC)-derived keratocytes (hiPSC-CSKs) can be used in cell-based therapy for treating corneal blindness. Our goal was to develop an effective small molecule-based technique for differentiating hiPSCs into keratocytes.

hiPSCs were cultured in chemically defined medium, and embryoid bodies (EBs) were generated; these EBs were induced into CSKs using keratocyte-differentiated medium. The expression of keratocyte-specific markers was assessed using quantitative RT-PCR, immunostaining and Western blotting.

We found that the expression of genes encoding keratocyte markers, including aldehyde dehydrogenase 1 family member A1 (ALDH1A1), lumican and keratocan, was upregulated. Immunostaining showed positive staining for ALDH1A1 and keratocan in the hiPSC-CSK samples. Similarly, western blot analysis indicated that ALDH1A1 and keratocan expression levels were significantly greater in the hiPSC-CSKs than in the control cells. In addition, hiPSC-CSKs were not transformed into fibroblasts or myofibroblasts.

We established an innovative and effective method to generate CSKs via the EB-based differentiation of hiPSCs, which might be employed for cell-based therapy of corneal stromal opacities.

The cornea acts as the eye foremost protective layer and is essential for its focusing power. Corneal blindness may arise from physical trauma or conditions like dystrophies, keratitis, keratoconus, or ulceration. While conventional treatments involve medical therapies and donor allografts-sometimes supplemented with keratoprostheses-these options are not suitable for all corneal defects. Consequently, the development of bioartificial corneal tissue has emerged as a critical research area, aiming to address the global shortage of human cornea donors. Bioengineered corneas hold considerable promise as substitutes, with the potential to replace either specific layers or the entire thickness of damaged corneas. This review first delves into the structural anatomy of the human cornea, identifying key attributes necessary for successful corneal tissue bioengineering. It then examines various corneal pathologies, current treatments, and their limitations. Finally, the review outlines the primary approaches in corneal tissue engineering, exploring cell-free, cell-based, and scaffold-based options as three emerging strategies to address corneal failure.

Dry eye disease (DED) is a growing public health concern affecting millions of people worldwide and causing ocular discomfort and visual disturbance. Developing its therapeutic drugs based on animal models suffer from interspecies differences and poor prediction of human trials. Here, we established long-term 3D human corneal epithelial organoids, which recapitulated the cell lineages and gene expression signature of the human corneal epithelium. Organoids can be regulated to differentiate ex vivo, but the addition of FGF10 inhibits this process. In the hyperosmolar-induced DED organoid model, the release of inflammatory factors increased, resulting in damage to the stemness of stem cells and a decrease in functional mucin 1 protein. Furthermore, we found that the organoids could mimic clinical drug treatment responses, suggesting that corneal epithelial organoids are promising candidates for establishing a drug testing platform ex vivo. In summary, we established a functional, long-term 3D human epithelial organoid that may serve as an ex vivo model for studying the functional regulation and disease modelling.

The eyes are one of the most important sensory organs in the human body. Currently, diseases such as limbal stem cell deficiency, cataract, retinitis pigmentosa and dry eye seriously threaten the quality of people's lives, and the treatment of advanced blinding eye disease and dry eye is ineffective and costly. Thus, new treatment modalities are urgently needed to improve patients' symptoms and suffering. In recent years, stem cell-derived three-dimensional structural organoids have been shown to mimic specific structures and functions similar to those of organs in the human body. Currently, 3D culture systems are used to construct organoids for different ocular growth and development models and ocular disease models to explore their physiological and pathological mechanisms. Eye organoids can also be used as a platform for drug screening. This paper reviews the latest research progress in regard to eye organoids (the cornea, lens, retina, lacrimal gland, and conjunctiva).

