Computer-controlled piezoactuator for cell manipulation

The development of a computer-controlled piezomanipulation system is presented for biomedical applications involving cells manipulation. An application of the developed system to intracytoplasmic sperm injection (ICSI) is presented. The hardware setup and the control strategies are described in full detail to illustrate the advantages of this approach compared to manual injection methods. The system developed offers the automated and efficient execution of the ICSI process, thus achieving an improved success rate in terms of the end objectives of the process.     

Immobilized MWCNT support osteogenic cell culture

The broad use of versatile, strong, lightweight multi-walled carbon nanotubes (MWCNT) for use in biomaterial applications is tempered by ongoing debate about their safety. Recent reports suggest that factors such as their diameter and surface coating affect their function and cytotoxicity. The cell culture surfaces used in the current study were made of MWCNT immobilized in a high-density polyethylene substrate, differentiating it from most studies of MWCNT cytotoxicity. The purity, chemical functionalization, and immobilization of MWCNT were evaluated to elucidate their effect on MWCNT behavior relative to controls. While purity was found not to be significant in determining the behavior of cells on MWCNT relative to standard controls, the presence of carboxyl functional groups was generally associated with reduced cell metabolic activity, proliferation, and differentiation as measured using the MTS assay, nucleic acid incorporation, and alkaline phosphatase expression, respectively. This study demonstrates that the culture of osteogenic cells on surfaces made of nonfunctionalized and immobilized MWCNT is associated with a level of cell growth and differentiation comparable to those of standard tissue culture controls.     

Human vascular endothelial cells in primary cell culture for the evaluation of nanoparticle bioadhesion

Nanoparticles (NP) are employed in various therapeutic approaches for innovative drug delivery strategies. Among them, there is drug delivery to the brain and sustained release forms for intravenous drug delivery. In order to optimize drug carriers and to elucidate involved mechanisms such as bioadhesion and cellular uptake, NP were surface modified and analyzed for their interaction with human endothelial cells in cell culture. Fluorescently labeled NP of different diameters (50 to 1000 nm) were surface modified either by simple adsorption of chitosan or by covalent binding to the lectin ulex europaeus agglutinin and thereafter applied to human endothelial cells for different incubation periods. After incubation with NP the binding of NP was quantified directly by the fluorescence emission signals from the cell layers. In order to visualize the binding behaviour, NP were localized three-dimensionally in the cell layer by confocal laser scanning microscopy. Cell binding experiments in phosphate buffer were observed to be particle size dependent with the 50 nm NP showing the highest binding percentage over all experiments. Binding decreased with increasing particle diameter and shorter incubation interval. The adhesion was further enhanced by NP surface modifications in the order blank < chitosan < lectin. The presence of plasma proteins enhanced the adhesiveness of chitosan coated NP, while the binding of lectin coated NP was inhibited. Experiments at 4 degrees C indicated the involvement of an active process in the binding of NP to endothelial cells.     

Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic products. The agar-overlay test is a short term semi-solid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell free zones. The aim of this study was to develop a more sensitive cytotoxicity test system for biomaterials, using methylcellulose as a culture gel, mixed with human fibroblasts. The main advantage of the test system is the possibility of evaluating cytotoxicity for a period of up to seven days without renewal of the culture gel. Furthermore it is possible to both quantitatively evaluate by counting absolute cell numbers and to qualitatively evaluate by studying cell morphology with light- and/or electron microscopy. Processed dermal sheep collagen was selected as test material, since contradictory results concerning the cytotoxicity of its extracts have been reported by others [2, 15, 18, 19]. Using our test system, both primary and secondary cytotoxic effects were found. Primary cytotoxicity is due to direct leakage of products from the material, detected by testing, extracts of the collagen or the collagen itself. Secondary cytotoxicity is due to release of cytotoxic products resulting from cell-biomaterial interactions. We conclude that our test system is extremely useful to test materials which are suspected of primary and/or secondary cytotoxicity, either with slow release of cytotoxic products or release of products with late cytotoxic effects.     

