The Effects of Prolonged Storage on ARPE-19 Cells Stored at Three Different Storage Temperatures.

This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures (4 °C, 16 °C, and 37 °C) for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 °C compared to 4 °C and 16 °C. During storage, morphology started to deteriorate first at 4 °C, then at 37 °C, and was maintained the longest at 16 °C. Viability of the cells after three weeks of storage was best preserved at 16 °C, while cells stored at 4 °C and 37 °C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 °C reduces metabolic activity while protecting viability and morphology.

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins.

We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch's membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

Effect of limbal marking prior to laser ablation on the magnitude of cyclotorsional error.

PURPOSE: To evaluate the residual registration error after limbal-marking-based manual adjustment in cyclotorsional tracker-controlled laser refractive surgery. METHODS: Two hundred eyes undergoing custom surface ablation with the iVIS Suite (iVIS Technologies) were divided into limbal marked (marked) and non-limbal marked (unmarked) groups. Iris registration information was acquired preoperatively from all eyes. Preoperatively, the horizontal axis was recorded in the marked group for use in manual cyclotorsional alignment prior to surgical iris registration. During iris registration, the preoperative iris information was compared to the eye-tracker captured image. The magnitudes of the registration error angle and cyclotorsional movement during the subsequent laser ablation were recorded and analyzed. RESULTS: Mean magnitude of registration error angle (absolute value) was 1.82°±1.31° (range: 0.00° to 5.50°) and 2.90°±2.40° (range: 0.00° to 13.50°) for the marked and unmarked groups, respectively (P<.001). Mean magnitude of cyclotorsional movement during the laser ablation (absolute value) was 1.15°±1.34° (range: 0.00° to 7.00°) and 0.68°±0.97° (range: 0.00° to 6.00°) for the marked and unmarked groups, respectively (P=.005). Forty-six percent and 60% of eyes had registration error >2°, whereas 22% and 20% of eyes had cyclotorsional movement during ablation >2° in the marked and unmarked groups, respectively. CONCLUSIONS: Limbal-marking-based manual alignment prior to laser ablation significantly reduced cyclotorsional registration error. However, residual registration misalignment and cyclotorsional movements remained during ablation.

Retinal Pigment Epithelium Transplantation: Past, Present, and Future.

Retinal pigment epithelium (RPE) is a monolayer of cells situated between photoreceptors and the underlying choroid. It is essential for normal retinal function. Damaged RPE is associated with diseases such as age-related macular degeneration, Stargardt's macular dystrophy, and retinitis pigmentosa. RPE cells can easily be visualized in vivo, sustainable in vitro, and differentiated from stem cells with a relatively straightforward protocol. Due to these properties and the clinical significance of this epithelium in various retinal diseases, RPE transplantation as a treatment modality has gained considerable interest in the last decade. This paper presents the main techniques for RPE transplantation and discusses recent clinically relevant publications.

Seasonal variations in presenting symptoms and signs of dry eye disease in Norway.

The study investigated the seasonal variations of presenting symptoms and signs of dry eye disease (DED) in Norway. 652 consecutive DED patients examined between August 2012 and May 2015 in Oslo, Norway, were included. Presenting symptoms and signs were related to the season according to when each patient was examined. Weather report data from the examination day were compared with the presenting symptoms and signs. Oslo's mean seasonal temperatures during spring, summer, fall, and winter were 6.4 °C, 15.6 °C, 9.3 °C, and - 2.1 °C, respectively. Dry eye severity level and self-reported symptoms measured by the Ocular surface disease index questionnaire did not differ between seasons. Schirmer I was lower during summer than in other seasons (P < 0.01). The percentage of patients with a pathological tear meniscus height (< 0.2 mm) was higher during fall (P < 0.01) and lower during winter (P < 0.05) compared to the other seasons. Signs and symptoms of DED generally did not correlate with weather report data, although intraocular pressure was weakly associated with mean daily air temperature (r = - 0.22; P < 0.001). Neither dry eye severity level nor dry eye symptoms differ between seasons in Oslo, Norway. However, some parameters for assessing DED show seasonal variations (Schirmer I and tear meniscus height), which are essential to consider when examining patients with DED.

Successful treatment with repeated dexamethasone implant injections for recurrent macular edema after acute retinal necrosis.

BACKGROUND: The treatment of recurrent cystoid macular edema associated with acute retinal necrosis is challenging due to the concern that treatment with intravitreal steroids may reactivate the retinitis. CASE REPORT: An immunocompetent patient diagnosed with acute retinal necrosis was treated with oral valacyclovir and intravitreal injections of foscarnet. Giant tears in her retina necessitated a vitrectomy with silicone oil. She developed cystoid macular edema after the removal of the silicone oil. The edema responded to high-dose prednisolone but recurred when the dose was tapered to 20 mg daily. Under close surveillance and increased antiviral medication, she was treated with a dexamethasone implant with complete resolution of the edema. Unfortunately, the edema recurred, and the treatment had to be repeated. Over 18 months, she received five dexamethasone implants without recurrence of the viral retinitis. CONCLUSIONS: This case shows successful treatment of recurring cystoid macular edema following acute retinal necrosis with repeated intravitreal dexamethasone implants in a patient receiving valacyclovir maintenance treatment.