Single-cell RNA sequencing (scRNA-seq) has enabled the identification of novel gene signatures and cell heterogeneity in numerous tissues and diseases. Here we review the use of this technology for Fuchs' Endothelial Corneal Dystrophy (FECD). FECD is the most common indication for corneal endothelial transplantation worldwide. FECD is challenging to manage because it is genetically heterogenous, can be autosomal dominant or sporadic, and progress at different rates. Single-cell RNA sequencing has enabled the discovery of several FECD subtypes, each with associated gene signatures, and cell heterogeneity. Current FECD treatments are mainly surgical, with various Rho kinase (ROCK) inhibitors used to promote endothelial cell metabolism and proliferation following surgery. A range of emerging therapies for FECD including cell therapies, gene therapies, tissue engineered scaffolds, and pharmaceuticals are in preclinical and clinical trials. Unlike conventional disease management methods based on clinical presentations and family history, targeting FECD using scRNA-seq based precision-medicine has the potential to pinpoint the disease subtypes, mechanisms, stages, severities, and help clinicians in making the best decision for surgeries and the applications of therapeutics. In this review, we first discuss the feasibility and potential of using scRNA-seq in clinical diagnostics for FECD, highlight advances from the latest clinical treatments and emerging therapies for FECD, integrate scRNA-seq results and clinical notes from our FECD patients and discuss the potential of applying alternative therapies to manage these cases clinically.

The limited availability of a healthy donor cornea and the incidence of allograft failure led researchers to seek other corneal substitutes via tissue engineering. Exploring the trend of clinical trials of the cornea with the vision of tissue engineering provides an opportunity to reveal future potential corneal substitutes. The results of this clinical trial are beneficial for future study designs to overcome the limitations of current therapeutic approaches. In this study, registered clinical trials of bio-based approaches were reviewed for corneal regeneration on March 22, 2024. Among the 3955 registered trials for the cornea, 392 trials were included in this study, which categorized in three main bio-based scaffolds, stem cells, and bioactive macromolecules. In addition to the acellular cornea and human amniotic membrane, several bio-based materials have been introduced as corneal substrates such as collagen, fibrin, and agarose. However, some synthetic materials have been introduced in recent studies to improve the desired properties of bio-based scaffolds for corneal substitutes. Nevertheless, new insights into corneal regenerative medicine have recently emerged from cell sheets with autologous and allogeneic cell sources. In addition, the future perspective of corneal regeneration is described through a literature review of recent experimental models.

Organoid technology provides a versatile platform for simulating organogenesis, investigating disease pathogenesis, and exploring therapeutic interventions. Among various types of organoids that have been developed, corneal limbal organoids, the three-dimensional miniaturized corneas which are derived from either pluripotent stem cells or limbal epithelial stem cells, are particularly promising for clinical translation. This narrative review summarized the state-of-the-art in corneal limbal organoids research including the cultivation methods, clinical relevance and its limitations and challenges. The potential of corneal limbal organoids in mimicking corneal development, disease modelling, drug screening, and regenerative medicine was discussed. Technical improvements in cultivation techniques, imaging modalities, and gene editing tools are anticipated to overcome current limitations and further promote its clinical potential. Despite challenges and difficulties, the development of corneal limbal organoids opens a new era of regenerative medicine and provides a potential source of stem cell replacement therapies for challenging corneal diseases with the establishment of an in vitro corneal limbal organoid bank.

The cornea, as a dynamic and responsive tissue, constantly interacts with mechanical forces in order to maintain its structural integrity, barrier function, transparency and refractive power. Cells within the cornea sense and respond to various mechanical forces that fundamentally regulate their morphology and fate in development, homeostasis and pathophysiology. Corneal cells also dynamically regulate their extracellular matrix (ECM) with ensuing cell-ECM crosstalk as the matrix serves as a dynamic signaling reservoir providing biophysical and biochemical cues to corneal cells. Here we provide an overview of mechanotransduction signaling pathways then delve into the recent advances in corneal mechanobiology, focusing on the interplay between mechanical forces and responses of the corneal epithelial, stromal, and endothelial cells. We also identify species-specific differences in corneal biomechanics and mechanotransduction to facilitate identification of optimal animal models to study corneal wound healing, disease, and novel therapeutic interventions. Finally, we identify key knowledge gaps and therapeutic opportunities in corneal mechanobiology that are pressing for the research community to address especially pertinent within the domains of limbal stem cell deficiency, keratoconus and Fuchs' endothelial corneal dystrophy. By furthering our understanding corneal mechanobiology, we can contextualize discoveries regarding corneal diseases as well as innovative treatments for them.

