Francesc Gòdia

Improvement of CHO Cell Culture Medium Formulation: Simultaneous Substitution of Glucose and Glutamine

The formulation of the culture medium for a Chinese hamster ovary (CHO) cell line has been investigated in terms of the simultaneous replacement of glucose and glutamine, the most commonly employed carbon and nitrogen sources, pursuing the objective of achieving a more efficient use of these compounds, simultaneously avoiding the accumulation of lactate and ammonium in the medium. The key factor in this process is the selection of compounds that are slowly metabolized. Among the different compounds studied, galactose and glutamate provide the best results, allowing support of cell growth with an optimal balance between nutrient uptake and cell requirements and the generation of minimal quantities of lactate and ammonium. The attained results also highlight the capacity of the cells to redistribute their metabolism as a response to the changes in medium composition.

Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells

Whether aging modifies mesenchymal stem cell (MSC) properties is unknown.

MELISSA: a loop of interconnected bioreactors to develop life support in space.

The development of a loop of interconnected continuous bioreactors, aimed to provide life support in space, is reported. The complete loop concept consists of four bioreactors and one higher plant compartment. For its realization the continuous and controlled operation of the bioreactors is characterized, up to the pilot scale level, first for each individual reactor, second for the interconnected reactor operation. The results obtained with the two more advanced bioreactors in the Micro Ecological Life Support System Alternative (MELISSA) loop are described more specifically. These reactors consist of a packed-bed reactor working with an immobilized co-culture of Nitrosomonas and Nitrobacter cells, and an external loop gas-lift photobioreactor for the culture of the cyanobacteria Spirulina platensis. Their individual operation for long duration runs has been achieved and characterized, and their interconnected operation at pilot scale is reported.

Recovery and treatment of Spirulina platensis cells cultured in a continuous photobioreactor to be used as food

The development of auto-regenerative biological life support systems for men in Space will be based on photosynthetic organisms, such as higher plants and algae, providing edible material. In this work, Spirulina platensis grown in a continuous photobioreactor was used to design a process for its recovery and further treatment to be used as food. Two different possibilities are studied (liquid or dry food). In each case, different steps are considered in the design of the process and further characterised: cell harvesting, washing, pasteurisation and spry-drying. Special emphasis is made on biomass quality, both in terms of potential microbial contamination and changes in its composition during the different steps of the process. Cell harvesting was conducted with a net recovery of solids and water higher than 95% with a solids concentration factor about 20–30. Biomass quality was shown as satisfactory in all the treatments tested.

Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering

The present work describes the genetic modification of a hybridoma cell line with the aim to change its metabolic behaviour, particularly reducing the amounts of ammonia and lactate produced by the cells. The cellular excretion of ammonia was eliminated by transfection of a cloned glutamine synthetase gene. The metabolic characterisation of the transformed cell line includes the analysis of the changes introduced in its intracellular metabolic fluxes by means of a stoichiometric model. Furthermore, the reduction of lactate accumulation was attempted through an antisense mRNA approach, aiming to generate a rate limiting step in the glycolytic pathway, thus lowering the glucose consumption rate. The physiological results obtained with the transformed cells are discussed. A maximum reduction of about 47% in the glucose consumption rate was obtained for one of the transformations. However a main drawback was the lack of stability of the transformed cells

FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 ± 0.2 vs. 4.1 ± 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.

Determination of glucose and ethanol effective diffusion coefficients in Ca-alginate gel

Glucose and ethanol diffusion coefficients in 2% Ca-alginate gel were measured using the experimental technique based on solute diffusion into or out of gel beads in a well-stirred solution. The aim of the study was to make the measurements under typical conditions found in alcoholic fermentations, such as the concentrations of glucose (100 g l-1) and ethanol (50 g l-1), the simultaneous counter-diffusion of glucose and ethanol, and the presence of cells in the gel beads at a level of 10(9) cells g-1 of beads. Previously, an evaluation of the error associated with the methodology used indicated how the experimental procedure would minimize the error. The individual measurement of glucose and ethanol coefficients in 2% Ca-alginate with no cells gave values of 5.1 and 9.6 x 10(-6) cm2 s-1, respectively, which are lower than those in water. When the effect of counter-diffusion was investigated, both coefficients decreased: glucose by 14% and ethanol by 28%. When cells were incorporated into the beads, only the ethanol coefficient decreased significantly, while the glucose coefficient apparently increased its value to 6.9 10(-6) cm2 s-1.

Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium

Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8μg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively.

Metabolic engineering of apoptosis in cultured animal cells: implications for the biotechnology industry.

Animal cells have been widely used to obtain a wide range of products for human and animal healthcare applications. However, the extreme sensitivity of these cells in respect to changes experienced in their environment is evidenced by the activation of a gene-encoded program known as apoptosis, resulting in their death and destruction. From the bioprocess angle, losses in cell viability bring lower productivities and higher risks of product degradation. Consequently, many research efforts have been devoted to the development of apoptosis protective mechanisms, including the metabolic engineering of apoptosis pathways, that has proven effective in diminishing programmed cell death in a variety of biotechnological relevant cell lines. This review is focused especially in the encouraging initial results obtained with the over-expression of cloned anti-apoptosis genes, from both endogenous and viral origin interfering at mitochondrial and initiator caspases levels.

Immobilized cells: behaviour of carrageenan entrapped yeast during continuous ethanol fermentation

SummaryData of cell concentration, viability and microscopic observation of cell distribution inside carrageenan immobilized yeast beads are reported. Results were obtained from a continuous packed-bed reactor performing alcoholic fermentation and the main observations made on cell activity are in agreement with the fermentation profiles inside the fermenter.