Biomedical subjects

M J Carrondo

Publications and source records attributed to M J Carrondo.

At least 19 recordsLinked to original sources

PubMed2001

Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression.

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

Animals↗

PubMed2001

Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)P-NMR spectroscopy.

Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line (31)P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol.

Adenine Nucleotides↗

PubMed2000

Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells.

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.

Animals↗

PubMed2000

Metabolically optimised BHK cell fed-batch cultures.

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.

Animals↗

PubMed2000

In vitro response of the brown bullhead catfish (BB) and rainbow trout (RTG-2) cell lines to benzo[a]pyrene.

Established cell lines from rainbow trout (RTG-2) and brown bullhead catfish (BB) were evaluated as bioindicators of benzo[a]pyrene (B[a]P) toxicity with 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytochrome P450 1A1 (CYP1A1) enzymatic activity was also evaluated, and taken as a biological indicator of the B[a]P induction power by ethoxyresorufin O-deethylase (EROD) and ethoxycoumarin O-deethylase (ECOD) assays. The BB and RTG-2 cells were compared after 1 and 6 days of exposure to B[a]P. The photoactivation of the compound (B[a]PUV) was another parameter taken into consideration. Cytotoxicity was not observed after 1 day of incubation with B[a]P in both cell lines, although the enzymatic activities of ECOD and EROD presented an induction. Apparently, after 1 day, cells did not metabolise sufficient amounts of B[a]P to cytotoxic metabolites. After 6 days of exposure to this compound a significant reduction in cell viability was observed, this reduction being superior to 50% at the highest B[a]P concentrations for the RTG-2 cell line. These results are in agreement with the values observed for the ECOD and EROD induction. The B[a]P cytotoxicity determined in both cell lines could be ascribed to the significant increase of EROD activity by 6 days of exposure. The photoactivation of B[a]P showed marked differences in both cytotoxic assays and CYP1A1 enzymatic activities, for both cell lines. After 1 day of exposure there was a significant reduction in cell viability, superior to 50% for the RTG-2 cell line. However, it was observed that no induction occurred but rather a decrease in ECOD and EROD activities. Six days of incubation with B[a]PUV showed a decrease in cell viability at the highest concentrations for the BB cells and at the lowest concentrations for the RTG-2 cell line, and the CYP1A1 enzymatic activity presented a significant induction. These results and those observed after 1 day of exposure suggest that B[a]PUV acts as a direct-acting toxicant as well as a metabolism-mediated toxicant-like B[a]P. The RTG-2 cells were more sensitive to B[a]P and its toxic metabolites as well as to the photoactivation of the compound, in both exposure times tested. The finding that the cell lines responded to the CYP1A1 induction in a very efficient way gives proof of the applicability of this system to environmental biomonitoring and toxicology.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2000

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia.

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.

Animals↗

PubMed1999

Proteolytic activity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles.

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing.

Adenosine Triphosphate↗

PubMed1999

Processing of extracellular lipase of Lactobacillus plantarum: involvement of a metalloprotease.

The hypothesis of a proteolytic involvement in the extracellular lipase processing of a strain of Lactobacillus plantarum was considered and tested, in vitro assays with acid proteases, cathepsin D and renin revealed that both did affect lipolytic activity positively. In vivo assays with growth in the presence of the protease inhibitors pepstatin, phenylmethylsulfonyl fluoride, transepoxysuccinyl-L-leucylamido-(4-guanidino)butane and ethylenediaminetetraacetic acid showed that ethylenediaminetetraacetic acid did affect the pattern of the proteins that possess lipolytic activity. Therefore, it is suggested that a metalloprotease is involved in the processing of the extracellular lipases of L. plantarum, although other proteases can also be important.

Lactobacillus↗

PubMed1999

Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells.

The present work aims at characterizing the regulatory mechanisms of metabolism and product formation of BHK cells producing a recombinant antibody/cytokine fusion protein. This work was carried out through the achievement of several steady-states in chemostat cultures, corresponding to different glucose and glutamine levels in the feed culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on residual glucose and glutamine concentrations, respectively. Similar dependence was also observed for lactate and ammonia productions. K(Glc)(Glc) and K(Gln)(Gln) were estimated to be 0.4 and 0.15 mM, respectively, while q(max)(Glc) and q(max)(Gln) were estimated to be 1.8 and 0.55 nmol 10(-6)cells min(-1), respectively. At very low glucose concentrations, the glucose-to-lactate yield decreased markedly showing a metabolic shift towards lower lactate production; also, the glucose-to-cells yield was increased. At very low-glutamine concentrations, the glutamine-to-ammonia and glutamine-to-cells yields increased, showing a more efficient glutamine metabolism. Overall, amino acid consumption was increased under low glucose or glutamine concentrations. Metabolic-flux analysis confirmed the metabolic shifts by showing increases in the fluxes of the more energetically efficient pathways, at low-nutrient concentrations. No effect of glucose or glutamine concentrations on the cell-specific productivity was observed, even under metabolically shifted metabolism; therefore, it is possible to confine the cells to a more efficient metabolic state maintaining the productivity of the recombinant product of interest, and consequently, increasing final product titers by increasing cell concentration and culture length. This work is intended to be a model approach to characterize cell metabolism in an integrated way; it is highly valuable for the establishment of operating strategies in mammalian cell fermentations in which cell metabolism is to be confined to a desired state.

Amino Acids↗

PubMed1999

Influence of environmental factors on lipase production by Lactobacillus plantarum.

A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, "chouriço", was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30 degrees C and was kept at a significant level over a wide range of dilution rates (0.05-0.4 h-1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture.

Bioreactors↗

PubMed1999

Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture.

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (KLacGlc) was 1.4 +/- 0.1 mM and for the dependence of ammonia production on glutamine (KAmmGln) was 0.25 +/- 0.11 mM and 0.10 +/- 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was highly dependent on glucose concentration, the oxygen consumption also increased with the increase in glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism.

Ammonia↗

PubMed1999

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells.

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. "mono-", "bi-" and "tri-cistronic" vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in antitumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation-time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

Animals↗

PubMed1998

Optimization of the production of virus-like particles in insect cells.

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.

Animals↗

PubMed1997

Cell-dislodging methods under serum-free conditions.

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml-1). equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity.

Animals↗

PubMed1996

Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR.

Immobilization of primary astrocytes and cerebellar granule neurons in basement membrane gel threads and on porous microcarriers, respectively, is described. Five different porous microcarriers were examined for their capabilities to support maturation of neurons, as evaluated by determinations of protein content and glucose uptake. These systems proved suitable for perfusion in the NMR tube for long periods of time. The energetic status of the cells was monitored by 31P-NMR, and a very good temporal resolution (less than 30 min) was achieved with superfused astrocytes; thus, this provides a valuable method for real time NMR investigation of biochemical processes in primary astrocytes. Significantly longer acquisition times (approximately 8 h) were required in order to obtain a spectrum of neurons with a reasonably good signal to noise ratio, even so, the results presented here and obtained with this difficult system are very encouraging.

Animals↗

PubMed1994

Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

Animals↗

PubMed1994

Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics.

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (mu). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as mu decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in mu. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers. Qsproduct decreased as mu increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to mu are correlated with changes in cell morphology. The relationship between Qsproduct and mu is also affected by the choice of cell line.

Alkaline Phosphatase↗

PubMed1994

Changes in animal cell natural aggregates in suspended batch cultures.

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical dissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing in size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1.2 x 10(6) cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 x 10(8) cells/ml, with a porosity of 55%. Viability was consistently in the range 85-90%, both for aggregate and suspended cells.

Animals↗