Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts.

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.

Application of "Tissueoid Cell Culture System" Using a Silicate Fiber Scaffold for Cancer Research.

BACKGROUND: We developed a 3-dimensional (3D) culture system using a high-purity silica fiber scaffold of unwoven sheets called CellbedTM. METHODS: We used adherent colon and esophagogastric junction adenocarcinoma cells, tongue squamous cell carcinoma (SqCC) cells, and nonadherent gastric cancer cells. These cells were subjected to staining with various substances and observed by electron microscopy. To evaluate the effects of extracellular matrix in carcinoma tissues, SqCC cells were cultured in Cellbed coated with collagens I, III, and IV. RESULTS: Especially well-differentiated carcinoma cells cultured in this 3D system showed their own unique characteristics: luminal formation in adenocarcinoma cells and cell stratification and keratinization in SqCC cells. Scanning electron microscopy revealed the proliferation of cancer cells with cytoplasm entwined in Cellbed. Intercellular desmosomes in squamous epithelia were detected by transmission electron microscopy of vertical cross sections. SqCC cells cultured in Cellbed coated with collagen IV showed enhanced invasive and proliferative abilities. CONCLUSION: Because the morphology of cancer cells cultured in this 3D culture system is similar to that in living organisms, we called the system a "tissueoid cell culture system." Coating with collagen IV enables the modification of cell-matrix interactions as well as recapitulation of the in vivo microenvironment.

A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells.

Insulin-producing and -secreting cells derived from mouse pluripotent stem cells (PSCs) are useful for pancreatic development research and evaluating drugs that may induce insulin secretion. Previously, we have established a differentiation protocol to derive insulin-secreting cells from mouse embryonic stem cells (ESCs) using a combination of growth factors, recombinant proteins, and a culture substratum with net-like fibers. However, it has not been tested which materials and diameters of these fibers are more effective for the differentiation. Therefore, the present study aimed to produce net-like culture substratum formed from polyamide (PA) and polyacrylonitrile (PAN) fibers. Substrata were delineated into PA100, 300, 600, PAN100, 300, and 600 groups based on fiber diameters. The differentiation efficiencies of mouse ESCs cultured on the substrata were then examined by insulin 1 (Ins1) expression. Expression was found to be highest in PA300 differentiated cells, indicating the potential to produce high levels of insulin. To understand any differences in substratum properties, the adsorption capacities of laminin were measured, revealing that PA300 had the highest for it. We next examined the stage of differentiation affected by incubation with PA300. This showed that Sox17- and Pdx1-GFP-positive cells increased during the first step of differentiation. To show the production of insulin without absorption from the medium, we confirmed the expression of insulin C-peptide after differentiation. Finally, we tested the effects of PA300 on the differentiation of human-induced PSC, and found more Sox17-positive cells with the PA300 substratum at the definitive endoderm stage. Furthermore, these cells expressed insulin C-peptide and had glucose-responsive C-peptide secretion. In summary, our study identified and validated a novel substratum which is suitable for pancreatic differentiation of mouse and human PSCs.

Improvement of Hepatic Functions by Spheroids Coculture with Fibroblasts in 3D Silica Nonwoven Fabrics.

In order to realize organ-on-a-chip as an effective tool for regenerative medicine and drug development, tissue-mimic cell culture methods which promote liver-specific function for long period have been developed. We have previously demonstrated that coculture of hepatocyte spheroids on fibroblasts using micropatterned substrate improved the hepatic functions due to the heterotypic cell-cell interactions and paracrine signaling from each other. In addition, hepatocyte function cultured as monolayer was also promoted in separately coculture with fibroblasts cultured as monolayer, and it is more improved in separately coculture with fibroblasts in 3D silica nonwoven fabrics. In this study, separately coculture of hepatocyte spheroids with fibroblasts cultured on 3D silica nonwoven fabrics was estimated for further improvement of hepatocyte functions. The hepatic function cocultured with fibroblast was more promoted than mono spheroids culture. The functional enhancement was significantly most improved in separately coculture with fibroblast in 3D silica nonwoven fabrics. Thus, these results were suggested that 3D culture of fibroblasts in 3D silica nonwoven fabrics increased the heterotypic secretion of paracrine factors, and it is essential for improved hepatic performance.

Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells in Electrospun Silica Nonwoven Fabrics.

