Microsupport with Two-Dimensional Geometry (2D-MS).

CHO-K1 and VERO cells have been grown on MicroHextrade mark, a polystyrene-based microsupport with two-dimensional geometry and the consistency of their growth kinetics were established. These cells have been detached by exposure to buffered ethylenediaminetetraacetic acid (EDTA) followed by controlled mechanical shear to yield well-isolated cells suspended in EDTA. Neutralisation of the EDTA followed by restoration of the chemical composition of the growth medium causes detached CHO-K1 cells to display unaltered growth kinetics.

Microsupport with two-dimensional geometry (2D-MS) 4. Temperature-induced detachment of anchorage-dependent CHO-K1 cells from cryoresponsive MicroHex((R)) (CryoHex).

Tissue culture flasks were activated by electron beam irradiation and subsequently treated with N-isopropylacrylamide to make them cryoresponsive. Leaving such 'cryoflasks' unattended for 10 minutes at room temperature sufficed to almost completely detach the anchorage-dependent cells. MicroHex((R)), a polystyrene-based tissue culture microsupport with two-dimensional geometry, was handled in the same way to obtain CryoHex, i.e. a cryoresponsive MicroHex from which anchorage-dependent cells could be detached by exposure to low temperature (4-20 degrees C). Experimental conditions were determined allowing one to detach the cells from small and large microsupport culture volumes. Cells detached from CryoHex by exposure to low temperature displayed a high cell viability and, upon subcultivation on MicroHex((R)), did not show any alteration of their growth kinetics.

Microsupport with two-dimensional geometry (2D-MS). In situ determination of the growth kinetics of anchorage-dependent cells by Laser Diffraction Particle Sizing (LDPS).

The procedure used to establish in situ (without cell trypsinization) the growth kinetics characteristics of anchorage-dependent cells propagated on microcarriers by Aperture Impedance Pulse Spectroscopy can be replaced by a novel method based on the time-dependent shifts of the size distribution histograms of cell-laden microcarriers. This we have named Laser Diffraction Particle Sizing.

Simple blood-spot sampling with nested polymerase chain reaction detection for epidemiology studies on Brugia malayi.

In the absence of a suitable Brugia malayi antigen detection assay, PCR remains one of the more sensitive alternatives to Giemsa-stained thick blood films for B. malayi detection. The need for refrigerated storage and transportation of blood has limited the use of PCR for large-scale epidemiology studies in remote endemic areas. Here we report simple finger-prick blood-spot collection, a one-tube DNA template extraction method and the development of a B. malayi-specific nested PCR assay. The assay was tested on 145 field samples and was positive for all 30 microscopy-positive samples and for an additional 13 samples which were microscopy-negative.

Detection of malaria in Malaysia by nested polymerase chain reaction amplification of dried blood spots on filter papers.

A modified nested polymerase chain reaction (PCR) method for detection of Plasmodium falciparum, P. vivax and P. malariae was combined with a simple blood collection and deoxyribonucleic acid (DNA) extraction method and evaluated in Malaysia. Finger-prick blood samples from 46 hospital patients and 120 individuals living in malaria endemic areas were spotted on filter papers and dried. The simple Chelex method was used to prepare DNA templates for the nested PCR assay. Higher malaria prevalence rates for both clinical (78.2%) and field samples (30.8%) were obtained with the nested PCR method than by microscopy (76.1% and 27.5%, respectively). Nested PCR was more sensitive than microscopy in detecting mixed P. falciparum and P. vivax infections, detected 5 more malaria samples than microscopy on the first round of microscopical examination, and detected malaria in 3 microscopically negative samples. Nested PCR failed to detect parasite DNA in 2 microscopically positive samples, an overall sensitivity of 97.4% compared to microscopy. The nested PCR method, when coupled with simple dried blood spot sampling, is a useful tool for collecting accurate malaria epidemiological data, particularly in remote regions of the world.

Fastin situ determination of the biomass of anchorage-dependent cells.

The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.

Dielectric spectroscopy of mammalian cells. 2. Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3.

Low-frequency dielectric spectroscopy has been used in situ, i.e. while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells. This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is characterized by a somewhat lower degree of resolution. Suggestions are made on how to determine the capacitance of the spent growth medium alone, still keeping the probe inserted in the bioreactor. This will make dielectric spectroscopy the first truly in situ, on-line, in real time, non-invasive measure of the biomass.

Dielectric spectroscopy of mammalian cells. 1. Evaluation of the biomass of HeLa- and CHO cells in suspension by low-frequency dielectric spectroscopy.

The capacitance of suspensions of CHO and HeLa cells (0.5-3 x 10(6) cells/ml) has been measured between 0.2 and 10 MHz. As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes. At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only. The intensity of this signal varies linearly with the biomass and cell size. At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5 x 10(6) cells/ml can be accurately measured. Suggestions are made how to make these measures on-line, non-invasive and in real time.

Microbeads and anchorage-dependent eukaryotic cells: the beginning of a new era in biotechnology.

