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 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 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.
Highlights
Cultured epidermal cell sheets (CES) have been used as a life-saving treatment for patients with severe large area burns since 1984 [1]
Trypsin-ethylenediaminetetraacetic acid (EDTA), soybean trypsin inhibitor, sodium bicarbonate, goat serum, sodium azide, Tween-20, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Triton X-100, gentamicin, bovine serum albumin (BSA), fetal bovine serum (FBS), penicillin-streptomycin, human recombinant insulin, hydrocortisone, adenine, 3,3,5-triiodo-L-thyronine (T3), dispase II, and trypan blue were purchased from Sigma Aldrich (St Louis, MO)
Gibco low-glucose Dulbecco’s modified Eagle’s medium (DMEM), Gibco 1:1 DMEM/F12 nutrient mix, minimal essential medium (MEM), phosphate buffered saline (PBS), Hank’s balanced salt solution, routine plastics, pipettes and Nunclon Δ surface multi-dishes were purchased from Thermo Fisher Scientific (Life Technologies) (Waltham, MA)
Summary
Cultured epidermal cell sheets (CES) have been used as a life-saving treatment for patients with severe large area burns since 1984 [1]. They have since been applied to treat skin ulcers and recent studies indicate that CES have potential for use in regenerative medicine applications such as urethral reconstruction [2] and corneal regeneration in limbal stem cell deficiency [3]. Short-term storage and transportation of CES could be key to meeting heightened clinical demand and provide worldwide access to cell-based regenerative medicine treatments [5]. Short-term storage aims to preserve the proliferative potential and stem cell function of CES and extend the transplantation window providing flexibility. The storage period provides a window for testing CES quality, phenotype and sterility
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