Corneal organoids are useful tools for disease modeling and tissue transplantation; however, they have not yet been well studied during maturation. We characterized human iPSC-derived corneal organoids at 1, 2, 3, and 4 months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1 month; early corneal epithelial, endothelial, and stromal cell markers at 2 months; keratocytes as the largest cell population at 3 months; and a large epithelial cell population at 4 months. We compared organoid to fetal corneal development at different stages and found that 4-month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity trajectory analysis, we found that less differentiated cells appear to give rise to corneal epithelial cells during development.

The human corneal epithelial barrier plays a crucial role in drug testing studies, including drug absorption, distribution, metabolism, and excretion (ADME), as well as toxicity testing during the preclinical stages of drug development. However, despite the valuable insights gained from animal and current in vitro models, there remains a significant discrepancy between preclinical drug predictions and actual clinical outcomes. Additionally, there is a growing emphasis on adhering to the 3R principles (refine, reduce, replace) to minimize the use of animals in testing. To tackle these challenges, there is a rising demand for alternative in vitro models that closely mimic the human corneal epithelium. Recently, remarkable advancements have been made in two key areas: microphysiological systems (MPS) or organs-on-chips (OoCs), and stem cell-derived organoids. These cutting-edge platforms integrate four major disciplines: stem cells, microfluidics, bioprinting, and biosensing technologies. This integration holds great promise in developing powerful and biomimetic models of the human cornea.

To explore the advances and future research directions in image analysis and computational modelling of human stem cells (hSCs) for ophthalmological applications.

hSCs hold great potential in ocular regenerative medicine due to their application in cell-based therapies and in disease modelling and drug discovery using state-of-the-art 2D and 3D organoid models. However, a deeper characterisation of their complex, multi-scale properties is required to optimise their translation to clinical practice. Image analysis combined with computational modelling is a powerful tool to explore mechanisms of hSC behaviour and aid clinical diagnosis and therapy.

Many computational models draw on a variety of techniques, often blending continuum and discrete approaches, and have been used to describe cell differentiation and self-organisation. Machine learning tools are having a significant impact in model development and improving image classification processes for clinical diagnosis and treatment and will be the focus of much future research.

Keratoconus is a corneal ectatic disease caused by stromal thinning leading to astigmatism and progressive loss of vision. Loss of the keratocytes and excessive degradation of collagen fibres by matrix metalloproteinases are the molecular signatures of the disease. Despite several limitations, corneal collagen cross-linking and keratoplasty are the most widely used treatment options for keratoconus. In the pursuit of alternative treatment modalities, clinician scientists have explored cell therapy paradigms for treating the condition.

Articles pertaining to keratoconus cell therapy with relevant key words were used to search in PubMed, Researchgate, and Google Scholar. The articles were selected based on their relevance, reliability, publication year, published journal, and accessibility.

Various cellular abnormalities have been reported in keratoconus. Diverse cell types such as mesenchymal stromal cells, dental pulp cells, bone marrow stem cells, haematopoietic stem cells, adipose-derived stem cells apart from embryonic and induced pluripotent stem cells can be used for keratoconus cell therapy. The results obtained show that there is a potential for these cells from various sources as a viable treatment option.

There is a need for consensus with respect to the source of cells, mode of delivery, stage of disease, and duration of follow-up, to establish a standard operating protocol. This would eventually widen the cell therapy options for corneal ectatic diseases beyond keratoconus.

