Maximum Viable Cell Research Articles

Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient nonenzymatic cell harvesting during 3D culturing.

Enzymatic detachment of cells might damage important features and functions of cells and could affect subsequent cell-based applications. Therefore, nonenzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, we prepared thermosensitive PNIPAm-co-AAc-b-PS and PNIPAm-co-AAm-b-PS copolymers and low critical solution temperature (LCST) was tuned near to body temperature. Then, spin coated polymer films were prepared for cell adhesion and thermal-induced cell detachment. The alpha-step analysis and scanning electron microscope image of the films suggested that the thickness of the films depends on the molecular weight and concentration which ranged from 206 to 1330 nm for PNIPAm-co-AAc-b-PS and 97.5-497 nm for PNIPAm-co-AAm-b-PS. The contact angles of the films verified that the polymer surface was moderately hydrophilic at 37°C. Importantly, RAW264.7 cells were convincingly proliferated on the films to a confluent of >80% within 48 h and abled to detach by reducing the temperature. However, relatively more cells were grown on PNIPAm-co-AAm-b-PS (5%w/v) films and thermal-induced cell detachment was more abundant in this formulation. As a result, PNIPAm-co-AAm-b-PS (5%w/v) was further used to coat commercial cytodex 3 microcarriers for 3D cell culturing and interestingly enhanced cell detachment with preserved potential of recovery was observed at a temperature of below LCST. Thus, surface modification of microcarriers with thermosensitive PNIPAm-co-AAm-b-PS could be vital strategy for nonenzymatic cell detachment and to achieve adequate number of cells with maximum cell viability and functionality.

Fermentation Profile and Probiotic-Related Characteristics of Bifidobacterium longum MC-42

This article presents new data on Bifidobacterium longum MC-42—a strain that has been actively used for the preparation of commercial dairy products in Russia for almost 40 years. It was demonstrated that this strain possesses high activities of β-galactosidase, α-glucosidase, and leucine arylaminidase; inhibits the growth of pathogens such as Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli; and can efficiently remove cholesterol from the cultural medium. The resistance of B. longum MC-42 determined for 15 commonly used antibiotics was in agreement with those previously reported for Bifidobacterium spp. The absence of frequently transmittable antibiotic resistance genes in the genome and the lack of undesirable activity of β-glucuronidase proved the safe use of B. longum MC-42 as a probiotic and starter culture. Additionally, the impact of two growth-promoting additives—yeast extract or milk protein hydrolysate containing supplementation—on the B. longum MC-42 fermentation profile was assessed. The introduction of these additives increases the maximum attainable viable cell count by orders of magnitude, significantly changed the profile of aminopeptidase activities in extracellular extracts, and influenced the antioxidant and antihypertensive properties of the obtained fermented products.

Quality Characteristics of Kimchi Prepared with Dioscorea batatas

In this study, we produced kimchi with cooked Dioscorea batatas yam based on the its outstanding nutritional value, biological activity, and pharmacological action. Sliced kimchi cabbage, processed white radish kimchi (kkakdugi), and whole kimchi cabbage were prepared with 3% Dioscorea batatas, and the physicochemical and bioactivity characteristics were analyzed. In three kinds of Dioscorea batatas. The pH of the kimchi decreased and its acidity increased as the storage period was extended. The period of the maximum total viable cell count of the sliced cabbage and the processed white radish kimchi (kkakdugi) was 14 days, while the period for whole kimchi cabbage was 21 days. The period of maximum lactic acid bacteria count was 14 days for all three kinds. For physiological activities, polyphenol and flavonoid contents and DPPH elimination were highest immediately after production of the kimchi. Also, anthocyanin content increased as the storage period extended. The pH, acidity, total viable cell count, lactic acid bacteria count, and physiological activities were shown to be different according to the type of kimchi and the storage period.

Dioscin-loaded zein nanoparticles alleviate lipopolysaccharide-induced acute kidney injury via the microRNA-let 7i signalling pathways.

