Intracellular characterization of Gag-GFP VLP production upon PEImediated transient transfection of HEK 293 cells

Transient Gene Expression is a fast, flexible, and cost-effective approach to produce high-quality products that circumvents the time and cost required for the generation of stably transfected cell lines. However, the levels of recombinant protein produced by TGE, tend to be significantly lower than those of stable cell lines. Despite the continued interest in transient gene expression approaches, little is known about the transfection process at intracellular level, particularly for complex products such as VLPs. The kinetics of PEI-mediated transient transfection was studied with the aim of characterizing and understanding the complete process leading to VLP generation, and identifying important events to drive process improvement. For this purpose, DNA/PEI polyplexes were tracked using Cy3 DNA staining and the production of Gag-GFP VLPs was monitored by flow cytometry, confocal microscopy, and fluorometry. Flow cytometry and confocal microscopy assays show that using a standard transfection protocol DNA:PEI polyplexes interact with the cell membrane from time point zero. A linear increase in transfection efficiency is observed until 60 minutes of contact between cells and polyplexes. No change in transfection efficiency (percentage of GFP positive cells) or VLP production levels is obtained when additional contact time is allowed, reaching a maximum transfection efficiency of 60% and VLP production of 10x109 VLPs/mL harvested at 48 hours post transfection (hpt). After 1.5 hpt, polyplexes are detected in the cytoplasm of transfected cells and reach the nucleus around 4 hpt. Of note, all cells show the presence of DNA/PEI complex in the cytoplasm after transfection but only a fraction of cells express the fluorescent Gag protein. By flow cytometry analysis of isolated nuclei, it was determined that polyplexes are only present in 60% of the nuclei at 6 hpt (concomitant with the GFP expressing cells), suggesting that the entrance of polyplexes to the nucleus is one of the limiting steps of the transfection process. After 10 hpt, GFP fluorescence is detected homogenously inside the cells, but generalized budding of VLPs is not observed until 48 hpt. As mentioned before, a unique population of cells Cy3+ (with a polyplex inside) appears from the very beginning of the transfection. A new population of cells that do not contain any polyplex inside (Cy3-) and do not express the protein (GFP-) appears at 24 hpt suggesting plasmid loss after this time point. The VLP production kinetics was also studied, observing that fluorescence in the supernatant is always 40% less than total fluorencense (supernatant plus pellet). Maximum VLP levels in the cell culture supernatant, while keeping cell culture viability still high, are observed at 72 hpt, which was determined to be the optimal harvest time. Three bottlenecks in VLP production could be identified in this work: polyplexes entry into the nucleus, plasmid loss during the production phase and VLP buddin

A continuous loop of bioreactors to provide for life support in space

MELiSSA project is developing Life Support technologies for long-term Space missions. The goals of the MELiSSA loop are the recovery of food, water and oxygen from wastes, i.e. CO2 and organic wastes, using light as a source of energy. It is conceived as a series of compartments, each one performing a specific function within this cycle, inspired in the terrestrial ecological systems. Each one of the compartments is colonized with specific bacteria or higher plants depending on its dedicated function. The MELiSSA Pilot Plant is a facility conceived for the demonstration of this technology, the development of the MELiSSA compartments and its integration to build the complete MELiSSA loop. It uses laboratory rats as a demonstrator of the crew. The experimental results from the connection of a 100L air-lift photobioreactor culturing the cyanobacteria Arthrospira platensis, producing oxygen with an isolator with rats as the mock crew, as oxygen consumers, is presented. Several experiments of continuous connection with duration of 4-5 weeks have shown the robustness of the system and the ability of the control system to adjust the dynamics of the oxygen production compartment to that of the oxygen consumption compartment, while maintaining a desired percentage of oxygen in the gas phase of the animal compartment. The key variable used to adjust the production of the photosynthetic compartment to the consumer’s compartment is the illumination intensity in the photobioreactor, governed by the control system of the MELiSSA Pilot Plant. To note, the demand of oxygen by the consumers is changing following 12 hours day/night periods. The results show a very precise adjustment of the operation of the integrated system and a fast capacity of the control system to drive the oxygen level to a given set point. The evolution and mathematical modeling of the different variables of the integration, such as oxygen production and CO2 consumption in the photobioreactor, the oxygen consumption and CO2 production in the animal compartment, the illumination intensity, percentage of oxygen in the gas phase and operational conditions of the system at several experimental conditions will be discusse

