Cyclopore Membrane Research Articles

Estimation of effective thickness of Cyclopore polycarbonate membrane by scanning electrochemical impedance microscopy

• O impedance related to radial diffusion and restricted planar diffusion; for tip closely to hole surface-Cyclopore membrane. • Equivalent circuits including Cole-Cole diffusion impedance (Z M ), Cole-Davidson type impedance (Z W ) and O impedance (Z O ) proposed for different separation distances. • Difference in diffusion impedance on hole and that on flat surface on Cyclopore membrane for estimating membrane thickness. A new, feasible, on-line method was proposed to determine Cyclopore polycarbonate membrane thickness by scanning electrochemical impedance microscopy (SEIM) technique. The thickness is 8.4 µm, which close to the report technical data. Three effective impedances for equivalent circuit model including Cole-Cole diffusion impedance, Cole-Davidson type impedance and O impedance were firstly proposed for radial diffusion and restricted planar diffusion mode when tip electrode on flat or hole surface in Cyclopore membrane. Data analysis results showed that restricted planar diffusion emerged when tip electrode approach tightly to hole surface. Additionally, SEIM responses on the flat and hole surface were compared and could be applied for topographic imaging in the future work.

Endothelial cell activation of the smooth muscle cell phosphoinositide 3-kinase/Akt pathway promotes differentiation

Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, atherosclerosis, and restenosis. We aimed to determine the intracellular signaling mechanisms by which ECs promote a differentiated SMC phenotype. Bovine thoracic aorta ECs and SMCs were isolated and cultured. For co-culture studies, ECs were grown to confluence on one side of a semi-permeable Cyclopore membrane. SMCs were then plated on the opposite side of the membrane and cultured for 24 to 48 hours. For adenovirus experiments, SMCs were infected prior to plating opposite ECs. For conditioned media studies, SMCs cultured alone on plastic were treated with media harvested from EC/SMC in co-culture. SMC phenotype was assayed by microscopy and measurement of two-dimensional area, or by western blotting for contractile protein markers of differentiation. Akt activation was measured by western blotting for phospho-Serine 473. Although SMCs cultured alone exhibit a dedifferentiated synthetic phenotype, we report that bilayer co-culture with ECs induced a differentiated SMC phenotype as measured by morphology and cell area and expression of protein markers of differentiation, including contractile proteins and the cyclin-dependent kinase inhibitor p27 kip . The EC/SMC bilayer co-culture resulted in activation of the SMC protein kinase Akt, with no effect on total Akt expression. Similarly, conditioned media from co-cultured EC/SMC promoted rapid Akt phosphorylation and subsequent expression of differentiation protein markers in SMCs cultured alone. Adenoviral overexpression of constitutively active Akt in SMCs cultured alone mimicked the ability of ECs to induce SMC differentiation. Notably, inhibition of phosphoinositide 3 (PI 3)-kinase activity with wortmannin or adenoviral overexpression of a dominant-negative Akt prevented the EC-mediated effect on SMC morphology and differentiation protein marker expression. ECs direct SMCs towards a differentiated phenotype through activation of the SMC PI 3-kinase/Akt pathway. Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, collateral blood vessel development, atherosclerosis, and restenosis. Alterations in SMC phenotype occur in each of these processes. Endothelial denudation has been suggested to contribute to the SMC proliferative response to vessel injury by angioplasty or other catheterization procedures. We have employed a co-culture approach to dissect the molecular signals that are dependent on the spatial relationship between ECs and SMCs, and have identified the importance of the PI3K/Akt pathway in EC-induced SMC differentiation. This pathway may suggest targets for therapeutic interventions for intimal hyperplasia and restenosis.

Atomic force microscope study of the rejection of colloids by membrane pores

An atomic force microscope (AFM) in conjunction with the colloid probe technique has been used to study the electrical double layer interactions between a 0.75 μm silica sphere and a polymeric microfiltration track etch Cyclopore membrane (nominally 1 μm) in aqueous solutions. The silica colloid probe was used to image the membrane surface (using the double layer mode) at different imaging forces in high purity water and at constant imaging force in sodium chloride solutions of different ionic strengths at pH 8. Force-distance measurements show clearly how the sphere detects the membrane surface. Quality of images produced from scanning the 0.75 μm silica particle across the surface deteriorates with increasing distance between the silica sphere and membrane surface. Such images were compared with those obtained from scanning a sharp silicon nitride tip over the membrane surface.