The present study investigates the potential role of dioscin (DIO) in the lipopolysaccharide (LPS)‐induced kidney injury. For this purpose, DIO‐loaded zein nanoparticles (DIO‐ZNPs) were formulated and evaluated for physicochemical parameters. The DIO‐ZNPs exhibited a controlled release of drug compared with that of the free drug suspension. Results showed that the cell viability of NRK‐52E consistently decreased with the increase in LPS from 0.01 µg/ml to 2 µg/ml. When compared with LPS, DIO‐induced NPs showed 1.10‐, 1.32‐, 1.57‐ and 1.92‐fold increase in the cell viability for concentrations of 20 µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml, respectively. DIO‐ZNPs exhibited the most remarkable recovery in the cell proliferation compared with free DIO as shown by the cellular morphology analysis. Furthermore, Annexin‐V staining analysis showed that the LPS‐treated cells possess the lowest green fluorescence indicating fewer viable cells, whereas DIO‐ZNPs exhibited the maximum green fluorescence comparable with that of the non‐treated cells indicating maximum cell viability. Furthermore, the results show that DIO‐ZNPs significantly increased the expression of miR‐let‐7i in the epithelial kidney cells, whereas the expression levels of TLR4 were significantly downregulated compared with that of the LPS‐treated cells. In conclusion, miR‐let‐7i could be an interesting therapeutic target and nanoparticle‐based DIO could be a potential candidate in the management of acute kidney injury

Synthesis and characterization of nano-hydroxyapatite from Sardinella longiceps fish bone and its effects on human osteoblast bone cells

Organic debris in the form of fish bone wastes account to several thousand tons annually. In recent years, researchers have turned attention towards the bioconversion of organic debris into materials with biomedical applications. Accordingly, the present study synthesized nano-Hydroxyapatite (n-HAP) from bones of discarded Sardinella longiceps by the alkaline hydrolysis method. The synthesized n-HAP was characterized by using the scanning electron microscope (SEM), X-ray diffraction (XRD), atomic force microscope (AFM), and Fourier transform infrared spectroscopy (FTIR). Crushed fish bone demonstrated an agglomerate of fine and rod-like crystals as observed in SEM, whereas n-HAP exhibited a structure of dense thick particles. FTIR spectral data confirmed the functional groups such as alkanes, esters, saturated aliphatic, and aromatic groups. XRD analysis exhibited strong diffraction peaks of HAP confirming its presence in synthesized n-HAP. AFM analysis affirmed that the synthesized particles had an average size of 19.65 nm. Cell viability was tested at different concentrations (10, 50, 100, 250 μg/mL) against human osteoblast bone cells (MG-63).The maximum cell viability (141.3 ± 3.1%) was observed at 100 μg/mL (24 h). Mineralization was evaluated using Alizarin red staining of osteoblast MG-63 cells treated with n-HAP at the concentration of 50 and 100 μg/mL (0.54 ± 0.03 and 0.99 ± 0.05%) which exhibited red color indicating good results. The size, morphology, functional groups, viability and mineralization of the synthesized n-HAP are favorable for its use in bone tissue engineering and other potential osteo and dental applications.

<p><strong>Carbohydrate fraction of <em>Aloe vera </em>ameliorates inflammation through suppression of pro-inflammatory mediators and oxidative stress <em>in vitro</em> and rats with Freund’s adjuvant induced paw edema</strong></p>

Aloe vera (L.) Burm. F (Asphodelaceae) is a traditional medicinal plant having multiple biological activities such as wound healing, anticancer and antidiabetic activity, etc. These properties have been attributed to the constituents present in Aloe vera. One of the major constituents is carbohydrate, which has not been explored much for its antioxidant and anti-inflammatory activity. Here, we evaluated the antioxidant and anti-inflammatory potential of carbohydrate fraction (CF) of Aloe vera extract using in vitro and in vivo model for its therapeutic application in the treatment of inflammation. CF extract was prepared using hot water extraction followed by ethanolic precipitation. The CF extract was tested for free radical scavenging assays, lipopolysaccharide-stimulated RAW264.7 cells in vitro and in vivo by Freund’s adjuvant induced rat paw edema model. The free radical scavenging activity of CF extract significantly increased in a concentration-dependent manner with maximum inhibition observed at the concentration of 2 mg/mL. CF extract (20 and 60 µg/mL) showed their maximum cell viability in RAW264.7 cells by MTT assay. CF extract shows a considerable decrease of inflammatory mediators in both in vitro and as well as in vivo studies. The increase of superoxide dismutase and catalase levels in CF treated adjuvant induced rats decreases ROS/RNS levels and inflammatory mediators. It may be primarily activated by NF-κB and AP-1 indicating its potent antioxidant activity that promotes modulation in cellular redox state. The results suggest that carbohydrate fraction of Aloe vera could be useful for the management of oxidative stress and inflammatory diseases.

