Medium Replenishment Research Articles

Enhanced production yields of rVSV-SARS-CoV-2 vaccine using Fibra-Cel® macrocarriers.

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.

A technique for enhancing tight oil recovery by multi-field reconstruction and combined displacement and imbibition

A seepage-geomechanical coupled embedded fracture flow model has been established for multi-field coupled simulation in tight oil reservoirs, revealing the patterns of change in pressure field, seepage field, and stress field after long-term water injection in tight oil reservoirs. Based on this, a technique for enhanced oil recovery (EOR) combining multi-field reconstruction and combination of displacement and imbibition in tight oil reservoirs has been proposed. The study shows that after long-term water flooding for tight oil development, the pressure diffusion range is limited, making it difficult to establish an effective displacement system. The variation in geostress exhibits diversity, with the change in horizontal minimum principal stress being greater than that in horizontal maximum principal stress, and the variation around the injection wells being more significant than that around the production wells. The deflection of geostress direction around injection wells is also large. The technology for EOR through multi-field reconstruction and combination of displacement and imbibition employs water injection wells converted to production and large-scale fracturing techniques to restructure the artificial fracture network system. Through a full lifecycle energy replenishment method of pre-fracturing energy supplementation, energy increase during fracturing, well soaking for energy storage, and combination of displacement and imbibition, it effectively addresses the issue of easy channeling of the injection medium and difficult energy replenishment after large-scale fracturing. By intensifying the imbibition effect through the coordination of multiple wells, it reconstructs the combined system of displacement and imbibition under a complex fracture network, transitioning from avoiding fractures to utilizing them, thereby improving microscopic sweep and oil displacement efficiencies. Field application in Block Yuan 284 of the Huaqing Oilfield in the Ordos Basin has demonstrated that this technology increases the recovery factor by 12 percentage points, enabling large scale and efficient development of tight oil.

Effect of exposure conditions on chemical properties of materials for surgical endodontic procedures.

This study investigated the role of aging and changes in environmental conditions on selected properties of a prototype radiopacified calcium silicate-based cement (TZ-base) with or without incorporation of silver nanoparticles or bioactive glass, and two commercial materials, Biodentine and intermediate restorative material. Materials were immersed in ultrapure water or fetal bovine serum for 28 days and were characterized with scanning electron microscopy and energy dispersive x-ray analysis. Immersion media were either replaced weekly or not replenished at all and were assessed for alkalinity and calcium release after 1, 7, 14, 21, and 28 days; antibacterial effect against 2-day monospecies biofilms; and cytotoxicity by the 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide assay after 1, 7, or 28 days. Alkalinity, calcium release, antibacterial activity, and cell cytotoxicity increased over time when the medium was not changed but decreased with medium replenishment. Immersion in fetal bovine serum resulted in lower alkalinity, less bactericidal properties, and lower cytotoxicity of prototype cements and Biodentine than did water immersion. Biodentine and 20% bioactive glass-containing cement had overall lower alkalinity, calcium release, and antibacterial activity than TZ-base, and Biodentine was less cytotoxic than TZ-base. In conclusion, exposure conditions and cement modifications significantly affected materials' leaching properties. Exposure conditions warrant consideration when evaluating cements' clinical properties.

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

A superhydrophobic chip integrated with an array of medium reservoirs for long-term hanging drop spheroid culture

Hanging drop (HD) is one of the most popular methods used for forming three-dimensional (3D) cell spheroids. However, conventional hanging drop systems are only applicable for short-term spheroid culture due to their inconvenience in exchanging cell culture media. Here we present a medium-reservoir-integrated superhydrophobic (MRI-SH) chip for long-term HD spheroid cultures. The device consists of two main components: i) a patterned superhydrophobic (SH) surface containing an array of wettable spots which anchor arrays of droplets of cell suspension, and ii) an array of chambers that serve as medium reservoirs, both interconnected via an array of thru-holes. This configuration provides two distinct advantages over conventional HD configurations: i) the high wettability contrast of the SH pattern on the chip leads to the formation and adhesion of nearly spherical hanging droplets on its surface, which minimizes interactions between the liquid and the substrate; ii) the integrated chambers provide large volumes of medium to maintain longer culture durations. Using this device, spheroids of MHCC97H cells were successfully formed, and the cultured spheroids could maintain high viability for up to 30 days and exhibited enhanced spheroid morphology compared to those cultured in the conventional HD systems. Statement of significanceThis paper presents a medium-reservoir-integrated superhydrophobic hanging drop (HD) platform for the long-term culture of spheroids with enhanced morphology. By monolithically integrating medium reservoirs and a patterned SH surface into a single device, this HD platform can not only produce high-quality spheroids, but also permit them to sustain high viability for up to 30 days without the need for tedious medium replenishment. We believe that such a platform will be valuable in a wide range of biological or biomedical applications, including tissue engineering, regenerative medicine, and drug discovery.