Ceramic support for cell cultures

Research was carried out on the use of ceramics as supports to host mammalian cells. The research was part of a programme whose priority was to study the possibility of using ceramics in the non-traditional sector of the biomedical field. The aim of the study was also to verify the suitability of particular types of ceramics dealt with in the literature for these applications. Among the different samples tested the cordieritic one proved to be very interesting, at least in relation to the cellular cultures considered. The chemical composition of the material is not, however, the only important aspect, since other parameters concur to make the hosting of cells highly acceptable. Of particular importance is the rugosity and porosity of the surface and its flaking, not only externally, but also in the pores.This paper was originally accpeted after the 1993 Conference of the European Society for Biomaterials.     

A semi-automated cell culture evaluation system for cytotoxicity testing of dental materials

A rapid and quantitative determination of viable cells in the cytotoxicity testing of dental materials is desirable to evaluate large numbers of samples in a short time. For this purpose a new and semi-automated cell culture evaluation system was developed using the fluorogenic dye fluorescein diacetate (FDA) for vital staining and microtiter plates for culture vessels. Our methodological experiments showed that in this system the fluorescence intensity was linearily related to the cell number from 500 to 20 000 cells per culture^–1 and that fluorescence recording was stable for between at least 1 to 2 h using a quencher solution for absorbing extracellular fluorescence. The results from toxicity testing of different dental materials (two glass ionomer cements, a phosphate cement, a composite resin and monomeric methylmethacrylate) corresponded to those derived from other, standard test methods. Because of the ease of performance, the quantitative, rapid evaluation system and the small culture vessels requiring only few cells per culture, the test method presented may be an interesting alternative to other cell culture techniques for cytotoxicity testing.     

Simple multifrequency and phase-shifting fringe-projection system based on two-wavelength lateral shearing interferometry for three-dimensional profilometry

We propose a simple multifrequency spatial-carrier and phase-shifting fringe-projection system based on two-wavelength lateral shearing interferometry (LSI). In this system a wedge-shaped plate lateral shearing interferometer is used and, owing to the presence of tilt, a finite number of fringes parallel to the direction of the shear appears; hence a significant spatial-carrier frequency is generated at the focus position. We further enhance the spatial-carrier frequency either by changing the wavelength of the laser light or by slight defocusing. A synthetic interferogram with low spatial-carrier frequency is obtained by use of laser light of two wavelengths simultaneously in the lateral shear interferometer. We obtain the phase-shifted fringe patterns from the same setup by simply moving the wedge plate in an in-plane parallel direction, using a linear translator. The fringe projection system was tested for measurement of the three-dimensional shape of a discontinuous object. The present system has many advantages; e.g., it is a common-path interferometry and hence is insensitive to external vibrations, is compact in size, and is relatively inexpensive.     

Computer-controlled system for surface resistance measurements ofHTc superconducting films

A measurement system capable of determining the microwave surface resistance of high-temperature superconducting films is described. The measurement is based on evaluating the resonant curve of a circular cylindrical waveguide transmission resonator. The cylindrical wall, and the top plane of the resonator are made of copper. The test sample is the ground plane of the resonator. The surface resistance of the test sample can be directly evaluated from the quality factor of the resonator when the surface resistance of copper is used as a reference. The computer-controlled measurement system consists of a backward-wave oscillator, the cylindrical resonator, a spectrum analyzer for the detection of power and frequency, and a refrigerator for cooling the resonator and the test sample. Several measurements have been made. Surface resistances of different thin-film samples have been determined. The lowest values measured so far approximate 25 mΩ at 66.8 GHz and 77 K     

Fibrillized peptide microgels for cell encapsulation and 3D cell culture

One of the advantages of materials produced by self-assembly is that in principle they can be formed in any given container to produce materials of predetermined shapes and sizes. Here, we developed a method for triggering peptide self-assembly within the aqueous phase of water-in-oil emulsions to produce spherical microgels composed of fibrillized peptides. Size control over the microgels was achieved by specification of blade type, speed, and additional shear steps in the emulsion process. Microgels constructed in this way could then be embedded within other self-assembled peptide matrices by mixing pre-formed microgels with un-assembled peptides and inducing gelation of the entire composite, offering a route towards multi-peptide materials with micron-scale domains of different peptide formulations. The gels themselves were cytocompatible, as was the microgel fabrication procedure, enabling the encapsulation of NIH 3T3 fibroblasts and C3H10T-1/2 mouse pluripotent stem cells with good viability.     

