Preclinical assessment of transplantable human nasal mucosal epithelial cell sheets.

INTRODUCTION: We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF). METHODS: Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated. RESULTS: The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials. CONCLUSIONS: The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

A stable protocol for the fabrication of transplantable human oral mucosal epithelial cell sheets for clinical application.

INTRODUCTION: Cultured stratified epithelial cell sheets have been clinically utilized as transplantable grafts for the regeneration of epithelial tissues, such as the esophagus, cornea, skin, and intraoral cavity. These cell sheets are expected to gain widespread use as regenerative medicine products and save many patients. For this purpose, establishing and disseminating the stale protocol of fabricating the cell sheet is crucial. The fabrication of cultured stratified epithelial cell sheets consists of many important steps, and since the patients' epithelial cell conditions vary widely and are sometimes unstable, the qualities of the epithelial cell grafts are likewise potentially unstable. Therefore, in this paper, we report the stable protocol for fabrication of the transplantable cell sheet particularly from patient-derived oral mucosal tissues. METHODS: Serum extracted from blood and buccal mucosal tissue were collected in Nagasaki University and transported to Tokyo Women's Medical University. Oral mucosal epithelial cells were collected by minimum trypsin method, and this treatment was studied whether to be a critical procedure. After 14 days cultivation, cultured cells were examined whether to be transplantable as cell sheets. RESULTS: We successfully transported buccal mucosal tissue and serum without damage and contamination. Oral mucosal epithelial cells were collected with high viability by minimum trypsin method. Finally, we succeeded to stably fabricate oral mucosal epithelial cell sheets in all 10 patients. CONCLUSIONS: We established a stable protocol for the fabrication of human oral mucosal epithelial cell sheets and their transportation in clinical settings in this study. These methodologies could also be basis for transplantation therapy using cultured cell sheets of various types other than oral mucosal epithelial cell and will contribute largely to the future development of regenerative medicine.

Human keratinocyte stem cells: From cell biology to cell therapy.

Human keratinocyte cultures contain keratinocyte stem cells, and have been involved in significant progress regarding stem cell biology as well as keratinocyte biology. Such cultures have also been applied in cell therapy for extensive severe burns for more than three decades, and in genetic disorders of the skin recently. Human keratinocyte stem cells were firstly characterized as holoclones by ex post clonal analysis, but in situ identification of keratinocyte stem cells is required for clinical applications. Recently, it was demonstrated that human keratinocyte stem cells display a unique rotational motion at early stages of culture, with subsequent dynamic collective motion at later stages. This finding enables image-based identification of keratinocyte stem cells, and noninvasive evaluation of their proliferative capacity, which can be applied for the quality assurance of human keratinocyte cultures. This review summarizes the historical development of human keratinocyte cultures and its applications for cell biology and cell therapy. This article also introduces recent advances in keratinocyte stem cell research with medical relevance and discusses the next-generation of regenerative medicine using human keratinocyte stem cells.

Keratinocyte sheets prepared with temperature-responsive dishes show enhanced survival after in vivo grafting on acellular dermal matrices in a rat model of staged bi-layered skin reconstruction.

INTRODUCTION: Bi-layered skin reconstruction can be achieved by staged grafting of acellular dermal matrices (ADMs) and cultured epithelial keratinocyte sheets (KSs). Both KSs and ADMs have been used for long; yet, their combined use has shown poor effectiveness. This outcome has been related to the enzymatic treatment used in the preparation of KSs, which impairs their adhesion potential to ADMs and the formation of a basement membrane (BM). Temperature-responsive (TR) culture dishes allow for enzyme-free preparation of KSs with preservation of BMs and intercellular adhesion proteins; yet, their use has not been previously applied to staged bi-layered skin reconstruction. Using an in vivo rat model, we tested the hypothesis that TR cultures enhance KSs survival and BM preservation after sequential grafting on ADMs. METHODS: In nude rats (n = 9/group), a 9-cm [2] full-thickness dorsal skin defect was repaired with a commercial ADM. At 2 weeks after surgery, we grafted the ADM with KSs (circular, 25 mm diameter), prepared from human cells either by enzymatic Dispase treatment (DT control group) or a TR culture dish (TR experimental group). KSs survival and BMs preservation was assessed one week later by digital imaging, histology (hematoxylin & eosin), immunohistochemistry (collagen IV, pancytokeratins) and immunofluorescence (cytokeratin 1-5-6, laminin). RESULTS: The TR group showed a significantly higher KSs survival (120 ± 49 vs. 63 ± 42 mm2; p < 0.05) and epidermal thickness (165 ± 79 vs. 65 ± 54 µm; p < 0.01) compared with the control DT group, as well as higher epidermal maturation (cytokeratin) and a denser laminin and Collagen IV expression in the BMs in vitro and in vivo. CONCLUSION: These findings suggest that KSs prepared with TR culture dishes have significantly enhanced survival when grafted on ADMs; these outcomes could help improve current clinical strategies in wound care by skin reconstruction.

