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.

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.

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.

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.

Collagen Vitrigel Membrane-Coated Nylon Line Prevents Stenosis After Conization of the Cervix Uteri.

Cervical stenosis is a postoperative complication of conization for uterine cervical malignancy, but a standard method of preventing this complication has yet to be established. Collagen vitrigel is a collagen-based biomaterial that has antifibrotic and epithelization promoting actions. We evaluated the antistenotic effect of an indwelling collagen vitrigel membrane-coated nylon line (CVNL) after cervical conization in rabbits. In one group of rabbits, a CVNL was placed in the cervical canal after conization. In another group, a nylon line without a collagen coating was placed in the cervical canal after conization. The control group underwent cervical conization without placement of a device. The control (conization alone) and nylon (conization plus indwelling nylon line) groups exhibited cervical swelling. Rabbits in the CVNL group (cervical conization plus indwelling CVNL in the xerogel state) had a normal cervical surface. The cervical canal in the control group was enlarged and showed cystic changes attributed to cervical stenosis. The nylon group exhibited a trend toward cervical canal dilatation. In the CVNL group, the cervical canal was normal and did not show cystic dilatation. Fibrosis occurred to a lesser degree in the nylon group than in the control group, and the CVNL group exhibited minimal interstitial fibrosis. The control and nylon groups showed increased numbers of myofibroblasts in the regenerated cervix, but few myofibroblasts were observed in the CVNL group. Abundant collagen type III was observed in regenerated cervical tissue in the control and nylon groups but not in the CVNL group. The number of proliferative mesenchymal cells in the regenerated cervix was lowest in the CVNL group. The expressions of connective tissue growth factor (CTGF, a regulator of fibroblast growth and extracellular matrix secretion), extracellular signal-regulated protein kinases 1 and 2, and c-Jun N-terminal kinase (which are involved in the induction of CTGF by transforming growth factor-ß) were lower in the CVNL group than in the control or nylon groups. This study describes an indwelling CVNL that prevents cervical stenosis and cystic changes after conization. These effects were likely mediated by inhibition of fibrosis, myofibroblast emergence, CTGF expression, and collagen type III deposition in regenerating cervix. Impact statement Collagen vitrigel is a high-density collagen material that promotes epithelization, inhibits fibrosis, and suppresses inflammation in regenerating tissue. We evaluated whether a collagen vitrigel membrane-coated nylon line would prevent cervical stenosis after conization in the rabbit. We found that an indwelling collagen vitrigel membrane-coated nylon line prevented cervical canal stenosis and cystic changes after cervical conization by inhibiting fibrosis, myofibroblast emergence, connective tissue growth factor expression, and collagen type III deposition in the regenerating cervix. Our device has potential as a new method of preventing cervical canal fibrosis and stenosis after conization for cervical cancer.

Novel microvascular endothelial model utilizing a collagen vitrigel membrane and its advantages for predicting histamine-induced microvascular hyperpermeability.

INTRODUCTION: The evaluation of microvascular permeability is crucial for drug development. Nonetheless, there are few reliable test methods in vitro due to the lack of vascular endothelial models suitable for quantitative analyses. The purpose of this study is to construct a novel microvascular endothelial model with the high endothelial barrier function and sensitivity to physiological stimuli utilizing a collagen vitrigel membrane (CVM) composed of high-density collagen fibrils. METHODS: Human microvascular endothelial cells (HMVECs) were cultured for 6 days in a CVM chamber with or without human dermal fibroblasts (HDFs) cocultured on the reverse surface of the CVM. The endothelial barrier function was evaluated by measurement of transendothelial electrical resistance (TEER) and macromolecular permeation. RESULTS: The TEER value of a monolayer of HMVECs cultured on the CVM was 15-20 Ωï½¥cm2 during the culture period while it reached over 60 Ωï½¥cm2 by coculturing with HDFs. The TEER value was decreased from 5.7 to 3.4 Ωï½¥cm2 by 100 µM histamine in the monolayer model and from 50 to 32 Ωï½¥cm2 by 1 nM histamine in the coculture model, respectively. Interestingly, the permeability coefficient of the compound with a molecular weight of not 376 and 40,000 but 4000 was selectively increased in the histamine-treated coculture model. DISCUSSION: HMVECs cocultured with HDFs via a CVM formed the tight endothelial barrier and showed high responsivity to histamine. The model might be useful for exploring molecules that modulate microvascular permeability and pass through the microvascular endothelial barrier.

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.

Transplantation of multiciliated airway cells derived from human iPS cells using an artificial tracheal patch into rat trachea.

Tracheal resection is often performed for malignant tumours, congenital anomalies, inflammatory lesions, and traumatic injuries. There is no consensus on the best approach for the restoration of tracheal functionality in patients with tracheal defects. Artificial grafts made of polypropylene and collagen sponge have been clinically used by our group. However, 2 months are required to achieve adequate epithelialization of the grafts in humans. This study aimed to investigate the feasibility of transplantation therapy using an artificial trachea with human-induced pluripotent stem cell (hiPSC)-derived multiciliated airway cells (hiPSC-MCACs). Collagen vitrigel membrane, a biocompatible and absorbable material, was used as a scaffold to cover the artificial trachea with hiPSC-MCACs. Analyses of hiPSC-MCACs on collagen vitrigel membrane were performed by immunocytochemistry and electron microscopy and by assessing ciliary beat frequency. Along with the artificial trachea, hiPSC-MCACs were transplanted into surgically created tracheal defects of immunodeficient rats. The survival of transplanted cells was histologically evaluated at 1 and 2 weeks after the transplantation. The hiPSC-MCACs exhibited motile cilia on collagen vitrigel membrane. The surviving hiPSC-MCACs were observed in the endotracheal epithelium of the tracheal defect at 1 and 2 weeks after transplantation. These results suggest that hiPSC-MCAC is a useful candidate for tracheal reconstruction.