Corrigendum: Concise Review: Considering Optimal Temperature for Short-Term Storage of Epithelial Cells.

[This corrects the article DOI: 10.3389/fmed.2021.686774.].

Concise Review: Considering Optimal Temperature for Short-Term Storage of Epithelial Cells.

Transplantation of novel tissue-engineered products using cultured epithelial cells is gaining significant interest. While such treatments can readily be provided at centralized medical centers, delivery to patients at geographically remote locations requires the establishment of suitable storage protocols. One important aspect of storage technology is temperature. This paper reviews storage temperature for above-freezing point storage of human epithelial cells for regenerative medicine purposes. The literature search uncovered publications on epidermal cells, retinal pigment epithelial cells, conjunctival epithelial cells, corneal/limbal epithelial cells, oral keratinocytes, and seminiferous epithelial cells. The following general patterns were noted: (1) Several studies across different cell types inclined toward 4 and 16°C being suitable short-term storage temperatures. Correspondingly, almost all studies investigating 37°C concluded that this storage temperature was suboptimal. (2) Cell death typically escalates rapidly following 7-10 days of storage. (3) The importance of the type of storage medium and its composition was highlighted by some of the studies; however, the relative importance of storage medium vs. storage temperature has not been investigated systematically. Although a direct comparison between the included investigations is not reasonable due to differences in cell types, storage media, and storage duration, this review provides an overview, summarizing the work carried out on each cell type during the past two decades.

The acute phase response protein SERPINA3 is increased in tear fluid from the unaffected eyes of patients with unilateral acute anterior uveitis.

BACKGROUND: To identify candidate tear fluid biomarkers in patients with unilateral acute anterior uveitis (AAU) that can aid in the differentiation between these patients and patients with bacterial keratitis or healthy controls. METHODS: Thirteen patients (40.1 ± 16.2 years of age) with unilateral AAU, seven patients with unilateral bacterial keratitis (40.2 ± 15.3 years of age), and 14 healthy subjects (41.1 ± 11.6 years of age) were included. The tear proteome of affected eyes was compared with that of the unaffected eye or healthy controls. Proteins were identified by liquid chromatography tandem mass spectrometry and enzyme-linked immunosorbent assay. RESULTS: Relative protein ratios were detected and calculated for 272 unique proteins. Compared with healthy controls and the unaffected eye, the top upregulated proteins in AAU eyes were submaxillary gland androgen regulated protein 3B (SMR3B) and SMR3A. Similarly, the top upregulated proteins in bacterial keratitis were S100 calcium-binding protein A9 and orosomucoid 2. The acute phase response protein Serpin Family A Member 3 (SERPINA3) was increased in the healthy eye of AAU patients (P = 0.019) compared with healthy controls. Laser flare measurements in affected eyes of AAU patients showed positive logarithmic correlation with SERPINA3 in tear samples of the unaffected eye (P = 0.022). The use of SERPINA3 as a tear biomarker yielded a sensitivity of 85% and a specificity of 71% in detecting patients with AAU in the study population. CONCLUSIONS: The acute phase response protein SERPINA3 was increased in tear samples of unaffected eyes of patients with unilateral AAU compared with healthy controls. This study highlights SERPINA3 as a potential biomarker for AAU. Future research should explore the dynamic properties of SERPINA3 in the tear fluid of active and quiescent uveitis eyes.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro.

PURPOSE: Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). METHODS: ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin's effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. RESULTS: Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. CONCLUSIONS: This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin's viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Unilateral acute anterior uveitis is associated with ipsilateral changes in the tear fluid proteome that involves the LXR/RXR pathway.

PURPOSE: To determine whether unilateral acute anterior uveitis (AAU) induces ipsilateral changes in the tear fluid proteome. METHODS: Five patients (25-77 years old) with unilateral AAU were included. Tear fluid samples were obtained using Schirmer's test strips. The healthy eye served as control. Proteins were identified by liquid chromatography tandem mass spectrometry. RESULTS: Two hundred forty-two tear fluid sample proteins were identified, of which 75 were present in at least three patients. Nine proteins were at least 1.5-fold increased, whereas eight were at least 1.5-fold decreased in tears from the diseased eye compared with the healthy eye. APOBEC3A was significantly increased (1.43-fold; P = 0.04), whereas TGM2 was significantly decreased (- 1.21-fold; P = 0.03) in tears from the diseased eye relative to the healthy eye. Ingenuity Pathway Analysis identified LXR/RXR (P < 1.02E-4) as a top canonical pathway. CONCLUSION: Unilateral AAU induced detectable changes in the ipsilateral tear fluid proteome and involvement of the inflammation-associated LXR/RXR pathway.

Molecular layer deposition builds biocompatible substrates for epithelial cells.

