Multi-laboratory Validation Study of the Vitrigel-Eye Irritancy Test Method as an Alternative to In Vivo Eye Irritation Testing.

Collagen vitrigel membranes (CVMs) comprising high-density collagen fibrils equivalent to in vivo connective tissues have been widely used in cell culture applications. A human corneal epithelium (hCE) model was previously developed by the Takezawa group, by culturing HCE-T cells (derived from hCE cells) on a CVM scaffold in a chamber that provided an air-liquid interface culture system. This hCE model was used to establish a new test method, known as the Vitrigel-Eye Irritancy Test (Vitrigel-EIT) method, which can be used to estimate the ocular irritation potential of test chemicals by analysing relative changes in transepithelial electrical resistance (TEER) over time. The current study was conducted in order to assess the reliability and relevance of the Vitrigel-EIT method at three participating laboratories by determining the method's within-laboratory reproducibility and between-laboratory reproducibility, as well as its capacity for distinguishing non-irritants from irritants in a bottom-up approach. The initial test sample size was found to be too low to evaluate the predictive capacity of the test method, and so it was evaluated with additional in-house data for a total of 93 test chemicals. The results showed 80-100% within-laboratory reproducibility and an excellent between-laboratory reproducibility that met the acceptance criteria of 80%. However, the method's predictive capacity for distinguishing non-irritants (test chemicals not requiring classification and labelling for eye irritation or serious eye damage, i.e. United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS) No Category) from irritants (GHS Categories 1 and 2) in a bottom-up approach was unacceptable because of false negative rates as high as 16.7%. After considerable review of the data with a view to using the method for regulatory purposes, it was determined that a more defined applicability domain, excluding test chemical solutions with a pH of 5 or less and solid test chemicals, improved the false negative rate to 4.2%. These results suggested that, within this carefully defined applicability domain, the Vitrigel-EIT method could be a useful alternative for distinguishing test chemicals that are ocular non-irritants from those that are irritants as part of a bottom-up approach.

Fabrication of a Corneal Model Composed of Corneal Epithelial and Endothelial Cells via a Collagen Vitrigel Membrane Functioned as an Acellular Stroma and Its Application to the Corneal Permeability Test of Chemicals.

A collagen vitrigel membrane (CVM) we developed can function as both a scaffold for cells and a pathway for chemicals. To extrapolate the corneal permeability of chemicals in vivo, we proposed six corneal models using the CVM. Thin and thick CVMs were used as models for Bowman's membrane (BM) and an acellular stroma (AS), respectively. Models for a corneal epithelium (CEpi), a CEpi-AS, a CEpi-endothelium (Endo), and a CEpi-AS-Endo were fabricated by culturing corneal epithelial cells and/or corneal endothelial cells on the surface of CVMs. Subsequently, the permeability coefficient (Papp) value of each model was calculated using five chemicals with different molecular radii; cyanocobalamin and four fluorescein isothiocyanate-dextrans (FD) (FD-4, FD-10, FD-20, and FD-40). The slopes of Papp versus molecular radii of those chemicals in the both BM and AS models were almost similar to data using an excised rabbit corneal stroma. The ratios of Papp values in models for BM, CEpi, and CEpi-Endo against those in data using an excised rabbit cornea were calculated as 75.4-fold, 6.4-fold, and 4.5-fold for FD-4, and 38.7-fold, 10.0-fold, and 4.2-fold for FD-10, respectively. Similarly, those in models for AS, CEpi-AS, and CEpi-AS-Endo were calculated as 26.1-fold, 2.5-fold, and 0.6-fold for FD-4, and 26.1-fold, 1.5-fold, and 0.6-fold for FD-10, respectively. These results suggest that the CEpi-AS-Endo model with both the barrier function of corneal cell layers and the diffusion capacity of chemicals in thick CVM is most appropriate for extrapolating the corneal permeability of chemicals in vivo.

High-density collagen patch prevents stricture after endoscopic circumferential submucosal dissection of the esophagus: a porcine model.

