Synergistic Effect of Sarocladium sp. and Cryptococcus sp. Co-Culture on Crude Oil Biodegradation and Biosurfactant Production

This study was conducted to evaluate the co-culture ability of two yeast (Sarocladium sp. and Cryptococcus sp.) isolates as compared to their individual cultures in surfactant production and oil degradation. The results showed that individual culture of each strain was capable of producing surfactant, degrading oil, and pyrene; also, a synergistic effect was observed when a co-culture was applied. Oil removal and biomass production were 28 and 35% higher in the co-culture than in individual cultures, respectively. To investigate the synergistic effects of mix culture on oil degradation, the surface tension, emulsification activity (EA), and cell surface hydrophobicity of individual and co-culture were studied. A comparison between the produced biosurfactant and chemical surfactants showed that individual culture of each yeast strain could reduce the surface tension like SDS and about 10% better than Tween 80. The results showed that the microbial consortium could reduce the surface tension more, by 10 and 20%, than SDS and Tween 80, respectively. Both individual cultures of Sarocladium sp. and Cryptococcus sp. showed good emulsification activity (0.329 and 0.412, respectively) when compared with a non-inoculated medium. Emulsification activity measurement for the two yeast mix cultures showed an excellent 33 and 67% increase as compared to the individual culture of Sarocladium sp. and Cryptococcus sp., respectively. The cell surface hydrophobicity of Sarocladium sp. and Cryptococcus sp. increased (38 and 85%) when the cells were treated with pyrene as a hydrophobic substrate for four generations. Finally, a 40% increase for pyrene degradation was measured in a co-culture of the two yeast mix culture. According to the results of the present study, the co-culture system exhibited better performance and this study will enhance the understanding of the synergistic effects of yeast co-culture on oil degradation.

     

Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils

Many biological processes, such as stem cell differentiation, wound healing and development, involve dynamic interactions between cells and their microenvironment. The ability to control these dynamic processes in vitro would be potentially useful to fabricate tissue engineering constructs, study biological processes, and direct stem cell differentiation. In this paper, we used a parylene-C microstencil to develop two methods of creating patterned co-cultures using either static or dynamic conditions. In the static case, embryonic stem (ES) cells were co-cultured with fibroblasts or hepatocytes by using the reversible sealing of the stencil on the substrate. In the dynamic case, ES cells were co-cultured with NIH-3T3 fibroblasts and AML12 hepatocytes sequentially by engineering the surface properties of the stencil. In this approach, the top surface of the parylene-C stencil was initially treated with hyaluronic acid (HA) to reduce non-specific cell adhesion. The stencil was then sealed on a substrate and seeded with ES cells which adhered to the underlying substrate through the holes in the membrane. To switch the surface properties of the parylene-C stencils to cell adhesive, collagen was deposited on the parylene-C surfaces. Subsequently, a second cell type was seeded on the parylene-C stencils to form a patterned co-culture. This group of cells was removed by peeling off the parylene-C stencils, which enabled the patterning of a third cell type. Although the static patterned co-culture approach has been demonstrated previously with a variety of methods, layer-by-layer modification of microfabricated parylene-C stencils enables dynamic patterning of multiple cell types in sequence. Thus, this method is a promising approach to engineering the complexity of cell-cell interactions in tissue culture in a spatially and temporally regulated manner.     

Photocontrol of cell adhesion and proliferation by a photoinduced cationic polymer surface

