Long-term survival of cultivated oral mucosal epithelial cells in human cornea: generating cell sheets using an animal product-free culture protocol.
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ABSTRACT:
SUBMITTER: Ma DH
PROVIDER: S-EPMC8496076 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe loss of limbal stem cells owing to either corneal burn or inflammation leads to the repopulation of opaque skin over the raw surface of the cornea. It has been proposed that reconstitution of oral mucosal stem cells over this raw surface will mimic the limbal stem cells and restore vision. The efficacy and safety of applying a sheet of cultivated oral mucosal cells as an autologous graft for corneal replacement were evaluated.Case presentationThe study was conducted during 2014-2015 and involved a total of six patients, of whom three had suffered a chemical burn and three had Stevens-Johnson Syndrome (SJS). Oral mucosal tissue was dissected from each patient, seeded onto irradiated J2 fibroblast feeder cells for 14 days, and analyzed for quality and safety 1 day before being transplanted onto the cornea of the affected eyes. After transplantation, topical antibiotic and anti-inflammatory eye drops were instilled four times daily, and the patients wore contact lenses. Subjects were clinically followed for visual acuities and adverse effects at 2, 4, and 6 weeks, 3 and 6 months, and 1 year post-transplantation. Data were presented descriptively. Visual acuities in patients improved at 2 weeks post-surgery. However, two patients with SJS had corneal ulcer at 2 weeks postoperatively. At the 1-year postoperative examination, the eyes of two patients were in good condition with decreased vascularization and epithelial defect.ConclusionsCultivated oral mucosal epithelial sheet transplantation in limbal stem cell deficiency had a favorable efficacy. In this study, patients with chemical burn had more clinical benefit than those with SJS. Trial registration ClinicalTrials.gov: NCT02415218. Registered retrospectively 4 Apr 2015 ( https://clinicaltrials.gov/ct2/show/NCT02415218 ).
| S-EPMC9351062 | biostudies-literature
Project description: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.
| S-EPMC6346716 | biostudies-literature
Project description:Introduction:Carrier-free autologous mucosal epithelial cell sheets have been clinically utilized as a cell therapy for various epithelial disorders. Fabrication of a transplantable oral mucosal epithelial cell sheet without mouse feeder layers requires a higher seeding density than that of a sheet with mouse feeder layer culture; therefore, a large amount of donor mucosal tissue is needed. However, cell grafts co-cultured with mouse feeder layers are classified by the US Food and Drug Administration (FDA) as xenogeneic products. The goal of this study was to evaluate the utility of oral mucosal epithelial cells expanded by primary explant culture for the fabrication of an adequate number of transplantable epithelial cell sheets without mouse feeder layers. Methods:Small fragments derived from minced oral mucosal tissue were placed into culture dishes for primary explant culture in keratinocyte culture medium. After primary explant culture, the outgrown cells were treated with trypsin-EDTA and were seeded on a temperature-responsive cell culture insert. After subculture, the cultured cells were harvested as a confluent cell sheet from the culture vessel by temperature reduction. Results:Carrier-free human oral mucosal epithelial cell sheets were fabricated in all human cases, and autologous transplantation of the harvested cell sheets showed rapid epithelial regeneration to cover epithelial defects in a rabbit model. The explant culture method, involving the use of small fragments for primary culture, was sufficient for preparing a large number of mucosal epithelial cells without mouse feeder layers. Moreover, oral mucosal epithelial cells derived from the primary explant culture after cryopreservation allowed for the fabrication of cell sheets. Conclusions:This method for fabricating transplantable oral mucosal epithelial cell sheets is an attractive technique for regenerative medicine. It offers a patient-friendly manufacturing method in which a small amount of biopsy material from the patient represents a sufficient epithelial cell source, and a manufacturing plan for preparing cell grafts can be easily tailored.
| S-EPMC6298907 | biostudies-other
Project description:BackgroundAutologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization.MethodsRat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs).ResultsCOMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs.ConclusionsLNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.
| S-EPMC6448320 | biostudies-literature
Project description:Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4-5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.
