Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures.

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.

Lactate and glucose concomitant consumption as a self-regulated pH detoxification mechanism in HEK293 cell cultures.

One of the most important limitations of mammalian cell-based processes is the secretion and accumulation of lactate as a by-product of their metabolism. Among the cell lines commonly used in industrial bioprocesses, HEK293 has been gaining importance over the last years. Up recently, HEK293 cells were known to consume lactate in late stages of cell culture usually when glucose and/or glutamine were depleted from media. Remarkably, in both scenarios, no significant cell growth was reported. However, we have observed a different metabolic behavior regarding lactate production and consumption in HEK293 cultures. HEK293 cells were able to co-metabolize glucose and lactate simultaneously, even in exponentially growing cell cultures. Our deep study of the effects of environmental conditions on lactate metabolism revealed that pH was the key to trigger the metabolic shift from lactate production to lactate and glucose concomitant consumption. Remarkably, this shift could be triggered at will when pH was set at 6.8. Even more interesting was the fact that lowering pH to 6.6 and supplementing media with exogenous lactate resulted in co-consumption of glucose and lactate from the beginning of cell culture, without affecting cell growth or protein productivity. On the contrary, cell growth was clearly hampered at this low pH if extracellular lactate was lacking. From our results, we hypothesize that HEK293 cells metabolize extracellular lactate as a strategy for pH detoxification, by means of co-transporting extracellular protons together with lactate into the cytosol. This novel hypothesis for unraveling lactate metabolism in HEK293 cells could open a door to re-direct genetic engineering strategies in order to obtain more efficient cell lines and also to further develop animal cell technology applications.

HEK293 Cell-Based Bioprocess Development at Bench Scale by Means of Online Monitoring in Shake Flasks (RAMOS and SFR).

The platforms for bioprocess development have been developed in parallel to the needs of the manufacturing industry of biopharmaceuticals, aiming to ensure the quality and safety of their products. In this sense, Quality by Design (QbD) and Process Analytical Technology (PAT) have become the pillars for quality control and quality assurance.A new combination of Shake Flask Reader (SFR) and Respiration Activity Monitoring System for online determination of OTR and CTR (RAMOS) allows online monitoring of main culture parameters needed for bioprocess development (pH, pO2, OTR, CTR, and QR) as presented below. Eventually, a case study of the application of the combination of SFR-RAMOS system is presented. The case study discloses the optimization of HEK293 cells cultures through the manipulation of their metabolic behavior.

Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab.

BACKGROUND: Monoclonal antibodies (mAbs) and their derivatives have become one of the most important classes of therapeutic drugs. Their multiple applications increased the interest for understanding their complex structure. In vivo, animal cells are able to fold mAbs correctly (Song et al, J Biosci Bioeng 110:135-40, 2010), whereas previous in vitro approaches were scarce and mostly unsuccessful. RESULTS: In this work, we compared in vitro assembly characteristics of trastuzumab, produced either by A) physical separation and refolding of its sub-units or B) direct joining of individually produced heavy and light chains. Native and denatured structures of trastuzumab were determined by SEC-HPLC, HIC-HPLC and SDS-PAGE. CONCLUSIONS: Our results demonstrate the requirement of correctly folded HC, forming disulfide-bonded dimers, in order to form a fully functional mAb. Otherwise, the unfolded HC tend to precipitate. We were able to assemble trastuzumab in this fashion by only mixing them to LC in pH-buffered conditions, while monomeric HC structure was too unstable to render a functional mAb. This approach has been used in the generation of homogeneous ADC, with results pending to be published.

Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly.

Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reduction - oxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG1), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays).

Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes.

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein-Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0-G1 state and this is translated in lower numbers of dead cells.

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum.

BACKGROUND: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. METHODS: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. RESULTS: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. CONCLUSIONS: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.

Structural changes of Arthrospira sp. after low energy sonication treatment for microalgae harvesting: Elucidating key parameters to detect the rupture of gas vesicles.

The buoyancy suppression by low energy sonication (LES) treatment (0.8W·mL-1, 20kHz, 10s) has recently been proposed as an initial harvesting step for Arthrospira sp. This paper aims to describe the structural changes in Arthrospira sp. after LES treatment and to present how these structural changes affect the results obtained by different analytical techniques. Transmission electron microscopy (TEM) micrographs of trichomes evidenced the gas vesicles rupture but also revealed a rearrangement of thylakoids and more visible phycobilisomes were observed. Differences between treated and untreated samples were detected by confocal microscopy, flow cytometry and optical microscopy but not by electrical impedance spectroscopy (EIS). After LES treatment, 2-fold increase in autofluorescence at 610/660nm was measured (phycocyanin/allophycocyanin emission wavelengths) and a ten-fold decrease in side scatter light intensity (due to a reduction of trichome's inner complexity). This was further confirmed by optical microscopy showing changes on trichomes appearance (from wrinkled to smooth).

