Application of on-line OUR measurements to detect actions points to improve baculovirus-insect cell cultures in bioreactors.

The continuous monitoring of a process based on the culture of Sf9 insect cells and infection by a baculovirus as a vector to obtain recombinant VP2 protein is studied. On-line OUR determination is based on the direct oxygen measurement in the cell culture vessel and the application of the dynamic method. This approximation allows a proper description of cell growth, with precise identification of the balanced cell growth end and the most important action times in the process, as virus infection time and final cell harvesting. A detailed study of the OUR profiles allows on-line monitoring of the effects of infection and expression protein process, a tool enabling the automatisation of the protein production process in a baculovirus-insect cell system. These parameters have been defined as time of action (TOAs), and include the most relevant actions to take in these type of processes: time of infection (TOI), time of feeding (TOF) and time of harvesting (TOH).

Four versus two-electrode measurement strategies for cell growing and differentiation monitoring using electrical impedance spectroscopy.

The aim of this work is to provide optimization tools for cell and tissue engineering processes through continuous monitoring of cell cultures. Structural cell properties can be obtained from non-destructive electrical measurements by using electrical impedance spectroscopy (EIS). EIS measurements on monolayer animal cell cultures are usually performed using a two-electrode strategy. Because of this, the measurement is very sensitive to the electrode covering ratio and to the degree of adherence of cells. Of course, these parameters give useful information but can mask the behaviour of the cell layer above the electrodes. In a previous work, preliminary measurements with commercial microelectrode structures were performed with simulated grow processes using the settlement of cell suspensions with two and four microelectrode strategies to validate the technique. In this work, real cell growths of Vero cells are described and the resulting EIS biomass density estimators are compared to cell counts. The four-electrode impedance spectra are fitted to the Cole-Cole impedance model and the time course of their parameters are extracted and studied.

Multiple automated minibioreactor system for multifunctional screening in biotechnology.

The current techniques applied in biotechnology allow to obtain many types of molecules that must be tested on cell cultures (high throughput screening HTS). Although such tests are usually carried out automatically on mini or microwell plates, the procedures in the preindustrial stage are performed almost manually on higher volume recipients known as bioreactors. The growth conditions in both stages are completely different. The screening system presented in this work is based on the multiwell test plates philosophy, a disposable multiple minibioreactor that allows reproduction of industrial bioreactor culture conditions: aeration, stirring, temperature, O2, pH and visible range optical absorbance measurements. It is possible to reproduce the growth conditions for both suspended and adherent animal cell types using 1 to 10 ml vol. bioreactors. In the case of bacteria or yeast, it is not possible to achieve a high biomass concentration, due to the reduced head volume air supply.

On-line monitoring of yeast cell growth by impedance spectroscopy.

The application of impedance spectroscopy to estimate on-line cell concentration was studied. The estimation was based on the relative variation between electrical impedance measured at low (10 kHz) and high frequencies (10 MHz). Studies were carried out to characterise the influence of changes in physical and chemical parameters on the impedance measurement. Two different possibilities to perform on-line measurements were tested: a simple set-up, based on an in situ probe, gave good results but was not suitable for high agitation and aeration rates. An ex situ flow-through on-line measuring cell was used to overcome these problems, showing a better performance. The use of this set-up for the growth monitorisation of a Saccharomyces cerevisiae culture showed an efficient performance, having the correlation between estimated and measured S. cerevisiae a Pearson coefficient of 0.999.

The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity.

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins. (Blood. 2001;97:3820-3828)

Salivary gland lymphomas in patients with Sjögren's syndrome may frequently develop from rheumatoid factor B cells.

OBJECTIVE: Patients with Sjögren's syndrome (SS) have an increased risk of developing monoclonal B cell non-Hodgkin's lymphomas (MNHL), which frequently occur in the salivary glands (SG). The transition from the benign lymphocyte infiltrate of the gland that characterizes SS to MNHL is not well understood. Previous sequence analyses of the expressed variable (V) region genes have supported the theory that the surface Ig (sIg) plays an important role in the initial expansion of nonmalignant B cell clones and in lymphomagenesis. However, the antigenic specificities of these B cells were unknown. We describe the specificities of the Ig expressed by 2 cases of MNHL that developed in the SG of 2 patients with SS. METHODS: The expressed V genes were amplified by polymerase chain reaction from biopsy specimens, sequenced, and subcloned into eukaryotic expression vectors. The constructs were transfected into P3X63-Ag8.653 cells to obtain 2 monoclonal cell lines, each secreting 1 of the sIg expressed by the MNHL. These IgM were tested by enzyme-linked immunosorbent assay and immunofluorescence against a panel of antigens potentially implicated in SS. RESULTS: Our main finding was that the Ig products of the neoplastic B cells were rheumatoid factors (RF). Contrary to expectations, they did not react with nuclear or cytoplasmic antigens, double-stranded DNA, self antigens commonly bound by natural autoantibodies, or SG tissue. CONCLUSION: Previous analyses of V gene use have provided indirect evidence that SG MNHL may frequently express RF. We demonstrate that this hypothesis is true in the 2 patients we studied. Large-scale studies will be needed to establish the exact frequency of RF specificity among SS-associated MNHL.

Dissociation of telomere dynamics from telomerase activity in human thyroid cancer cells.

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by the in vitro "TRAP" assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before "plateauing" and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.