A high-density collagen xerogel thread prevents the progression of peritoneal fibrosis.

Peritoneal fibrosis is often provoked by peritoneal dialysis and is an essential precursor condition to the development of encapsulating peritoneal sclerosis. This study aimed to determine the efficacy of a high-density collagen xerogel thread (CXT) for the prevention of peritoneal fibrosis. Female ICR mice received intraperitoneal injections of chlorhexidine gluconate (CG) every other day to induce peritoneal fibrosis. For evaluation, the insertion of CXT or infusion of atelocollagen gel into the peritoneal cavity was conducted on the day before CG injection. For comparison, no collagen treatment after CG injection, and abdominal puncture without CG injection were also performed. Peritoneal fibrosis and inflammation were significantly suppressed by CXT for a long period. CXT prevented mesothelial epithelial-mesenchymal transition, myofibroblast emergence, and inflammatory cell invasion in the peritonitis tissue. In the early phase, atelocollagen gel modulated the expression of the fibrosis-associated protein transforming growth factor (TGF)-ß, connective tissue growth factor (CTGF), and CD105 in the peritoneum under CG-induced inflammation, while CXT did not. In contrast, CXT regulated the expression of CTGF and CD105 in the late phase and maintained antimicrobial protein REG3G at the same level as the Sham group in the early and late phases. Although the precise mechanism remains to be clarified, these findings suggest that CXT may have the potential to be developed as a simple therapeutic device to prevent peritoneal fibrosis, a severe complication in patients undergoing long-term peritoneal dialysis.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

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.

Transplantation of Human Corneal Endothelial Cells Cultured on Bio-Engineered Collagen Vitrigel in a Rabbit Model of Corneal Endothelial Dysfunction.

PURPOSE: To evaluate the effect of transplanting bioengineered corneal endothelial grafts in a rabbit model of corneal endothelial failure. METHODS: Human corneal endothelial cells (HCECs) were seeded on a vitrigel carrier. After Descemet's membrane was removed from the eyes of rabbits, transplantation was done with a vitrigel/HCEC graft or vitrigel alone without cells, or the eyes were left untreated. Slit lamp examinations and measurement of the central corneal thickness (CCT) were performed for 14 days postoperatively. RESULTS: HCECs cultured on vitrigel were strongly positive for ZO-1 and Na+/K+ ATPase. On day 14, the cornea showed mild edema and the pupil margins were visible through the grafts in the vitrigel/HCEC graft group. HCECs completely covered the grafts on day 14. In contrast, there was severe corneal edema and the pupil margins were undetectable on day 14 after transplantation of the vitrigel carrier alone or no transplantation. Proliferation of host cells was not observed in these groups. On day 14, the mean CCT was significantly thinner in the vitrigel/HCEC graft group than in the other two groups (p = 0.0008). CONCLUSIONS: Transplantation of a vitrigel/HCEC graft was effective for reducing the corneal thickness and restoring corneal transparency, suggesting the usefulness of vitrigel as a carrier for corneal endothelial cells.

Effects of artificial tracheal fixation on tracheal epithelial regeneration and prevention of tracheal stenosis.

CONCLUSION: Tight fixation of the artificial trachea is important for epithelialization and tracheal stenosis. OBJECTIVE: The authors have developed an artificial trachea and have used it for tracheal reconstruction. Although various studies on tracheal reconstruction have been conducted, no studies have examined the effect of artificial tracheal fixation on tracheal stenosis and regeneration. Therefore, the purpose of the present study was to evaluate the effect of artificial tracheal fixation. STUDY DESIGN: Preliminary animal experiment. METHODS: Artificial tracheae were implanted into rabbits with partial tracheal defects. Tracheal stenosis and regeneration of the tracheal epithelium on the artificial tracheae were evaluated by endoscopic examination, scanning electron microscopic analysis, and histological examination. The artificial tracheae fixed to the tracheal defects were classified into three groups (0-point, 4-point, and 8-point) by the number of fixation points. RESULTS: At 14 and 28 days post-implantation, the luminal surface of the implantation area was mostly covered with epithelium in all fixation groups. However, a small amount of granulation tissue was observed in the 0-point fixation group at 14 days post-implantation. Moreover, tracheal stenosis did not occur in the 8-point fixation group, but stenosis was detected in the other groups.

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.

Effects of a cell-free method using collagen vitrigel incorporating TGF-ß1 on articular cartilage repair in a rabbit osteochondral defect model.

We studied the ability of collagen vitrigel material to repair cartilage in vivo when used alone or with transforming growth factor-ß (TGF-ß). We measured the time course and quantity of TGF-ß1 released from the collagen vitrigel in vitro to quantify the controlled release of TGF-ß1. Over 14 days, 0.91 ng of TGF-ß was released from the collagen vitrigel. Osteochondral defects were made in the femoral trochlear groove in 36 Japanese white rabbits, which were divided into three groups: untreated group (group A), collagen vitrigel-implanted group (group B), and TGF-ß1-incorporated collagen vitrigel-implanted group (group C). The weight distribution ratio between the affected and unaffected limbs served as an indicator of pain. Animals were sacrificed at 4 and 12 weeks after surgery, and their tissues were assessed histologically. The weight distribution ratio increased in all groups and did not differ significantly between groups at 12 weeks. Group A needed 6 weeks to attain maximum improvement, and groups B and C showed near-maximum improvement at 4 and 2 weeks, respectively. The International Cartilage Repair Society II score improved significantly in group C relative to the other groups. These findings suggest that sustained, slow release of TGF-ß caused early pain mitigation and cartilage repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2592-2602, 2017.

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.