BACKGROUND AND AIMS: Extensive excision of the esophageal mucosa by endoscopic submucosal dissection (ESD) frequently evokes a luminal stricture. This study aimed to determine the efficacy of a high-density collagen patch for the prevention of esophageal stricture in extensive ESD. METHODS: Six pigs underwent circumferential esophageal ESD under general anesthesia. In 3 pigs, artificial ulcers were covered by 2 collagen patches. The other 3 pigs underwent circumferential ESD only. RESULTS: The 2 collagen patches were settled onto the ulcer surface using a general endoscope and instruments. The collagen patch-treated group showed significantly better patency rates on both the oral and anal sides of the wound area compared with the control group at day 14. The mucosal re-epithelization ratio was significantly promoted, and the extent of mucosal inflammation and fibrosis was significantly decreased with the collagen patch treatment in the wound area. The frequency of cells positive α-smooth muscle actin was significantly reduced in the collagen patch-treated group compared with the control group. CONCLUSIONS: We have established a high-density collagen device that can reduce the esophageal stricture associated with extensive ESD. This easy-to-handle device would be useful during superficial esophageal cancer treatment by ESD.

Progress in cell culture systems for pathological research.

Cell culture is a well-established standard technique and a fundamental tool in biology and medicine. Establishment of a novel culture method by meeting various challenges can sometimes open up new fields of cell biology and medicine. An artificial microenvironment for cultured cells is made up of complicated factors, including cytokines, scaffold material type, cell-cell interactions, and physical stress. To replicate the tissue architecture, cell-cell interactions, and specific physical microenvironment, we previously demonstrated the effectiveness of a three-dimensional culture system, and further established two simple culture systems: air-liquid interface (ALI) and fluid flow stress (FFS). A three-dimensional collagen gel culture system can replicate cell-cell interactions in vitro. As skin is constantly exposed to air, the ALI system closely mimicked the skin microenvironment and maintained the homeostasis of the epidermis and dermis. The ALI culture system also revealed the possibility of skin regeneration through ectopic mesenchymal cell involvement. Fluid streaming and shear stress were recently demonstrated to constitute the critical microenvironment for various cell types. The FFS system demonstrated that fluid streaming induced epithelial-mesenchymal transition of mesothelial cells, leading to peritoneal fibrosis. Our novel culture systems will hopefully open up new fields of regenerative medicine and pathological research.

Increase of ß2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane.

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. ß-actin, ß-catenin, and ß1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but ß2-integrin expression markedly increased.

Predictive performance of the Vitrigel-eye irritancy test method using 118 chemicals.

We recently developed a novel Vitrigel-eye irritancy test (EIT) method. The Vitrigel-EIT method is composed of two parts, i.e., the construction of a human corneal epithelium (HCE) model in a collagen vitrigel membrane chamber and the prediction of eye irritancy by analyzing the time-dependent profile of transepithelial electrical resistance values for 3 min after exposing a chemical to the HCE model. In this study, we estimated the predictive performance of Vitrigel-EIT method by testing a total of 118 chemicals. The category determined by the Vitrigel-EIT method in comparison to the globally harmonized system classification revealed that the sensitivity, specificity and accuracy were 90.1%, 65.9% and 80.5%, respectively. Here, five of seven false-negative chemicals were acidic chemicals inducing the irregular rising of transepithelial electrical resistance values. In case of eliminating the test chemical solutions showing pH 5 or lower, the sensitivity, specificity and accuracy were improved to 96.8%, 67.4% and 84.4%, respectively. Meanwhile, nine of 16 false-positive chemicals were classified irritant by the US Environmental Protection Agency. In addition, the disappearance of ZO-1, a tight junction-associated protein and MUC1, a cell membrane-spanning mucin was immunohistologically confirmed in the HCE models after exposing not only eye irritant chemicals but also false-positive chemicals, suggesting that such false-positive chemicals have an eye irritant potential. These data demonstrated that the Vitrigel-EIT method could provide excellent predictive performance to judge the widespread eye irritancy, including very mild irritant chemicals. We hope that the Vitrigel-EIT method contributes to the development of safe commodity chemicals. Copyright © 2015 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.

Fluid dwell impact induces peritoneal fibrosis in the peritoneal cavity reconstructed in vitro.