Formation of multilayers in the caco-2 cell culture model: a confocal laser scanning microscopy study.

To introduce confocal laser scanning microscopy (CLSM) combined with digital image restoration to characterise Caco-2 cells under different culture conditions, and thus to define additional valid criteria for the optimisation of culture models. Growth curves were established and transepithelial electrical resistance (TEER) measured for cells grown in EMEM or DMEM medium on Cyclopore membranes. Cytoskeleton, cell nuclei and tight junctions (TJ) were investigated by CLSM. Cultures reached a plateau of approximately 4.5 x 10(5) cells/cm2 after approximately 10 days. At the same time TEER reached 750 omega cm2. An irregular, fairly complete network of TJ was present at confluence (approximately 2 d). Between 15 and 30 days a regular TJ network was established. Cells formed mixed mono- and multilayers under most conditions with two exceptions: flat monolayers were observed on polycarbonate filters with EMEM and with the Biocoat intestinal epithelium differentiation environment system. In multilayers TJ were found in the upper as well as in the lower cell layers although the regular vertical polarity was disturbed. CLSM represents an important tool to investigate the cytoarchitecture of Caco-2 cells. 3D-analysis of confocal data gives important clues on the characteristics of cell layers and thus helps to validate optimisation strategies.

Co-stimulation of human decidual natural killer cells by interleukin-2 and stromal cells.

At the late secretory phase of the menstrual cycle and in early pregnancy, the uterine mucosa is infiltrated by large numbers of natural killer (NK) cells with a distinctive phenotype (CD56bright CD16- CD3-) and large granular lymphocyte (LGL) morphology. Circulating CD56bright NK cells generally proliferate in the presence of interleukin-2 (IL-2), but it is clear that cofactors besides IL-2 are required for optimal response. In the bone marrow, this co-stimulating signal is provided by stromal cells. In the present study we observe that uterine CD56+ cells from early pregnancy decidua similarly proliferate vigorously when cultured with decidual stromal cells and a suboptimal dose of IL-2. This response is dependent on cell-cell contact, as no proliferation of decidual NK cells was observed when they were separated from stromal cells by a permeable cyclopore membrane. In addition, we have studied the expression of Bcl-2 by decidual CD56+ cells. Our results show that the microenvironment of the uterus is likely to have a significant influence on the proliferation and survival of uterine CD56+ cells.

Charge Adsorption and Zeta Potential in Cyclopore Membranes

The surface electrochemistry of several microporous membranes is studied. Concretely, we deal here with six types of track-etched microporous filters from Cyclopore whose surface charges are obtained, within the framework of the so-called microcapillary pore model, from typical streaming potential experiments when bathed in diluted LiCl solutions. The dependence of the surface charge densities and the zeta potentials on the bulk concentration is studied by assuming that an adsorption process of anions is the cause of these charges. The adsorption isotherm data are treated according to two adsorption mechanisms, a heterogeneous Freundlich adsorption followed by a homogeneous Langmuir adsorption for higher bulk concentrations. Both stages allow us to obtain important parameters concerning the membrane material: the molar free energy of specific adsorption, the maximum number of sites accessible to anions, and the actual surface charge density at the solid surface (or proper surface charge density). All these parameters are quite similar for all the membranes analyzed, showing significative differences depending on the adsorption step; in particular, fewer adsorption sites and higher adsorption free energies appear for high concentrations while the proper charge is positive but low and acts only for very low bulk concentrations. This two-step adsorption model gives a very approximate prediction of actual zeta potentials as well.