Silica nonwoven fabrics (SNFs) with enough mechanical strength are candidates as implantable scaffolds. Culture of cells therein is expected to affect the proliferation and differentiation of the cells through cell-cell and cell-SNF interactions. In this study, we examined three-dimensional (3D) SNFs as a scaffold of mesenchymal stem cells (MSCs) for bone tissue engineering applications. The interconnected highly porous microstructure of 3D SNFs is expected to allow omnidirectional cell-cell interactions, and the morphological similarity of a silica nanofiber to that of a fibrous extracellular matrix can contribute to the promotion of cell functions. 3D SNFs were prepared by the sol-gel process, and their mechanical properties were characterized by the compression test and rheological analysis. In the compression test, SNFs showed a compressive elastic modulus of over 1 MPa and a compressive strength of about 200 kPa. These values are higher than those of porous polystyrene disks used for in vitro 3D cell culture. In rheological analysis, the elastic modulus and fracture stress were 3.27 ± 0.54 kPa and 25.9 ± 8.3 Pa, respectively. Then, human bone marrow-derived MSCs were cultured on SNFs, and the effects on proliferation and osteogenic differentiation were evaluated. The MSCs seeded on SNF proliferated, and the thickness of the cell layer became over 80 µm after 14 days of culture. The osteogenic differentiation of MSCs on SNFs was induced by the culture in the commercial osteogenic differentiation medium. The alkaline phosphatase activity of MSCs on SNFs increased rapidly and remained high up to 14 days and was much higher than that on two-dimensional tissue culture-treated polystyrene. The high expression of RUNX2 and intense staining by alizarin red s after differentiation supported that SNFs enhanced the osteogenic differentiation of MSCs. Furthermore, permeation analysis of SNFs using fluorescein isothiocyanate-dextran suggested a sufficient permeability of SNFs for oxygen, minerals, nutrients, and secretions, which is important for maintaining the cell viability and vitality. These results suggested that SNFs are promising scaffolds for the regeneration of bone defects using MSCs, originated from highly porous and elastic SNF characters.

Silicate fiber-based 3D cell culture system for anticancer drug screening.

BACKGROUND: Three-dimensional (3D) in vitro cultures can recapitulate the physiological in vivo microenvironment. 3D Modeling techniques have been investigated and applied in anticancer drug screening. MATERIALS AND METHODS: A silicate fiber scaffold was used for 3D cell cultures, and used to model the efficacy of anticancer drugs, such as mytomicin C and doxorubicin. RESULTS: A unique 3D structure was observed in 13 human tumor cell lines on scaffold, and these cells exhibited higher drug resistance than cells in two-dimensional (2D) cultures. Furthermore, the production of lactate and expression of the nuclear factor-kappa B (NF-κB)-regulated genes B cell lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) were higher in 3D cultures than in 2D cultures. CONCLUSION: These findings suggest that a 3D model using a silicate fiber scaffold can mimic features of cancer, and is also a suitable model for the evaluation of anticancer drugs in vitro.

Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun polyacrylonitrile fibers.

Butyl-biodiesel production using electrospun polyacrylonitrile fibers with Pseudomonas cepacia lipase immobilized through physical adsorption was studied. About 80% conversion to butyl-biodiesel was achieved after 24h by suspending the catalyst at 2.4 mg/mL in a mixture of rapeseed oil and n-butanol at a molar ratio of 1:3, containing water at 8000 ppm at 40 degrees C. A further 24h of operation resulted in 94% conversion. The initial reaction rate detected for this process was 65-fold faster than those detected for Novozym 435 on a total catalyst mass basis. The immobilized lipase continued to work as a catalyst for 27 d, within a 15% reduction in conversion yield at the outlet of the reactor compared with the average value detected during the first 3d of operation in a continuous butyl-biodiesel production system.

Immobilization of Pseudomonas cepacia lipase onto electrospun polyacrylonitrile fibers through physical adsorption and application to transesterification in nonaqueous solvent.

The lipase of Pseudomonas cepacia was immobilized onto electrospun polyacrylonitrile (PAN) fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane. The rate of reaction with the adsorbed lipase was 23-fold higher than the initial material. After 10 recyclings, the initial reaction rate was 80% of the original rate. This system of enzyme immobilization is therefore suitable for carrying out transesterification reactions in nonaqueous solvents.