In this study photoinduced cation generation, based on the photochemical properties of malachite green (MG), was used for the surface design and in vitro photochemical control of cell adhesion and proliferation. The MG-derivatized surface was prepared by coating a photoreactive polymer as a substrate onto a poly(ethylene terephthalate) (PET) sheet. The photoreactive polymer was radical copolymer of styrene with the MG-derivatized monomer diphenyl(4-vinylphenyl)methane leucohydroxide (degree of substitution of MG unit: 12.4 mol%). Water contact angle measurements and X-ray photoelectron spectroscopy revealed high hydrophobicity and homogeneous distribution of the MG groups on the outermost surface of the coated film, respectively. When the coated film was exposed to ultraviolet light (UV) irradiation at wavelengths of 290-410 nm, a time-dependent color change of the film was observed from pale yellow, before irradiation, to green. These results indicated generation of cations on the film surface by photochemical cation generation of the MG groups, which was quantitatively characterized by force versus distance curves measurements in atomic force microscopic (AFM) observation using a carboxylated AFM tip. The seeding and culture of endothelial cells showed a marked reduction in adhesion on the nonirradiated coated film surface, whereas the UV-irradiated surface promoted cell adhesion and proliferation except for incubation in serum-free medium, which was similar to commercial tissue culture PET sheet. These observations may be due to adsorption of cell adhesive proteins, typified by fibronectin, in serum-containing medium onto the cationized photoreactive copolymer surface by electrostatic interactions.     

Surface characterization of the extracellular matrix remaining after cell detachment from a thermoresponsive polymer

The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.     

Investigation of the Effective Action Distance Between Hematopoietic Stem/Progenitor Cells and Human Adipose-Derived Stem Cells During Their In Vitro Co-culture

The in vitro suitable action distance between umbilical cord blood-derived hematopoietic stem/progenitor cells and its feeder cell, human adipose-derived stem cells, during their co-culture, was investigated through a novel transwell co-culture protocol, in which the distance between the two culture chambers where each cell type is growing can be adjusted from 10 to 450 μm. The total cell number was determined with a hemacytometer, and the cell morphology was observed under an inverted microscope each day. After 7 days of co-culture, the fold-expansion, surface antigen expression of CD34(+) and CFU-GM assay of the hematopoietic mononuclear cells (MNCs) were analyzed. The results showed that there was an optimal communication distance at around 350 μm between both types of stem cells during their in vitro co-culture. By using this distance, the UCB-MNCs and CD34(+) cells were expanded by 15.1?±?0.2 and 5.0?±?0.1-fold, respectively. It can therefore be concluded that the optimal action distance between stem cells and their supportive cells, when cultured together for 7 days, is of around 350 μm.     

A Simple Method for Cell Sheet Fabrication Using Mica Surfaces Grafted with Peptide Detergent A6K

Cell sheet technology is a very important strategy for scaffold‐free tissue engineering. In order to fabricate cell sheets by a simple method, peptide detergent A₆K was grafted on mica surfaces by dropping its aqueous solution at different concentrations on the surface. As revealed by surface topographical observation and water contact angle measurement, the most hydrophobic surface was obtained using peptide solution at the concentration of 0.2 mg · mL^?1. The peptide‐grafted mica surface was used to culture mouse preosteoblast cell MC3T3‐E1. After the cells reached confluence and the peptide was biodegraded, an intact cell sheet was peeled from the mica. This simple method does not need any non‐biological reagents or complicated procedures, and may have great potential in tissue engineering based on cell sheet technology.

     

Highly robust protein production by co-culture of CHO spheroids layered on feeder cells in serum-free medium

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.     

Biological Response of Osteoblastic and Chondrogenic Cells to Graphene-Containing PCL/Bioactive Glass Bilayered Scaffolds for Osteochondral Tissue Engineering Applications

Graphene-containing 13-93 bioactive glass and poly(ε-caprolactone)-based bilayer, electrically conductive scaffolds were prepared for osteochondral tissue repair. Biological response of osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells to the composite scaffolds was assessed under mono-culture and co-culture conditions. Cytotoxicity was investigated using MTT assay, cartilage matrix production was evaluated by Alcian blue staining, and mineralization of both types of cells in the different culture systems was observed by Alizarin red S staining. Results showed that osteoblastic and chondrogenic cells utilized in the study did not show toxic response to the prepared scaffolds under mono-culture conditions and higher cell viability rates were obtained in co-culture conditions. Larger mineralized areas were determined under co-culture conditions and calcium deposition amount significantly increased compared with that in control group samples after 21 days. Additionally, the amount of glycosaminoglycans synthesized in co-culture was higher compared to mono-culture conditions. Electric stimulation applied under mono-culture conditions suppressed the viability of MC3T3-E1 cells whereas it enhanced the viability rates of ATDC5 cells. The study suggests that the designed bilayered osteochondral constructs have the potential for osteochondral defect repair.     