| S-EPMC5107917 | biostudies-literature
Project description:Tissue engineering has become an essential tool in the development of regenerative medicine. We have developed cell sheet-based techniques for use in regenerative medicine that have already been successfully used in clinical applications. Native corneal epithelium is produced from limbal stem cells located in the transition zone between the cornea and the bulbar conjunctiva. Limbal stem cell deficiency (LSCD) is a severe defect of the limbal stem cells leading to vision loss due to conjunctival epithelial invasion and neovascularization. Rabbit LSCD models were treated with transplantable autologous oral mucosal epithelial cell (OEC) sheets fabricated on temperature-responsive cell culture surfaces, after which, the ocular surfaces were clear and smooth with no observable defects. The central part of the reconstructed ocular surface was scraped and wounded, after which proliferating epithelial cells covered the scraped area within a few days. The ocular surfaces were clear and smooth even after repeated scrapings and consisted of only OECs or heterogeneously mixed with corneal epithelial cells. This study demonstrates that transplanted cell sheets containing oral mucosal epithelial stem cells could reconstruct the ocular surface to maintain cornea homeostasis; moreover, they provide an ideal microenvironment to support the proliferation of remaining native limbal stem cells.
| S-EPMC4435588 | biostudies-literature
Project description:Cellular Phenotype and Apoptosis: The function of epithelial tissues is the protection of the organism from chemical, microbial, and physical challenges which is indispensable for viability. To fulfill this task, oral epithelial cells follow a strongly regulated scheme of differentiation that results in the formation of structural proteins that manage the integrity of epithelial tissues and operate as a barrier. Oral epithelial cells are connected by various transmembrane proteins with specialized structures and functions. Keratin filaments adhere to the plasma membrane by desmosomes building a three-dimensional matrix. Cell-Cell Contacts and Bacterial Influence: It is known that pathogenic oral bacteria are able to affect the expression and configuration of cell-cell junctions. Human keratinocytes up-regulate immune-modulatory receptors upon stimulation with bacterial components. Periodontal pathogens including P. gingivalis are able to inhibit oral epithelial innate immune responses through various mechanisms and to escape from host immune reaction, which supports the persistence of periodontitis and furthermore is able to affect the epithelial barrier function by altering expression and distribution of cell-cell interactions including tight junctions (TJs) and adherens junctions (AJs). In the pathogenesis of periodontitis a highly organized biofilm community shifts from symbiosis to dysbiosis which results in destructive local inflammatory reactions. Cellular Receptors: Cell-surface located toll like receptors (TLRs) and cytoplasmatic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) belong to the pattern recognition receptors (PRRs). PRRs recognize microbial parts that represent pathogen-associated molecular patterns (PAMPs). A multimeric complex of proteins known as inflammasome, which is a subset of NLRs, assembles after activation and proceeds to pro-inflammatory cytokine release. Cytokine Production and Release: Cytokines and bacterial products may lead to host cell mediated tissue destruction. Keratinocytes are able to produce diverse pro-inflammatory cytokines and chemokines, including interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-?. Infection by pathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) can induce a differentiated production of these cytokines. Immuno-modulation, Bacterial Infection, and Cancer Cells: There is a known association between bacterial infection and cancer. Bacterial components are able to up-regulate immune-modulatory receptors on cancer cells. Interactions of bacteria with tumor cells could support malignant transformation an environment with deficient immune regulation. The aim of this review is to present a set of molecular mechanisms of oral epithelial cells and their reactions to a number of toxic influences.
| S-EPMC6383680 | biostudies-literature
Project description:BackgroundWith the recent development of endoscopic submucosal dissection (ESD), large oesophageal cancers can be removed with a single procedure, with few limits on the resectable range. However, after aggressive ESD, a major complication that arises is postoperative inflammation and stenosis that can considerably affect the patient's quality of life.AimsTo examine a novel treatment combining ESD and the endoscopic transplantation of tissue-engineered cell sheets created using autologous oral mucosal epithelial cells, in a clinically relevant large animal model.MethodsOral mucosal epithelial cells, harvested from beagle dogs, were cultured under normal conditions at 37 degrees C, on temperature-responsive dishes. After ESD (5 cm in length, 180 degrees in range), cell sheets were harvested by a simple reduction in temperature to 20 degrees C, and transplanted by endoscopy.ResultsThe transplanted cell sheets were able to adhere to and survive on the underlying muscle layers in the ulcer sites, providing an intact, stratified epithelium. Four weeks after surgery, complete wound healing, with no observable stenosis, was seen in the animals receiving autologous cell sheet transplantation. By contrast, noticeable fibrin mesh and host inflammation, consistent with the intermediate stages of wound healing, were observed in the control animals that received only ESD.ConclusionsThese findings in a clinically relevant canine model show the effectiveness of a novel combined endoscopic approach for the potential treatment of oesophageal cancers that can effectively enhance wound healing and possibly prevent postoperative oesophageal stenosis.
| S-EPMC1856478 | biostudies-literature
Project description: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.
| S-EPMC7283219 | biostudies-literature
Project description:Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes ?Np63? and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-V) and protection of the ocular surface (cadherin-1) had ~3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.
| S-EPMC6955856 | biostudies-literature