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

BACKGROUND: Whether aging modifies mesenchymal stem cell (MSC) properties is unknown. AIM: To compare the differentiation capacity of human CD105(+) MSCs obtained from young and elderly donors. METHODS AND RESULTS: Cells were obtained from young (n=10, 24+/-6.4 years) and elderly (n=9, 77+/-8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca(2+) dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1+/-6.4% and 40.3+/-3.6%, p=0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105(+) MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca(2+) between neighbouring cells. Spontaneous Ca(2+) raises did not significantly increase after 5-AZ treatment in both age groups. CONCLUSION: Age does not influence the adipogenic and myogenic differentiation potential of human CD105(+) MSCs.

Considerations on the lactate consumption by CHO cells in the presence of galactose.

A CHO cell line producing t-PA was cultured using glutamate and glucose or galactose to decrease the formation of metabolic end-products and therefore improving the process. In batch cultures using glutamate (6 mM) with glucose at two different levels (5 and 20 mM) or with glucose and galactose (5 and 20 mM, respectively) a remarkable difference in cell culture parameters was evidenced. For 20 mM glucose, a usual cell pattern was observed with lactate built-up in the medium. For 5 mM glucose, cell growth was arrested due to glucose depletion and only a limited use of the excreted lactate could be observed, not supporting cell growth sufficiently. However, when glucose 5 mM and galactose 20 mM were used together, cells consumed the glucose first and, interestingly, in a second phase they continued growing on galactose with the simultaneous consumption of the endogenous lactate. Under these conditions, cell growth was even improved with respect to growth on 20 mM glucose, used as a control. This metabolic behavior is further investigated by using metabolic flux analysis, suggesting that the lactate produced is not used in the oxidative metabolism through the TCA cycle. Metabolic fate of the lactate consumed is discussed.

Optimization of ferric chloride concentration and pH to improve both cell growth and flocculation in Chlorella vulgaris cultures. Application to medium reuse in an integrated continuous culture bioprocess.

Combined effect of ferric chloride and pH on Chlorella vulgaris growth and flocculation were optimized using DoE. Afterwards, an integrated bioprocess for microalgae cultivation and harvesting conceived as a sole step was run in continuous operation mode. Microalgae concentration in a 2L-photobioreactor was about 0.5gL(-1) and the efficiency of flocculation in the coupled sedimentation tank was about 95%. Dewatered microalgae reached a biomass concentrations increase about 50-fold, whereas it was only about 0.02gL(-1) in the clarified medium. Then, the reuse of the clarified medium recovered was further evaluated. The clarified medium was reused without any further nutrient supplementation, whereas a second round of medium reuse was performed after supplementation of main nutrients (phosphate-sulfate-nitrate), micronutrients and ferric chloride. The medium reuse strategy did not affect cell growth and flocculation. Consequently, the reuse of medium reduces the nutrients requirements and the demand for water, and therefore the production costs should be reduced accordingly.

Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide.

Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested. Secondly, 5 different signal peptides were assessed in stable transfected cell lines. Spectrofluorometry was used to determine intra- and extracellular protein expression levels in terms of fluorescence, and the results were further confirmed by SDS-PAGE. The highest secretion efficiency was found for human IFNα2 signal peptide, achieving up to 2-fold increase in the amount of secreted protein compared to other signal peptides. The results showed that the combination of CMV promoter and IFNα2 signal peptide resulted highly efficient for recombinant protein production in HEK293 cells.

BHRF1 exerts an antiapoptotic effect and cell cycle arrest via Bcl-2 in murine hybridomas.

