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

Utheim, Tor Paaske; Ræder, Sten; Utheim, Øygunn Aass; Paz, María de la; Roald, Borghild; Lyberg, Torstein

doi:10.1136/bjo.2008.149591

Cited by 22 publications

(18 citation statements)

References 19 publications

(24 reference statements)

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“…We previously investigated the effect of 5°C, 23°C and 31°C storage on the viability of cultured human limbal epithelial cells, concluding that 5°C and 31°C storage yielded inferior viability. 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.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to the PI viability analysis, the percentage of remaining viable and dead cells following four-day storage at 4°C, 12°C and 24°C was measured using a CAM/EH-1 Live/Dead viability kit as described previously, 16 with some modifications. Calcein-acetoxymethyl enters the cytosol where it is enzymatically cleaved to the green fluorescent calcein by esterases that are present in living cells.…”

Section: Methodsmentioning

confidence: 99%

“…9 Furthermore, a reliable storage technology is mandatory to enable transportation of tissue, 10 which will become increasingly important in regenerative medicine as various technologies leave the experimental stage to be used in mainstream medicine. Studies on the preservation of cultured ocular epithelia are still limited, however.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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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“…Previous studies from our research group have shown that cultured HLEC sheets can be stored in a closed container for 7 days at 23°C in bovine serum-containing storage medium [7,8]. Storage technology offers several advantages, including flexibility for both the patient and the surgeon in scheduling of surgery [9], sufficient time for sterility testing and other quality assessments [7], and time to transport cultured tissue e.g.…”

Section: Introductionmentioning

confidence: 99%

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

et al. 2015

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Aim/Purpose of the StudyTo 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 MethodsHuman 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).ResultsThe 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).ConclusionLimbal 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.

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“…With continuing improvement of RPE tissue engineering approaches, and with more than 20 million patients suffering from AMD worldwide, 4 an upcoming need for improved storage and transportation methods for cultured RPE is anticipated. A storage period of seven days is considered to be sufficient time to allow both phenotypical and microbiological assessment of the cultured transplants prior to surgery, 5 increased flexibility for scheduling surgeries, as well as transportation of transplants to eye clinics worldwide.…”

Section: Introductionmentioning

confidence: 99%

Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells

Pasovic¹,

Eidet²,

Olstad³

et al. 2016

Current Eye Research

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

Abstract: 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.

Cited by 22 publications

References 19 publications

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

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

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells

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