Peritoneal fluid dwell impacts the peritoneum by creating an abnormal physiological microenvironment. Little is known about the precise effects of fluid dwell on the peritoneum, and no adequate in vitro models to analyze the impact of fluid dwell have been established. In this study, we developed a peritoneal fluid dwell model combined with an artificial peritoneal cavity and fluid stirring generation system to clarify the effects of different dwelling solutions on the peritoneum over time. To replicate the peritoneal cavity, we devised a reconstructed peritoneal cavity utilizing a mesothelial layer, endothelial layer, and collagen membrane chamber. The reconstructed peritoneal cavity was infused with Dulbecco's modified Eagle's medium, saline, lactated Ringer's solution or peritoneal dialysis solution with repeated 4-h dwells for 10 or 20 consecutive days. The above-described solutions induced epithelial-mesenchymal transition (EMT) and hyperplasia of mesothelial cells. All solution types modulated nitric oxide synthase activities in mesothelial and endothelial cells and nitric oxide concentrations in dwelling solutions. Inhibition of nitric oxide synthase activity acted synergistically on mesothelial EMT and hyperplasia. The present findings suggest that solutions infused into the peritoneal cavity are likely to affect nitric oxide production in the peritoneum and promote peritoneal fibrosis. Our newly devised peritoneal cavity model should be a promising tool for understanding peritoneal cellular kinetics and homeostasis.

A new cell-free bandage-type artificial skin for cutaneous wounds.

Engineered skin substitutes are widely used in skin wound management. However, no currently available products satisfy all the criteria of usability in emergency situations, easy handling, and minimal scar formation. To overcome these shortcomings, we designed a cell-free bandage-type artificial skin, named "VitriBand" (VB), using adhesive film dressing, silicone-coated polyethylene terephthalate film, and collagen xerogel membrane defined as a dried collagen vitrigel membrane without free water. We analyzed its advantages over in-line products by comparing VB with hydrocolloid dressing and collagen sponge. For evaluation, mice inflicted with full-thickness skin defects were treated with VB, hydrocolloid dressing, and collagen sponge. A plastic film group treated only with adhesive film dressing and silicone-coated polyethylene terephthalate film, and a no treatment group were also compared. VB promoted epithelization while inhibiting the emergence of myofibroblasts and inflammation in the regenerating tissue more effectively than the plastic film, hydrocolloid dressing, and collagen sponge products. We have succeeded in establishing a cell-free bandage-type artificial skin that could serve as a promising first-line medical biomaterial for emergency treatment of skin injuries in various medical situations.

Epithelial-to-mesenchymal transition and slit function of mesothelial cells are regulated by the cross talk between mesothelial cells and endothelial cells.

Peritoneal dysfunction is a major factor leading to treatment failure of peritoneal dialysis (PD). However, the precise mechanism of the peritoneal diffusion changes related to PD remains to be elucidated. To this end, we have established a novel peritoneal diffusion model in vitro, which consists of a three-dimensional culture system using a collagen vitrigel membrane chamber and a fluid-stream generation system. This artificial peritoneal model revealed that high-glucose culture medium and fluid flow stress promoted the epithelial-mesenchymal transition (EMT) process of mesothelial cells and that endothelial cells inhibited this mesothelial EMT process. Mesothelial cells in the EMT state showed high expression of connective tissue growth factor and low expression of bone morphogenic protein-7, while non-EMT mesothelial cells showed the opposite expression pattern of these two proteins. In addition, these protein expressions were dependent on the presence of endothelial cells in the model. Our model revealed that the endothelial slit function was predominantly dependent on the covering surface area, while the mesothelial layer possessed a specific barrier function for small solutes independently of the surface area. Notably, a synergic barrier effect of mesothelial cells and endothelial cells was present with low-glucose pretreatment, but high-glucose pretreatment abolished this synergic effect. These findings suggest that the mesothelial slit function is not only regulated by the high-glucose-induced EMT process but is also affected by an endothelial paracrine effect. This peritoneal diffusion model could be a promising tool for the development of PD.

Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery.

Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.

Comparative Study of Immunodeficient Rat Strains in Engraftment of Human-Induced Pluripotent Stem Cell-Derived Airway Epithelia.

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.

Adipose tissue behavior is distinctly regulated by neighboring cells and fluid flow stress: a possible role of adipose tissue in peritoneal fibrosis.