The demand for novel biocompatible materials as surface coating in the field of regenerative medicine is high. We explored molecular layer deposition (MLD) technique for building surface coatings and introduced a new group of substrates consisting of amino acids, or nucleobases, and the biocompatible metal titanium. The substrates were built from titanium tetraisopropoxide (TTIP) with l-lysine, glycine, l-aspartic acid, l-arginine, thymine, uracil, and adenine. Substrates based on zirconium chloride and terephthalic acid were also included. Titanium oxide (TiO2 ) substrates made by atomic layer deposition and uncoated cover slips served as controls. Rat conjunctival epithelial goblet cells were grown in RPMI 1640 and RT-PCR, immunofluorescence, cell attachment, proliferation, and viability were analyzed. Cells cultured on MLD and uncoated substrates were proliferating (positive for Ki67). Cell attachment after 3 h of culture on MLD substrates was similar to uncoated coverslips (p > 0.05). Compared to uncoated coverslips, cell proliferation assayed with alamarBlue® after 4 days was significantly higher on all MLD substrates (p < 0.05), whereas terephthalic acid-containing MLD substrates reduced proliferation (p < 0.01). Viability assessed by LIVE/DEAD® was high (>85%) for all substrates after 5 days. The novel MLD technique is promising for building biocompatible substrates that direct epithelial cell growth. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3090-3098, 2018.

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets.

There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7-11 days at 12°C in media supplemented with various additives. Effects were evaluated by calcein staining of live cells as well as morphology. Twenty-six additives were tested using (1) a two-level factorial design in which 10 additives were added or omitted in 64 different combinations and (2) a mixture design with 5 additives at 5 different concentrations in a total of 64 different mixtures. Automated microscopy and cell counting with Fiji enabled efficient processing of data. Significant regression models were identified by Design-Expert software. A calculated maximum increase of live cells to 37 ± 6% was achieved upon storage of cell sheets for 11 days in the presence of 6% glycerol. The beneficial effect of glycerol was shown for epidermal cell sheets from three different donors in two different storage media and with two different factorial designs. We have thus developed a high throughput screening system enabling robust assessment of live cells and identified glycerol as a beneficial additive that has a positive effect on epidermal cell sheet upon storage at 12°C. We believe this method could be of use in other cell culture optimization strategies where a large number of conditions are compared for their effect on cell viability or other quantifiable dependent variables.

Tissue Harvesting Site and Culture Medium Affect Attachment, Growth, and Phenotype of Ex Vivo Expanded Oral Mucosal Epithelial Cells.

Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.

Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases.

PURPOSE: Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. METHODS: We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. RESULTS: IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA). CONCLUSION: The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies.

Corneal collagen cross-linking (CXL) in thin corneas.

Corneal collagen cross-linking (CXL) is a therapeutic procedure aiming at increasing the corneal stiffness in the keratoconus eyes by induction of cross-links within the extracellular matrix. It is achieved by ultraviolet-A (370 nm) irradiation of the cornea after saturation with the photosensitizer riboflavin. In the conventional CXL protocol, a minimum de-epithelialized corneal thickness of 400 µm is recommended to avoid potential irradiation damage to the corneal endothelium. In advanced keratoconus, however, stromal thickness is often lower than 400 µm, which limits the application of CXL in that category. Efforts have been undertaken to modify the conventional CXL procedure to be applicable in thin corneas. The current review discusses different techniques employed to achieve this end and their results. The overall safety and efficacy of the modified CXL protocols are good, as most of them managed to halt the progression of keratectasia without postoperative complications. However, the evidence of safety and efficacy in the use of modified CXL protocols is still limited to few studies with few patients involved. Controlled studies with long-term follow-up are required to confirm the safety and efficacy of the modified protocols.

Concise Review: Comparison of Culture Membranes Used for Tissue Engineered Conjunctival Epithelial Equivalents.

The conjunctival epithelium plays an important role in ensuring the optical clarity of the cornea by providing lubrication to maintain a smooth, refractive surface, by producing mucins critical for tear film stability and by protecting against mechanical stress and infectious agents. A large number of disorders can lead to scarring of the conjunctiva through chronic conjunctival inflammation. For controlling complications of conjunctival scarring, surgery can be considered. Surgical treatment of symblepharon includes removal of the scar tissue to reestablish the deep fornix. The surgical defect is then covered by the application of a tissue substitute. One obvious limiting factor when using autografts is the size of the defect to be covered, as the amount of healthy conjunctiva is scarce. These limitations have led scientists to develop tissue engineered conjunctival equivalents. A tissue engineered conjunctival epithelial equivalent needs to be easily manipulated surgically, not cause an inflammatory reaction and be biocompatible. This review summarizes the various substrates and membranes that have been used to culture conjunctival epithelial cells during the last three decades. Future avenues for developing tissue engineered conjunctiva are discussed.

Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells.

Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels.

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.

AIM/PURPOSE OF THE STUDY: To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. MATERIALS AND METHODS: Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). RESULTS: The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 µm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). CONCLUSION: Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.