Study of cell transmigration using a co-culture microsystem under shear stress

This study reports a novel cell co-culture technique using micro-molding in capillaries (MIMIC) technology that was utilized to observe the transmigration conditions of two types of cells with and without fluidic shear stress. Besides, the gap size of co-culture device could achieve shortest and not mixture. Endothelial cells (ECs) and smooth muscle cells (SMCs) were used in our experiment. In addition, concentrations of two cell are 8000 cells/μL (ECs) and 9000 cells/μL (SMCs), respectively, the shear stress is 7 dyne/cm₂, and the isolation distance between two types of cell are 50 and 200 μm. It is found that in the smaller culture space (50 μm) condition, ECs and SMCs would induce mutually, which would further make cell migration; in larger culture space (200 μm) condition, no inducing reaction took place between ECs and SMCs. It will have more advantages in bio-manipulation and tissue repair engineering.     

Tools for micropatterning epithelial cells into microcolonies on transwell filter substrates

Despite the importance of epithelial tissue in most major organs there have been limited attempts to tissue engineer artificial epithelium. A key feature of mature epithelium is the presence of an apical-basal polarization, which develops over 7-20 days in culture. Currently, the most widely used 2D system to generate polarized epithelium in vitro involves the filter insert culture system, however this system is expensive, laborious and requires large numbers of cells per sample. We have developed a set of micropatterning techniques to spatially control the organization of epithelial cells into microsheets on filter inserts under the culture conditions necessary to induce epithelial cell polarization. Micropatterning improves cell uniformity within each microsheet, allows multiple sheet analysis on one filter insert, and reduced cell number requirements. We describe an agarose patterning method that allows maintenance of cell patterns for over 15 days, the time necessary to induce apical-basal polarization. We also describe a Parafilm? patterning method that allows patterning for 5 to 15 days depending on cell type and only allows the generation of stripes and circular microsheets. The parafilm? method however is extremely straightforward and could be easily adopted by any laboratory without the need of access to specialized microfabrication equipment. We also demonstrate that micropatterning epithelial cells does not alter the localization of the apical-basal marker ZO-1 or the formation of cilia, a marker of epithelium maturation. Our methods provide a novel tool for studying epithelial biology in polarized epithelium microsheets of controlled size.     

Using a co-culture microsystem for cell migration under fluid shear stress

We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering.     

Formation and recovery of a cell sheet by a particle monolayer with the surface roughness

We studied the topographical effect of roughness displayed by a closely packed particle monolayer on formation of a cell monolayer (cell sheet). Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527nm (SA053) and 1270nm (SA127) in diameter. Human umbilical vein endothelial cells (HUVECs) were seeded at a high density (2.0 x10(5)cells/cm(2)) onto particle monolayers. It was found that cells gradually became into contact with adjacent cells on the SA053 monolayer and the formed cell sheet could be readily detached from the particle monolayer by gentle pipetting. On the other hand, cells adhering onto the tissue culture polystyrene (TCPS) and the SA127 particle monolayer were difficult to peel off. At a low cell seeding density (5.0x10(4)cells/cm(2)), pre-coating with bovine plasma fibronectin (FN) allowed cell growth on an SA053 particle monolayer, and a confluent monolayer was able to be peeled as a cell sheet from the particle monolayer just by pipetting. By immunostaining of human fibronectin, we found that fibronectin was secreted and concentrated onto the substrate side of a cell sheet. The obtained cell sheet adhered and grew on the TCPS again within 20min.     