Effect of Light Intensity and Wavelength on Biomass Growth and Protein and Amino Acid Composition of Dunaliella salina

Dunaliella salina is a halotolerant, photoautotrophic marine microalga and one of the richest sources of natural carotenoids but also shows potential as a novel food source with high protein quality. This study sought to optimise the production of biomass, protein and amino acids from D. salina, alongside carotenoids using a two-stage cultivation approach based on the use of light of different intensities and quality, i.e., white, red and blue LED light. In stage 1, four white LED light intensities were tested. In stage 2, the same four light intensities from either blue or red LEDs were applied once exponential growth ceased and cells reached the stationary phase under white LED light in stage 1. Remarkably, both biomass concentration and biomass productivity showed a 1.3–1.7-fold increase in stage 2, without medium replenishment, while protein concentration and protein productivity showed an ~1.1-fold increase. The amino acid content and amino acid index remained unchanged from stage 1 to stage 2, and minimum difference was found across different light intensities. Overall, D. salina delivered so-called high protein quality, with an essential amino acid index (EAAI) of 0.99, and red light, which has previously been shown to increase carotenoid production, boosted further biomass production over and above white light, at all light intensities tested.

Primary Human Colonic Mucosal Barrier Crosstalk with Super Oxygen-Sensitive Faecalibacterium prausnitzii in Continuous Culture

The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

Translation of small-scale CAR-T cell manufacturing methods to a clinical-scale production platform

Background & Aim CAR-T cells have displayed potent antitumor effects in liquid but not in solid tumors. Variables like technical and immuno-oncological factors contribute to the modest clinical effect of CAR-Ts in solid tumors. To improve their function, manufacturing conditions have been optimized to yield a favorable CAR-T cell phenotype. A product enriched in stem cell- and central memory T cells (TCM) is believed to improve durability of antitumor responses and engraftment of the adoptive cells, where highly differentiated T cell products may be prone to exhaustion and senescence. Finding optimal manufacturing conditions requires testing of media components and selection of appropriate bioreactor system for scale-up. To avoid the substantial costs of multiple bioreactor runs, screening typically relies on flask scale. We set out to determine the predictive ability of the flask-scale screening on the process performance in clinical scale. Glioblastoma-specific CAR-Ts were produced from six healthy donors in various medium compositions and compared to those produced in closed-system bioreactors. Methods, Results & Conclusion Lentivirally-transduced CAR-T cells were cultured in flasks in Miltenyi TexMACS medium with or without human AB serum (HABS) and either IL2 or IL7 and IL15. In the bioreactor process, T cells were purified and transduced in the Miltenyi CliniMACS Prodigy system, then further expanded in IL7 and IL15 containing HABS-free media in a suspension bioreactor. We characterized the cells by CAR expression level, viability, expansion rate, memory phenotype and in vitro functionality. All CAR-T products were highly viable and specifically cytotoxic, though biological differences between donors can cause variation. The main difference between culture conditions was in expansion rates. The phenotype was substantially effector memory T cell-biased in all flask cultures. The presence or absence of HABS rather than cytokine selection appeared to be the main determinant of the product characteristics. In our bioreactor scale pilot the lead media composition (IL7, IL15, no HABS) yielded a markedly different product, seen as superior expansion and significant enrichment of the TCM fraction. Our results suggest that small-scale screening may translate poorly to bioreactor systems. Such factors as medium replenishment, oxygenation, cell density and culture vessel rocking may have an impact on the product. Early-stage scale-up and testing using the final platform is essential for product success.

