Improved Cell Growth Research Articles

Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli

Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression along with the deciphering of regulation of the related enzymes and an analogue transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli expressing the ALA biosynthetic pathway. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5′-untranslated regions (5′-UTRs). Then, the tricarboxylic acid (TCA) cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. Although the resulting strain showed a severe metabolic imbalance and low ALA production, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production. Thus, this precise tuning of the glyoxylate cycle in a quantitative manner should also enable efficient production of other value-added products derived from the TCA cycle.

Highly defined 3D printed chitosan scaffolds featuring improved cell growth

The augmented demand for medical devices devoted to tissue regeneration and possessing a controlled micro-architecture means there is a need for industrial scale-up in the production of hydrogels. A new 3D printing technique was applied to the automation of a freeze-gelation method for the preparation of chitosan scaffolds with controlled porosity. For this aim, a dedicated 3D printer was built in-house: a preliminary effort has been necessary to explore the printing parameter space to optimize the printing results in terms of geometry, tolerances and mechanical properties of the product. Analysed parameters included viscosity of the starting chitosan solution, which was measured with a Brookfield viscometer, and temperature of deposition, which was determined by filming the process with a cryocooled sensor thermal camera. Optimized parameters were applied to the production of scaffolds from solutions of chitosan alone or with the addition of raffinose as a viscosity modifier. Resulting hydrogels were characterized in terms of morphology and porosity. In vitro cell culture studies comparing 3D printed scaffolds with their homologous produced by solution casting evidenced an improvement in biocompatibility deriving from the production technique as well as from the solid state modification of chitosan stemming from the addition of the viscosity modifier.

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors.

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s−1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).

Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae

The aim of this study was to metabolically construct Saccharomyces cerevisiae for achievement of direct methanol utilization and value added product (mainly pyruvate) production. After successful integration of methanol oxidation pathway originated from Pichia pastoris into the chromosome of S. cerevisiae, the recombinant showed 1.04g/L consumption of methanol and 3.13% increase of cell growth (OD600) when using methanol as the sole carbon source. Moreover, 0.26g/L of pyruvate was detected in the fermentation broth. The supplementation of 1g/L yeast extract could further improve cell growth with increase of 11.70% and methanol consumption to 2.35g/L. This represents the first genetically modified non-methylotrophic eukaryotic microbe for direct methanol utilization and would be of great value concerning the development of biotechnological processes.

Key analogs of a uniquely potent synthetic vinblastine that contain modifications of the C20′ ethyl substituent

A key series of vinblastine analogs 7–13, which contain modifications to the C20′ ethyl group, was prepared with use of two distinct synthetic approaches that provide modifications of the C20′ side chain containing linear and cyclized alkyl groups or added functionalized substituents. Their examination revealed the unique nature of the improved properties of the synthetic vinblastine 6, offers insights into the origins of its increased tubulin binding affinity and 10-fold improved cell growth inhibition potency, and served to probe a small hydrophobic pocket anchoring the binding of vinblastine with tubulin. Especially noteworthy were the trends observed with substitution of the terminal carbon of the ethyl group that, with the exception of 9 (R=F vs H, equipotent), led to remarkably substantial reductions in activity (>10-fold): R=F (equipotent with H)>N3, CN (10-fold)>Me (50-fold)>Et (100-fold)>OH (inactive). This is in sharp contrast to the maintained (7) or enhanced activity (6) observed with its incorporation into a cyclic C20′/C15′-fused six-membered ring.

Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation

BackgroundMicrocarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types.MethodsA screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs.ResultsFirstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation.ConclusionsThis is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy.

Catan-ionic hybrid lipidic nano-carriers for enhanced bioavailability and anti-tumor efficacy of chemodrugs.