Dry eye disease (DED) is characterized by ocular dryness, irritation and blurred vision and has a significant impact on the patient's quality of life. This condition can be particularly severe in patients with aqueous deficient dry eye disease (ADDE) due to Sjögren's syndrome (SS), an autoimmune disease that affects the lacrimal and salivary glands. Current treatments for ADDE are often limited to symptomatic relief. A literature review was conducted to explore the current surgical interventions used or tested in humans with ADDE (I). These interventions include procedures involving the eyelids and tear ducts, transplantation of amniotic membrane or salivary glands, injections around the tear ducts and cell-based injections into the lacrimal gland (LG). Each treatment has its advantages and disadvantages; however, treating dry eyes in patients with SS presents a particular challenge due to the systemic nature of the disease. Moreover, there is a need for new therapeutic options. Mesenchymal stem cells (MSCs) are a type of stem cell that have shown promise in regenerating damaged tissue and reducing inflammation in various diseases. Previous studies in animal models have suggested that MSCs could be effective in treating ADDE. Thus, this thesis aims to investigate the safety and efficacy of injecting MSCs into the LG as a treatment option for patients with ADDE secondary to SS. The study also aims to see this treatment in light of existing and novel investigational treatment options. The clinical studies conducted for this thesis are the first of their kind in humans. MSCs derived from healthy donors' adipose tissue (ASCs) were cultured in a laboratory, frozen and thawed ready for use. In the safety study, we performed the first human trial involving the administration of a single injection of ASCs into the LG of one eye in seven patients suffering from severe ADDE (II). The primary objective was to test the safety of this treatment, while the secondary objective was to assess improvements in subjective and objective signs of dry eye. The results of the trial showed no serious side effects within 4 months of follow-up after treatment. On average, there was a 40% reduction in dry eye symptoms assessed with the Ocular Surface Disease Index (OSDI) questionnaire. Additionally, in the treated eye, there was a significant decrease in tear osmolarity, an increase in tear film stability and an increase in tear production. To further investigate the efficacy of this treatment, our research group performed a clinical, randomized study aiming to compare the ASC injection into the LG with the injection of a vehicle (the excipient in which the ASCs are dissolved) and observation (no intervention) (III). The study involved 20 subjects receiving ASC injection, 20 subjects receiving vehicle injection and 14 patients being observed without intervention. The subjects were examined to assess the outcomes with a 12-month follow-up after treatment. Both intervention groups showed a significant reduction in subjective dry eye symptoms of approximately 40%. This improvement was evident at the 1-week follow-up and persisted until the 12-month follow-up. The observation group did not experience any change in OSDI score. The ASCs group exhibited a significant mean increase in non-invasive tear break-up time (NIKBUT) of 6.48 s (149%) at the four-week follow-up, which was significantly higher than that in the vehicle group (p = 0.04). Moreover, the ASCs group showed a significant increase in NIKBUT compared to that in the observation group at the 12-month follow-up (p = 0.004). In both the ASCs and vehicle group, a significant increase in Schirmer test scores at the 4-month follow-up and the 12-month follow-up was observed. In conclusion, this thesis contributes valuable findings with a new treatment option for patients with dry eye disease. Injection of ASCs into the LG was shown to be safe and to improve subjective dry eye symptoms and specifically the tear film stability in patients with ADDE due to SS. Compared to other treatment modalities of ADDE, this treatment has greater potential, as ASCs could potentially be used as an anti-inflammatory therapeutic option for