[J-TEC's efforts to industrialize regenerative medicine in Japan].

Japan Tissue Engineering Co., Ltd., J-TEC, was launched in 1999 to industrialize regenerative medicine in Japan. We developed the first regenerative medicine product, JACE (autologous cultured epidermis), which received PMDA approval for treating serious burns in 2007. Then, JACC (autologous cultured cartilage), the second product, was approved in 2012 for efficacy on traumatic cartilage defects. In 2014, the Pharmaceutical Affairs Law was revised to the Pharmaceutical and Medical Device Act, and regenerative medicine products, including gene therapies, were newly classified to accelerate productization. Subsequently, Nepic (autologous cultured corneal epithelium) and Ocural (autologous cultured oral mucosal epithelium) for epithelialization of limbal stem cell deficiencies in ophthalmology were approved in 2020 and 2021, respectively. Furthermore, a new product, JACEMIN (autologous cultured epidermis maintaining melanocyte) for vitiligo treatment was approved in 2023. We have developed five products of regenerative medicine that construct human tissues to graft rather than injectable cell suspensions like drugs. To develop regenerative medicine products, it is necessary to ensure the safety of raw materials, standardize the cultivation process, examine cell characteristics on GLP tests, construct transportation methods, build GCTP facilities, and conduct clinical trials on GCP. Re-examinations of JACE for serious burns and JACC for cartilage defects were completed after 7 years of all-case postmarketing surveillance. The commercialization of these products has become a benchmark for domestic regulation and has induced the development of a regenerative medicine industry promoted by Japan.

Treating epidermolytic ichthyosis and ichthyosis with confetti with epidermal autografts cultured from revertant skin.

BACKGROUND: No efficient treatment has been established yet for epidermolytic ichthyosis (EI) caused by pathogenic variants in KRT1 or KRT10. Patients with ichthyosis with confetti (IWC) show multiple normal-appearing spots, caused by the revertant somatic recombination of pathogenic variants that occurs at each spot independently. Additionally, some patients with EI have large areas of normal skin due to revertant postzygotic mosaicism. OBJECTIVE: To assess the feasibility transplanting cultured epidermal autografts (CEAs) produced from revertant epidermal keratinocytes in patients with EI and IWC. METHODS: We performed a clinical trial of treatment with CEAs produced from each patient's own revertant epidermal keratinocytes as a proof-of-concept study. This is a single-arm, open (masking not used), uncontrolled, single-assignment, treatment purpose study. The primary outcome was the rate of areas without the recurrence of ichthyosis lesions 4 weeks after the final transplant (%). The secondary outcome was the rate of areas without the recurrence of ichthyosis lesions 24 weeks after initial transplantation (%). RESULTS: We successfully produced CEAs from the genetically confirmed revertant skin of the two mosaic EI patients and one IWC patient and genetically confirmed that CEAs mainly consist of revertant wild-type cells by amplicon sequencing and droplet digital PCR analysis. Single-cell RNA sequencing analysis confirmed the normal proliferation and safety profiling of CEAs. CEAs were transplanted to desquamated lesional sites of the patients. Four weeks after this transplantation, the rate of areas without the recurrence of ichthyosis lesions in the three cases was 39.52%, 100.0%, and 100.0% respectively, although the recurrence of ichthyosis lesions was seen at the site of CEA transplantation in all three patients at 24 weeks after transplantation. CONCLUSION: CEAs from normal skin have the potential to be a safe and local treatment option for EI and IWC. TRIAL REGISTRATION: jRCTb041190097.

Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet.

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

Dried human-cultured epidermis accelerates wound healing in a porcine partial-thickness skin defect model.

