Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets

Jackson, Catherine; Reppe, Sjur; Eidet, Jon Roger; Eide, Lars; Tønseth, Kim Alexander; Bergersen, Linda Hildegard; Dartt, Darlene A.; Griffith, May; Utheim, Tor Paaske

doi:10.1038/s41598-017-08586-7

Cited by 5 publications

(11 citation statements)

References 60 publications

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“…Y-27632 inhibits Rho-associated, coiled-coil containing protein kinase (ROCK) by competing with ATP for binding to the catalytic site [ 18 ], thereby preventing apoptosis and increasing cell survival. Jackson et al found the accumulation of mitochondrial reactive oxygen species (ROS) increased differentiation, DNA damage and cell death after storing human ECSs at 4 degrees for 1 week [ 19 ]. Although some protective ingredients can be added, the cell activity of ECSs preserved at low temperature is easy to decrease.…”

Section: Discussionmentioning

confidence: 99%

“…Generally, the undifferentiated cells expressing p63 in the basal layer of the skin are self-renewing and play a vital role in the development and differentiation of epithelial tissue [ 27 ]. Retention of the p63-positive cell phenotype in cultured and stored ECSs can improve the success rate of transplantation in the clinic [ 19 ]. During the freezing/thawing process, the differentiated epithelial cells on the surface of ECSs were more likely to die and fall off than undifferentiated epithelial cells, which explained that the proportion of P63-positive cells in the frozen group was slightly higher than that in the fresh control group.…”

Section: Discussionmentioning

confidence: 99%

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Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model

et al. 2021

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Background Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. As a widely used material in clinic, there is little information on the biobanking of ECSs and its repair effect after storage. Results Two methods for biobanking foreskin ECSs were compared in a short term (7 days): 4-degree storage and programmed cryopreservation. Cell sheet integrity, viability, apoptosis, immunogenicity, mechanical properties and function were evaluated. In vivo, ECSs were directly transplanted to skin defect models and histological examination was performed at 1 week postoperatively. We successfully extracted human foreskin-derived primary epithelial cells and fabricated them into ECSs. Compared with 4-degree storage, programmed cryopreservation preserved the ECS structural integrity, enhanced the mechanical properties, decreased HLA-I expression, and increased cell viability and survival. An increased proportion of melanocytes with proliferative capacity remained in the cryopreserved sheets, and the undifferentiated epithelial cells were comparable to those of the fresh sheets. In vivo, cryopreserved ECSs could reduce inflammatory cell infiltration and promote connective tissue remodeling, epithelial cell proliferation and vascular regeneration. Conclusions Programmed cryopreservation of ECSs was superior and more feasible than 4-degree storage and the cryopreserved ECSs achieved satisfying skin wound healing in vivo. We anticipate that the off-the-shelf ECSs could be quickly used, such as, to repair human epithelial defect in future. Graphical abstract

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model

et al. 2021

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“…Local ethical committee approval was obtained for the use of tissue from the Department of Plastic Surgery, Oslo University Hospital, Oslo, Norway [2013/815/REK]. Cells were obtained as described [8], seeded (6000 cells/well) in serum-free CnT-Prime medium on Collagen IV coated 96-well Nunclon Δ surface multidishes, and cultured in a 5% CO 2 incubator at 37°C for 5–7 days to obtain a confluent monolayer. Culture medium was changed every two days.…”

Section: Methodsmentioning

confidence: 99%

“…Earlier studies have shown that storage of CES at 12°C is optimal for preserving undifferentiated epidermal cell characteristics and provides close to 100% viability over a one-week storage period [8, 9]. However, viability falls to ~60% over a two-week storage period [9, 10].…”

Section: Introductionmentioning

confidence: 99%

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets

Reppe

Jackson

Ringstad

et al. 2018

Stem Cells International

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There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7–11 days at 12°C in media supplemented with various additives. Effects were evaluated by calcein staining of live cells as well as morphology. Twenty-six additives were tested using (1) a two-level factorial design in which 10 additives were added or omitted in 64 different combinations and (2) a mixture design with 5 additives at 5 different concentrations in a total of 64 different mixtures. Automated microscopy and cell counting with Fiji enabled efficient processing of data. Significant regression models were identified by Design-Expert software. A calculated maximum increase of live cells to 37 ± 6% was achieved upon storage of cell sheets for 11 days in the presence of 6% glycerol. The beneficial effect of glycerol was shown for epidermal cell sheets from three different donors in two different storage media and with two different factorial designs. We have thus developed a high throughput screening system enabling robust assessment of live cells and identified glycerol as a beneficial additive that has a positive effect on epidermal cell sheet upon storage at 12°C. We believe this method could be of use in other cell culture optimization strategies where a large number of conditions are compared for their effect on cell viability or other quantifiable dependent variables.

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“…We have established that storage of CES at the optimum temperature of 12˚C can extend their useful period for at least 7 days [8][9][10]. The present study aimed to investigate important indicators of CES quality in the interval between 7-15 days to establish the optimal storage period.…”

Section: Introductionmentioning

confidence: 96%

Stem cell function is conserved during short-term storage of cultured epidermal cell sheets at 12°C

et al. 2020

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Transplantation of cultured epidermal cell sheets (CES) can be life-saving for patients with large area burns. CES have also been successfully used to regenerate eye and urethral epithelia in animal models. Short-term storage aims to extend the transplantation window, offers flexibility in timing surgery and allows testing of CES quality, phenotype and sterility. This study investigated extended CES storage and explored the effect of additional re-incubation recovery time following storage. The proliferative quality of stored confluent versus pre-confluent CES was also investigated using functional testing. CES were stored at 12˚C and results compared to non-stored control CES. Investigation of timepoints during 15 days storage revealed that viability began to deteriorate by day 11 and was associated with increased lactate in the storage medium. The percentage of apoptotic cells also significantly increased by day 11. Flow cytometry analysis of integrin β1 expression and cell size indicated best retention of stem cells at 7 days of storage. Functional testing of pre-confluent and confluent cells following 7 days storage showed that pre-confluent cells responded well to 1-day re-incubation after storage; they became highly prolific, increasing in number bỹ 67%. Conversely, proliferation in stored confluent cells declined by~50% with 1-day reincubation. Pre-confluent stored CES also had far superior stem cell colony forming efficiency (CFE) performance compared to the confluent group. Re-incubation improved CFE in both groups, but the pre-confluent group again out-performed the confluent group with significantly more colonies. In conclusion, a maximum storage period of 7 days is recommended. Use of pre-confluent cells and one day recovery incubation greatly improves viability, colony-forming ability and proliferation of cells stored for 7 days at 12˚C. Thus, these

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Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets

Cited by 5 publications

References 60 publications

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets

Stem cell function is conserved during short-term storage of cultured epidermal cell sheets at 12°C

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