managing DED of other causes as well. RESUMÉ (DANISH SUMMARY): Tørre øjne, karakteriseret ved tørhedsfornemmelse og irritation af øjnene samt sløret syn, har en betydelig indvirkning på patientens livskvalitet. Denne tilstand kan vaere saerligt alvorlig hos patienter med nedsat tåreproduktion (ADDE) som følge af Sjögrens syndrom (SS), en autoimmun sygdom, der påvirker tårekirtlerne og spytkirtlerne. Nuvaerende behandlinger for ADDE er ofte begraenset til symptomlindring. Vi gennemførte en litteraturgennemgang for at undersøge, hvilke nuvaerende kirurgiske behandlingsmetoder, der anvendes eller testes hos patienter med ADDE (I). Disse interventioner inkluderer procedurer, der involverer øjenlåg og tårekanaler, transplantation af amnionhinde eller spytkirtler, injektioner omkring tårekanalerne samt cellebaserede injektioner i tårekirtlen. Hver behandling har sine fordele og ulemper, men behandling af tørre øjne hos patienter med SS udgør en saerlig udfordring på grund af sygdommens systemiske udbredning, og der er behov for nye behandlingsmuligheder. Mesenkymale stamceller (MSCs) er en type stamcelle, der har vist lovende resultater med hensyn til at regenerere beskadiget vaev og reducere inflammation i forskellige sygdomme. Tidligere undersøgelser i dyremodeller har indikeret, at MSCs kan vaere en effektiv behandling af ADDE. Denne afhandling har til formål at undersøge sikkerheden og effekten af injektion af MSCs i tårekirtlen som en mulig behandling til patienter med ADDE som følge af SS. Afhandlingen sigter også mod at sammenligne denne behandling med andre eksisterende, kirurgiske behandlingsmuligheder af ADDE. Som led i dette projekt udførte vi de første kliniske forsøg af sin art i mennesker. MSCs fra raske donorers fedtvaev (ASCs) blev dyrket i et laboratorium, frosset ned og er optøet klar til brug. Det første mål var at teste sikkerheden ved denne behandling og sekundaert at undersøge behandlingens effekt. For at undersøge dette modtog syv forsøgspersoner med svaer ADDE én injektion med ASCs i tårekirtlen på det ene øje (II). Resultaterne af forsøget viste ingen alvorlige bivirkninger inden for fire måneders opfølgning efter behandlingen. I gennemsnit fandt vi yderligere en 40% reduktion i symptomer på tørre øjne vurderet med et spørgeskema, og en markant stigning i tåreproduktionen og af tårefilmens stabilitet i det behandlede øje. For yderligere at undersøge effekten af denne behandling udførte vi et klinisk, randomiseret forsøg med det formål at sammenligne injektion af ASCs i tårekirtlen med injektion af en kontrolopløsning (vaesken, hvor stamcellerne var opløst) og observation (ingen intervention) (III). Studiet omfattede 20 forsøgspersoner, der modtog ASC-injektion, 20 forsøgspersoner, der modtog injektion af kontrolopløsningen, og 14 forsøgspersoner i observationsgruppen. Forsøgspersonerne blev undersøgt med en opfølgningstid på 12 måneder efter behandling. Begge interventionsgrupper viste en betydelig reduktion på ca. 40% i subjektive symptomer på tørre øjne. Denne forbedring var betydelig allerede ved opfølgning efter en uge og varede ved 12 måneder efter behandling. Observationsgruppen oplevede ingen betydelig aendring i symptomer. ASCs gruppen viste desuden en signifikant stigning i tårefilmsstabiliteten (NIKBUT) på 6,48 sekunder (149%) ved opfølgning efter fire uger, hvilket var markant højere end efter injektion af kontrolopløsning (p = 0,04). Desuden viste ASCs gruppen en betydelig stigning i NIKBUT sammenlignet med observationsgruppen ved opfølgning efter 12 måneder (p = 0,004). Både injektion af ASCs og kontrolopløsning medførte en betydelig stigning i tåreproduktionen ved opfølgning fire måneder og 12 måneder efter behandling. Denne afhandling bidrager med vigtige resultater inden for en ny behandlingsmulighed af tørre øjne. Injektion af ASCs i tårekirtlen viste sig at vaere sikker, forbedrede subjektive symptomer på tørre øjne og øgede saerligt tårfilmens stabilitet hos patienter med ADDE på grund af SS. Sammenlignet med andre behandlingsmuligheder for ADDE har denne behandling vist et stort potentiale. ASCs kan muligvis også bruges som en anti-inflammatorisk behandling af tørre øjne af andre årsager i fremtiden.

