Culture Duration Research Articles

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

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

P–146 Differential impact of three embryo culture media for IVF on in vitro development and perinatal outcome: a single-center RCT

Abstract Study question This report provides updated data from an RCT determining which embryo culture medium yields optimal IVF outcomes. Summary answer Embryo culture systems used for IVF differentially affected preimplantation development and resultant obstetric and perinatal outcomes, including birthweights of live-born singletons. What is known already Currently, multiple embryo culture medium systems are in use for IVF, raising questions regarding which is optimal. However, the ability of a medium to yield preimplantation embryos is not necessarily indicative of embryo viability. For example, supplementation of medium with serum was commonly used to increase animal blastocyst yields, but this impaired embryonic, fetal, and offspring health. In humans, medium composition and culture duration can influence IVF efficacy and offspring phenotype. Given the importance of culture systems in determining clinical outcomes, existing data regarding differential culture system impacts are insufficient and additional well-designed studies are required. Study design, size, duration Between February 2016 and August 2017, 795 couples undergoing their first autologous clinical IVF cycle and freeze-all strategy were recruited. Participants were randomized via computer-generated tables into three groups. Following standard oocyte retrieval and IVF/ICSI procedures, embryos were cultured using three different culture media, G1 Plus/G2 Plus (G1/G2; Vitrolife), Global Total (GT; LifeGlobal), or Sequential Cleav/Sequential Blast (SC/SB; Origio). Thirty-eight patients exhibiting no 2PN oocytes following insemination or those undergoing fresh embryo transfers were excluded. Participants/materials, setting, methods For patients yielding a single good-quality cleavage-stage (day–2 or day–3) embryo, that cleavage-stage embryo was vitrified. For patients yielding two or more good-quality cleavage-stage embryos, two or less good-quality cleavage-stage embryos were vitrified. The culture period of the remaining embryos was extended, and all good-quality blastocyst-stage (day–5 or day–6) embryos were vitrified. This report presents data for vitrified embryo transfer performed until the end of December 2020. Main results and the role of chance The mean per-cycle vitrified embryo yield (± SD) was comparable between groups for cleavage-stage embryos, but significantly different for blastocyst-stage embryos (G1/G2: 1.69 ± 2.2, GT: 2.53 ± 3.01, SC/SB: 2.04 ± 2.42; P = 0.001). Following vitrified cleavage- or blastocyst-stage embryo transfers, biochemical pregnancy rates were significantly different between groups (G1/G2: 55.6%, GT: 59.1%, SC/SB: 46.2%; P = 0.011). Furthermore, a between-group trend towards different live birth rates was observed (G1/G2: 41.7%, GT: 42.1%, SC/SB: 33.1%; P = 0.063). Of 382 live births, data for first-borns (n = 323; 295 singletons and 14 twin-pairs) are reported here. Perinatal data did not differ significantly between groups for both cleavage- and blastocyst-stage embryo transfers, including gestational age- and gender-adjusted singleton birthweight (z-score). Following multiple linear regression (including selected covariates), adjusted mean singleton birthweights were significantly lower in the G1/G2 and GT groups than in the SC/SB group (by 131 g; P = 0.011 and 110 g; P = 0.032, respectively) and tended to be lower for cleavage-stage embryo transfers than for blastocyst-stage embryo transfers (by 102 g; P = 0.053). Limitations, reasons for caution A larger cohort size and longer-term follow-up are required to verify and further elucidate the impact of embryo culture methods on child health. Such studies will raise awareness regarding the sensitivity of in vitro-cultured human embryos to their environment, ultimately resulting in practices that decrease IVF risks to offspring. Wider implications of the findings: Pregnancy outcome of the medium yielding fewer blastocysts was comparable or superior to that of other media, highlighting the importance of differentiating between the ability to support preimplantation development versus the ability to yield viable embryos. Embryo culture medium had a greater impact than embryo transfer stage on live birthweight. Trial registration number UMIN000020910

Influence of sweet whey permeate utilization on Tetradesmus obliquus growth and β‐galactosidase production

AbstractThe practicability of culturing microalgae in mixotrophic mode using sweet whey permeate (SWP), a highly perishable by‐product stream from cheese manufacturing, is evaluated for microalgal growth and β‐galactosidase production. This study is conducted with Tetradesmus obliquus, species known for their ability to adapt to various conditions with different carbon sources. The influence of pure lactose, the primary carbon source of SWP, is first surveyed. Partial substitutions of the basal culture medium with SWP in various proportions are then conducted. Results show that substituting 60% (v/v) of the culture medium with SWP significantly enhances T. obliquus growth (up to 2.1 g L−1) and total enzyme production (up to 208.5 U L−1). Besides, a molecule expected to be a galactooligosaccharide (GOS) is detected in the presence of high SWP proportions. Overall results suggest a short‐term culture duration (around 8 days) for producing the β‐galactosidase enzyme and a long‐term culture duration (over 15 days) for biomass production. However, the use of 100% (v/v) SWP seems to be non‐profitable due to a lack of some essential nutrients.