Degradation behavior and application of recycled PVC sheet made of floor sheet for railway vehicle

A floor sheet made of plasticized poly(vinyl chloride) (PVC) is used as interior material for railway vehicle. Some of the floor sheets mainly used in Express and Shinkansen vehicles were layered by vulcanized surface to protect against the fire. Therefore, these floor sheets have been considered as inappropriate material for recycling. In this paper, some varieties of recycled sheets made of these floor sheets have been studied with respect to a long-term stability which is necessary for practical application. Through the weathering test, these recycled sheets indicated suitable mechanical properties for long-term outdoor use; however, at the beginning of weathering test, the elongation was reduced by the rapid flow out of plasticizer. The mechanical strength was closely related to the plasticizer concentration. Based on the plasticizer concentration, the tensile strength could be estimated without dependence of specimen preparations and the slope of the elongation was related to the content of the floor sheet. To prevent flowing out of plasticizer, virgin PVC was laminated to the surface of recycled sheet. The laminated sheet was set up at the outdoor to be used as an anti-weed sheet. For more than five years, the laminated sheet has kept on preventing overgrowth of weed and flow out speed of plasticizer has decreased by the effect of virgin PVC lamination. It is presumable that the laminated sheet was estimated to be used as the anti-weed sheet for more years.     

A Facile Steam Reforming Strategy to Delaminate Layered Carbon Nitride Semiconductors for Photoredox Catalysis

The delamination of layered crystals that produces single or few‐layered nanosheets while enabling exotic physical and chemical properties, particularly for semiconductor functions in optoelectronic applications, remains a challenge. Here, we report a facile and green approach to prepare few‐layered polymeric carbon nitride (PCN) semiconductors by a one‐step carbon/nitrogen steam reforming reaction. Bulky PCN, obtained from typical precursors including urea, melamine, dicyandiamide, and thiourea, are exfoliated into few‐layered nanosheets, while engineering its surface carbon vacancies. The unique sheet structures with strengthened surface properties endow PCNs with more active sites, and an increased charge separation efficiency with a prolonged charge lifetime, drastically promoting their photoredox performance. After an assay of a H₂ evolution reaction, an apparent quantum yield of 11.3 % at 405 nm was recorded for the PCN nanosheets, which is much higher than those of PCN nanosheets. This delamination method is expandable to other carbon‐based 2D materials for advanced applications.     

基于聚N-异丙基丙烯酰胺的细胞智能分离材料

聚 N -异丙基丙烯酰胺(PNIPAm)在水中是具有温度响应性的智能高分子材料,可用于细胞培养和自动脱附。本文从材料的制备方法出发,介绍了电子束照射接枝、等离子体处理接枝、表面活性自由基聚合、水凝胶等方法制备的材料对细胞培养及脱附的影响;阐述了细胞的脱附机理;讨论了加快细胞脱附的方法,包括共聚改性PNIPAm、PNIPAm接枝多孔膜、聚乙二醇(PEG)共聚PNIPAm接枝多孔膜、聚偏氟乙烯(PVDF)膜辅助细胞转移。从PNIPAm温敏性材料表面智能分离得到的细胞片因结构完整并保留了细胞外基质成分,在组织修复中得到了应用。     

Characterization of hydrogen production by the co-culture of dark-fermentative and photosynthetic bacteria

Apprehension over exhaustion of fossil fuels and global warming, due to increasing amounts of CO₂, has generated a lot of attention for the subject of renewable energy. Renewable energy has an intermittency problem and its output fluctuates depending on natural conditions. Biohydrogen is one of the promising renewable energy sources. Hydrogen produced by photosynthetic bacteria depends on the intensity of light irradiation and also fluctuates with the daily variation of sunlight. The co-culture system of dark-fermentative and photosynthetic bacteria is one solution for reducing the dependency of hydrogen production on light intensity. Because these two strains of bacteria have different processes of hydrogen production, it is possible to combine different outputs so far as the co-culture system works well. This study performed hydrogen production by the co-culture system composed of agar gels embedded with both dark-fermentative bacteria, Clostridium butyricum MIYAIRI, and photosynthetic bacteria, Rhodobacter sphaeroides RV, under a fluctuating light-irradiation. The time-course of hydrogen production was determined for the different conditions of co-culture in the mixing ratios of the two bacterial strains and light-irradiation patterns. As a result, the co-culture system succeeded in producing hydrogen exceeding that in the case of a single culture system and improved its stability against light fluctuation. Hydrogen production by the co-culture system would be applicable to the reduction of intermittency in renewable energies.     