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications.

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.

A Method for the Immortalization of Newborn Mouse Skin Keratinocytes.

Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-ras(Ha) infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation.

Pathogenic activity of circulating anti-desmoglein-3 autoantibodies isolated from pemphigus vulgaris patients.

INTRODUCTION: There are scarce data on immunochemical properties of pemphigus antibodies detected in clinical remission in pemphigus vulgaris (PV) patients. The aim of the study was to compare biological activity of anti-Dsg3 autoantibodies purified from the sera of PV patients in active stage and in clinical remission. MATERIAL AND METHODS: The effect of purified antibodies on expression of procaspase-3, Bax, Bcl-2, uPAR, IL-1ß, IL-6, and TNF-α mRNAs in the HaCaT keratinocytes was evaluated by Western blot and RT-PCR method. RESULTS: Incubation of HaCaT cells with anti-Dsg-3 autoantibodies caused their binding to cell membranes surfaces. Anti-Dsg3 autoantibodies isolated from the patients in active stage and clinical remission showed proapoptotic effect, caused enhanced expression of analyzed proinflammatory cytokines' mRNAs and uPAR mRNA. CONCLUSIONS: Our data revealed similar pathogenic activity of anti Dsg-3 autoantibodies isolated from active and clinical remission PV patients.

Human dermal fibroblasts as a feeder layer promote the growth of human keratinocytes / 基础医学与临床

Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P

2D and 3D structural study of rete ridge in oral mucosa and skin paddle of various free flaps

OBJECTS: With the advancement of tissue engineering techniques, the effort to develop bioartificial mucosa have been actively delivered. The problem we met with this technique is the lack of mechanical strength between kerationocyte layer and dermal layer, where in the normal skin and mucosa, they are tightly bound with rete ridge structure. The purpose of this study is to understand the 2D and 3D structure of rete ridge of mucosa and skin paddle for rendering more biomimetic structure to the artificial mucosa. MATERIALS AND METHODS: Oral mucosa and skin from the patients who received the oral surgery and maxillofacial reconstruction were harvested. The epidermis was separated from the dermis after treating with dispase for 12-16 hours. H and E staining was performed for 2D(dimensional) structure study and confocal LASER and SEM study were performed for 3D structure. Mean height(Sc) and arithmetic mean deviation(Sa) of all surface height were calculated. RESULTS: The average height of rete ridge of skin flap was between 67.14micrometer and 194.55micrometer. That of oral mucosa was between 146.26micrometer and 167.51micrometer. Pressure bearing area and attached gingiva of oral mucosa showed deeper rete ridges. CONCLUSION: To obtain the adequate strength of artificially cultured keratinocyte skin and mucosa flap, it is necessary to imitate the original skin and mucosa structure, especially rete ridge. Through this study, 2D and 3D rete ridge structure of normal mucosa and skin was obtained. These results can be used as basis for substrate morphology for keratinocytes culture.

Antifungal Susceptibility Testing in Three-dimensional Keratinocyte Culture Model / 대한피부과학회지

BACKGROUND: Standardization of antifungal susceptibility testing for dermatophytes is important and several variables, such as inoculum size, length and temperature of incubation, media, and end point determination has recently been established. However, a more improved test model which can evaluate bioavailability and has clinical relevance is still needed. OBJECTIVE: We performed antifungal susceptibility testing by using three-dimensional keratinoctyte culture model, living skin equivalent (LSE), as an in vitro model. METHODS: LSE was prepared and various concentrations of antifungals were added to media. Microconidia of Trichophyton mentagrophytes was inoculated onto LSE and incubated for 6 days at 35 degrees C. RESULTS: Inhibition of fungal proliferation and invasion were observed at 0.1microgram/ml of terbinafine, 0.01 microgram/ml of itraconazole solution, 0.1 microgramg/ml of itraconazole powder, and 10 microgram/ml of fluconazole, respectively. CONCLUSION: Our culture model is similar to in vivo situation and the results were relatively well accordant to those of other previous studies. Our LSE model is considered as a promising in vitro system for evaluating the activity and safety of antifungal agents. However, further study using more various species of dermatophytes and more strains is still needed.