Apoptosis has been widely studied in order to find methods to increase the life-span and production performance in large-scale animal cell cultures. The use of anti-apoptotic genes has emerged as an efficient method to reduce apoptosis in a variety of biotechnological relevant cell lines, including CHO and hybridomas, alternatively to small molecule inhibitors. It is already known that expression of BHRF1, an Epstein-Barr virus-encoded early protein homologous to the anti-apoptotic protein Bcl-2, protects hybridoma cells from apoptosis in batch and continuous operation modes resulting in a delay in the cell death process under glutamine starvation conditions. In the present study, the mechanism of action of BHRF1 was investigated in a murine hybridoma cell line. BHRF1 protein was found in the mitochondrial cell fraction both under normal growing conditions and apoptosis-inducing conditions. Remarkably, the expression of the anti-apoptotic gene bcl2 in BHRF1-expressing cells was up-regulated 25-fold compared to mock-transfected controls under apoptosis triggering conditions and its expression correlated with survival of transgenic cultures and cell cycle arrest in G1. Bcl-2 activity was revealed to be crucial for the BHRF1-mediated effect since the addition of specific inhibitors of Bcl-2 (namely HA14-1 and YC-137) resulted in a loss of function of BHRF1-expressing cells under glutamine starvation conditions. Moreover, the interaction of BHRF1 with the pro-apoptotic BH3-only Bim conferred mitochondrial stability to BHRF1 expressing cells under apoptosis-triggering conditions.

The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions.

Programmed cell death (PCD) or apoptosis process in a hybridoma cell line induced by the deprivation of one of the main nutrients, glutamine, has been studied. The use of caspase inhibitors has enabled maintenance of cell viability during a significant period of time, when glutamine depletion was maintained in the culture. Two caspase inhibitors partially suppressed the progress of PCD under glutamine deprivation: Ac-DEVD-cho and z-VAD-fmk. Indeed, as a consequence of this protection, the number of viable cells decreased by 10% (for z-VAD-fmk) and by 80% (for Ac-DEVD-cmk) after 36 h of culture, while it decreased by 90% for a control culture in the absence of protective compounds. However, when the culture was exposed to non-apoptotic conditions after this period of time under apoptosis protection conditions, a normal growth pattern was not recovered. Interestingly, the simultaneous use of both inhibitors made the recovery of the cell culture possible even after a period of 36 h under glutamine depletion, indicating that the inhibition of the effector caspases occurs upstream of the point in which hybridoma cells enter into the commitment step of the death programme.

Protective effect of viral homologues of bcl-2 on hybridoma cells under apoptosis-inducing conditions.

Targets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl-2 gene and also with viral Bcl-2 homologues, particularly ksbcl-2 and bhrf-1 genes. When cells were exposed to apoptosis-inducing conditions (i.e., glutamine-free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl-2 and ksbcl-2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf-1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine-deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine-deprived conditions during a significant time window, up to 72 h.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization.

Use of a chronic model of articular cartilage and meniscal injury for the assessment of long-term effects after autologous mesenchymal stromal cell treatment in sheep.

Regenerative therapies using adult stem cells have attracted great interest in the recent years and offer a promising alternative to current surgical practices. In this report, we evaluated the safety and efficacy of an autologous cell-based treatment of osteoarthritis using mesenchymal stromal cells expanded from bone marrow aspirates that were administered intra-articularly. Ten 2-year old ewes were divided in two groups (for analysis at 6 and 12 months, respectively). Full thickness articular cartilage defects of approximately 60mm(2) were created arthroscopically in the medial femorotibial condyles and a meniscal tear in the anterior horn of the medial meniscus in the 20 hind legs. Intra-articular injection of 4 mL of either treatment (a suspension of cells) or control (same as treatment, without cells) were applied one month after generating a chronic condition similar to human pathology. Animals were monitored radiographically, by MRI and ultrasound scanning; and macroscopic and histological analyses were conducted at 6 and 12 months. Furthermore a full necropsy was performed at 12 months post-treatment. The intra-articular injection of autologous MSC was safe, as judged by the lack of local or systemic adverse effects during the clinical follow-up and by a full necropsy performed at 12 months post-treatment. Evidence of regeneration of articular cartilage and meniscus was case-dependent but statistically significant improvement was found in specific macroscopic and histological parameters. Such parameters included colour, rigidity, cell distribution and hyaline quality of the refill tissue as well as the structure of subchondral bone.

Mummified fetus in the thoracic cavity of a domestic short-haired cat.

This short communication describes the diagnosis, treatment, and clinical course of a domestic short-haired cat with diaphragmatic hernia in which the herniated structure in the thoracic cavity contained a mummified fetus. The cat was pregnant when rescued from the street and, days later, gave birth without abnormalities. Some months later, during an ovariohysterectomy, an abnormal localization of the uterus was observed, and at that time the case was referred to our center. A thoracic radiograph showed an abnormal thoracic mass cranial to the heart. The main suspicion was the presence of a thoracic hernia with the uterus herniated and containing a mummified fetus. A thoracotomy was performed to confirm the nature of the mass and reduce the diaphragmatic hernia. Although this clinical case is quite rare, a mummified fetus can be observed in thoracic hernias.

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.

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.