The structure and major cell types of the multi-layer human cornea have been extensively studied. However, various cell states in specific cell types and key genes that define the cell states are not fully understood, hindering our comprehension of corneal homeostasis, related diseases, and therapeutic discovery. Single-cell RNA sequencing is a revolutionary and powerful tool for identifying cell states within tissues such as the cornea. This review provides an overview of current single-cell RNA sequencing studies on the human cornea, highlighting similarities and differences between them, and summarizing the key genes that define corneal cell states reported in these studies. In addition, this review discusses the opportunities and challenges of using single-cell RNA sequencing to study corneal biology in health and disease.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

The eyes possess sophisticated physiological structures, diverse disease targets, limited drug delivery space, distinctive barriers, and complicated biomechanical processes, requiring a more in-depth understanding of the interactions between drug delivery systems and biological systems for ocular formulation development. However, the tiny size of the eyes makes sampling difficult and invasive studies costly and ethically constrained. Developing ocular formulations following conventional trial-and-error formulation and manufacturing process screening procedures is inefficient. Along with the popularity of computational pharmaceutics, non-invasive in silico modeling & simulation offer new opportunities for the paradigm shift of ocular formulation development. The current work first systematically reviews the theoretical underpinnings, advanced applications, and unique advantages of data-driven machine learning and multiscale simulation approaches represented by molecular simulation, mathematical modeling, and pharmacokinetic (PK)/pharmacodynamic (PD) modeling for ocular drug development. Following this, a new computer-driven framework for rational pharmaceutical formulation design is proposed, inspired by the potential of in silico explorations in understanding drug delivery details and facilitating drug formulation design. Lastly, to promote the paradigm shift, integrated in silico methodologies were highlighted, and discussions on data challenges, model practicality, personalized modeling, regulatory science, interdisciplinary collaboration, and talent training were conducted in detail with a view to achieving more efficient objective-oriented pharmaceutical formulation design.

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4-7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.

Aniridia is a pan-ocular genetic developmental eye disorder characterized by complete or partial iris and foveal hypoplasia, for which there is no treatment currently. Progressive sight loss can arise from cataracts, glaucoma, and aniridia-related keratopathy, which can be managed conservatively or through surgical intervention. The vast majority of patients harbor heterozygous mutations involving the PAX6 gene, which is considered the master transcription factor of early eye development. Over the past decades, several disease models have been investigated to gain a better understanding of the molecular pathophysiology, including several mouse and zebrafish strains and, more recently, human-induced pluripotent stem cells (hiPSCs) derived from aniridia patients. The latter provides a more faithful cellular system to study early human eye development. This review outlines the main aniridia-related animal and cellular models used to study aniridia and highlights the key discoveries that are bringing us closer to a therapy for patients.

The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease, and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here, we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma, and endothelium, with subpopulations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a 3D model to study corneal diseases and integrated responses of different cell types.

Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors.

Organs-on-chips are microfluidic devices for cell culturing to simulate tissue-level or organ-level physiology and recapitulate their microenvironment, providing new and significant solutions other than traditional animal tests. In vitro testing platforms for ocular biological studies have been increasingly used in preclinical efficacy and toxicity prediction. Here, we developed a microfluidic platform consisting of human corneal cells and porous membrane, replicating the multi-scale structural organization and biological phenotype. We verified the fully integrated human cornea's barrier effects on the chip. Moreover, we found that extracellular vesicles derived from bone marrow-derived mesenchymal stem cells can significantly accelerate the mild corneal epithelial wound healing, and the decreased expression of matrix metallopeptidase-2 protein indicated that this method effectively inhibits corneal inflammation and angiogenesis. This work improves our ability to simulate the interaction between the human cornea and the external world in vitro and contributes to the future development of new screening platforms for biopharmaceuticals.

Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal transplantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine.

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

Mucins are an ancient group of glycoproteins that provide viscoelastic, lubricating and hydration properties to fluids bathing wet surfaced epithelia. They are involved in the protection of underlying tissues by forming a barrier with selective permeability properties. The expression, processing and spatial distribution of mucins are often determined by organ-specific requirements that in the eye involve protecting against environmental insult while allowing the passage of light. The human ocular surface epithelia have evolved to produce an extremely thin and watery tear film containing a distinct soluble mucin product secreted by goblet cells outside the visual axis. The adaptation to the ocular environment is notably evidenced by the significant contribution of transmembrane mucins to the tear film, where they can occupy up to one-quarter of its total thickness. This article reviews the tissue-specific properties of human ocular mucins, methods of isolation and detection, and current approaches to model mucin systems recapitulating the human ocular surface mucosa. This knowledge forms the fundamental basis to develop applications with a promising biological and clinical impact.