Impedance endothelial cell biosensor for lipopolysaccharide detection

It is important to analyse endothelial cell adherence for the development of biomedical devices of antithrombogenic vascular grafts. Endothelial cells must be firmly attached to the biomaterials when cells are seeded in order to create a natural lining.

Polystyrene (PS) is presented as a reproducible implant model substrate for studying cell–material interactions. Polystyrene was deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting the endothelial cell adhesion was then adsorbed on PS surface. The different steps of this multilayer assembly were characterized by Electrochemical Impedance Spectroscopy (EIS). The charge transfer resistance and the capacitance of the total layer were modified at each step in agreement with the electrical properties of each layer. The electrical properties of the confluent layer of endothelial cells were determined: (i) a charge transfer resistance of 2 kΩ cm^− 2 shows no large defects in the cell layer, (ii) as the cells attach and spread on the gold electrode, the impedance increases.

EIS was used for testing behaviour of endothelial cells on substrate coated by fibronectin layer and in presence of cytotoxicants such as lipopolysaccharide (LPS). The impedance measurement may be a valuable method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. The results indicate that LPS causes a dose-dependent decrease in impedance of the endothelial cell monolayer, indicating widening of the paracellular pathways and increasing vascular endothelial permeability. This study is an increasing trend towards the development of impedimetric biosensors and designing cell sensor arrays for toxic and drug detection.     

细胞培养用温度敏感凝胶的合成与性能研究

用丙烯酸（ARc）对壳聚糖（CS）进行化学改性，合成反应中问体壳聚糖衍生物CS-ARc，进一步合成不同配比的CS-ARc与N-异丙基丙烯酰胺（NIPA）的共聚凝胶P（CS-AAc-NIPA），通过红外光谱和元素分析等表征了产物的结构和组成，并研究了P（CS—ARc-NIPA）凝胶在水中和细胞培养基中的溶胀性能．结果表明共聚凝胶在水中和培养基中均显示较好的温度敏感性．对P（CS-ARc-NIPA）共聚凝胶进行细胞培养研究发现，其表面可成功种植成纤维细胞（L929），细胞贴附生长情况良好，表明材料具有很好的细胞相容性．当环境温度降低后，共聚凝胶发生疏水．亲水变化，导致其表面细胞自动脱附，从而避免了使用酶解法脱附细胞造成的细胞功能损伤．’     

Microstructuring ceramic scaffolds for hepatocyte cell culture

Both extracorporeal liver support devices and tissue engineering of liver for transplantation require the maintenance of functionality of liver cells (hepatocytes) in cell culture for a long time. One approach to achieve this is to optimize hepatocyte in vitro environment by using a scaffold with topographic structure at sub-millimeter scale which controls cell distribution. Therefore, a set of new type of titania ceramic scaffolds, containing cavities of several sizes, has been produced for deducing the best choice of cavity dimensions for culturing hepatocytes. The aim of this paper is to describe in detail the production methods and characterization of such ceramic scaffolds. Experimental production of the scaffolds consists of microfabrication of silicon templates as well as preparation and molding of titania ceramics. The templates, containing arrays of conical protrusions arranged in close-packed hexagonal order, have been achieved using microfabrication methods of photolithography and anisotropic etching in KOH at 50 °C. Protrusion dimensions and overall quality of the templates has been evaluated by scanning electron microscopy. The microfabricated templates have resulted in well-defined and reproducible cavities of corresponding dimensions on the titania ceramic surface after injection-molding. Alternatively, simple embossing of the plastified green ceramics with the silicon templates attached to a metal plate also creates cavities on the ceramic surface. While both methods yield good results, they have different advantages: the injection-molding provides a higher quality of imprints while embossing is quicker and less complicated, and is not limited by dimensions of specific molding equipment. © 2001 Kluwer Academic Publishers     