Layered MoS2@graphene functionalized with nitrogen-doped graphene quantum dots as an enhanced electrochemical hydrogen evolution catalyst

Layered MoS₂@graphene functionalized with nitrogen-doped graphene quantum dots (MoS₂@NGQDs-GR) was obtained by one-pot hydrothermal method, as an enhanced electrochemical hydrogen evolution catalyst.     

纳米细菌纤维素膜的表征与生物相容性研究

利用木醋杆菌静态培养法制备的由纳米纤维组成的细菌纤维素膜具有超细的三维网络结构和适当的孔隙率. 利用光镜、扫描电镜和原子力显微镜对其进行结构表征发现, 细菌纤维素膜具有极为精细的纳米网络结构, 冻干膜的孔径约为0.6~2.8 μm; 纤维素带宽度约为50~80 nm. 采用湿重与浮重结合法测定烘干膜和冻干膜的孔隙率分别约为70%和90%. 由于细菌纤维素含有大量的羟基, 故烘干膜表现出极好的透湿性. 将细菌纤维素膜分别与成纤维细胞和软骨细胞进行复合培养, 并将成纤维细胞和细菌纤维素膜的复合物进行裸鼠皮下移植实验. 结果显示, 移植的复合物很好地融入了裸鼠正常皮肤, 成纤维细胞和软骨细胞在细菌纤维素表面形成连续的细胞层, 绿色荧光蛋白表达正常. 以上结果表明, 细菌纤维素膜非常适合细胞贴附和增殖, 表现出较好的生物相容性, 有望成为新型组织工程支架材料.     

A Robust Method to Generate Mechanically Anisotropic Vascular Smooth Muscle Cell Sheets for Vascular Tissue Engineering

In arterial tissue engineering, mimicking native structure and mechanical properties is essential because compliance mismatch can lead to graft failure and further disease. With bottom‐up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve the necessary macroscale properties in the final implant. This study develops a thermoresponsive cell culture platform for growing aligned vascular smooth muscle cell (VSMC) sheets by photografting N‐isopropylacrylamide (NIPAAm) onto micropatterned poly(dimethysiloxane) (PDMS). The grafting process is experimentally and computationally optimized to produce PNIPAAm–PDMS substrates optimal for VSMC attachment. To allow long‐term VSMC sheet culture and increase the rate of VSMC sheet formation, PNIPAAm–PDMS surfaces were further modified with 3‐aminopropyltriethoxysilane yielding a robust, thermoresponsive cell culture platform for culturing VSMC sheets. VSMC cell sheets cultured on patterned thermoresponsive substrates exhibit cellular and collagen alignment in the direction of the micropattern. Mechanical characterization of patterned, single‐layer VSMC sheets reveals increased stiffness in the aligned direction compared to the perpendicular direction whereas nonpatterned cell sheets exhibit no directional dependence. Structural and mechanical anisotropy of aligned, single‐layer VSMC sheets makes this platform an attractive microstructural building block for engineering a vascular graft to match the in vivo mechanical properties of native arterial tissue.

     

Imitation of drug metabolism in cell co-culture microcapsule model using a microfluidic chip platform coupled to mass spectrometry

In this work,a multi-functional analysis platform by coupling a microfluidic chip to a mass spectrometry(MS) detector was described.We constructed a three-dimensional tumor-endothelial co-culture model for simulating drug resistance during tumor treatment.On this specially designed integrated platform,the first step was to prepare heterogeneous cell-encapsulated alginate microcapsules for threedimensional co-culture,and the second step was to achieve on-line perfusion culture and continuous drug stimulation on chip.It facilitates cell proliferation analysis and the collection of metabolism medium.After micro solid phase extraction column(SPE) pretreatment,subsequent mass spectrometry could detect drug metabolism.The high activity of two kinds of cells(A549 and HUVEC) shows the biocompatibility of the platform.Paclitaxel was used as a model drug,the distinctions of drug absorption between the mono-culture group and co-culture group were clearly observed by electrospray ionization quadrupole time-of-flight mass spectrometry(ESI-Q-TOF MS).Therefore,the integrated platform has shown promise as a high throughput,low cost for cell metabolism research and drug screening processes.