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Cell damage, one of critical issues in the bioreactor design for animal cell culture, is caused mainly from the bubble bursting at the free surface subjected to strong extensional flows. In this work, extensive computational studies are performed to investigate bubble bursting process in great details. Extensive numerical simulations are performed for a wide range of bubble diameters (from 0.5 to 6 mm) and the surface tension values (from 0.03 to 0.072 N/m), with which effects of the bubble size and surfactant (PF68) concentration on the hydrodynamic stress are investigated. For all the cases, the maximum extensional stress appears at the instance when receding films impact each other at the bottom of the bubble. A model equation based on numerical simulations is presented to predict the maximum extensional stress as a function of the bubble diameter and the surface tension. The bubble diameter has turned out to contribute significantly the maximum hydrodynamic extensional stress. In addition, the bubble collapsed time and the affected volume around a bubble subjected to the critical extensional stress are investigated. The extensional stress estimation is reported as a function of the bubble size and the surface tension. The influence of the bubble size on the maximum stress dominates and extensional stress reaches up to the order of 10 Pa for bubble size of 0.5 mm. [ABSTRACT FROM AUTHOR]

Conventional USP optimization concepts have led to increasing product titers and significantly raised impurity profiles. Varying compositions of cultivation broth present additional challenges in downstream processing. This work addresses the resulting problems by combining methodical and technological approaches. The methodical approach is based on the integration of upstream and downstream process development by extending the range of USP optimization. In addition to high product titer, cell numbers and product quality, the composition and concentration of host cell proteins were taken into account. The feasibility of this approach was demonstrated within this work. A model CHO cell line producing IgG was used to develop a medium, which generates higher titers and cell numbers as well as a lower amount of HCP. The new composition of HCP was designed to simplify subsequent purification steps. Experiments were statistically planned and the results were validated, all resulting in higher product titers but lower HCP levels. In the next step, the influence of the cultivation and cell harvest operations on the impurities was investigated. Centrifugation, filtration and aqueous two phase extraction were evaluated regarding their ability to separate cells and HCP without product loss. ATPE distinguished itself as the cell harvest operation of choice by high cell separation efficiency, high yields, and a substantial removal of HCP which could not be met by centrifugation and filtration. [ABSTRACT FROM AUTHOR]

In vitro -derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture. [ABSTRACT FROM AUTHOR]

In this paper an overview of bioreactors for animal cell cultivation is presented. Their major characteristics and mode of action are described together with potential (dis)advantages considering biotech production. In the final part of the paper we discuss strategies of bioreactor selection regarding various issues and requirements for setting up a bioprocess. Certain trends and prospects of bioreactor development are also pointed out [ABSTRACT FROM AUTHOR]

We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in the cell culture medium. Focusing on the fact that animal cells consume glucose and produce lactate under anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays were integrated into the PAD. The PAD was designed to have three laminated layers. By using a double-sided adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s reaction, the colorless chromophore is converted to a blue-colored product, generating concentration-dependent signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the smartphone-embedded camera. Images of the detection zones were acquired using a mobile application and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format, we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD (0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to 0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose that the optical sensing system developed is feasible for practical monitoring of animal cell culture. [ABSTRACT FROM AUTHOR]

In this work, two external filtration devices for cell retention, the commercial alternating tangential flow (ATF) system and a tangential flow filtration (TFF) setup driven by a bearingless centrifugal pump were compared. Physical characterization of the bioreactor hydrodynamics revealed significantly smaller maximum stress values in the ATF at similar mixing and oxygen mass transfer rates. Suitable operating parameters in terms of aeration, stirring and operation of the retention device were determined. Steady culture of Chinese hamster ovary (CHO) cells at 20, 40 and 60 × 10 6 cells/mL for at least one week was achieved in perfusion at a fixed harvest rate of one reactor volume per day for both setups. Contrary to the ATF, considerable retention (up to 50%) of the produced monoclonal antibody in the TFF was observed, however without significant difference in product quality. The integration of physical and cell culture characterization allows the comprehensive assessment of cell culture performance. The presented approach can serve as a general procedure to develop a scalable mammalian cell perfusion culture. [ABSTRACT FROM AUTHOR]

Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made from different polymers. Since the microcapsules can be easily retained in the bioreactor without the need for a cell separation device, high cell densities were achieved with a maximum of 4 × 107 cell/mlmicrocapsules, corresponding to a colonisation of 5% of the internal capsule volume. Measurement of microcapsule integrity and mechanical resistance showed that alginate-PLL microcapsules are not suitable for perfusion cultures since they are very sensitive to media composition, mainly the presence of non-gelling ions that have a higher affinity for alginate than PLL and Ca2+, leading to the leakage of PLL and Ca2+, and to microcapsule rupture. [ABSTRACT FROM AUTHOR]

