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

ABSTRACT: 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 ?1 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 by ~67%. Conversely, proliferation in stored confluent cells declined by ~50% with 1-day re-incubation. 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 recommendations should be considered under culture and storage of high-quality CES for clinical use.

SUBMITTER: Ringstad H

PROVIDER: S-EPMC7239464 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Ringstad Håkon H Reppe Sjur S Schøyen Tine Hiorth TH Tønseth Kim Alexander KA Utheim Tor Paaske TP Jackson Catherine Joan CJ

PloS one 20200520 5

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 ...[more]

PMID: 32433698

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Project description:BACKGROUND:Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2?8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. METHODS:Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. RESULTS:A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 106 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. DISCUSSION:In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 106 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

Dataset Information

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

Publications

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

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