Evaluation of safety and efficacy of autologous oral mucosa-derived epithelial cell sheet transplantation for prevention of anastomotic restenosis in congenital esophageal atresia and congenital esophageal stenosis.

BACKGROUND: We performed the first autologous oral mucosa-derived epithelial cell sheet transplantation therapy in a patient with refractory postoperative anastomotic stricture in congenital esophageal atresia (CEA) and confirmed its safety. In this study, patients with CEA and congenital esophageal stenosis were newly added as subjects to further evaluate the safety and efficacy of cell sheet transplantation therapy. METHODS: Epithelial cell sheets were prepared from the oral mucosa of the subjects and transplanted into esophageal tears created by endoscopic balloon dilatation (EBD). The safety of the cell sheets was confirmed by quality control testing, and the safety of the transplantation treatment was confirmed by 48-week follow-up examinations. RESULTS: Subject 1 had a stenosis resected because the frequency of EBD did not decrease after the second transplantation. Histopathological examination of the resected stenosis revealed marked thickening of the submucosal layer. Subjects 2 and 3 did not require EBD for 48 weeks after transplantation, during which time they were able to maintain a normal diet by mouth. CONCLUSIONS: Subjects 2 and 3 were free of EBD for a long period of time after transplantation, confirming that cell sheet transplantation therapy is clearly effective in some cases. In the future, it is necessary to study more cases; develop new technologies such as an objective index to evaluate the efficacy of cell sheet transplantation therapy and a device to achieve more accurate transplantation; identify cases in which the current therapy is effective; and find the optimal timing of transplantation; and clarify the mechanism by which the current therapy improves stenosis. TRIAL REGISTRATION: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

Allogeneic transplantation of epidermal cell sheets followed by endoscopic submucosal dissection to prevent severe esophageal stricture in a porcine model.

Introduction: Endoscopic submucosal dissection (ESD) is a minimally invasive treatment for early esophageal cancer. However, large mucosal defects after esophageal ESD result in refractory strictures. In the present study, we histologically evaluated the endoscopic transplantation of allogeneic epidermal cell sheets (ECSs) as a feasible therapy for preventing esophageal stricture after circumferential ESD in a porcine model. Methods: Epidermal cells were isolated from the skin tissue of allogeneic pigs and cultured on temperature-responsive cell culture inserts for 2 weeks. Transplantable ECSs were harvested by reducing the temperature and endoscopically transplanting the sheets to ulcer sites immediately after esophageal ESD. The engraftment of transplanted ECSs was then evaluated in two pigs at 7 days after transplantation. Next, ten pigs were divided into two groups to evaluate the endoscopic transplantation of allogeneic ECSs for the prevention of esophageal strictures after ESD. Allogeneic ECSs were transplanted immediately after esophageal ESD in the transplantation group (n = 5), whereas the control group (n = 5) did not undergo transplantation. Results: Most of the transplanted allogeneic ECSs were successfully engrafted at the ulcer sites in the early phase. Fluorescence in situ hybridization analysis revealed that several allogeneic cells were present in the transplanted area at 7 days after ESD. At 14 days after ESD, significant differences in body weight loss, dysphagia scores, and mucosal strictures were observed between the control and transplantation groups. Transplanting allogeneic ECSs after esophageal ESD promotes mucosal healing and angiogenesis and prevents excessive inflammation and granulation tissue formation. Conclusions: Endoscopic and histological analyses revealed that allogeneic ECSs promoted artificial ulcer healing after ESD, preventing esophageal strictures after ESD.

First-in-human autologous oral mucosal epithelial sheet transplantation to prevent anastomotic re-stenosis in congenital esophageal atresia.

BACKGROUND: Congenital esophageal atresia postoperative anastomotic stricture occurs in 30-50% of cases. Patients with severe dysphagia are treated with endoscopic balloon dilatation (EBD) and/or local injection of steroids, but many patients continue to experience frequent stricture. In this study, we investigated the transplantation of autologous oral mucosa-derived cell sheets (epithelial cell sheets) as a prophylactic treatment for congenital esophageal atresia postoperative anastomotic stricture. METHODS: Epithelial cell sheets were fabricated from a patient's oral epithelial tissue, and their safety was confirmed by quality control tests. The epithelial cell sheets were transported under controlled conditions from the fabrication facility to the transplantation facility and successfully transplanted onto the lacerations caused by EBD using a newly developed transplantation device for pediatric patients. The safety of the transplantation was confirmed by follow-up examinations over 48 weeks. RESULTS: The dates that EBD was performed were recorded for one year before and after epithelial cell sheet transplantation, and the intervals (in days) were evaluated. For about 6 months after transplantation, the intervals between EBDs were longer than in the year before transplantation. The patients were also aware of a reduction in dysphagia after transplantation. CONCLUSIONS: These results suggest that cell sheet transplantation may be effective in preventing anastomotic stricture after surgery for congenital esophageal atresia, but the effect was temporary and limited in this case. Although we chose a very severe case for the first human clinical study, it may be possible to obtain a more definitive effect if the transplantation is performed before the disease becomes so severe. Future studies are needed to identify cases in which cell sheet transplantation is most effective and to determine the appropriate timeframes for transplantation. TRIAL REGISTRATION: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