There is an urgent critical need for the development of clinically relevant tissue-engineered large bone substitutes that can promote early vascularization after transplantation. To promote rapid blood vessel growth in the engineered tissue, we preincubated aortic fragments, as well as, co-cultures of aortic fragments and osteoblast-like cells in matrigel-filled PLGA scaffolds before implantation into the dorsal skinfold chambers of balb/c mice. Despite an acceptable and low inflammatory response, preincubated aortic fragments accelerate early angiogenesis of tissue-engineered constructs; the angiogenesis was found to occur faster than that observed in previous studies. Thus, the time-period for achieving a denser microvascular network could be reduced to half. In addition, co-culture with osteoblasts enhances this angiogenic effect significantly (against preincubated aortic fragments alone). During the preincubation period, aortic fragments begin to form a network of vessel-like structures additionally supported by osteoblast-like cells. After transplantation, further development of a dense microvasculature continues rapidly. Therefore, preincubation of aortic fragments, especially in co-culture with osteoblast-like cells, in 3D extracellular matrices supports the rapid vascularization of tissue-engineered constructs. This method is a promising approach to establish a dense microvascular network in these constructs. [ABSTRACT FROM AUTHOR]

Different techniques have been employed to produce chitosan nanoparticles; thus, suitable alternatives and easy-handling production methods are highly desired. We used a tank reactor with baffles and mechanical stirring with Cowles impellers that can feasibly be up-scaled and allows for the production of homogenous chitosan nanoparticles. First, we explored the effects of temperature on the production of chitosan (CHI)/pentasodium tripolyphosphate (TPP) nanoparticles. We compared the effect of gradual temperature decrease between 40 to 55 °C with the effect of isothermal processes (25, 45 and 55 °C). CHI/TPP nanoparticles produced by the gradual temperature decrease technique had polydispersity indices (PDIs) that were significantly smaller than the nanoparticles produced by isothermal processes. To further control the production process, we employed an experimental design to determine the effects of CHI and TPP concentrations and CHI/TPP mass ratios in the gradual temperature decrease technique. Under all conditions, the sizes of the CHI/TPP nanoparticles were on the nanoscale. Gene delivery capacities of the nanoparticles were evaluated; the plasmid pEGFP-N1 was incorporated (10 and 40% of the chitosan weight) into the nanoparticles and incubated with 293A cells. The in vitro transfection efficiencies were evaluated over 120 h and indicated sustained gene delivery at 10% pDNA. The polyplexes demonstrated some cytotoxicity after 120 h. These findings will contribute to the development of processes for generating CHI/TPP nanoparticles for gene delivery applications. [ABSTRACT FROM AUTHOR]

Compared to other systems biology tools, genomic microarrays represent a mature platform that allows for facile access to the internal biological mechanisms of cell culture. While the large datasets generated by microarrays are a potential goldmine of information, ironically, it is their large size that also makes data-mining a cumbersome task. This can get further complicated by unavoidable batch effects generated when different datasets are combined. Furthermore, gene expression profiles are dependent on combinations of various complex intracellular events and as such identifying the signals primarily related to the phenotype of interest poses a substantial challenge. In this study we addressed these issues by utilizing a workflow that allows adjustment of time-course gene expression datasets for batch effects and incorporates the use of sparse partial least squares analysis to identify specific genes of interest. We were able to identify a set of relevant genes that displayed a strong correlation with cell growth in fed-batch bioreactors under different nutrient compositions. By conducting further biological network analysis, we identified four transcriptional regulators, namely ATP7B, SREBP1, SCAP and INSIG2 that are responsible for regulation of these genes and are likely important drivers for cell growth differences in response to change in nutrient composition. [ABSTRACT FROM AUTHOR]

Vaccine manufacturing processes are designed to meet present and upcoming challenges associated with a growing vaccine market and to include multi-use facilities offering a broad portfolio and faster reaction times in case of pandemics and emerging diseases. The final products, from whole viruses to recombinant viral proteins, are very diverse, making standard process strategies hardly universally applicable. Numerous factors such as cell substrate, virus strain or expression system, medium, cultivation system, cultivation method, and scale need consideration. Reviewing options for efficient and economical production of human vaccines, this paper discusses basic factors relevant for viral antigen production in mammalian cells, avian cells and insect cells. In addition, bioreactor concepts, including static systems, single-use systems, stirred tanks and packed-beds are addressed. On this basis, methods towards process intensification, in particular operational strategies, the use of perfusion systems for high product yields, and steps to establish continuous processes are introduced. [ABSTRACT FROM PUBLISHER]

