Biomedical subjects

M J Carrondo

Publications and source records attributed to M J Carrondo.

24 records · Page 2Linked to original sources

PubMed1994

Repeated-batch cultures of baby hamster kidney cell aggregates in stirred vessels.

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate characteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this 'selection' procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.

Alkaline Phosphatase↗

PubMedDate not supplied

Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing.

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.

Bioreactors↗

PubMedDate not supplied

Comparison of different bioreactor systems for the production of high titer retroviral vectors.

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).

Bioreactors↗

PubMedDate not supplied

Effect of viscosity upon hydrodynamically controlled natural aggregates of animal cells grown in stirred vessels.

The effect of medium viscosity upon cell growth and aggregate characteristics of baby hamster kidney (BHK) cells cultivated in stirred tanks was evaluated. Two thickening agents were tested, 9300 MW dextran and a low-viscosity sodium (carboxymethyl)-cellulose; both were used in two different sets of experiments: (i) 250 cm3 Wheaton spinner flasks with a ball impeller operated at 45 rpm; (ii) 500 cm3 Corning spinner flasks with a paddle impeller, operated at constant power dissipation (88 cm2 s-3). Aggregate diameter and the fraction of cells in aggregates increased with the increase in viscosity. Power laws were applied to the experimental results. A dependence of aggregate size upon power dissipation of the order of -0.19 and kinematic viscosity of 0.34 and 0.49 for the constant agitation and constant power dissipation tests were obtained, respectively. A model based upon the entire universal-equilibrium range (i.e., the entire spectrum of isotropic eddies) was used to predict theoretical relationships between the variables studied. The model leads to a power dependence of -0.25 for the energy dissipated in the entire universal-equilibrium range and between 0.25 and 0.5 for the kinematic viscosity in the viscous dissipation subrange, depending on the energy correlation used; it also gives a good explanation for the dependence of aggregate size on the hydrodynamics of the vessel.

Animals↗

PubMedDate not supplied

Modeling retrovirus production for gene therapy. 1. Determination Of optimal bioreaction mode and harvest strategy.

Although retroviruses are a promising tool for gene therapy, there are two major problems limiting the establishment of viable industrial processes: retrovirus stability and low final yield in the supernatant. This fact emphasizes the need for an effective process optimization, not only at a genetic level but also at a bioprocess engineering level. In part 1 of this paper a mathematical model was developed to optimize the bioreaction yield by determining the best retrovirus harvest strategy in perfusion cultures. PA317 cells producing recombinant retroviruses were used to develop and test this model. Cell culture was performed in stirred tanks using porous supports. The parameters of the proposed model were experimentally determined for batch and perfusion cultures at 32 and 37 degrees C both with and without additives to enhance production; the model was then validated. This model allowed the determination of the optimal values of all operational variables included: batch and perfusion duration and perfusion rate. The highest productivity (2682 virus cm(-)(3) h(-)(1)) was obtained under the following conditions: batch at 37 degrees C for 53 h followed by perfusion at 32 degrees C for 23 h with a perfusion rate of 0.107 h(-)(1). This value was 3.5-fold higher than the best result obtained in batch cultures for the same conditions of titer and quality. A sensitivity analysis of the parameters showed that the parameters that affect most the final productivity depend on the bioreaction phase: cell growth in batch culture and production and product degradation in perfusion culture. In part 2 of this paper, this model is extended to the first step of downstream processing, and the addition of further steps to the process is discussed in order to achieve global process optimization.

Animals↗

PubMedDate not supplied

Metabolism of 3-(13)C-malate in primary cultures of mouse astrocytes.

Malate, specifically labeled with carbon 13 on C(3), was synthesized by chemical means and used to study malate metabolism by primary cultures of mouse cortical astrocytes. 3-(13)C-Malate in combination with glucose as well as 3-(13)C-malate alone were used as substrates; the effect of 3-nitropropionic acid, an inhibitor of succinate dehydrogenase and fumarase was also examined. The consumption of malate was only 0.26 micromol/mg of protein, approx. 25-fold lower than the consumption of glucose. Besides lactate, glutamine and fumarate were the two major metabolites released to the medium. Very low and similar levels of isotopic enrichment were detected on C(2) and C(3) of lactate; glutamine was labeled on C(2) and C(3) to a similar extent as well and labeling on C(4) was only detected when glucose was not added. These labeling studies suggest that cytosolic malic enzyme is not active in primary astrocytes and support the occurrence of pyruvate recycling in astrocytes.

Animals↗