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.

Oral keratinocyte-derived exosomes regulate proliferation of fibroblasts and epithelial cells.

Extracellular vesicles (EVs), including exosomes, are small membrane-bound particles released by cells. From a therapeutic point of view, EVs can often convey similar biological function as their parent cell. Grafts originating from oral mucosa have frequently been used in regenerative medicine, and we have previously described the use of oral cell sheets to prevent stricture formation of the esophagus. Further, we recently found that exosomes derived from these cell sheets have pro-regenerative effect on skin wound healing. Here, we have isolated exosomes from conditioned media from oral keratinocyte ("OKEx") and dermal fibroblast ("FEx") cultures. The exosomes were probed for classical EV-markers by western blot (CD9, annexin V and Flotillin-1), FEx were positive for all markers while OKEx were positive only for CD9. Tunable resistive pulse sensing indicated a mean size of around 110 nm and transmission electron microscopy showed a spherical morphology, for both groups. After fluorescent labelling, we studied the uptake of exosomes co-cultured with fibroblasts or keratinocytes. Signal from OKEx could be detected after 90 min, and signal could be detected in all groups after 16 h. Finally we studied the exosomes' modulation of cell proliferation. Both groups suppressed proliferation of healthy keratinocyte and fibroblasts, at some doses to similar levels as dexamethasone (a drug commonly used to prevent stricture formation). In contrast, the exosomes also suppressed the proliferation of the carcinoma cell line TR146, while dexamethasone had no effect. In conclusion, we believe that exosome-signaling might be one of the mode-of-actions of cell sheet-therapy for stricture prevention.

Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing.

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

Esophageal tissue engineering: from bench to bedside.

For various esophageal diseases, the search for alternative techniques for tissue repair has led to significant developments in basic and translational research in the field of tissue engineering. Applied to the esophagus, this concept is based on the in vitro combination of elements judged necessary for in vivo implantation to promote esophageal tissue remodeling. Different methods are currently being explored to develop substitutes using cells, scaffolds, or a combination of both, according to the severity of lesions to be treated. In this review, we discuss recent advances in (1) cell sheet technology for preventing stricture after extended esophageal mucosectomy and (2) full-thickness circumferential esophageal replacement using tissue-engineered substitutes.

Endoscopic Transplantation of Mesenchymal Stem Cell Sheets in Experimental Colitis in Rats.

Owing to the recent progress in regenerative medicine technology, clinical trials that harnessed the regeneration and immune modulation potentiality of stem cells for treating IBD have shown promising results. We investigated the feasibility and utility of intraluminal endoscopic transplantation of rat MSC sheets in murine models of experimental colitis for targeted delivery of stem cells to lesions. We isolated adipose-derived mesenchymal stem cells (AD-MSC) and bone marrow-derived mesenchymal stem cells (BM-MSC) from EGFP-transgenic rats and fabricated the cells in sheet forms using temperature-responsive culture dishes. The MSC sheets were endoscopically transplanted to the inflamed area in electrocoagulation and DNBS colitis model. The effect of the transplantation was verified using endoscopic scoring and histological analysis. In the electrocoagulation model, the AD-MSC group showed significantly decreased ulcer size in the transplanted regions. In the DNBS colitis model, the AD-MSC group showed decreased inflammation and colitis in the transplanted regions. Histologic analysis showed that the MSC sheets had successfully attached to the inflamed mucosa in both the electrocoagulation and DNBS colitis model. Our results show that endoscopic transplantation of MSC sheets could be a new effective mode of stem cell therapy for IBD treatment.

Oral epithelial cell sheets engraftment for esophageal strictures after endoscopic submucosal dissection of squamous cell carcinoma and airplane transportation.