Adipose tissue, together with the mesothelial layer and microvessels, is a major component of the mesenteric peritoneum, and the mesenterium is a target site for peritoneal fibrosis. Adipose tissue has been speculated to play a role in peritoneal dialysis (PD)-related fibrosis, but the precise cellular kinetics of adipose tissue during this process remain to be determined. To clarify this critical issue, we analyzed the kinetics of adipose tissue using a novel peritoneal reconstruction model in which the effects of mesothelial cells or endothelial cells could be identified. Adipose tissue was co-cultured with mesothelial cells or endothelial cells in a combined organ culture and fluid flow stress culture system. Spindle mesenchymal cells and immature adipocytes derived from adipose tissue were characterized by immunohistochemistry. Adipose tissue fragments cultured in this system yielded many spindle mesenchymal cells in non-co-culture conditions. However, the number of spindle mesenchymal cells emerging from adipose tissue was reduced in co-culture conditions with a covering layer of mesothelial cells. Mesothelial cells co-cultured in the separated condition did not inhibit the emergence of spindle mesenchymal cells from adipose tissue. Interestingly, endothelial cells promoted the emergence of lipid-laden immature adipocytes from adipose tissue under fluid flow stress. We have demonstrated that adipose tissue behavior is not only regulated by mesothelial cells and endothelial cells under fluid flow stress, but is also involved in fibrosis and fat mass production in the peritoneum. Our findings suggest that adipose tissue is a potential source of cells for peritoneal fibrosis caused by PD therapy.

Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis.

E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning.

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.

Regeneration of tracheal epithelium using a collagen vitrigel-sponge scaffold containing basic fibroblast growth factor.

OBJECTIVES: Our group has had good results in tracheal mucosal regeneration using a collagen vitrigel-sponge scaffold in an animal model. In this study, the effectiveness of this scaffold with the application of basic fibroblast growth factor (b-FGF) was investigated. METHODS: A collagen vitrigel-sponge scaffold was fabricated with simultaneous addition of b-FGF. Three types of collagen vitrigel-sponge scaffolds were made: no b-FGF, 10 ng of b-FGF, and 100 ng of b-FGF. At 3, 5, 7, and 14 days after implantation in rats, the tracheas were removed and histologically evaluated. The regeneration of mucosal epithelium and the subepithelial layer was evaluated. RESULTS: Mucosal epithelium, including pseudostratified epithelium and ciliated cells, regenerated earlier in the scaffolds when b-FGF was applied than when b-FGF was not applied. Regeneration of the subepithelial layer, infiltration of inflammatory cells and fibroblasts, and angiogenesis were promoted earlier in the scaffolds with b-FGF application. CONCLUSIONS: Our technique for tracheal reconstruction using collagen vitrigel-sponge scaffolds with b-FGF application affords a feasible approach for accelerating the regeneration of the intraluminal surface and subepithelial layer of tracheal tissue.

HepG2-NIAS cells, a new subline of HepG2 cells that can enhance not only CYP3A4 activity but also expression of drug transporters and form bile canaliculus-like networks by the oxygenation culture via a collagen vitrigel membrane.

We reported the enhanced liver-specific function and structure of HepG2 cells by the oxygenation culture via a collagen vitrigel membrane (CVM). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. Here, we clarified the features of HepG2 cells with three different culture histories by analyzing their morphology and viability, CYP3A4 activity, the potential to form bile canaliculus-like structures, and the expression of drug transporters. On plastic, HepG2-NIAS cells grew as a monolayer without the formation of large aggregates involving dead cells that were observed in HepG2-RCB1648 cells and HepG2-RCB1886 cells. In the oxygenation culture via a CVM, the CYP3A4 activity of HepG2-NIAS cells increased to almost half level in direct comparison to that of differentiated HepaRG cells cultured on a collagen-coated plate; however, that of HepG2-RCB1648 cells and HepG2-RCB1886 cells was almost not detected. HepG2-NIAS cells formed bile canaliculus-like networks in which fluorescein was accumulated after the exposure of fluorescein diacetate, although HepG2-RCB1648 cells and HepG2-RCB1886 cells did not possess the potential. Also, immunohistological observations revealed that HepG2-NIAS cells remarkably enhanced the expression of drug transporters, NTCP, OATP1B1, OATP1B3, BSEP, MDR1, MRP2, and BCRP. These results suggest that HepG2-NIAS cells are a new subline of HepG2 cells useful for drug development studies. HepG2-NIAS cells were registered in RCB with the number of 4679.