The widely used immortalised human meibomian gland epithelia cell (iHMGEC) line has made possible extensive studies of the biology and pathophysiology of meibomian glands (MG). Tissue culture protocols for iHMGEC have been revised and modified to optimise the growth conditions for cell differentiation and lipid accumulation. iHMGEC proliferate in serum-free medium but require serum or other appropriate exogenous factors to differentiate. Several supplements can enhance differentiation and neutral lipid accumulation in iHMGEC grown in serum-containing medium. In serum-free medium, rosiglitazone, a peroxisome proliferator activator receptor-γ (PPARγ) agonist, is reported to induce iHMGEC differentiation, neutral lipid accumulation and expression of key biomarkers of differentiation. iHMGEC cultured in serum-containing medium under hypoxia or with azithromycin increases DNAse 2 activity, a biomarker of terminal differentiation in sebocytes. The production of lipids with composition similar to meibum has not been observed in vitro and this remains a major challenge for iHMGEC culture. Innovative methodologies such as 3D ex vivo culture of MG and generation of MG organoids from stem cells are important for further developing a model that more closely mimics the in vivo biology of human MG and to facilitate the next generation of studies of MG disease and dry eye.

Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated.

A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes.

In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix.

This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease.

The 21st century has seen a steadily increasing social awareness of animal suffering, with increased attention to ethical considerations. Developing new integrated approaches to testing and assessment (IATA) strategies is an Organisation for Economic Co-operation and Development (OECD) goal to reduce animal testing. Currently, there is a lack of alternative models to test for ocular surface toxicity (aside from irritation) in lieu of the Draize eye irritation test (OECD guideline No. 405) performed in rabbits. Five alternative in vitro or ex vivo methods have been validated to replace this reference test, but only in combination. However, pathologies like Toxicity-Induced Dry Eye (TIDE), cataract, glaucoma, and neuropathic pain can occur after exposure to a pharmaceutical product or chemical and therefore need to be anticipated. To do so, new models of lacrimal glands, lens, and neurons innervating epithelia are required. These models must take into account real-life exposure (dose, time, and tear film clearance). The scientific community is working hard to develop new, robust, alternative, in silico, and in vitro models, while attempting to balance ethics and availability of biological materials. This review provides a broad overview of the validated methods for analyzing ocular irritation and those still used by some industries, as well as promising models that need to be optimized according to the OECD. Finally, we give an overview of recently developed innovative models, which could become new tools in the evaluation of ocular surface toxicity within the scope of IATAs.

The prevalence of chronic fundus diseases is increasing with the aging of the general population. The treatment of these intraocular diseases relies on invasive drug delivery because of the globular structure and multiple barriers of the eye. Frequent intraocular injections bring heavy burdens to the medical care system and patients. The use of topical drugs to treat retinal diseases has always been an attractive solution. The fast development of new materials and technologies brings the possibility to develop innovative topical formulations. This article reviews anatomical and physiological barriers of the eye which affect the bioavailability of topical drugs. In addition, we summarize innovative topical formulations which enhance the permeability of drugs through the ocular surface and/or extend the drug retention time in the eye. This article also reviews the differences of eyes between different laboratory animals to address the translational challenges of preclinical models. The fast development of in vitro eye models may provide more tools to increase the clinical translationality of topical formulations for intraocular diseases. Clinical successes of topical formulations rely on continuous and collaborative efforts between different disciplines.

The cornea is an important barrier to consider when developing ophthalmic formulations, but proper modeling of this multilayered tissue remains a challenge. This is due to the varying properties associated with each layer in addition to the dynamics of the tear film. Hence, the most representative models to date rely on animals. Animal models, however, differ from humans in several aspects and are subject to ethical limitations. Consequently, in vitro approaches are being developed to address these issues. This review focuses on the barrier properties of the cornea and evaluates the most advanced three-dimensional cultures of human corneal equivalents in literature. Their application potential is subsequently assessed and discussed in the context of preclinical testing along with our perspective toward the future. Impact statement Most ocular drugs are applied topically, with the transcorneal pathway as the main administration route. Animal corneas are currently the only advanced models available, contributing to the drug attrition rate. Anatomical and physiological interspecies differences might account for a poor translatability of preclinical results to clinical trials, urging researchers to devise better corneal equivalents. This review elaborates on the emerging generation of three-dimensional in vitro models, which comprises spheroids, organoids, and organs-on-chips, which can serve as a stepping stone for advancements in this field.

Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4-5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.