Endoscopic submucosal dissection (ESD) permits en bloc removal of superficial oesophageal squamous cell carcinoma (ESCC). However, post-procedure stricture is common after ESD for widespread tumours, and multiple endoscopic balloon dilation (EBD) procedures are required. We aimed to evaluate the safety and effectiveness of endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets that had been transported by air over a distance of 1200 km in controlling postprocedural oesophageal stricture. Ten patients who underwent complete circular or semicircular ESD for ESCC were transplanted with cell sheets. The safety of the entire process including cell sheet preparation, transport, ESD and cell sheet transplantation was assessed. The incidence of oesophageal stricture, number of EBD sessions, and time until epithelialization were investigated. Each ESD was successfully performed, with subsequent cell sheet engrafting carried out safely. Following cell sheet transplantation, the luminal stenosis rate was 40%, while the median number of EBD sessions was 0. The median post-ESD ulcer healing period was rather short at 36 days. There were no significant complications at any stage of the process. Cell sheet transplantation and preparation at distant sites and transportation by air could be a safe and promising regenerative medicine technology.

Approaches for stricture prevention after esophageal endoscopic resection.

BACKGROUND AND AIMS: Endoscopic resection of extensive esophageal lesions has become more common as endoscopic resection techniques and equipment have developed. However, extensive esophageal endoscopic resections can cause postoperative esophageal strictures, which have a negative impact on the quality of life of patients. We aimed to review current treatments and innovative approaches to prevent esophageal strictures after widespread endoscopic resection of esophageal lesions. METHODS: We performed a comprehensive literature search from 2000 to 2016 using predetermined search terms to identify relevant articles and summarized their results as a narrative review. RESULTS: A total of 21 original articles and case series were identified. A circumferential mucosal defect involving more than three fourths of the esophageal luminal circumference was the primary risk factor for developing an esophageal stricture after endoscopic resection. Oral and injectable steroid therapy demonstrated promise in preventing post-endoscopic submucosal dissection esophageal strictures, with both strategies significantly reducing the number of required endoscopic balloon dilations. More data are needed on prophylactic self-expandable metal stents, local botulinum toxin injection, and oral tranilast as a strategy to prevent post-endoscopic submucosal dissection esophageal strictures. Although preliminary studies of tissue-shielding resection sites with polyglycolic acid sheets and fibrin glue and autologous cell sheet transplantation have demonstrated promising results, additional larger validation studies are needed. CONCLUSIONS: Oral and locally injected/administered steroids are first-line options for the prevention of esophageal strictures, but additional innovative solutions are being developed.

Autologous adipose-derived stem cell sheets enhance the strength of intestinal anastomosis.

OBJECTIVE: Adipose-derived stem cells (ASCs) are capable of multiple differentiation pathways, imparting immunomodulatory effects, and secreting factors that are important for wound healing. These characteristics can be exploited to decrease the incidence of anastomotic leakage. METHODS: In order to delay local wound healing at the anastomotic site, we induced ischemia in a portion of porcine small intestine by ligating vessels. Then, we injected mitomycin C into the serosa of the small intestine above the ligated vessels. Anastomotic sites were created by 2 cm incisions made in the opposite mesenteric area. ASCs were isolated from the porcine subcutaneous fat tissues and expanded under culture conditions. ASCs were trypsinized and seeded on temperature-responsive dishes and cultured to form confluent sheets. Three ASC sheets were transplanted onto the serous membrane after suturing. The extent of anastomotic wound healing was evaluated by bursting pressure, hydroxyproline content, and mRNA expression of collagen-1 alpha1 and collagen-3 alpha1. RESULTS: We found that transplantation of ASC sheets increased anastomotic site bursting pressure. Additionally, transplantation of ASC sheets increased the hydroxyproline content of the anastomoses. Furthermore, transplantation of ASC sheets increased mRNA expression of collagen-1 alpha1 and collagen-3 alpha1. CONCLUSIONS: Our findings showed that transplantation of autologous ASC sheets enhanced collagen synthesis and anastomotic strength. Further studies are necessary to identify substances that, in combination with ASC sheets, might enhance collagen synthesis and healing in sites of anastomosis.

Treatment of chemically induced oral ulcer using adipose-derived mesenchymal stem cell sheet.

