Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

Biopsy harvesting site and distance from the explant affect conjunctival epithelial phenotype ex vivo.

The purpose of the study was to investigate if the number of goblet cells expanded ex vivo from a conjunctival explant is affected by the biopsy harvesting site on the conjunctiva and the distance from the explant. Conjunctival explants from six regions: superior and inferior bulbus, fornix, and tarsus of male Sprague-Dawley rats were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for eight days. Histochemical and immunofluorescent staining of goblet (CK-7/UEA-1/MUC5AC), stratified squamous, non-goblet (CK-4), proliferating (PCNA) and progenitor (ABCG2) cells were analyzed by epifluorescence and laser confocal microscopy. Outgrowth was measured with NIH ImageJ. For statistical analysis the Mann-Whitney test and Spearman's rank-order correlation test were used. Cultures from superior and inferior fornix contained the most goblet cells as indicated by the presence of CK-7+, UEA-1+ and MUC5AC+ cells. Superior and inferior forniceal cultures displayed 60.8% ± 9.2% and 64.7% ± 6.7% CK-7+ cells, respectively, compared to the superior tarsal (26.6% ± 8.4%; P < 0.05), superior bulbar (31.0% ± 4.0%; P < 0.05), inferior bulbar (38.5% ± 9.3%; P < 0.05) and inferior tarsal cultures (27.7% ± 8.3%; P < 0.05). While 28.4% ± 6.3% of CK-7+ goblet cells co-labeled with PCNA, only 7.4% ± 1.6% of UEA-1+ goblet cells did (P < 0.01). CK-7+ goblet cells were located at a lower concentration close to the explant (39.8% ± 3.1%) compared to near the leading edge (58.2% ± 4.5%; P < 0.05). Both markers for goblet cell secretory product (UEA-1 and MUC5AC), however, displayed the opposite pattern with a higher percentage of positive cells close to the explant than near the leading edge (P < 0.05). The percentage of CK-4+ cells was higher near the explant compared to near the leading edge (P < 0.01). The percentage of CK-7+ goblet cells in the cultures did not correlate with the outgrowth size (r(s) = -0.086; P = 0.435). The percentage of UEA-1+ goblet cells correlated negatively with outgrowth size (r(s) = -0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (r(s) = 0.473; P < 0.05). We conclude that forniceal explants yield the highest number of goblet cells ex vivo and thereby seem to be optimal for goblet cell transplantation. We also suggest that CK-7+/UEA-1- cells represent highly proliferative immature goblet cells. These cells could be important during conjunctival migration as they are mostly located close to the leading edge and their density does not decrease with increasing outgrowth size.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

Dry eye disease treatment: the role of tear substitutes, their future, and an updated classification.

OBJECTIVE: The aim of this review is to summarize the results of a consensus meeting held by a group of experts in dry eye disease (DED) to discuss the importance of tear substitutes in the treatment of DED. The meeting focused especially on the main characteristics of lacrimal substitutes, the development of in vitro models to investigate DED pathophysiology and treatment, the importance of conducting rigorous clinical trials, the requirements of the upcoming European Legislation on medical devices, the advances in the formulation of safer preservatives, the peculiarities of treatment in younger subjects, and the importance of an updated terminology for lacrimal substitutes. MATERIALS AND METHODS: A literature search was conducted using MEDLINE, with different combinations of pertinent keywords, depending on the subject under discussion, such as "dry eye disease"; "tear substitutes"; "in vitro models"; "ocular surface"; "clinical trials"; "European Regulation"; "preservatives" "younger patients". Also, each author included in the discussion selected articles from their personal library. Using a consensus-based method called nominal group technique to reach a conclusion and proposal for a new classification of eye drops used to improve the tear film and ocular surface epithelia, the experts also conducted a round table meeting. RESULTS: The new terms proposed by the authors are "wetting agents", "multiple-action tear substitutes" or "ocular surface modulators". The new classification is needed to distinguish eye drops used to improve the tear film and ocular surface epithelia, in line with the new definition of DED, which recognizes the loss of ocular homeostasis, and the creation of a vicious circle of chronic inflammation and ocular damage as fundamental aspects of DED pathophysiology. CONCLUSIONS: Although tear substitutes have been historically used to provide eye lubrication to the ocular surface, recent advances in the pathophysiology of dry eye disease (DED) clarified that treatment should not just focus on tear film quality or quantity, but address the loss of homeostasis of the ocular surface, blocking the vicious circle of chronic inflammation and ocular damage. Given the scant comparative evidence on tear substitutes currently on the market, further studies should focus on developing new agents, considering the advantages provided by in vitro models, importance of conducting rigorous clinical trials, availability of less harmful preservatives and obligations related to the new European legislation on medical devices. Based on the discussion of these topics, a group of experts held a consensus meeting to identify new and more appropriate terms for different tear substitutes. The proposed terms are wetting agents, multiple-action tear substitutes and ocular surface modulators. Regardless of the agent used, it is important to note that tear substitutes represent one of many options for DED treatment, which should not overlook the psychological aspects of the disease and the peculiarities of younger subjects, who seem to have a higher risk for DED, possibly related to digital devices excessive use.

Patients with non-Sjögren's sicca report poorer general and oral health-related quality of life than patients with Sjögren's syndrome: a cross-sectional study.

Understanding the impact of the disease on quality of life is crucial in patient management. In this cross-sectional study, general and oral health-related quality of life questionnaires, and thorough examinations of oral and ocular dryness were performed in age- and sex-matched patients with primary Sjögren's syndrome (pSS group), non-Sjögren's syndrome sicca (non-SS group) and healthy controls. General and oral health-related quality of life were investigated with the 36-Item Short Form Health Survey and the 14-Item Oral Health Impact Profile questionnaires, respectively. Subjective symptoms of xerostomia and ocular dryness were recorded using the Summated Xerostomia Inventory and Ocular Surface Disease Index, respectively. Clinical examinations included evaluation of clinical oral dryness scores, candida counts, unstimulated and stimulated saliva secretory rates, tear osmolarity, tear film break-up time, Schirmer I test and ocular surface staining. Both patient groups had pronounced signs and symptoms of xerostomia and ocular dryness. Even though the non-SS patients had less severe clinical signs than the pSS patients, they demonstrated much poorer general and oral health-related quality of life. In conclusion, non-SS patients require more attention in order to improve their quality of life.

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells.

PURPOSE: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. METHODS: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. RESULTS: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin ß1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. CONCLUSION: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

Effect of Transportation on Cultured Limbal Epithelial Sheets for Worldwide Treatment of Limbal Stem Cell Deficiency.

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation.

BACKGROUND/AIMS: To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage. METHODS: HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23 degrees C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry. RESULTS: No microbial contamination was observed after 1 week's storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained. CONCLUSION: This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week's storage was unsatisfactory.