Introduction: Autologous cultured epidermis (CE) is an effective approach for overcoming the deficiency of donor sites to treat extensive burns. However, the production of autologous CE takes 3-4 weeks, which prevents its use during the life-threatening period of severe burns. In contrast, allogeneic CE can be prepared in advance and used as a wound dressing, releasing several growth factors stimulating the activity of recipient cells at the application site. Dried CE is prepared by drying CEs under controlled temperature and humidity conditions until all the water is completely removed and no viable cells are present. Dried CE accelerates wound healing in a murine skin defect model and is potentially a new therapeutic strategy. However, the dried CE safety and efficacy have not yet been studied in large animal models. Therefore, we studied the safety and efficacy of human-dried CE in wound healing using a miniature swine model. Methods: Human CE was manufactured using Green's method from donor keratinocytes. Three types of CEs (Fresh, Cryopreserved, and Dried) were prepared, and the ability of each CE to promote keratinocyte proliferation was confirmed in vitro. Extracts of the three CEs were added to keratinocytes seeded in 12-well plates, and cell proliferation was evaluated using the WST-8 assay for 7 days. Next, we prepared a partial-thickness skin defect on the back of a miniature swine and applied three types of human CE to evaluate wound healing promotion. On days 4 and 7, the specimens were harvested for hematoxylin-eosin, AZAN, and anti-CD31 staining to assess epithelialization, granulation tissue, and capillary formation. Results: The conditioned medium containing dried CE extract significantly enhanced keratinocyte proliferation compared to the control group (P < 0.05). In vivo experiments revealed that human-dried CE significantly accelerated epithelialization at day 7 to the same extent as fresh CE, compared to the control group (P < 0.05). The three CE groups similarly affected granulation formation and neovascularization. Conclusions: Dried CE accelerated epithelialization in a porcine partial-thickness skin defect model, suggesting that it may be an effective burn treatment alternative. A clinical study with a long-term follow-up is needed to assess the applicability of CEs in clinics.

Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency.

PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Establishment of a keratinocyte and fibroblast bank for clinical applications in Japan.

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.

A novel approach for wound treatment using dried cultured epidermal allograft: A phase I/II, single-center, open-label clinical trial.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Dried human cultured epidermis accelerates wound healing in diabetic mouse skin defect wounds.

Cryopreserved allogeneic cultured epidermis (CE) is used for treating second-degree burn wounds and diabetic foot ulcers; however, the need for cryopreservation limits its use. We have previously reported that CE accelerates wound healing irrespective of its viability and hypothesized that dehydrated CEs lacking living cells may act as an effective wound dressing. We prepared dried CE and investigated its morphological and physical properties and wound-healing effects and compared them with those of cryopreserved CE. Hematoxylin-eosin staining, immunostaining for basement membrane, and electron microscopy revealed that the morphologies of dried CE and cryopreserved CE were comparable and that the membrane structure was not damaged. The breaking strength, modulus of elasticity, and water permeability of dried CE were comparable with those of the cryopreserved CE. Furthermore, the levels of various active cytokines and chemokines in dried CE were comparable with those in cryopreserved CE. Dried CE applied to skin defect in diabetic mice significantly reduced the wound area and increased the new epithelium length 4 and 7 days after implantation, similar to that observed for cryopreserved CE. Consequently, dried CE had similar morphological and physical properties and wound-healing effects compared with those of cryopreserved CE and can be a physiological and versatile wound-dressing.

Cultured epidermal autografts for treatment of stable vitiligo: Quantitative analysis of color matching with surrounding normally pigmented skin.

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.

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

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<0.05) and accelerated wound closure on day 7 (p<0.01) as fresh CEs compared with the control group. In conclusion, the promoting effect of allo-CE on wound healing does not depend on cell viability. Lyophilized CEs may be a suitable wound dressing with a long storage period at room temperature.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets.

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.

Cultured Epidermal Autografts from Clinically Revertant Skin as a Potential Wound Treatment for Recessive Dystrophic Epidermolysis Bullosa.

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.

Gene Expression and Methylation Analysis in Melanomas and Melanocytes From the Same Patient: Loss of NPM2 Expression Is a Potential Immunohistochemical Marker for Melanoma.

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.

Cultured Human Epidermis Combined With Meshed Skin Autografts Accelerates Epithelialization and Granulation Tissue Formation in a Rat Model.

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-ß1, 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.

The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization.

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.