BACKGROUND: This study investigated the effects of mesenchymal stem cell (MSC) sheet transplantation on healing of chemically induced oral ulceration in a rabbit animal model. METHODS: Oral mucosal ulcers were induced by topical application of filter paper soaked with 70% acetic acid to the anterior gingiva and buccal mucosa of 12 New Zealand white rabbits. The animals were randomly assigned to two groups: with (treatment group, n = 6) or without (control group, n = 6) cell sheets applied to ulcers. Gross findings were sequentially evaluated, and histologic examination was performed on day 7. RESULTS: Based on gross inspection, ulceration resolved before day 5 in the treatment group; however, in the control group, healing was incomplete on day 7. In the treatment group, the total area of the ulcer decreased significantly from day 2 to day 5 (P < 0.001) and from day 5 to day 7 (P = 0.020), whereas the area decreased significantly from day 5 to day 7 in the control group (P < 0.001). Histologic and immunofluorescence examination revealed full-thickness mucosa healing and complete basal cell coverage in the treatment group; in contrast, only partial healing was observed on day 7 in the control group. CONCLUSIONS: Cell sheet technology using MSC can be an alternative treatment for oral ulcerations in that it can decrease healing time without invasive properties.

Transplantation of epidermal cell sheets by endoscopic balloon dilatation to avoid esophageal re-strictures: initial experience in a porcine model.

Background and study aims: Epidermal cell sheet (ECS) transplantation immediately after aggressive endoscopic submucosal dissection (ESD) has been shown to be safe and effective in the prevention of esophageal strictures. This study evaluated the feasibility of ECS transplantation after endoscopic balloon dilation (EBD) in a porcine model. Methods: Six pigs underwent circumferential esophageal ESD under general anesthesia. Two weeks later, two pigs underwent EBD and transplantation of an autologous ECS, two underwent EBD alone, and two underwent endoscopic observation only (control). Results: The two pigs in the transplantation group underwent six ECS transplants after EBD with five of the six (83 %) being successful, as shown by engraftment of transplanted ECSs after 7 days. No adverse events were observed. Stricture rates were lower in the two transplanted pigs (55 % and 60 %) than in the control (92.2 % and 87.7 %) and EBD-treated (71.7 % and 78.2 %) pigs. Infiltration of inflammatory cells was significantly lower in the transplanted pigs than in the control and EBD-treated pigs. Conclusion: Preliminary results indicate the stability of the ECS transplantation procedure and the engraftment of transplanted ECS in the tears after EBD. This proof-of-concept study suggests that covering tears with ECSs after EBD may avoid re-strictures.

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products.

The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is needed. This forum concluded that the continued collective actions are expected to promote international convergence of regulatory approaches of the products. The Pharmaceuticals and Medical Devices Agency and Japanese Society for Regenerative Medicine jointly convened the forum with support from the National Institutes of Biomedical Innovation, Health and Nutrition. Participants at the forum include 300 experts in and outside of Japan.

Transplantation of tissue-engineered cell sheets for stricture prevention after endoscopic submucosal dissection of the oesophagus.

BACKGROUND AND OBJECTIVE: Endoscopic mucosal dissection (ESD) is a treatment option for oesophagus tumours localized to the mucosa enabling en bloc removal of large lesions. The resulting larger mucosal defects have resulted in an increase in the occurrence of post-treatment strictures. Transplantation of autologous cell sheets, cultured from oral mucosa, has been shown to prevent post-ESD strictures. The aim of the study was to assess the efficacy and safety of cell sheet transplantation after oesophageal ESD in a Western patient population where reflux-associated pre-malignant and malignant conditions predominate. METHODS: Patients with Barrett's oesophagus associated high-grade dysplasia or early adenocarcinoma where ESD entailed a resection >3 cm in length and ≥75% of the circumference were eligible for treatment under hospital exemption. Cell sheets were cultured from buccal mucosa according to Good Manufacturing Practice and were endoscopically applied to the post-ESD defect directly after resection. Patients were followed with weekly endoscopy examinations, including confocal laser microscopy, for a total of four weeks. RESULTS: Five patients were treated. ESD was extensive with resections being circumferential in three patients and 9-10 cm in length in two. The number of transplanted cell sheets ranged from two to six. Three patients developed strictures requiring two to five dilatation sessions. CONCLUSIONS: Cell sheet transplantation shows to be safe and feasible in a Western population. Results suggest that transplantation has a protective effect on the mucosal defect after ESD, decreasing both the risk for and extent of stricture formation.

Novel isolated cecal pouch model for endoscopic observation in rats.