Activation of toll-like receptor signaling in endothelial progenitor cells dictates angiogenic potential: from hypothesis to actual state.

Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000µg/ml, for 48h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

Construction of tissue engineering bone with the co‑culture system of ADSCs and VECs on partially deproteinized biologic bone invitro: A preliminary study.

Scaffold-based bone tissue engineering has therapeutic potential in the regeneration of osseous defects. The present study aimed to explore the adhesion and cell viability of a co-culture system composed of vascular endothelial cells PI−/Annexin V+ represents early apoptotic cells, and PI+/Annexin V+ represents late apoptotic cells (VECs) and adipose-derived stem cells (ADSCs) on partially deproteinized biologic bone (PDPBB) in vitro, and determine the optimum time period for maximum cell viability that could possibly be used for standardizing the scaffold transplant into the in vivo system. VECs and ADSCs were isolated from pregnant Sprague-Dawley rats and confirmed by immunostaining with von Willebrand factor and CD90, respectively. PDPBB was prepared using standardized protocols involving coating partially deproteinized bone with fibronectin. PDPBB was incubated in a mono-culture with VECs or ADSCs, or in a co-culture with both of these cells at a ratio of 1:1. An MTT assay was used to assess the adhesion and cell viability of VECs and ADSCs on PDPBB in the three different cultures. Scanning electron microscopy was used to observe the adhesion, cell viability and morphology of the different types of cells on PDPBB. It was observed that the absorbance of each group increased gradually and peaked on the 10th day; the highest absorbance was found for the co-cultured cells group. The difference of cell viability between each cell group was statistically significant. On the 10th day, in the co-cultured cells group, several cells adhered on the PDPBB material and a nest-like distribution morphology was observed. Therefore, the adhesion and cell viability of the co-cultured cells was higher compared with the mono-cultures of VECs or ADSCs. As cell viability was highest on the 10th day, this could be the optimal length of time for incubation and therefore could be used for in vivo experiments.

Cell biophysical characteristics of PPy-GNPs and their application in photothermal therapy of SKOV-3 cell

The pursuit of multifunctional, stable, more efficient, and safer cancer treatment has gained increasing interest in the research of nanoparticle-mediated photothermal therapy. Many nanoparticles have good absorption chracteristics in the near-infrared region, because of the high light-to-heat conversion efficiency; they could destroy tumor cells by heat; thus, they are often treated as a potential photothermal therapy agent. In this work, a kind of polypyrrole (PPy)-coated gold nanoparticles particles (PPy-GNPs) with high photothermal conversion capability was prepared. Their particle sizes were about 90 ± 11.2 nm. The temperature of PPy-GNPs solution in a concentration of 0.25 mg/ml rises 13.6 °C, and the nanoparticles could significantly inhibit the proliferation of SKOV-3 cells under irradiation of 25 W·cm−2 laser (808 nm) for 10 min, with the maximum cell viability value is maintained at 87.6% by MTT assay. In the same photothermal and cultured conditions, the photothermal conversion efficiency and cytostatic effect of the gold nanoparticle are much lower than that of the PPy-GNPs group. The atomic force microscopy (AFM) was used to identify the function of nanoparticles for the structural changes of the cells. The SKOV-3 cell of adhesion forces decrease by 21%, and Young’s modulus increases by 26% compared with control. These results suggest that PPy-GNPs are effective targeted photothermal therapeutic agents for cancer treatment.

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production.

