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

Eidet, Jon Roger; Utheim, Øygunn Aass; Ræder, Sten; Dartt, Darlene A.; Lyberg, Torstein; Carreras, Elisa; Huynh, Trang; Messelt, Edward B.; Louch, William E.; Roald, Borghild; Utheim, Tor Paaske

doi:10.1016/j.exer.2011.11.015

Cited by 26 publications

(24 citation statements)

References 24 publications

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“…Different storage conditions, inherent differences in cell function found between intact skin tissue and CECS, and species differences may explain the different results. Comparable cell viability has been demonstrated after storage of cultured limbal and conjunctival epithelial cells at 23°C for seven days [30], [31]. A smaller peak in cell viability was seen at 12°C (∼60%) which coincided with highest PCNA expression.…”

Section: Discussionmentioning

confidence: 89%

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

et al. 2014

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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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Section: Discussionmentioning

confidence: 89%

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

et al. 2014

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“…19 Storage at 23°C, or ambient temperature, has proven suitable in several studies, 9, 18, 19, 28, 29 including a recent study on the storage of cultured HCjE. 30 Corneal epithelial cells encapsulated in alginate reportedly maintained a higher percentage of viable cells after storage at ambient temperature (18°C–22°C) than when stored at 4°C or 31°C. 18 Thus, the results of the present study concur with previous work on ocular cell storage and with a study examining ovarian cells by Hunt and colleagues, 31 which demonstrated that 17°C storage resulted in the highest cell viability and the lowest viability followed storage at 4°C and 37°C.…”

Section: Discussionmentioning

confidence: 99%

The Impact of Storage Temperature on the Morphology, Viability, Cell Number and Metabolism of Cultured Human Conjunctival Epithelium

Eidet

Utheim

Islam

et al. 2014

Current Eye Research

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Purpose To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjE). Materials & methods Three-day cultured HCjE were stored at nine different temperatures between 4°C and 37°C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer. Results Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19, and the proliferation marker PCNA. Cell morphology was best maintained following four-day storage between 12°C and 28°C, and following 12°C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12°C and 28°C, while it had decreased in all other groups (P<0.05; n=4). After seven days this percentage was maintained in the 12°C group, but it had decreased in all other groups, compared to the control (P<0.05; n=4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (P<0.05; n=4), except 12°C and 20°C. Following seven days this number had decreased in all groups (P<0.01; n=4), except 12°C. Four-day storage at 12°C demonstrated superior preservation of the number of calcein-stained viable cells (P<0.05) and the least accumulation of ethidium homodimer 1-stained dead cells (P<0.001), compared to storage at 4°C and 24°C (n=6). The total metabolism of glucose to lactate after four-day storage were higher in the 24°C group compared to 4°C and 12°C, as well as the control (P<0.001; n=3). Conclusions Storage at 12°C appears optimal for preserving morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12°C may be related to temperature-associated effects on cell metabolism.

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“…Positive controls included fixed sections of whole rat eyes with eyelids containing structures with known positive staining for each of the antibodies used. Semi-quantitative immunohistochemical analysis of the sections was carried out at a magnification of 630 × and scored as previously described (Eidet et al, 2012b). Positive staining in the sections was graded as 0 (undetectable), + (detectable in <l/4 of the cells), ++ (detectable in 1/4–1/2 of the cells), +++ (detectable in 1/2–3/4 of the cells) and ++++ (detectable in >3/4 of the cells).…”

Section: Methodsmentioning

confidence: 99%

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

Fostad

Eidet

Shatos

et al. 2012

Experimental Eye Research

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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-1/1MUC5AC), 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 (rs = −0.086; P = 0.435). The percentage of UEA-1 + goblet cells correlated negatively with outgrowth size (rs = −0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (rs = 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.

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

Cited by 26 publications

References 24 publications

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

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

The Impact of Storage Temperature on the Morphology, Viability, Cell Number and Metabolism of Cultured Human Conjunctival Epithelium

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

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