Atomic force microscope studies of membranes: Surface pore structures of Cyclopore and Anopore membranes

Non-contact atomic force microscopy has been used to investigate the surface pore structure of Cyclopore and Anopore microfiltration membranes in air. Three Cyclopore membranes and three Anopore membranes of different pore sizes were studied. Excellent high resolution images were obtained. Analysis of the images gave quantitative information on the surface pore structure, in particular the pore size distribution. Non-contact AFM is an excellent means of obtaining such information for microfiltration membranes.

Establishment of a human pro-B cell line (JKB-1) and its differentiation of preestablished bone marrow stromal cell layer.

A human pro-B cell line, named JKB-1, was established from the bone marrow of a 16-year-old girl with acute lymphoblastic leukemia (ALL) in relapse. The origin of the JKB-1 cell line was indicated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells. This cell line has been growing for more than 14 months in suspension culture medium and had a doubling time of about 24 hours. JKB-1 expressed terminal deoxynucleotide transferase (TdT) and early antigens (HLA-DR, CD19, CD24) of B cells, with heavy chain gene rearrangement. However, it did not express late antigens (CD10, CD20, CD21, CD22, CD23) of B cells, light chain gene rearrangement, and cytoplasmic mu-chain. These results suggested that JKB-1 is at the stage of "pro-B" cell or early B-cell precursors. This cell line was induced to differentiate after 7 days of co-incubation with irradiated bone marrow stromal cells because of the expression of pre-B cell antigens (CD10, CD20), cytoplasmic mu-chain, light chain gene rearrangement, and disappearance of TdT, JKB-1 cells adhered to a preestablished bone marrow stromal cell layer with string-like processes under scanning electron microscope. When JKB-1 cells were separated from the stromal layer by a cyclopore membrane with 0.45 micron pore size, they did not differentiate. Bone marrow stromal cell conditioned medium could not induce differentiation either. Thus it was suspected that direct contact between JKB-1 cells and stromal cells was required for differentiation. In methylcellulose semisolid medium, the colony size and number of JKB-1 cells were increased by stem cell factor (SCF), or interleukin (IL)-3, or IL-7, but they were decreased by IL-6. Moreover, SCF synergized with IL-3 or IL-7 to stimulate the proliferation of JKB-1 cells. Because there are very few reproducible models for examining early stages of human B-cell differentiation, the JKB-1 cell line would be useful for studying the relationship between human B-cell differentiation and bone marrow microenvironment, as well as leukemogenesis.

Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.

The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes.

An assay for the analysis of lymphocyte migration across cerebral endothelium in vitro

We describe a recently developed assay for the analysis of leukocyte migration across cerebral endothelium in vitro. The endothelium is grown as monolayers on Goretex or Cyclopore membranes coated with extracellular matrix proteins and supported on inserts. This system permits the recovery and phenotyping of cells which migrate down through the endothelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migration assay. We have compared the results from this system with those from conventional adhesion assays. Binding of cells to the endothelium is rapid, but is confined to a particular subpopulation of the applied lymphocytes. We have followed cell migration over 24 h in the system using normal and cytokine-activated endothelium and have found that whereas adhesion depends both on the state of lymphocyte activation and on the condition of the endothelium, the level of migration of stimulated lymphocytes is largely independent of endothelial activation. Moreover, whereas CD8 + cells bind well to the endothelium, it is the CD4 + cells which migrate most effectively. Comparison of brain and epididymal fat endothelium showed similar migration levels over 2 h, but migration was greater across epididymal fat endothelium at 24 h.

Mechanisms governing permeate flux and protein rejection in the microfiltration of beer with a Cyclopore membrane

Mechanisms which determine permeate flux and protein rejection for the microfiltration of a clarified beer using a Cyclopore membrane have been investigated. Experimental results were analysed in terms of existing membrane filtration model equations and membrane resistance. Flux decay follows the standard blocking model (SBM) assuming that pore volume decreases proportionally to filtrate volume. The actual mechanisms involved in membrane fouling are thought to be related both to the internal and external fouling by macrosolutes. Protein-membrane interactions in the form of protein adsorption produce a protein-membrane “composite”. The build-up of this also called “a second dynamic membrane” explains the characteristic rejection profiles.

Neutrophil migration through in vitro interstitial matrix

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.