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Abstract Background Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Elizabeth Yeu,1 Damien F Goldberg,2 Francis S Mah,3 Kenneth A Beckman,4,5 Jodi I Luchs,6,7 Jonathan D Solomon,8 Darrell E White,9 Preeya K Gupta101Eastern Virginia Medical School, Virginia Eye Consultants, Norfolk, VA, USA; 2Wolstan & Goldberg Eye Associates, Torrance, CA, USA; 3Scripps Clinic Torrey Pines, La Jolla, CA, USA; 4Ohio State University, Columbus, OH, USA; 5Comprehensive Eye Care of Central Ohio, Westerville, OH, USA; 6Hofstra Northwell School of Medicine, Hempstead, NY, USA; 7South Shore Eye Care, Wantagh, NY, USA; 8Bowie Vision Institute, Bowie, MD, USA; 9Skyvision Centers, Westlake, OH, USA; 10Duke University Eye Center, Durham, NC, USAPurpose: Evaluate the safety and efficacy of cryopreserved amniotic cytokine extract (ACE) in the treatment of subjects with dry eye disease (DED).Patients and methods: This was a retrospective, multicenter, chart review of adult patients with DED that instilled cryopreserved ACE drops twice-daily for 4 or 12 weeks. Patients had corneal fluorescein staining (0–20 range) and/or a lissamine green conjunctival staining score (0–18 range) of ≥3 and ≤9 in at least 1 eye and a score ≥40 (0–100 range) of eye dryness/irritation on a visual analog scale (VAS). Following completion of a treatment course, medical records were reviewed from the initiation of therapy (baseline), and at post-treatment visits (4 weeks, 8 weeks, and 12 weeks). Patient records for visual acuity, adverse events, corneal fluorescein staining, conjunctival lissamine green staining, and symptom scores of ocular dryness/irritation were reviewed for each visit, as available. Safety and tolerability were assessed through the evaluation of patient-reported adverse events recorded in the medical records.Results: A total of 54 eligible patients were identified at 7 clinical sites; 16 patients administered ACE drops for 4-weeks, and 38 patients instilled ACE drops for 12 weeks. Significant improvements in the mean changes from baseline were observed for corneal fluorescein staining, lissamine green staining, visual acuity (LogMar) and VAS ocular symptom scores at the 4-week post-treatment visit (p