Study on the Composite Graft with an Acellular Xeno-dermis and Cultured Keratinocyte Sheets / 中国医师杂志

Objective To study a graft method of an acellular dermis composited with cultured human keratinocyte sheets,and investigate the outcomes of the composite graft on full-thickness wounds.Methods Using porcine skins,we prepared the acellular dermis and cultured human keratinocyte sheets in vitro.Athymic null mice were divided into the experimental group and the control group,the acellular xeno-dermis was implanted into the subcutaneous of the dorsal skin in experimental group,one week after the implantation the cultured keratinocyte sheets were grafted on the full-thickness defective area of the dorsal skin in the experimental group,in control group only implanted human keratinocyte sheet on the defective area wound,healing status was regular assessed,and biopsies for histological analysis and immunohistochemistry test were performed in post-operation.Results Compared with the control group,the appearance of wound coverage in the experimental group was better.Moreover,the histologic analysis revealed that fully differentiated epidermis,organized proliferated collagen fibers and significant reconstruction of epidermis-dermis junction were seen in the experimental group which lack of the acute immuno-rejection response.Conclusion As a kind of dermal substitutes,the acellular xeno-dermis can be compositely transplanted with cultured human keratinocyte sheet and can improve the quality of wound healing.

Measurement of the Cytotoxicity of Several Solvents Using a Normal Human Keratinocytes Culture Model / 대한피부과학회지

BACKGROUND: As in vivo models such as animal and human tests have many potential problems the keratinocyte culture model has previously been used as an in vitro model for testing skin irritancy for common skin irritants. OBJECTIVE: To determine the skin irritant potency of several solvents, we employed cultured human keratinocytes as an in vitro model. METHODS: To evaluate the cytotoxicity of solvents, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test, lactic dehydrogenase(LDH) release test, and tritiated thymidine incorporation test were used. RESULTS: Dose dependent decrease of cell viability and DNA synthesis, and dose dependent increase in leakage of LDH liberation were observed in normal cultured human keratinocytes after exposure to several solvents. The cytotoxicity potency of several solvents measured by each method were slightly different. CONCLUSION: These observations suggest that the cultured human keratinocyte model would be useful in evaluating the cytotoxicity of solvents.

Measurement of the Cytotoxicity of Several Solvents Using a Normal Human Keratinocytes Culture Model / 대한피부과학회지

BACKGROUND: As in vivo models such as animal and human tests have many potential problems the keratinocyte culture model has previously been used as an in vitro model for testing skin irritancy for common skin irritants. OBJECTIVE: To determine the skin irritant potency of several solvents, we employed cultured human keratinocytes as an in vitro model. METHODS: To evaluate the cytotoxicity of solvents, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test, lactic dehydrogenase(LDH) release test, and tritiated thymidine incorporation test were used. RESULTS: Dose dependent decrease of cell viability and DNA synthesis, and dose dependent increase in leakage of LDH liberation were observed in normal cultured human keratinocytes after exposure to several solvents. The cytotoxicity potency of several solvents measured by each method were slightly different. CONCLUSION: These observations suggest that the cultured human keratinocyte model would be useful in evaluating the cytotoxicity of solvents.

In Vitro Effects of Several Irritants Using Human Keratinocyte Culture Model / 대한피부과학회지

Primary irritant dermatitis is one of the most common skin disease caused by various hazardous chemicals produced from the environment. For the detection of skin irritant potency, in vivo models such as human and animal patch test have been used, Keratinocyte culture method which has been set up very recently is another alternative in vivo method of detecting skin irritarlcy. LVe have investigated the effects of three skin irritants, phenol, benzoyl peroxide (BP), and sodium lauryl sulfate(SLS) on the keratinocyte culture system. Prostaglandin E(PGE) measurement, cell count and electron microscopic observation were performed after adding three irritants of different concentrations to the cultured keranocyte cells. The main results of this study were as follows : 1. There were statistically significant decreased cell number in concentration of 10 M phenol, 10 4M BP and SLS. The order of cytotoxic potency was SLS>BP >phenol. 2. In case of PGE production, decreased PGE production was observed 6 hours after addition of the irritants, except 10 M phenol and 10M BP groups. Decrea sing tendency sustained until 24 hours, however all were statistically nonsignificant comparing with control group. 3. Electron microscopic finding showed that dilatation of endoplasmic reticulums in 10 M phenol group, condensation and dilatation of mitochondrias in 10 4M BP group, and most of the cells were swollen in 10 4M SLS group. These results suggest that cell count is a useful model for performing cytotoxi city test in keratinocyte culture decreased PGE production represents cytotoxic effect in high concentration of primary irritants and ultrastructural changes may reflect the different pathomechanisms in cytotoxicity.

Ultrastructural Study of Human Epidermal Keratinocytes Cultured in Low Calcium Medium / 第二军医大学学报

A low calcium medium developed for epidermal keratinocytes were prepared according to the MCDB 153 modified formula and used in human epidermal keratinocyte culture compared with DMEM culture system. The observation by contrast microscopy and electron microscopy showed that in the low calcium medium keratinocytes grew as a monolayer of high proliferation and had many characteristics of basal cells, with a more rounded shape and large intercellular spaces. Increasing the calcium ion concentration in the medium or changing the other culture conditions the cells in these cultures could be induced stratification and terminal differentiation. The results suggest that the growth, proliferation and differentiation of cultured human epidermal keratinocytes can be controlled and regulated someway.