Effect of heat processing on the nutrient composition, colour, and volatile odour compounds of the long-horned grasshopper Ruspolia differens serville

Heat processing is commonly used to prepare edible insects for consumption. This study aimed at determining the effect of boiling and subsequent oven roasting on Ruspolia differens’ nutrient composition, colour and odor compounds. Boiling leads to: a significant increase in protein and decrease in fat content on a dry matter basis; a minimal influence on its amino and fatty acids profile; a significant reduction in its ash content due to leaching of phosphorus, potassium and sodium; a significant increase in iron, zinc, copper, manganese and calcium content; and a fivefold reduction in the amount of vitamin B12. Roasting leads to a relative increase in the amount of calcium and trace mineral elements but doesn’t affect other nutrients. Roasting results into a more uniform colour intensity when green and brown polymorphs are roasted together. Lipid oxidation is responsible for the colour and aroma of heat processed R. differens.status: Published onlin

A general artificial neural network for the modelization of culture kinetics of different CHO strains

Animal cell cultures are characterized by very complex nonlinear behaviors, difficult to simulate by analytical modeling. Artificial Neural Networks, while being black box models, possess learning and generalizing capacities that could lead to better results. We first trained a three-layer perceptron to simulate the kinetics of five important parameters (biomass, lactate, glucose, glutamine and ammonia concentrations) for a series of CHO K1 (Chinese Hamster Ovary, type K1) batch cultures. We then tried to use the same trained model to simulate the behavior of recombinant CHO TF70R. This was achieved, but necessitated to synchronize the time-scales of the two cell lines to compensate for their different specific growth rates.SCOPUS: cp.jinfo:eu-repo/semantics/publishe

Dynamics of long-term continuous culture of Limnospira indica in an air-lift photobioreactor

International audienceMELiSSA (Microecological Life Support System Alternative) is a developing technology for regenerative life support to enable long-term human missions in Space and has developed a demonstration Pilot Plant. One of the components of the MELiSSA Pilot Plant system is an 83L external loop air-lift photobioreactor (PBR) where Limnospira indica (previously named Arthrospira sp. PC8005) is axenically cultivated in a continuous operation mode for long-periods. Its mission is to provide O2 and consume CO2 while producing edible material. Biological and process characterization of this PBR is performed by analysing the effect of two main variables, dilution rate (D) and PFD (Photon Flux Density) illumination. A maximum oxygen productivity (urn:x-wiley:17517915:media:mbt213882:mbt213882-math-0001) of 1.35 mmol l−1 h−1 is obtained at a D of 0.025 h−1 and PFD of 930 µmol m−2 s−1. Photoinhibition can occur when a 1 g l−1 cell density culture is exposed to PFD higher than 1700 µmol m−2 s−1. This process is reversible if the illumination is returned to dim light (150 µmol m−2 s−1), proving the cell adaptability and capacity to respond at different illumination conditions. Influence of light intensity in cell composition is also described. Specific photon flux density (qPFD) has a direct effect on phycobiliproteins and chlorophyll content causing a decrease of 62.5% and 47.8%, respectively, when qPFD increases from 6.1 to 19.2 µmol g−1 s−1. The same trend is observed for proteins and the opposite for carbohydrate content. Morphological and spiral structural features of L. indica are studied by confocal microscopy, and size distribution parameters are quantified. A direct effect between trichome width and CDW/OD ratio is observed. Changes in size distribution are not correlated with environmental factors, further confirms the adaptation capacity of the cells. The systematic analysis performed provides valuable insights to understand the key performance criteria of continuous culture in air-lift PBRs

Development of a simple disposable six minibioreactor system for suspension mammalian cell culture