To date there has not been any report on catan-ionic hybrid lipidic nano-carriers, let alone a report on applying them to deliver insoluble anti-tumor drugs. Catan-ionic hybrid lipidic nano-carriers containing curcumin (CUR-C-HLN) inherit the merits of catan-ionic systems, hybrid lipidic systems and nano-structured carriers (the second-generation substitute of solid lipidic nano-systems). Catan-ionic surfactants increased microvesicle stabilization by producing unordered isometric clusters, enhanced absorptive amount as an inhibitor of enzyme and protein, improved tumor accumulation by cellular endocytosis and membranous fusion; hybrid lipids helped to obtain high drug content and low leakage by forming a less-organized matrix arrangement. CUR-C-HLN favorably changed absorptive and pharmacokinetic properties after oral and/or intravenous administrations; improved cell growth inhibition, apoptotic inducing and anti-invasion effects; enhanced antitumor efficiency and reduced cancerous growth. Catan-ionic hybrid lipidic nano-carriers provide an alternative good choice for effective delivery of anticancerous chemodrugs.

Polydeoxyribonucleotides (PDRNs) From Skin to Musculoskeletal Tissue Regeneration via Adenosine A2A Receptor Involvement.

Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production, and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo, and clinical studies (1990-2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area. A total of 29 studies were found by PubMed and www.webofknowledge.com searches: 20 were on skin (six in vitro, six in vivo, one vitro/vivo, seven clinical studies), while the other nine regarded bone (one in vitro, two in vivo, one clinical studies), cartilage (one in vitro, one vitro/vivo, two clinical studies), or tendon (one clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity, and reduced pain and inflammation, through the activation of adenosine A2A receptor. PDRNs are currently used for bone, cartilage, and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of three to a maximum of five times and they act trough the activation of A2A receptor. Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. J. Cell. Physiol. 232: 2299-2307, 2017. © 2016 Wiley Periodicals, Inc.

A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli

BackgroundEscherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.ResultsHere, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a “pre-defense” mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype.ConclusionsThe D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential “pre-defense” mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.

Control of cell proliferation in E-jet 3D-printed scaffolds for tissue engineering applications: the influence of the cell alignment angle.

The ideal 3D scaffold for biological applications has not yet been designed. Our aim is to better match the scaffold performance through fine control of the fabrication process. Here, we applied electro-hydrodynamic jet (E-jet) 3D printing technology using poly-(lactic-co-glycolic acid) (PLGA) solution to construct scaffolds for tissue engineering applications. We fabricated different scaffolds of 1 : 1, 1 : 2 and 1 : 3 aspect ratios and tested the biological compatibility of the 2D and 3D printed scaffolds with fibroblasts. The scaffolds were used to culture fibroblasts, the main cellular components of loose connective tissue. The results show that the E-jet printed scaffolds could guide and improve cell growth. These scaffolds were able to support cellular alignment and proliferation. The cell angle was consistent with the longitudinal direction of the scaffolds, potentially enhancing the wound healing performance. Thus, the potential of this flexible technology for the 3D printing of scaffolds for the engineering of tissues is extensive.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Evaluation of Anticancer Drugs with Kinetic and Static Alternating Cell Culture System

Variable bioreactors have been developed for the evaluation of anti-cancer drug efficacy. The Kinetic and Static Alternating Cell Culture System (KSACCS) combines the advantages of kinetic bioreactors and static cultures to improve cell growth by providing adequate metabolic support while minimizing shear-stress. In the current studies, the KSACCS in the ZYX Bioreactor could significantly increase the sensitivity of lung cancer cells (PLS008) and leukemia cells (HL60) to anticancer drugs Cisplatin and 5-FU by accelerating the apoptosis of cancer cells. It was also shown that excessive agitation of the cells could lead to severe cell damage, which resulted in a diminished sensitivity of anticancer drug evaluation, and co-culture systems tend to reduce the sensitivity of anticancer drug evaluation although it might better mimic in vivo conditions.

Preliminary Evaluation of Glyceric Acid-producing Ability of Acidomonas methanolica NBRC104435 from Glycerol Containing Methanol.

Some acetic acid bacteria produce large amounts of glyceric acid (GA) from glycerol in culture broth. However, methanol, which is a major contaminant of raw glycerol derived from the biodiesel fuel industry, sharply decreases cell growth and GA production [AMB Express, 3, 20, 2013]. Thus, we evaluated the methylotrophic acetic acid bacterium Acidomonas methanolica NBRC104435 for its ability to produce GA from glycerol containing methanol. This strain accumulated GA in its culture broth when 1-3 wt% glycerol was available as a carbon source. We observed improved cell growth and GA accumulation when 1 vol% methanol was added to the 3-5 wt% glycerol medium. The maximum concentration of GA was 12.8 g/L in medium containing 3 wt% glycerol plus 1 vol% methanol. In addition, the enantiomeric excess (ee) of the GA produced was revealed to be 44%, indicating that this strain converted glycerol to d-GA with a lower enantioselectivity than other acetic acid bacteria, which had 70-99% ee.

