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Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

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      Abstract

      Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.

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        THE METABOLISM OF TUMORS IN THE BODY

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          Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.

          Ocular trauma or disease may lead to severe corneal opacification and, consequently, severe loss of vision as a result of complete loss of corneal epithelial stem cells. Transplantation of autologous corneal stem-cell sources is an alternative to allograft transplantation and does not require immunosuppression, but it is not possible in many cases in which bilateral disease produces total corneal stem-cell deficiency in both eyes. We studied the use of autologous oral mucosal epithelial cells as a source of cells for the reconstruction of the corneal surface. We harvested 3-by-3-mm specimens of oral mucosal tissue from four patients with bilateral total corneal stem-cell deficiencies. Tissue-engineered epithelial-cell sheets were fabricated ex vivo by culturing harvested cells for two weeks on temperature-responsive cell-culture surfaces with 3T3 feeder cells that had been treated with mitomycin C. After conjunctival fibrovascular tissue had been surgically removed from the ocular surface, sheets of cultured autologous cells that had been harvested with a simple reduced-temperature treatment were transplanted directly to the denuded corneal surfaces (one eye of each patient) without sutures. Complete reepithelialization of the corneal surfaces occurred within one week in all four treated eyes. Corneal transparency was restored and postoperative visual acuity improved remarkably in all four eyes. During a mean follow-up period of 14 months, all corneal surfaces remained transparent. There were no complications. Sutureless transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface. Copyright 2004 Massachusetts Medical Society
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            Author and article information

            Affiliations
            [1 ]Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway
            [2 ]University of Oslo, Oslo, Norway
            [3 ]Ear, Nose and Throat Department, Division of Surgery, Akershus University Hospital, Lørenskog, Norway
            [4 ]Institute of Clinical Medicine, University of Oslo, Oslo, Norway
            [5 ]Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway
            [6 ]Centre for Clinical Research, LT Vastmanland, Uppsala University, Uppsala, Sweden
            University of Newcastle upon Tyne, United Kingdom
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: CJ TPU PA. Performed the experiments: CJ PA. Analyzed the data: CJ TPU PA JRE. Contributed reagents/materials/analysis tools: TPU TL EM MVU. Contributed to the writing of the manuscript: CJ TPU PA TL EM MVU JRE.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2014
            29 August 2014
            : 9
            : 8
            25170754
            4149437
            PONE-D-14-22083
            10.1371/journal.pone.0105808
            (Editor)

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Pages: 9
            Funding
            Funding was provided by (1) South East Norway Regional Health Authority, Norway, grant number 2012074 ( http://www.helse-sorost.no/omoss_/english_/) to CJ, and (2) University of Oslo, Norway. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Medicine and Health Sciences
            Critical Care and Emergency Medicine
            Trauma Medicine
            Burns
            Ophthalmology
            Visual Impairments
            Blindness
            Corneal Disorders
            Eye Diseases
            Inherited Eye Disorders
            Custom metadata
            The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

            Uncategorized

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