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Regenerative medicine products using allogeneic cells, such as allogeneic cultured epidermis (allo-CE), have become a more critical therapeutic method for the treatment of burns. However, there are no clinically available allo-CE products in Japan. Therefore, establishing a quality-controlled cell bank is mandatory to create regenerative medical products using allogeneic cells. In this study, we selected ten patients from the Department of Plastic Surgery of Kyoto University Hospital to become cell donors. We performed medical interviews and blood sampling for the donor to ensure virus safety. We examined the tissues and isolated cells by performing a nucleic acid test (NAT). To establish a master cell bank, quality evaluation was performed according to the International Conference of Harmonization (ICH) Q5A. Serological tests of the blood samples from the ten donors showed that two of them were ineligible. The cells registered in the cell bank were found to be compatible after virus testing was performed, and a master cell bank was constructed. Hence, we established a keratinocyte and fibroblast bank of clinically usable human cultured cells in Japan for the first time.

Autologous cultured epidermis (CE) is successfully used in burn care, but it requires a manufacturing time of three weeks and is very expensive owing to its custom-made nature of treatment. To compensate this disadvantage, dried allogeneic CE promises a novel therapeutic approach; and previous reports have demonstrated its efficacy in promoting wound healing using a murine skin defect model. Herein, a prospective clinical study was conducted to confirm the safety and efficacy of dried allogeneic CE for wound treatment.Dried CE was manufactured using donor keratinocytes obtained from excess surgical skin and applied to skin defects that were at least 3 cm in length and less than 10 % of the body surface area of the patients. The patients were observed for 14 days after CE application. The primary endpoint was the incidence of adverse events and the secondary endpoint was the percentage of wound healed since baseline, on days 7 and 14. Furthermore, as a stratified analysis, the percentage of wound healed, specified as deep dermal burns, was calculated.Six patients (five burns and one skin ulcer after necrotizing fasciitis) enrolled in the study. As a serious adverse event, a local infection was observed in one patient, which resolved by debridement and conventional skin grafting. Other adverse events that were potentially related to this treatment included two cases of skin erosion, and one case of systemic fever. No unresolved adverse events remained at the end of the study period. The percentage of wound healed was 73.4 ± 19.2 % on Day 7, and 92.2 ± 11.8 % on Day 14. When the targeted disease was restricted to deep dermal burns, the percentage of wound healed was 69.9 ± 28.9 % on Day 7 and 90.5 ± 13.2 % on Day 14.Treatment with dried CE was safely performed without any unresolved severe adverse effects. Dried CE is a new and promising modality for skin defect treatment, such as burns and ulcers, and is expected to compensate for the disadvantages of autologous CE. However, large-scale clinical trials are required to confirm their efficacy.

Cultured epidermal autografts (CEA) are surgical therapeutic alternatives for patients with stable vitiligo resistant to conventional medical treatments. In the present study, we assessed color matching before and at 12 months after CEA treatment. Eleven patients with 16 vitiligo lesions were included in this prospective study. The recipient sites were prepared by CO2 laser superficial ablation and subjected to CEA application. We clinically evaluated and categorized the color matching of the repigmented skin as well as the percentage of repigmentation. We also obtained three color values (L*a*b*) for the vitiligo lesions and surrounding normally pigmented skin. We then calculated the color differences between the two regions and compared them before and at 12 months after treatment. The mean percentage of repigmentation was 63.3% at 12 months. Six of the 16 lesions were categorized as “same as” and had color difference values of ≤5 at 12 months after treatment. Clinical evaluation of the color matching coincided well with the calculated color difference values. CEA application after CO2 laser superficial ablation was useful for treating vitiligo assessed by the percentage of repigmentation and color matching. Quantification of color differences may be a useful parameter for evaluating color matching in vitiligo.