Development of a longevous two-species biophotovoltaics with constrained electron flow

Microbial biophotovoltaics (BPV) offers a biological solution for renewable energy production by using photosynthetic microorganisms as light absorbers. Although abiotic engineering approaches, e.g., electrode modification and device optimization, can enhance the electrochemical communication between living cells and electrodes, the power densities of BPV are still low due to the weak exoelectrogenic activity of photosynthetic microorganisms. Here, we develop a BPV based on a d-lactate mediated microbial consortium consisting of photosynthetic cyanobacteria and exoelectrogenic Shewanella. By directing solar energy from photons to d-lactate, then to electricity, this BPV generates a power density of over 150 mW·m−2 in a temporal separation setup. Furthermore, a spatial-temporal separation setup with medium replenishment enables stable operation for over 40 days with an average power density of 135 mW·m−2. These results demonstrate the electron flow constrained microbial consortium can facilitate electron export from photosynthetic cells and achieve an efficient and durable power output.

Implementation of UV-based advanced oxidation processes in algal medium recycling

Algae show great potential as sustainable feedstock for numerous bioproducts. However, large volume of water consumption during algal biomass production makes that the culture media recycling is a necessity due to economic and environmental concern. To avoid the negative effect of enriched organic matters in the harvested culture media, pre-treatment prior to medium replenishment and reuse is required. In this study, degradation of algenitic organic matters (AOM) in the culture media by UV-based photolysis processes (i.e., direct UV, UV/peroxydisulfate (PDS), UV/H2O2, and UV/NH2Cl) was explored. The results showed that UV, UV/PDS, UV/H2O2 and UV/NH2Cl caused a decrease of SUVA for 29.9%, 35.4%, 40.45%, and 22.6%, respectively, though the organic matter was almost not mineralized. Fluorescence excitation-emission matrix combined with parallel factor analysis indicated that UV/PDS and UV/H2O2 degraded 47.26%–56.31% of the fulvic-like and humic-like fractions in AOM. Powder activated carbon absorption and growth evaluation for the AOPs-treated media indicated that UV/PDS and UV/H2O2 processes not only could remove the growth inhibitors in the media, but were also beneficial to the algae growth. These results suggested that UV/PDS and UV/H2O2 could effectively degrade the hydrophobic components in AOM and converted the growth inhibition fraction of AOM in the recycled media into nutrient source for algal growth. Different from the general application of UV-based AOP in the wastewater treatment, this study provided an innovative idea about how to pre-treat AOM in the media recycling: utilization rather than removal, which was a more sustainable and environment-friendly technology.

On the need for more realistic experimental conditions in laboratory-based microbiologically influenced corrosion testing

Microbiologically influenced corrosion (MIC) encompasses a complex suite of processes that can result in costly and dangerous acceleration of corrosion rates. However, the complexity of MIC, and the wide range of conditions in which it occurs in the natural environment, has resulted in researchers performing tests under a wide range of laboratory conditions. This scattergun approach can potentially lead to ambiguous results and can compromise the ability to compare data from different tests. Therefore there is a need for the corrosion community to develop more realistic and standardised approaches to laboratory-based testing. This paper seeks to illustrate the challenges by examining a range of test parameters that can influence the results of MIC studies, using Escherichia coli and carbon steel as a model system. The experimental parameters considered here include test media composition, immersion temperature, immersion time and medium replenishment. It is shown that each of these parameters can exert a significant effect on the outcome of the study, thus supporting the contention that more realistic testing procedures are required to develop a better understanding of MIC processes and ultimately allow predictive management of MIC.

Analysis of Tubespins as a suitable scale-down model of bioreactors for high cell density CHO cell culture.

High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490-499, 2017.

Development of an optimal light-feeding strategy coupled with semi-continuous reactor operation for simultaneous improvement of microalgal photosynthetic efficiency, lutein production and CO2 sequestration

In addition to leveraging some environmental and energy benefits, microalgae can also be used as a potentially sustainable source for carotenoids like lutein for healthcare applications. As light influences biosynthesis of lutein in microalgae, it would be interesting to investigate the role of incremental illumination in enhancing photosynthetic efficiency and consequent lutein production, while sequestering CO2 simultaneously. Thus, in this study, an optimal light feeding strategy coupled with a semi-continuous reactor operation was developed for achieving the above-mentioned goals. At a light intensity of 260μmolm−2s−1 as optimized in batch mode, baseline lutein productivity of 4.32mgL−1d−1 was obtained, when culturing Chlorella minutissima under optimal process conditions. On switching over to incremental illumination modes, the linear light-feeding (Strategy-III) resulted in higher lutein productivity of 5.35mgL−1d−1 with photosynthetic efficiency of 8.38%. When Strategy-III was integrated with semi-continuous mode involving 20% medium replenishment, lutein productivity, photosynthetic efficiency and CO2 sequestration rate were further enhanced by 19%, 41% and 34% respectively. Moreover, on this process optimization and integration, light-energy consumption was significantly reduced by 32%, in comparison with constant-illumination. Thus, this optimal production strategy resulted in significantly higher lutein productivity, content and photosynthetic efficiency, as compared to the relevant studies reported in literature.

