Wound-Healing Potential of Cultured Epidermal Sheets Is Unaltered after Lyophilization: A Preclinical Study in Comparison to Cryopreserved CES

Abstract

Lyophilized Cultured Epidermal Sheets (L-CES) have been reported to be as effective as the cryopreserved CES (F-CES) in treating skin ulcers. However, unlike F-CES, no preclinical study assessing wound-healing effects has been conducted for L-CES. The present study was set out to investigate the microstructure, cytokine profile, and wound-healing effects of L-CES in comparison to those of F-CES. Keratinocytes were cultured to prepare CES, followed by cryopreservation at −70°C and lyophilization. Under microscopic observation, intact cells with apparent intracellular junctions were observed in L-CES. The L-CES, like fresh CES, consisted of three to four well-maintained epidermal layers, as shown by the expression of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES and F-CES, and both CES were comparable to fresh CES. TGF-α, EGF, VEGF, IL-1α, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson's trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment.

Human cultured epidermis accelerates wound healing regardless of its viability in a diabetic mouse model

“…However, if living cells are necessary to promote wound healing, the result of this study has no consistency. Biologically active substances stored in the keratinocytes, such as interleukin-1, vascular endothelial growth factor, and transforming growth factor-α, may be released from the keratinocytes regardless of cell viability and may contribute to wound healing [26]. Besides, some authors have mentioned that the membrane structure of the CE contributes to creating a comfortable environment for recipient cells in the wound bed to proliferate and migrate [17,20].…”

“…For example, lyophilized CEs may be a suitable wound dressing, given its long storage period at room temperature. In fact, Jang et al reported that lyophilized CEs accelerated wound healing at the same level as cryopreserved CEs [26]. Besides, CEs sterilized by gamma irradiation would have less probability of contamination (e.g., with bacteria and viruses) to be a much safer product.…”

Ginsenoside Rd Ameliorates High Fat Diet‐Induced Obesity by Enhancing Adaptive Thermogenesis in a cAMP‐Dependent Manner

“…Therefore, by combining in vivo and in vitro experiments, we revealed that Rd activated the cAMP/PKA signaling pathway to enhance adaptive thermogenesis in Rd‐treated HFD mice. In addition, a previous study found that Rd, the most abundant active ingredient in ginseng leaves, increased cAMP levels in keratinocytes to activate Creb and promote skin regeneration . Although we found that Rd also increased cAMP levels in brown adipocytes, the molecular mechanisms by which it achieves this requires further study.…”

“…Ginsenosides as the main active ingredient of ginseng can be classified into protopanaxadiol (PPD) and protopanaxatriol groups according to the position of the sugar moieties at carbon‐3 and ‐6 . Ginsenoside Rd, a PPD‐type compound, is regarded as one of the major active components in ginseng leaves . It has been reported that Rd exhibits some pharmacological effects, including protective effects against transient focal cerebral ischemia and apoptosis induced by glutamate in neurons, protective effects against tumors and inflammation in many kinds of diseases such as colitis and Alzheimer disease, and an inhibitory effect on the formation of reactive oxygen species in cells.…”

Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells

“…Human mucosal epithelial cells were serially cultured for the skin epithelial sheets, following a previously described method (Rheinwald and Green, , ; Jang et al , ) with modifications. Briefly, the mucosal epithelial cells were co‐cultured on lethally irradiated 3T3 murine fibroblasts to form cell sheets, with or without fibrin, for 10 days.…”