新型辐射制冷器用纤维支撑系统的设计和分析

设计了1种新型辐射制冷器级间纤维支撑系统,介绍了其结构的设计,支撑材料的选择以及纤维接头的研制。利用MSC．Nastran有限元软件对系统进行准静态结构强度和纤维预紧力进行了分析。级间漏热的理论分析结果表明此纤维支撑系统为研制更大冷量的辐射制冷器提供了可能。     

Development of a mini 3D cell culture system using well defined nickel grids for the investigation of cell scaffold interactions

A bioreactor system was developed using a series of fine mesh nickel grids as free standing scaffolds to investigate the behaviours of fibroblasts and keratinocytes in tissue culture. It was found that the mesh size of the suspended grids, but not of the grids that attached to tissue culture surface, had significant influences on cell behaviour and there was a maximum size for fibroblast to span within the defined culture period. Time lapse video microscopy demonstrated fibroblasts cultured on these grids initially migrated onto the struts but then worked together to fill in the voids between struts with a membranous sheet of tissue. In contrast keratinocytes barely migrated from the initial site of cell deposition and when they moved (to a modest extent) it was as an integrated sheet of cells. Similar results were observed when both types of cells were co-cultured in the system.     

Optimized allylamine deposition for improved pluripotential cell culture

Deposition of allylamine (ALL) by plasma enhanced chemical vapor deposition has been optimized on silicon based models. Simultaneous energy recoil detection analysis and Rutherford backscattering spectra show that 100 W deposition is ideal in terms of polymerized film formation and H content while, lower or higher power induce reduced film retention or excessive cross linking, respectively. Surface composition of the ALL film was further probed by X-ray photoelectron spectroscopy revealing a monocomponent N 1s spectrum compatible with the presence of primary amines. Optimized ALL films were applied to polycaprolactone (PCL) surfaces after Ar plasma activation with implications in the chemistry and wettability of this biocompatible polymer. Human mesenchymal stem cells (hMSCs) were cultured on ALL coated PCL surface and controls. ALL functionalized PCL was found to be especially attractive for the formation of confluent monolayers of hMSCs after 72 h of culture.     

A multi-agent system for chemical supply chain simulation and management support

Modern chemical production is customer-driven and the desired delivery time for the products is often shorter than their campaign length. In addition, the raw materials supplying time is often long. These features make it desirable to provide tools to support collaborative supply chain decision making, preferably over the Internet, and where there are conflicts, compromise decisions can be quickly reached and the effects of the decisions can be quantitatively simulated. This paper des cribes such a multi-agent system (MAS) that can be used to simulate the dynamic behaviour and support the management of chemical supply chains over the Internet. Geographically distributed retailers, logistics, warehouses, plants and raw material suppliers are modelled as an open and re-configurable network of co-operative agents, each performing one or more supply chain functions. Communication between agents is made through the common agent communication language KQML (knowledge query message language). A t the simulation layer, the MAS allows distributed simulation of the chain behaviour dynamically, so that compromise decisions can be rapidly and quantitatively evaluated. Because in a chemical supply chain the scheduling of the plant often dominates the chain performance, an optimum scheduling system for batch plants is integrated into the MAS. The functions of the system are illustrated by reference to a case study for the supply and manufacture using a multi-purpose batch plant of paints and coatings.     

A thixotropic nanocomposite gel for three-dimensional cell culture

Thixotropic materials, which become less viscous under stress and return to their original state when stress is removed, have been used to deliver gel-cell constructs and therapeutic agents. Here we show that a polymer-silica nanocomposite thixotropic gel can be used as a three-dimensional cell culture material. The gel liquefies when vortexed--allowing cells and biological components to be added--and resolidifies to trap the components when the shear force from spinning is removed. Good permeability of nutrients and gases through the gel allows various cell types to proliferate and be viable for up to three weeks. Human mesenchymal stem cells cultured in stiffer gels developed bone-like behaviour, showing that the rheological properties of the gel can control cell differentiation. No enzymatic, chemical, or photo-crosslinking, changes in ionic strength or temperature are required to form or liquefy the gel, offering a way to sub-culture cells without using trypsin-a protease commonly used in traditional cell culture techniques.