Bi-layered carboxymethyl cellulose-collagen vitrigel dual-surface adhesion-prevention membrane.

During wound regeneration, both cell adhesion and adhesion-inhibitory functions must be controlled in parallel. We developed a membrane with dual surfaces by merging the properties of carboxymethyl cellulose (CMC) and collagen using vitrification. A rigid membrane was formed by vitrification of a bi-layered CMC and collagen hydrogel without using cross-linking reagents, thus providing dual functions, strong cell adhesion-inhibition with the CMC layer, and cell adhesion with the collagen layer. We referred to this bi-layered CMC-collagen vitrigel membrane as "Bi-C-CVM" and optimized the process and materials. The introduction of the CMC layer conferred a "tough but stably wet" property to Bi-C-CVM. This enables Bi-C-CVM to cover wet tissue and make the membrane non-detachable while preventing tissue adhesion on the other side. The bi-layered vitrification procedure can expand the customizability of collagen vitrigel devices for wider medical applications.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Airway Cells on Vitrigel Membrane into Rat Nasal Cavity.

The nasal mucosa functions as a frontline biological defense against various foreign substances and pathogens. Maintaining homeostasis of the nasal epithelium is necessary to promote good health. Nasal epithelia are constantly replaced under normal conditions. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Since there is no treatment for this underlying condition, extrinsic manipulation is necessary to recover and maintain nasal epithelia in cases of hereditary diseases. In this study, we explored the use of airway epithelial cells (AECs), including multiciliated airway cells, derived from human induced pluripotent stem cells (iPSCs) on porcine atelocollagen vitrigel membranes, as a candidate of a therapeutic method for irreversible nasal epithelial disorders. To confirm the regenerative capacity of iPSC-derived AECs, we transplanted them into nasal cavities of nude rats. Although the transplanted cells were found within cysts isolated from the recipient nasal respiratory epithelia, they survived in some rats. Furthermore, the surviving cells were composed of multiple cell types similar to the human airway epithelia. The results could contribute to the development of novel transplantation-related technologies for the treatment of severe irreversible nasal epithelial disorders. Impact Statement Nasal respiratory epithelia are important for the functions of nasal cavity, including humidifying the air and filtering various toxic substances. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Our novel method to transplant airway epithelial cells derived from human induced pluripotent stem cells will be a candidate method to replace malfunctioned nasal respiratory epithelia in such a situation. To secure our method's safety, we used porcine atelocollagen vitrigel membranes, which prevent the immune response and bovine spongiform encephalopathy, as a scaffold.

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming.

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.

Culture period-dependent changes in the uptake of transporter substrates in sandwich-cultured rat and human hepatocytes.

Sandwich-cultured hepatocytes (SCH) are a useful tool for evaluating hepatobiliary drug transport in vitro. Some studies have investigated the in vitro-in vivo correlations of the biliary clearance of drugs using SCH. In most cases, the biliary clearance observed in vivo correlated well with the predicted clearance, but the predicted absolute values were underestimated when based on in vitro experiments with SCH. We hypothesized that the down-regulated function of uptake transporters is one of the causes of this underestimation. Therefore, the uptake of taurocholate, digoxin, pravastatin, and rosuvastatin was investigated in sandwich-cultured rat hepatocytes (SCRH) cultured for 5, 24, 48, and 96 h, and the predicted hepatic clearance from in vitro uptake clearance (CL(H, vitro)) was calculated with a dispersion model. In SCRH cultured for 96 h, the saturable uptake of taurocholate, digoxin, pravastatin, and rosuvastatin decreased to 7.5, 3.3, 64, and 23%, respectively, of their uptake in hepatocytes cultured for 5 h, and a better prediction of in vivo hepatic clearance (CL(H, vivo)) was achieved when based on CL(H, vitro) of 5-h-cultured hepatocytes. These results suggest that the uptake activity is considerably reduced in cell culture, even in a sandwich-culture format. In a similar study, we also examined taurocholate and rosuvastatin in sandwich-cultured human hepatocytes (SCHH). Unlike in SCRH, the saturable uptake of these compounds did not differ markedly in SCHH cultured for 5 or 96 h. Thus, the uptake activity in SCHH was maintained relatively well compared with that in SCRH.