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it’s presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

INTRODUCTION: As the take rate of cultured epidermal autografts in burn wound treatment is variable, widely expanded meshed auto skin grafts are often used in combination with cultured epidermal autograft to increase the take rate and achieve definitive wound coverage. However, a long time (3–4 weeks) required to prepare a cultured epidermis sheet is a disadvantage. Allogeneic cultured epidermis can be prepared in advance and cryopreserved to be used in combination with auto meshed skin grafts for treating third-degree burns. Nevertheless, the human cultured epidermis (hCE) has not been proved to accelerate wound healing after meshed skin grafting. Here, we investigated the effect of hCE on wound healing in a rat model of meshed skin grafting. MATERIALS AND METHODS: Human cultured epidermis was prepared from human neonatal foreskin and assessed by the release of growth factors into the culture medium using enzyme-linked immunosorbent assay. Skin wounds were inflicted on male F344 rats and treated by the application of widely meshed (6:1 ratio) autogenous skin grafts with or without hCE (n = 8 rats per group). Wound area, neoepithelium length, granulation tissue formation, and neovascularization were evaluated on day 7 postgrafting. RESULTS: Human cultured epidermis secreted IL-1α, Basic fibroblast growth factor, platelet-derived growth factor-AA, TGF-α, TGF-β1, and vascular endothelial growth factor in vitro. In rats, hCE accelerated wound closure (P = 0.003), neoepithelium growth (P = 0.019), and granulation tissue formation (P = 0.043), and increased the number of capillaries (P = 0.0003) and gross neovascularization area (P = 0.008) compared with the control group. CONCLUSIONS: The application of hCE with meshed grafts promoted wound closure, possibly via secretion of growth factors critical for cell proliferation and migration, suggesting that hCE can enhance the healing effect of widely expanded skin autografts.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Allogeneic cultured epidermis (allo-CE) is a cultured keratinocyte sheet manufactured from donor cells and promotes wound healing when used in deep dermal burns, donor sites, and chronic ulcers and serves as a wound dressing. Allo-CE is usually cryopreserved to be ready to use. However, the cryopreservation procedure will damage the cell viability, and the influence of Allo-CE, according to its viability or wound healing process, has not been evaluated sufficiently. In this study, we aimed to prove the influence of keratinocyte viability contained in allo-CEs on wound healing. We prepared CEs with Green’s method using keratinocytes obtained from a polydactyly patient and then prepared four kinds of CEs with different cell viabilities [fresh, cryopreserved, frozen, and FT (freeze and thaw)]. The cell viabilities of fresh, cryopreserved, frozen, and FT CEs were 95.7%, 59.9%, 16.7%, and 0.0%, respectively. The four CEs had homogeneous characteristics, except for small gaps found in the FT sheet by transmission electron microscopy observation. The four CEs were applied on the full-thickness skin defect of diabetic mice (BKS.Cg-Dock 7m +/+ Leprdb/Jcl), and the wound area and neoepithelium length were evaluated on days 4, 7, and 14. As a result, FT CEs without viable cells similarly promoted epithelialization on days 4 and 7 (p

DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. In order to compare the methylation status in melanoma tissues and melanocytes from the same individuals, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports. In many normal melanocytes, NPM2 showed distinct immunohistochemical staining, while its expression was lost in malignant melanoma cells. In particular, intraepithelial lesions of malignant melanoma, an important challenge in clinical practice, could be distinguished from benign nevi. The present findings indicate the importance of using fresh melanoma samples, not melanoma cell lines and melanocytes in epigenetic studies. In addition, NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease.

Inherited skin disorders have been reported recently to have sporadic normal-looking areas, where a portion of the keratinocytes have recovered from causative gene mutations (revertant mosaicism). We observed a case of recessive dystrophic epidermolysis bullosa treated with cultured epidermal autografts (CEAs), whose CEA-grafted site remained epithelized for 16 years. We proved that the CEA product and the grafted area included cells with revertant mosaicism. Based on these findings, we conducted an investigator-initiated clinical trial of CEAs from clinically revertant skin for recessive dystrophic epidermolysis bullosa. The donor sites were analyzed by genetic analysis, immunofluorescence, electron microscopy, and quantification of the reverted mRNA with deep sequencing. The primary endpoint was the ulcer epithelization rate per patient at 4 weeks after the last CEA application. Three patients with recessive dystrophic epidermolysis bullosa with 8 ulcers were enrolled, and the epithelization rate for each patient at the primary endpoint was 87.7%, 100%, and 57.0%, respectively. The clinical effects were found to persist for at least 76 weeks after CEA transplantation. One of the three patients had apparent revertant mosaicism in the donor skin and in the post-transplanted area. CEAs from clinically normal skin are a potentially well-tolerated treatment for recessive dystrophic epidermolysis bullosa.