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability

Production of ethanol from xylose by recombinant Saccharomyces cerevisiae is suboptimal with slow fermentation rate, compared with that from glucose. In this study, a strain-expressing Scheffersomyces stipitis xylose reductase-xylitol dehydrogenase (XR-XDH) pathway was subjected to adaptive evolution on xylose; this approach generated populations with the significantly improved cell growth and ethanol production rate. Mutants were isolated, and the best one was used for sporulation to generate eight stable mutant strains with improved xylose fermentation ability. They were used in a microarray assay to study the molecular basis of the enhanced phenotype. The enriched transcriptional differences among the eight mutant strains and the native strain revealed novel responses to xylose, which likely contributes to the improved xylose utilization. The upregulated vitamin B1 and B6 biosynthesis indicated that thiamine served as an important cofactor in xylose metabolism and may alleviate the redox stress. The increased expression of genes involved in sulfur amino acid biosynthesis and the decreased expression of genes related to Fe(II) transport may alleviate redox stress as well. Meanwhile, it was remarkable that several glucose-repressible genes, including genes of the galactose metabolism, gluconeogenesis, and ethanol catabolism, had a lower expression level after adaptive evolution. Concomitantly, the expression levels of two regulators of the glucose signaling pathway, Rgs2 and Sip4, decreased, indicating a reshaped signaling pathway to xylose after adaptive evolution. Our findings provide new targets for construction of a superior bioethanol producing strain through inverse metabolic engineering.

Enhancement of fructose utilization from sucrose in the cell for improved l-serine production in engineered Corynebacterium glutamicum

Microbial fermentative production of l-serine has attracted increasing attention, and in our previous study, Corynebacterium glutamicum-SYPS-062ΔSSA has been successfully screened and engineered to produce l-serine from sucrose. It was noticed that there was significant fructose accumulation during l-serine fermentation process using sucrose as substrates, which is the preferred carbon resource for this strain. Lack of fructokinase may be responsible for poor fructose utilization. In the present study, a shortcut pathway for fructose utilization has been constructed via heterologous expression of scrK (fructokinase). Furthermore, pfkA (phosphofructokinase) was over-expressed to avoid over accumulation of fructose-6-phosphate, the resulting strain showed significantly improved cell growth and l-serine accumulation. In a 5-L bioreactor, the engineered C. glutamicum strain produced 30.6g/L l-serine (0.99mol/mol sucrose yield, with a theoretical maximal yield of 4mol/mol sucrose), whereas the reference strain produced 20.54g/L l-serine (with a yield of 0.67mol/mol sucrose) at 120h. After two feedings of sucrose, the l-serine production increased up to 37.0g/L at 96h.

In silico profiling of cell growth and succinate production in Escherichia coli NZN111.

BackgroundSuccinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB, respectively. However, this double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and consequently disables proper growth under anaerobic conditions.ResultsIn this study, genome-scale metabolic core model of E. coli was reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strain E. coli NZN111. Non-optimized artificial centering hit-and-run (ACHR) method and metabolite flux-sum analysis were utilized to evaluate metabolic characteristics of strains. Thus, metabolic characteristics of the strains wild-type E. coli, ldhA mutant, pflB mutant, and NZN111 under anaerobic conditions were successfully unraveled.Conclusions We found a viewpoint contrary to the widespread realization that an NADH/NAD+ in NZN111 mainly resulted from the inactivation of PFL rather than the inactivation of LDH. In addition, the two alternative anaerobic fermentation pathways, lactate and ethanol production pathways, were blocked owing to the disruption of ldhA and pflB, resulting in insufficient NAD+ regeneration to oxidize or metabolize glucose for cell growth. Furthermore, we speculated reaction NADH16, the conversion of ubiquinone-8 (q8) to ubiquinol-8 (q8h2), as a potential amplification target for anaerobically improving cell growth and succinate production in NZN111.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0125-5) contains supplementary material, which is available to authorized users.