Modern methods for the mass cultivation of anchorage-dependent mammalian cells started with the advent of microcarrier technology. Largely for reasons pertaining to their mode of preparation and ease of cultivation, 150-230 microns microbeads have been overwhelmingly adopted and the technology around them developed. To meet high biomass, macroporous microbeads have been developed. Also, the chemistry of the microsupport has been adapted in order to afford better protection of fragile cells to mechanical wear while simultaneously reorienting their differentiation towards the sought aims (production of cytokines, enzymes etc. ...). Future progress depends upon solutions being brought to problems inherent to this new technology (maintenance of steady state conditions of growth etc. ...) as well as to requirements arising from animal cell culture in general (biosensors, bioreactor's design etc. ...). Besides such technical implementations, biology at large is also expected to benefit from the advent of microcarriers in fields as diverse as the preparation of metaphasic chromosomes in bulk, toxicity testing, organ reconstitution following cell transplantation etc.

One-step purification of mouse monoclonal antibodies by mass ion exchange chromatography on Zetaprep.

A rapid one-step purification procedure was developed to isolate mouse monoclonal antibodies present in the growth medium of hybridoma cultures. The procedure, based on a sulfopropyl mass ion exchange chromatography, yields considerable amounts of purified monoclonal antibody. The immunoglobulins are essentially free of transferrin and albumin, as determined by SDS-polyacrylamide gel electrophoresis.

Growth of animal cells on microbeads. II. Estimation of biomass in situ and observation of cytopathic effects after infection of McCoy cells with Chlamydia trachomatis.

The Channelyzer C256 together with the ZB Coulter Counter have been used to study in situ the growth kinetics of McCoy cells attached on Cytodex 3 microbeads. The number of cells per microbead varied linearly with the average size of the covered microbeads, showing that electronic particle sizing can be used to measure biomass or cell numbers. As the cells became confluent, an increase in cell volume with little accompanying increase in cell number was detected. The technique also enables the experimenter to follow in situ the development of cytopathic effects resulting from the infection by pathogens such as Chlamydia trachomatis serotype L1.

Growth of animal cells on microbeads. I. In situ estimation of numbers.

Evaluation of the number of anchorage-dependent cells growing on the surface of microbeads kept in suspension in a bioreactor is a tedious procedure fraught with many difficulties. Using the Coulter counter as a biomass probe, this article shows that the number of cells adhering to microbeads can be determined while the cells are still attached.

Stimulation of protein accumulation in HeLa cells by inhibitors of DNA replication. Ferritin.

Incubation of HeLa cells for 24 h with either hydroxyurea (HU), aphidicolin (APHI), thymidine (T) or butyrate (BU), substances used to inhibit replication and accumulate cells at the G1/S interphase, followed by the elimination of the inhibitor and the addition of iron to the growth medium, results in an immediate (HU, APHI, T) or slightly delayed (BU) increased accumulation (18-24-fold higher than the basal level) of ferritin. Under the same experimental circumstances, 5-azacytidine is without effect. As a result of the action of these inhibitors on the structure of DNA, it is proposed that ferritin genes remain accessible to RNA polymerase allowing the accumulation in the cytoplasm of mature ferritin mRNA ready to be mobilized by iron for the production of ferritin molecules.

Flow cytometric analysis of hepatoma tissue and HeLa cells grown on various types of microbeads using hydroxyurea, nocodazole and aphidicolin in succession.

Applying Hydroxyurea, Nocodazole and Aphidicolin in succession to obtain parasynchronous growth, the progression of HTC and HeLa cells through the cell cycle has been monitored by laser flow cytometry. The experimental results show that HTC cells behave identically whether grown in monolayer or attached to dextran-based microbeads but that the chemical nature of the micro-support itself plays an important role especially on the speed with which the cells pass from mitosis into G1, polyacrylamide-based microbeads being superior in this respect.

RBC-mediated microinjection of chromatin components into cultured mammalian cells.

Radiolabeled DNA fragments or nuclear proteins were encapsulated within human erythrocytes, and the erythrocytes were then fused with cultured mammalian cells using Sendai virus. Autoradiography revealed that 125I-labeled DNA fragments remained dispersed in the cytoplasm and disappeared with a half-life of 24 hours. In contrast, the nuclear proteins, HMG1, HMG2, HMG17 and histone H1, rapidly localized within HeLa nuclei and exhibited half lives greater than 80 hours. Several biochemical criteria indicate that the association of the injected nuclear proteins with chromatin faithfully mimics the behavior of their endogenous counterparts.

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin.

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.

Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.

Vaccinia virus: the possibilities of its oncogenicity in humans.

Contrary to the earlier generally accepted view that vaccinia virus replicates in the cytoplasm only of suitable target cells, steadily accumulating data show that the viral genome spends a limited period of time in the nucleus. This, together with the many cases where a close association has been suspected or established between skin cancer and vaccination, suggests that vaccinia virus may, under certain yet undefined physiological conditions, act as an oncogenic virus in humans.

Study of messenger-like RNA extracted from HeLa cells polysomes. II. Identification of the types of RNA eluted from methylated albumin kieselguhr columns.

Together with the elution pattern of pure messenger RNA molecules of various origin, the labelling kinetics of rapidly labelled heterogeneously sedimenting RNA (HSRNA) extracted from polysomes of HeLa cells have been studied by chromatogrphy on columns made of methylated bovine serum albumin adsorbed on kieselguhr. HSRNA is eluted within three peaks-IP, Q2P and TDP-following in that order the increase of NaC1 concentration in the eluting buffer. Besides peak TDP which results from an experimental artefact, our data suggest that the appearance of peaks IP and Q2P reflects the absence and presence respectively of polyadenylic acid stretches in these molecules. Within peak Q2P, the critical factor affecting the order of elution is the size of the polyadenylic acid stretch.