Hepatocyte transplantation is a potential therapy for treating various liver diseases. However, oxygen shortage leading to loss of hepatocyte function becomes a limitation following hepatocyte transplantation. To overcome this problem, we developed a hybrid organoid, consisting of growth factor (GF)-immobilizable gel particles combined with hepatocytes. Benefits of the hybrid organoid were evaluated in three groups: (i) hybrid organoid consisting of cells and GF-immobilizable gel particles (HG-C); (ii) hybrid organoid consisting of cells and gel particles (G-C); and (iii) cells suspended in collagen (C–C). We found liver-specific functions of HG-C were maintained longer than in the other conditions during in vitro culture. Furthermore, after transplantation, HG-C was effective in maintaining viability of transplanted hepatocytes and promoting angiogenesis around the hepatocytes. In summary, transplantation of HG-C is a potential method for future liver tissue engineering. [ABSTRACT FROM AUTHOR]

Stainless steel bioreactors increasingly fall behind to their disposable counterparts in pharma research as they do not require cleaning or sterilization. This led company ATMI LifeSciences to develop the “Nucleo™”. Original in design, this disposable bioreactor comprises a rectangular parallelepiped plastic bag stirred by a paddle revolving in elliptic pendulum motion. Studies covering this bioreactor showed good homogeneity of culture medium as well as good productivity for animal cell cultures. To further explain these good performances, the flow inside the “Nucleo™” had to be resolved. This paper focuses on the mean flow description, computed from stereo-PIV measurements performed in 20 verticals covering the whole volume of a 50 dm 3 Nucleo™ bioreactor. As the flow is already turbulent in the chosen agitation conditions, its dimensionless mean velocity field does not vary with the paddle rotational speed. Mean flow pattern exhibits an axial symmetry – same flow is observed in opposite quarters of the tank – and can be described as a three-dimensional helix coiled on itself to form a distorted horizontal torus which covers the whole tank volume. Mean velocity value is on average doubled in the cone swept by the paddle, and its two horizontal components are twice higher than its vertical ones. However, mean velocity remains significant everywhere and, in particular, no stagnant area is observed in tank corners. Our results thus confirm previous studies observations. [ABSTRACT FROM AUTHOR]

To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 μm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 μm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 μg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 μm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel. [ABSTRACT FROM AUTHOR]

The main aim of this study was to investigate the potential role of levan polysaccharide in developing polysaccharide-based anticancer therapeutics. Moreover, by periodate oxidation, chemically modified forms of levan harboring increasing amounts of aldehyde groups were obtained and characterized. All the samples were found to be biocompatible when treated with the mouse fibroblast cell line L929. Anti-cancer activities of levan and its derivatives were investigated in A549 (human lung adenocarcinoma), HepG2/C3A (human liver hepatocellular carcinoma), AGS (human gastric adenocarcinoma), and MCF-7 (human breast adenocarcinoma) cell lines and were found to depend on the dose as well as on the cell type. The observed decrease in cancer cell viability was shown to be due to apoptosis via luminogenic-based caspase-3/7 activity assay. This study established the high potential of these levan-based polymers in developing polysaccharide-based nanocarrier systems for anti-cancer drugs. [ABSTRACT FROM AUTHOR]

Since protein-protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named "split FAK", the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase. [ABSTRACT FROM AUTHOR]

Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 105.875 TCID50 (median tissue culture infective dose)ml-1. To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5mM glucose, 5mM fructose and 25mM sucrose. Spinner culture confirms these data; virus titer was 107.375 TCID50ml-1. In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3gl-1 Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 107.275 TCID50ml-1. This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM+2% FCS. [ABSTRACT FROM AUTHOR]

Highlights: [•] A surface-immobilized polyamidoamine dendrimer was used in culture of hMSCs. [•] hMSCs on dendrimer surface formed cell aggregates through migration and division. [•] Cell aggregation enhanced fate determination toward a cardiomyogenic lineage. [•] Aggregate passage without enzymatic digestion gave more cardiomyocyte-like cells. [•] Our method induces initiation of cardiomyogenic lineage commitment of hMSCs. [ABSTRACT FROM AUTHOR]

Highlights: [•] mESC were cultivated on microcarriers in a fully controlled stirred tank reactor. [•] The influence of dissolved oxygen (DO) on cell growth and pluripotency was investigated. [•] The influence of different residence times (12, 24, 32, 48 and 96h) was evaluated. [•] Continuous perfusion with cell retention under 20% DO maximized cell production/numbers. [•] Cells retained pluripotency and differentiation potential throughout the process. [Copyright &y& Elsevier]