High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

Immuno biocompatibility and anti-quorum sensing activities of chitosan-gum acacia gold nanocomposite (CS-GA-AuNC) against Pseudomonas aeruginosa drug-resistant pathogen

In this study, an eco-friendly green route strategy was adopted for the synthesis of highly stable, biocompatible polymer-polymer- metal-based composite with high potential anti-bacterial applications. The composite was prepared by modified ionic gelation of Chitosan –gum acacia copolymer with gold nanoparticles (Cs-GA-AuNC). Structural, functional characteristics of the synthesised nanocomposite were studied by spectroscopic, electron microscopic and X-ray diffraction (XRD) studies which reveal functionalised polymers coupled with the gold nanoparticles to form electron core dense nanostructural composite material with notable nanoarchitecture. The synthesised nanocomposite was evaluated for anti-quorum sensing activity against human pathogenic bacterial strain Pseudomonas aeruginosa by measuring the expression of Las R. Nanocomposite treatment brought about drastic reduction of Las R gene expression followed by the remarkable impact on Las R gene-based virulence factors like biofilm formation, pyocyanin production, swarming motility, and total proteolytic activity. Biocompatibility of the composite was tested on Raw cells 246.7 –murine macrophage cell line/The nanocomposite was not inducing any sign of toxicity on the tested Raw cells 246.7 by exhibiting maximum cell viability, lower level of ROS generation and reduced nitride concentration which revealed the noteworthy biocompatibility of synthesised nanocomposite. This selective biological activity shown by the nanocomposite might find its application in the drug delivery for precision medicine.

Effect of Integrin Binding Peptide on Vascularization of Scaffold-Free Microtissue Spheroids

Three-dimensional (3D) biomimetic models via various approaches can be used by therapeutic applications of tissue engineering. Creating an optimal vascular microenvironment in 3D model that mimics the extracellular matrix (ECM) and providing an adequate blood supply for the survival of cell transplants are major challenge that need to be overcome in tissue regeneration. However, currently available scaffolds-depended approaches fail to mimic essential functions of natural ECM. Scaffold-free microtissues (SFMs) can successfully overcome some of the major challenges caused by scaffold biomaterials such as low cell viability and high cost. Herein, we investigated the effect of soluble integrin binding peptide of arginine-glycine-aspartic acid (RGD) on vascularization of SFM spheroids of human umbilical vein endothelial cells. In vitro-fabricated microtissue spheroids were constructed and cultivated in 0mM, 1mM, 2mM, and 4mM of RGD peptide. The dimensions and viability of SFMs were measured. Maximum dimension and cell viability observed in 2mM RGD containing SFM. Vascular gene expression of 2mM RGD containing SFM were higher than other groups, while 4mM RGD containing SFM expressed minimum vascularization related genes. Immunofluorescent staining results indicating that platelet/endothelial cell adhesion molecule and vascular endothelial growth factor protein expression of 2mM RGD containing SFM was higher compared to other groups. Collectively, these findings demonstrate that SFM spheroids can be successfully vascularized in determined concentration of RGD peptide containing media. Also, soluble RGD incorporated SFMs can be used as an optimal environment for successful prevascularization strategies.

Phytochemical profiling and screening of protective effects of Artabotrys odoratissimus on H2O2 induced oxidative stress in HEK-293 cells and erythrocytes

ABSTRACT Artabotrys odoratissimus Blume commonly known as the ylang ylang vine is an aromatic woody shrub native to eastern Asia. The plant is used in traditional medicine systems; however, the bioactive potential of the plant remains unexplored. The present study focussed on the phytochemical profiling, anti-oxidative and cytoprotective effects of the stem bark ethanol extract (BEE) and fruit ethanol extract (FEE) on HEK-293 cells and erythrocytes. The preliminary group tests revealed the presence of alkaloids, phenols, flavonoids, lactones, glycosides, oils and fats. HR-LCMS analysis of bark ethanol extract revealed the presence of isocorydine, quercetrin, caffeic acid, juglone. In addition, GC-MS analysis of fruit ethanol extracts revealed the presence of furanone, pyrrole and long-chain methylated gamma-butyrolactone. BEE showed a total phenolic content of 573.30 ± 0.67 µg/mg and flavonoid content of 831.03 ± 1.61 µg/mg when compared to FEE which had total phenolic content and flavonoid content of 164.40 ± 0.24 µg/mg and 630.17 ± 2.79 µg/mg, respectively. Both the extracts showed protective effects on H2O2 induced oxidative damage on DNA, HEK-293 cells and erythrocytes. BEE showed an IC50 value of 2.23 ± 0.16 µg/ml in ABTS assay, similar results were observed in HEK-293 cells where the cells pre-treated with BEE showed a maximum cell viability of 56% under oxidative stress and the percentage inhibition of erythrocyte hemolysis was observed to be 86.39 ± 0.67% at a concentration of 250 µg/ml. The study clearly illustrates that the plant extracts from A. odoratissiumus possess potential antioxidative prospects against induced oxidative damage.