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization.

BackgroundAlkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168.ResultsThe activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#.ConclusionThe results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids.

In this demonstration, spheroids formed from the β-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Ethanol as complementary carbon source in Scenedesmus obliquus cultivation

AbstractBACKGROUNDEthanol may be an attractive carbon source for microorganism growth for the production of several biomolecules. Nonetheless, high amounts of ethanol may be harmful for microbial cells. This study evaluated the effect of adding ethanol to the cultivation of Scenedesmus obliquus, under batch and fed‐batch processes.RESULTSA single addition of ethanol up to 460.00 mg L−1 did not influence cell growth, but above this threshold, different concentrations of ethanol had positive effects on maximum cell concentration. On the other hand, when ethanol was added daily for a period of 10 days, even the smallest ethanol feed rate (5.75 mg L−1 d−1; total added ethanol concentration = 57.5 mg L−1) positively influenced maximum cell concentration. Daily addition of 368 mg L−1 (fed‐batch culture; total added ethanol concentration = 3680 mg L−1) allowed the best cell growth of this study, with maximum cell concentration of 1068.70 mg L−1 and cell productivity of 113 ± 7.13 mg L−1 d−1. Under this condition, the maximum specific growth rate and maximum cell concentration obtained with mixotrophic growth were 2.83 and 2.70 times higher, respectively, than those obtained with autotrophic growth.CONCLUSIONCultures supplied with ethanol significantly improved cell growth. Addition of ethanol by a fed‐batch process allowed the highest biomass production with a total added ethanol concentration that hindered cell growth by batch process. © 2016 Society of Chemical Industry

Optimization of Callus Induction and Cell Suspension Culture of Betula pendula Roth for Improved Production of Betulin, Betulinic Acid, and Antioxidant Activity

This study aimed to establish an in vitro protocol for callus induction and cell suspension cultures of silver birch (Betula pendula Roth) and to assess the antioxidant activity of cell extract and the accumulation of betulin (B) and betulinic acid (BA). Callus was induced from the inner bark of stems on Gamborg (B5) and Nagata-Takebe (NT) media, and 23 different concentrations of growth regulators were tested. NT medium with 2.5 mg L−1 2,4-dichlorophenoxyacetic acid and 0.5 mg L−1 6-benzylaminopurine was optimal for callus induction. Cell suspension cultures were developed to improve cell growth and metabolite production under different concentrations of NT vitamins. Methanolic cell extracts indicated that the BA content of callus increased from 1 to 9 mo, with a maximum 2.01 mg g−1 (dry weight [DW]) at 9 mo. The highest level of B observed in cell suspension cultures (0.96 mg g−1 DW) was obtained on medium supplemented with 4× NT vitamins. The highest antioxidant activities of the 1,1-diphenyl-2-picrylhydrazyl free radical, 77 and 76%, were found in callus extract at 9 mo and cell extract in the 4× NT vitamin treatment, respectively. In conclusion, the use of NT medium supplemented with 4× NT vitamins effectively increased cell growth and the accumulation of B, BA, and antioxidant activity in cell suspension cultures of B. pendula.

Proliferation of mouse fibroblast-like and osteoblast-like cells on pure titanium films manufactured by electron beam melting.

The physical characteristics and biological compatibility of surfaces produced by electron beam melting (EBM) are not well known. In particular, there are not many reports on biocompatibility qualities. In this study, pure Ti films were manufactured using EBM. While it is reported that moderately hydrophilic biomaterial surfaces display improved cell growth and biocompatibility, contact angle measurements on the EBM-produced pure Ti films showed slight hydrophobicity. Nonetheless, we found the cell count of both fibroblast-like cells (L929) and osteoblast-like cells (MC3T3-E1) increased on pure Ti films, especially the MC3T3-E1, which increased more than that of the control. In addition, the morphology of L929 and MC3T3-E1 was polygonal and spindle-shaped and the cytoskeleton was well developed in the pure Ti surface groups. Upon staining with Alizarin red S, a slight calcium deposition was observed and this level gradually rose to a remarkable level. These results indicate that pure Ti films manufactured by EBM have good biocompatibility and could be widely applied as biomedical materials in the near future.