We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP) higher than 200 MPa and that human cultured epidermis (hCE) engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa). Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM). hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting.

We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue. However, the preparation of decellularized or inactivated skin using HHP has not been reported. The objective of this study was thus to prepare inactivated skin from human skin using HHP, and to explore the appropriate conditions of pressurization to inactivate skin that can be used for skin reconstruction. Human skin samples of 8 mm in diameter were packed in bags filled with normal saline solution (NSS) or distilled water (DW), and then pressurized at 0, 100, 150, 200 and 1000 MPa for 10 minutes. The viability of skin after HHP was evaluated using WST-8 assay. Outgrowth cells from pressurized skin and the viability of pressurized skin after cultivation for 14 days were also evaluated. The pressurized skin was subjected to histological evaluation using hematoxylin and eosin staining, scanning electron microscopy (SEM), immunohistochemical staining of type IV collagen for the basement membrane of epidermis and capillaries, and immunohistochemical staining of von Willebrand factor (vWF) for capillaries. Then, human cultured epidermis (CE) was applied on the pressurized skin and implanted into the subcutis of nude mice; specimens were subsequently obtained 14 days after implantation. Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW. The basement membrane and capillaries remained intact in all groups according to histological and immunohistological evaluations, and collagen fibers showed no apparent damage by SEM. CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa. These results indicate that human skin could be inactivated after pressurization at more than 200 MPa, but skin pressurized at 1000 MPa had some damage to the dermis that prevented the taking of CE. Therefore, pressurization at 200 MPa is optimal for preparing inactivated skin that can be used for skin reconstruction.

Giant congenital melanocytic nevi are intractable lesions associated with a risk of melanoma. High hydrostatic pressure (HHP) technology is a safe physical method for producing decellularized tissues without chemicals. We have reported that HHP can inactivate cells present in various tissues without damaging the native extracellular matrix (ECM). The objectives of this study were to inactivate human nevus tissue using HHP and to explore the possibility of reconstructing skin using inactivated nevus in combination with cultured epidermis (CE). Human nevus specimens 8 mm in diameter were pressurized by HHP at 100, 200, 500, and 1000 MPa for 10 min. The viability of specimens just after HHP, outgrowth of cells, and viability after cultivation were evaluated to confirm the inactivation by HHP. Histological evaluation using hematoxylin-eosin staining and immunohistochemical staining for type IV collagen was performed to detect damage to the ECM of the nevus. The pressurized nevus was implanted into the subcutis of nude mice for 6 months to evaluate the retention of human cells. Then, human CE was applied on the pressurized nevus and implanted into the subcutis of nude mice. The viability of pressurized nevus was not detected just after HHP and after cultivation, and outgrowth of fibroblasts was not observed in the 200, 500, and 1000 MPa groups. Human cells were not observed after 6 months of implantation in these groups. No apparent damage to the ECM was detected in all groups; however, CE took on nevus in the 200 and 500 MPa groups, but not in the 1000 MPa group. These results indicate that human nevus tissue was inactivated by HHP at more than 200 MPa; however, HHP at 1000 MPa might cause damage that prevents the take of CE. In conclusion, all cells in nevus specimens were inactivated after HHP at more than 200 MPa and this inactivated nevus could be used as autologous dermis for covering full-thickness skin defects after nevus removal. HHP between 200 and 500 MPa will be optimal to reconstruct skin in combination with cultured epidermal autograft without damage to the ECM.