Investigation of cell line specific responses to pH inhomogeneity and consequences for process design.

With increasing bioreactor volumes, the mixing time of the reactor increases as well, which creates an inhomogeneous environment for the cells. This can result in impaired process performance in large‐scale production reactors. Particularly the addition of base through the reactor headspace can be problematic, since it creates an area, where cells are repeatedly exposed to an increased pH. The aim of this study is to simulate this large‐scale phenomenon at lab‐scale and investigate its impact. Two different cell lines were exposed to pH amplitudes of a maximal magnitude of 0.05 units (pH of 6.95). Both cell lines showed similar responses, like decreased viable cell counts, but unaffected lactate levels. However, cell line B showed an initially increased specific productivity in response to the introduced amplitudes, whereas cell line A showed a consistently lower specific productivity. Furthermore, the time point at which base addition is started influences the impact, which pH amplitudes have on process performance. When pH control was started earlier in the process, maximal viable cell counts decreased and the lactate metabolic shift was less pronounced. These results show that the potential negative impact of pH amplitudes can be minimized by strategic process design.

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.

Fabrication of bioactive hydroxyapatite/silk fibroin/gelatin cross-linked nanocomposite for biomedical application

Hydroxyapatite/Silk fibroin/Gelatin (HSG) composite is a promising artificial bone graft material, which mimics the properties of the natural extracellular matrix components that meet the current need for bone tissue engineering. In this study, HSG composite in different ratios (40:60:0, 40:50:10, 40:40:20, 40:30:30 and 40:20:40) were prepared using in-situ co-precipitation method. Here, glutaraldehyde was used as a chemical cross-linking agent to improve interactions and control degradation of the composite. The formation of carbonated hydroxyapatite on the cross-linked polymer matrix was evidenced from the optical analysis. The influence of the gelatin content on crystallinity, crystallite size and morphology of the composite were confirmed by XRD and FESEM spectroscopic techniques. The Physico-chemical properties of the composites such as in-vitro biodegradation, swelling ratio and biomineralization were evaluated by treating it with simulated body fluid. Cell viability of the nanocomposite was tested with MG-63 cell lines, which emphasized the biocompatibility of the composites. Moreover, all-composite exhibits good antibacterial ability against Staphylococcus aureus and Pseudomonas aeruginosa. Finally, HSG (40:30:30) composite with a swelling ratio of 69.30 ± 0.42% (day 15) and biodegradation of 18.51 ± 0.53% (day 15) reveals maximum cell viability (87.27 ± 0.62%) and significant antibacterial property indicating a potential candidate for orthopaedic application.

Long-term preserved and rapidly revived methanogenic cultures: Microbial dynamics and preservation mechanisms