AIM: To create a new rat model for drug administration, cell transplantation, and endoscopic examination for the treatment of intestinal diseases. METHODS: F344/NJc l-rnu/rnu rats (10-wk-old males, 350-400 g) were used in this study. The rats were anesthetized via 2% isoflurane inhalation. The rat's cecum was isolated from the intestines, and a pouch was created. The remainder of the intestines was rejoined to create an anastomosis. The "side-to-side" anastomosis (SSA) technique initially involves the creation of a 2-cm longitudinal incision into each intestinal wall. To create an anastomosis along the ileal and colonic walls, both intestines were cut, and a continuous suture procedure was performed that included all layers of both intestines. The serous membrane was sutured along the edge and on the anterior wall of the anastomosis. The "end-to-end" anastomosis (EEA) technique was compared with the SSA technique. In the EEA technique, the frontal surfaces of both cut intestinal lumens were joined together by continuous sutures. Additional sutures were made at the serosa. After the anastomotic intestine was successfully constructed, the two intestinal lumens that were cut at the isolated cecum were managed. In addition, one luminal side of the pouch remained open to create an artificial anus on the dorsum as a passage for the residual substances in the pouch. Finally, small animal endoscopy was used to observe the inside of the pouch. RESULTS: In this animal model, mucus and feces are excreted through the reconstructed passage. Accordingly, the cecal pouch mucosa was not obstructed or contaminated by feces, thus facilitating observations of the luminal surface of the intestine. The endoscopic observation of the cecal pouch provided clear visualization given the absence of feces. The membrane surface of the cecum was clearly observed. Two methods of creating an anastomotic intestine, the "SSA" and "EEA" techniques, were compared with regard to animal survival rate, complication rate, and operation time. The SSA technique resulted in a significantly increased survival rate and a lower incidence of complications in rat models compared with the EEA technique. The complications of stenosis and leakage resulted in death in the EEA technique. Thus, the EEA technique exhibited a lower survival rate compared with the SSA technique. However, the SSA technique required a significantly longer operation time compared with the EEA technique. CONCLUSION: Our new rat model is potentially useful for the development of a novel treatment for intestinal diseases.

Endoscopic cell sheet transplantation device developed by using a 3-dimensional printer and its feasibility evaluation in a porcine model.

BACKGROUND: To prevent severe esophageal stenosis after aggressive endoscopic submucosal dissection (ESD), our group previously reported an efficient treatment using cell sheets that had been fabricated from patient cells. However, this transplantation procedure had not been easy for every endoscopist and needed to be improved to derive the full effect of epithelial cell sheets. OBJECTIVE: To develop an endoscopic device that enables easy and effective cell sheet transplantation and to evaluate its performance and clinical feasibility. DESIGN: Animal study. SETTING: Animal experimentation laboratory. INTERVENTION: Three pigs underwent circumferential esophageal ESD while under general anesthesia. A total of 12 cell sheets were endoscopically transplanted to the ESD site; 6 cell sheets were transplanted by using an endoscopic device that we developed, and 6 cell sheets were transplanted by using the conventional method. MAIN OUTCOME MEASUREMENTS: Procedure time, transplanted area on the ESD site, transplantation success rate, and monitoring of adverse events or incidents. RESULTS: The device allowed successful transplantation of all cell sheets with a shorter procedure time than with the conventional method (4.8 ± 0.8 minutes vs 13.3 ± 5.7 minutes, respectively) (P = .005) and onto a larger area (111.3 ± 56.3 mm(2) vs 41.8 ± 4.2 mm(2), respectively) (P = .023) with a higher success rate (100% vs 83%, respectively). No adverse incidents were monitored in each method. LIMITATIONS: Animal study, small sample. CONCLUSION: A newly designed endoscopic cell sheet transplantation device would be useful.

How to prevent contamination with Candida albicans during the fabrication of transplantable oral mucosal epithelial cell sheets.

We have utilized patients' own oral mucosa as a cell source for the fabrication of transplantable epithelial cell sheets to treat limbal stem cell deficiency and mucosal defects after endoscopic submucosal dissection of esophageal cancer. Because there are abundant microbiotas in the human oral cavity, the oral mucosa was sterilized and 40 µg/mL gentamicin and 0.27 µg/mL amphotericin B were added to the culture medium in our protocol. Although an oral surgeon carefully checked each patient's oral cavity and although candidiasis was not observed before taking the biopsy, contamination with Candida albicans (C. albicans) was detected in the conditioned medium during cell sheet fabrication. After adding 1 µg/mL amphotericin B to the transportation medium during transport from Nagasaki University Hospital to Tokyo Women's Medical University, which are 1200 km apart, no proliferation of C. albicans was observed. These results indicated that the supplementation of transportation medium with antimycotics would be useful for preventing contamination with C. albicans derived from the oral mucosa without hampering cell proliferation.