Highlights: [•] Oxygen mass transfer performance of unbaffled stirred tanks is presented. [•] Unbaffled vessels appear able to satisfy the oxygen demand of animal cell cultures. [•] Influence of impeller geometry on oxygen mass transfer performance is investigated. [ABSTRACT FROM AUTHOR]

Abstract: Skin injuries, congenital lesions, melasma, Addison's disease and many pigment abnormalities prompt us to search for an effective whitening agent. Ideal whitening agent is a natural compound that can inhibit melanogenesis and has no cytotoxic effects. In a previous study, we have developed an optimum method for the production and characterization of ectoine from a halophilic bacterium isolated from a salt environment in Taiwan was identified as Marinococcus sp. In the present study, we screened the whitening properties of the biosynthesized ectoine using mouse and human melanoma cell lines, B16-F0 and A2058. Here, we examined the cell viabilities of melanoma cells after ectoine treatment at various concentrations up to 500μM. Also, we addressed the melanin synthesis of melanoma cells after treatment with ectoine. The inhibitory effects of ectoine on tyrosinase activity were assessed in both mushroom tyrosinase and cellular tyrosinase. Furthermore, we investigated the type of inhibition of mushroom tyrosinase using Lineweaver–Burk enzyme kinetic. The melanogenesis-related gene expression (tyrosinase, TRP1, TRP2 and MITF) and their protein secretion were determined by the assays of quantitative real-time PCR and western blots, respectively. Our results demonstrated that ectoine is a safe and effective whitening agent, inhibited melanin synthesis, reduced both mushroom tyrosinase and cellular tyrosinase, and had various inhibitory effects on the expressions of melanogenesis-related genes and secretion of proteins in mouse and human melanoma cell lines. Thus, we suggest that ectoine can serve as a useful and safe new agent in cosmetic and clinical applications. [Copyright &y& Elsevier]

Highlights: [•] Various cereals were used as substrates to enhance functional compound production. [•] These substrates enhanced the production of polyphenols and triterpenoids. [•] Cereals based substrates promoted the antitumor and antioxidant activities. [•] Oat-cultured mycelial extract showed stronger overall antioxidant properties. [•] The extract greatly reduced the viability of MCF-7 cancer cells and HepG2 cells. [ABSTRACT FROM AUTHOR]

BACKGROUND Improvement of the mammalian cell culture process is based on the implementation of culture strategies with higher productivities, such as fed-batch or perfusion processes. The efficient development of these culture strategies depends strongly on the availability of suitable monitoring tools to determine the activity of cell culture and adjust nutrient feeding correspondingly. RESULTS In this study oxygen uptake rate ( OUR), cell density and glucose concentration on-line monitoring systems are applied to control fed-batch processes. Different combinations of these monitoring systems were used to determine cell growth and metabolic activity of cell culture in order to adjust substrate feeding in a balanced manner. Application of these monitoring tools in fed-batch processes allowed extension of the exponential growth of hybridoma cells up to 2-3-fold and increased antibody productivity from 84 to 168% with respect to conventional batch culture. CONCLUSIONS The three control strategies tested have been successful in providing improvement of the fed-batch culture of the hybridoma cell line used. Among the different alternatives studied, the use of OUR on-line monitoring to simultaneously determine cell growth and metabolic activity appears to be the best method to adjust substrate feeding owing to its simplicity and robustness. © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Adherent A431, BHK-21, and C2C12 cells were cultured on a flexible interface formed between two immiscible liquid phases: (i) hydrophobic perfluorodecalin (PFD) and (ii) aqueous culture medium (DMEM). BHK-21 cells formed multicellular aggregates characterized by irregular shapes. A431, as well as C2C12 cells, grew as tight multicellular sheets of 3-D cells. Enhanced mass transfer and facilitated access of the cells to the O dissolved in PFD/DMEM by approx. 250 % and thereby increased the density of BHK-21 cells. Thus the liquid/liquid system is a simple, ready-to-use, and fully scalable (independent of vessel shapes); consequently it is a method for 3-D cultures of adherent animal cells in which the growth of anchorage-dependent cells is not limited by confluence effect. [ABSTRACT FROM AUTHOR]

Highlights: [•] The production of HIV-1 VLPs in mammalian suspension cultures is reported. [•] HEK 293 cell growth is optimized by addition of non-animal derived supplements using DoE. [•] VLP titers were increased 2.4-fold obtaining 2.7×109 VLPs/mL. [ABSTRACT FROM AUTHOR]