Cellular uptake and fate of fibroin microspheres loaded with randomly fragmented DNA in 3T3 cells.

Purified fibroin protein can be obtained in large quantities from silk fibers and processed to form microscopic particles as delivery vehicles for therapeutic agents. In this study, we demonstrated that fibroin microspheres were taken up by 3T3 cells, localized in the nonlysosomal compartment, and secreted from the cytoplasm after medium replenishment. DNA-loaded microspheres were taken up by >95% of 3T3 cells. DNA cargo had no influence on the intracellular trafficking of microspheres, while fluorescently labeled cargo DNA was observed in the lysosomal compartment and in the microspheres. These results indicate that fibroin microspheres can travel through 3T3 cells without making any contact with the lysosomal compartments. The amount of DNA loaded in the microspheres taken up by 3T3 cells was estimated up to 831.0 pg/cell. Thus, fibroin microspheres can deliver a large amount of randomly fragmented DNA (<10 kb) into the cytoplasmic compartment of 3T3 cells.

Impact of cyclic pH shifts on carbon monoxide fermentation to ethanol by Clostridium autoethanogenum

Fermentation medium and fermentation pH are among the main parameters that control acidogenesis and solventogenesis in clostridia during CO bioconversion. The approach of cyclic pH shifts from high (pH 5.75) to low (pH 4.75) levels, in continuous gas-fed bioreactors, allowed Clostridium autoethanogenum to consume and convert CO and the produced acetic acid from CO to alcohols using one single bioreactor. Increasing amounts of ethanol accumulate after each pH shift cycle, while acids are used up and converted during each cycle. In this study, a total of three cyclic pH shifts along with partial medium replenishment, resulted in a high ethanol production, reaching a concentration of 7143mg/L. The mixotrophic fermentation using xylose as additional carbon source besides carbon monoxide allowed to conclude that xylose is a preferred carbon source compared to CO and that it allows to increase the cell mass concentration (421mg/L).

Microalgal TAG production strategies: why batch beats repeated-batch.

BackgroundFor a commercially feasible microalgal triglyceride (TAG) production, high TAG productivities are required. The operational strategy affects TAG productivity but a systematic comparison between different strategies is lacking. For this, physiological responses of Nannochloropsis sp. to nitrogen (N) starvation and N-rich medium replenishment were studied in lab-scale batch and repeated-batch (part of the culture is periodically harvested and N-rich medium is re-supplied) cultivations under continuous light, and condensed into a mechanistic model.ResultsThe model, which successfully described both strategies, was used to identify potential improvements for both batch and repeated-batch and compare the two strategies on optimized TAG yields on light (amount of TAGs produced per mol of supplied PAR photons). TAG yields on light, for batch, from 0.12 (base case at high light) to 0.49 g molph−1 (at low light and with improved strain) and, for repeated-batch, from 0.07 (base case at high light) to 0.39 g molph−1 (at low light with improved strain and optimized repeated-batch settings). The base case yields are in line with the yields observed in current state-of-the-art outdoor TAG production.ConclusionsFor continuous light, an optimized batch process will always result in higher TAG yield on light compared to an optimized repeated-batch process. This is mainly because repeated-batch cycles start with N-starved cells. Their reduced photosynthetic capacity leads to inefficient light use during the regrowth phase which results in lower overall TAG yields compared to a batch process.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0475-4) contains supplementary material, which is available to authorized users.

Implementation of flocculation and sand filtration in medium recirculation in a closed microalgae production system

Recycling growth medium is a necessity to reduce production cost and ecological foot print of large scale microalgae production systems. To prevent contamination and/or enrichment with particulate matter, medium recycling requires pre-treatment of the centrifuge supernatant (centrate), prior to medium replenishment and re-use. In this study, we investigated the applicability of high pH induced flocculation and/or sand filtration to interface with an existing microfiltration setup in order to prepare recycled growth medium for the mass cultivation of marine microalgae. Sand filtration partly alleviated the burden on subsequent microfiltration, but proved to be particularly useful to remove high pH induced flocs from the centrate. Combination of both techniques resulted in a removal of 78±18% of particles, resulting in an improvement of ‘modified fouling indices’ by 75±19%. Despite a partial to complete removal of remaining nutrients such as phosphate, calcium and magnesium during treatment, a cost saving of 72% compared to a scenario with fresh medium can be achieved.