Bioaugmentation with specialized inocula has been proven a feasible way to remediate under-performing anaerobic digestion (AD) processes. However, a major bottleneck for successful and cost-effective bioaugmentation is the lack of ready-to-use, specialized methanogenic cultures when required. The reason is the slow growth of the anaerobic consortia and the high cost of maintaining them active in the necessary amounts for successful bioaugmentation applications. This study offers an effective procedure where customized AD inocula could be preserved and used on-demand for remediation of ammonia inhibited AD reactors. Additionally, it introduces the biological and physicochemical mechanisms that render the long-term preservation of the AD inocula possible. Specifically, two different preservation carriers (i.e. agar gel and liquid basic anaerobic medium) were assessed at two different temperatures (i.e. 4 °C and 24 °C) using an ammonia tolerant methanogenic consortium. The results from methane production, lag-phase, maximum methane production rate and cell viability indicate that the consortium preserved for 168 days in agar gel at 24 °C performed best compared to the other tested preservation conditions. Meanwhile, 16S rRNA sequencing analysis indicated that Methanosarcina soligelidi and Methanoculleus palmolei shown a high revival rate and metabolic activity after long-term preservation. Thus, this successful long-term preservation method of ready-to-use AD consortia could render the successful bioaugmentation in full-scale biogas reactors economically possible in the near future.

Bifidobacterium sp. 고농도 성장을 위한 환경인자 및 세포활성도 최적화

For the industrial use of Bifidobacterium sp., maintaining cell viability is crucial along with the high cell concentration through fermentation optimization. The effects of initial glucose concentration, lactic acid concentration, pH and culture temperature on cell growth were investigated to optimize the environmental factors. In addition, the batch and fed-batch cultivation were performed to evaluate the correlation between the cell concentration based on the absorbance and the viable cell number. Both B. infantis and B. bifidum showed the highest cell growth rates at low glucose levels and the specific growth rate decreased with increasing glucose concentrations. The growth rate of B. bifidum was reduced with increasing glucose concentration, but the maximum cell concentration was increased. However, B. infantis showed lower maximum cell concentration in the higher glucose concentration. The impacts of lactic acid, pH and temperature of both B. infantis and B. bifidum were similar to those of most lactic acid bacteria. However, B. bifidum showed a more resilience within the same range of each environmental factor showing continuous growing at low specific growth rate even when B. infantis stopped growing. The number of viable cells of B. infantis was dramatically reduced even though the absorbance remained constant. In particular, the number of viable cells and the number of viable cells per absorbance decreased to 1/7 and 1/10 as the fed-batch culture progressed. Meantime, once the maximum viable cell number was reached, B. bifidum maintained the constant viable cell number and viable cell number per absorbance were maintained.

The Long-Term Fate of the Sonoporated Pancreatic Cancer Cells is Uncorrelated With the Degree of Model Molecular Loading

Studies have determined that ultrasound-activated microbubbles can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve drug loading. However, because of the distinct cavitation events adjacent to each cell, the degree of drug loading appeared to be heterogeneous. The relationship between the long-term fate trend and the degree of drug loading remains unclear. To investigate the time-lapse viability of diversity loading cells, fluorescein isothiocyanate–dextran (FITC-dextrans) was used as a molecular model mixed with 2% v/v SonoVue microbubbles (Bracco, Milan, Italy) and exposed to various peak negative pressures (0.25 MPa, 0.6 MPa, 1.2 MPa), 1 MHz frequency and 300 μs pulse duration. To select a suitable parameter, the cavitation activity was measured, and the cell analysis was performed by flow cytometry under these acoustic pressures. The sonoporated cells were then categorized into 3 sub-groups by flow cytometry according to the various fluorescence intensity distributions to analyze their long-term fate. We observed that the stable cavitation occurred at 0.25 MPa and microbubbles underwent ultra-harmonic emission, and obvious broadband signals were observed at 0.6 MPa and 1.2 MPa, suggesting the occurs of inertial cavitation. The cell analysis further showed the maximum delivery efficiency and cell viability at 0.6 MPa, and it was selected for the following experiment. The categorization displayed that the fluorescence intensity of FITC-dextrans in sub-groups 2 and 3 were approximate 5.62-fold and 19.53-fold higher than that in sub-group 1, respectively. After separation of these sub-groups, the apoptosis and necrosis ratios in all 3 sub-groups of sonoporated cells gradually increased with increasing culture time and displayed no significant difference in either the apoptosis (p > 0.05) or necrosis (p > 0.05) ratio after 6 h and 24 h of culture, respectively. Further analysis using Western blot verified that the long-term fate of sonoporated cells involves the mitochondrial signaling proteins. These results provide better insight into the role of cavitation-enhanced permeability and a critical guide for acoustic cavitation designs.