A simple disposable six minibioreactor system has been developed in order to perform multiple cell culture experiments in parallel, as a tool to accelerate experimentation in cell culture optimization. The system consists of a fixed part containing all instrumentation, sensors and actuators, and a disposable part, a compact unit with six minibioreactors with 10–15 mL of working volume each. This single-use unit is made of transparent biocompatible plastic material (polystyrene). Each one of the minibioreactors is equipped with agitation, headspace aeration supply and two optical probes, one for total cells measurement and pH, and another for dissolved oxygen measurement (and consequently the evaluation of Oxygen Uptake Rate, OUR). As an example of application, the performance of the system is successfully demonstrated for the culture of hybridoma cells growing in suspension under different conditions. The results allowed confirming the reproducibility of the system, and the feasibility to follow-up continuously the differences in cell growth, pH, pO2 and OUR evolution when hybridoma cells are cultured in different experimental conditions. For this, three different sets of experiments are considered. First, the use of the same culture medium DMEM supplemented with 10% fetal calf serum in all six minibioreactors. Second, the use of DMEM supplemented with three different FCS percentages (0, 1 and 10%) in two minibioreactors each. Third, the use of two different media (DMEM supplemented with 10% FCS and a chemically defined medium) in three minibioreactors each.Peer Reviewe

Development of a simple disposable six minibioreactor system for suspension mammalian cell culture

A simple disposable six minibioreactor system has been developed in order to perform multiple cell culture experiments in parallel, as a tool to accelerate experimentation in cell culture optimization. The system consists of a fixed part containing all instrumentation, sensors and actuators, and a disposable part, a compact unit with six minibioreactors with 10–15 mL of working volume each. This single-use unit is made of transparent biocompatible plastic material (polystyrene). Each one of the minibioreactors is equipped with agitation, headspace aeration supply and two optical probes, one for total cells measurement and pH, and another for dissolved oxygen measurement (and consequently the evaluation of Oxygen Uptake Rate, OUR). As an example of application, the performance of the system is successfully demonstrated for the culture of hybridoma cells growing in suspension under different conditions. The results allowed confirming the reproducibility of the system, and the feasibility to follow-up continuously the differences in cell growth, pH, pO2 and OUR evolution when hybridoma cells are cultured in different experimental conditions. For this, three different sets of experiments are considered. First, the use of the same culture medium DMEM supplemented with 10% fetal calf serum in all six minibioreactors. Second, the use of DMEM supplemented with three different FCS percentages (0, 1 and 10%) in two minibioreactors each. Third, the use of two different media (DMEM supplemented with 10% FCS and a chemically defined medium) in three minibioreactors each.Peer Reviewe

Integration of Nitrifying, Photosynthetic and Animal Compartments at the MELiSSA Pilot Plant

International audienceMELiSSA (Micro Ecological Life Support System Alternative) is developing bioregenerative Life Support technologies for long-term Space missions. The MELiSSA concept is conceived as a loop with several compartments, each one performing a specific function, providing all together edible material production, atmosphere regeneration and water recovery with a concomitant use of wastes, i.e., CO2 and organic wastes. Each one of the compartments is colonized with specific bacteria or higher plants depending on its specific function. The MELiSSA Pilot Plant is a facility designed for the terrestrial demonstration of this concept, hosting laboratory rats as a crew mock-up mimicking the respiration of humans. Currently, the MELiSSA Pilot Plant focus on the integration of three compartments: Compartment 3 (nitrifying packed-bed bioreactor based on the co-culture of immobilized Nitrosomonas europaea and Nitrobacter winogradsky), compartment 4a (an air-lift photobioreactor for the culture of the edible cyanobacteria Limnospira indica with concomitant oxygen production) and Compartment 5 (an animal isolator with rats as mock-up crew). The output from these tests shows a high robustness and reliability and the performance of oxygen producing and oxygen consuming compartments is successfully demonstrated under transitory and steady-state conditions. This contribution reports on the current state of development of the MELiSSA Pilot Plant Facility and the most recent results of the integration work