Stratification of yeast cells during chronological aging by size points to the role of trehalose in cell vitality.

Cells of the budding yeast Saccharomyces cerevisiae undergo a process akin to differentiation during prolonged culture without medium replenishment. Various methods have been used to separate and determine the potential role and fate of the different cell species. We have stratified chronologically-aged yeast cultures into cells of different sizes, using centrifugal elutriation, and characterized these subpopulations physiologically. We distinguish two extreme cell types, very small (XS) and very large (L) cells. L cells display higher viability based on two separate criteria. They respire much more actively, but produce lower levels of reactive oxygen species (ROS). L cells are capable of dividing, albeit slowly, giving rise to XS cells which do not divide. L cells are more resistant to osmotic stress and they have higher trehalose content, a storage carbohydrate often connected to stress resistance. Depletion of trehalose by deletion of TPS2 does not affect the vital characteristics of L cells, but it improves some of these characteristics in XS cells. Therefore, we propose that the response of L and XS cells to the trehalose produced in the former differs in a way that lowers the vitality of the latter. We compare our XS- and L-fraction cell characteristics with those of cells isolated from stationary cultures by others based on density. This comparison suggests that the cells have some similarities but also differences that may prove useful in addressing whether it is the segregation or the response to trehalose that may play the predominant role in cell division from stationary culture.

3T3 세포에 의한 피브로인 마이크로스피어의 흡수

Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.

Abstract 4330: Everolimus-induced hyperglycemia: Dual efficacy of metformin

Abstract The anti-cancer drug and mTOR inhibitor everolimus often causes hyperglycemia in patients. This side-effect potentially reduces its efficacy and may cause complications in the long term. The diabetes drug metformin, a mitochondrial complex 1 inhibitor, may counteract hyperglycemia while enhancing the anti-tumor efficacy of everolimus. Here, we investigated the growth inhibitory properties of both drugs and their combination in vitro with an emphasis on their effects on glucose metabolism. The breast cancer cell lines MCF-7, T47D and MDA-MB-231 were cultured in media containing 11 mM (high), 5.5 mM or 2.75 mM (low) glucose and treated with 1-10 mM metformin or 1-10 nM everolimus for 2 or 4 days. Proliferation, colony formation and apoptosis were measured. Intracellular effects were analyzed using glucose transporter 4 (GLUT4) staining. The Seahorse XF analyzer was used to measure mitochondrial respiration and glycolysis. Metformin (5 mM) added to high glucose medium reduced proliferation by 40-60%, depending on the cell line. Under low glucose conditions metformin reduced proliferation by 70-80%. The efficacy of everolimus in high glucose conditions was comparable to its efficacy in low glucose conditions. Combining metformin and everolimus resulted in at least 40% growth inhibition of the three cell lines under all glucose conditions. After 48h of treatment with 5 mM metformin basal mitochondrial respiration of all cell lines decreased to 20-40% of control. Simultaneously, glycolysis rate increased to 150-270% of control. Everolimus treatment (10 nM) for 48 h decreased mitochondrial respiration to the same extent, but did not affect glycolysis rate. Decreased GLUT4 membrane expression by at least 40% compared to the untreated control may explain this observation. The effects of metformin and everolimus on glucose metabolism were independent of glucose concentration. Since metformin strongly increased glycolysis rate, the effect of glucose depletion and replenishment on metformin induced cell death and proliferation inhibition was determined. 5 mM metformin treatment in high glucose medium for 4 days did not induce cell death in any cell line. However, the same treatment in low glucose media resulted in up to 50% apoptotic cells. Daily renewal of low glucose media reversed this effect. Metformin-induced growth inhibition in high or low glucose concentrations was not affected by medium replenishment in short term survival assays and clonogenic assays. Our study provides evidence that everolimus anti-tumor efficacy is not affected by high glucose concentrations. Metformin, however, was more effective at low glucose concentrations due to its glycolysis inducing capacity. Addition of metformin to everolimus treatment may be beneficial, especially under low glucose conditions as found within tumours. Moreover, this combination is appealing because of their complementary effects on cellular metabolism. Citation Format: Gerke Ariaans, Steven de Jong, Elisabeth G.E. de Vries, Mathilde Jalving. Everolimus-induced hyperglycemia: Dual efficacy of metformin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2014-4330