Effect of different nucleus sizes and culture duration on the quality of half pearls (MABÉ) produced by the winged pearl oyster Pteria sterna (Gould, 1851) in Ecuador

This study aims to evaluate the influence of nucleus size and culture duration on the nacre deposition rates of half pearls produced by the winged oyster P. sterna. For the nucleus size study, three different sizes of hemispherical implants were tested; small (9 mm), medium (11 mm) and large (14 mm), each of these treatments was assigned three baskets, containing 7 oysters each (for a total of 21 oysters), while an additional 21 individuals were kept without implants as a control treatment. All groups were cultured for six months. At the end of this experimental period, the shell length, shell height, and shell thickness growth of the oysters implanted with the small and medium-sized nuclei were significantly higher than that of the oysters with large nuclei. However, significantly higher nacre deposition rates were observed in the oysters with large nuclei. Mortality reached only 2% in all treatments, and the results show a lower proportion of good quality hemispherical pearls (23.0%) in the oysters implanted with a large nucleus, while for those implanted with small and medium nuclei the percentage of good quality half pearls reached 71.4% and 69.8%, respectively. Regarding the annual nacre deposition study, three replicates were sampled monthly, along with the environmental variables of the site used, to examine the influence of these variables on their nacre deposition rates. Results showed that, in just 8 months of culture, nacre thickness exceeded the average of 1.0 mm. A principal component analysis (PCA) showed a direct and significant relationship between instantaneous nacre deposition rate (k), water temperature, and food variables. These results demonstrate the potential for the winged oyster P. sterna for the development of a productive alternative for aquaculture in Ecuador.

Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis

BackgroundThe ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes.ResultsWe identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated.ConclusionThis investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.).

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

Sirt3 promotes the autophagy of HK‑2 human proximal tubular epithelial cells via the inhibition of Notch‑1/Hes‑1 signaling.

Diabetic nephropathy (DN) is a predominant cause of end-stage renal disease. The impairment of the autophagy of human renal tubular epithelial cells (HK-2 cells) is involved in the pathogenic mechanisms of DN. Sirtuin (Sirt)3 regulates the scavenging of damaged organelles and maintains energy balance. The present study aimed to examine the protective effects of Sirt3 on HK-2 cells stimulated by high glucose (HG). HK-2 cells were cultured in normal glucose (NG), HG or hyperosmotic medium. The viability of the HK-2 cells was detected using a Cell Counting Kit-8 assay. The expression and localization of Sirt3 were detected via immunofluorescence. Following transfection with an overexpression plasmid, the expression levels of key components in the Notch homolog 1 (Notch-1)/hairy and enhancer of split-1 (Hes-1) pathway and those of the autophagy-related proteins, Beclin-1, LC-3II and p62, were measured by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR). As the Notch-1/Hes-1 pathway was inhibited, the expression levels of Beclin-1, LC-3II and p62 were also examined at transcriptional and translational level. It was found that prolonged culture in HG medium markedly reduced cell viability compared with the cells cultured in NG or in NG + mannitol, an effect that was aggravated with the increasing duration of culture. HG was capable of inhibiting the expression levels of Beclin-1, LC-3II and Sirt3, and upregulating p62 and the Notch-1/Hes-1 pathway, as verified by western blot analysis and RT-qPCR. The results of immunofluorescence staining revealed that HG decreased Sirt3 expression. Sirt3 reversed the HG-induced inhibition of the expression of Beclin-1 and LC-3II and the upregulation of p62. Moreover, Sirt3 reversed the HG-induced inhibition of the Notch-1/Hes-1 signaling pathway. However, this autophagy-promoting effect of Sirt3 was counteracted by the Notch-1/Hes-1 pathway activator. On the whole, the present study demonstrated that Sirt3 promoted the autophagy of HK-2 cells, at least partly, via the downregulation of Notch-1/Hes-1.

Accumulation of Nε-(carboxyethyl) lysine in Caenorhabditis elegans is correlated with the formation of ketone body.

Advanced glycation end-products (AGEs) are formed when proteins react with carbonyl compounds, and they gradually accumulate with age. However, AGE accumulation with ageing is not fully understood because longevity studies in mammals are time-consuming. Therefore, we used Caenorhabditis elegans to evaluate the correlation between ageing and AGE accumulation. Age-synchronized C.elegans nematodes were cultured for 3 and 12 days. The levels of Nε-(carboxymethyl) lysine, Nω-(carboxymethyl) arginine, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl) ornithine and Nε-(carboxyethyl) lysine (CEL) were compared. Glucose, methylglyoxal and acetol were incubated with human serum albumin, and CEL formation was evaluated. The levels of methylglyoxal and ketone bodies in C.elegans were quantified. CEL accumulation increased significantly with culture duration. Methylglyoxal and ketone bodies-possible forerunners of AGE accumulation-were also quantified with respect to culture duration. The levels of ketone bodies increased significantly during culture, and correlated closely with CEL accumulation (R2 = 0.72, P = 0.0008), whereas the levels of methylglyoxal did not increase over time. CEL was formed in vitro in a time-dependent manner from methylglyoxal and acetol when incubated with human serum albumin (HSA) at the same temperature as C.elegans culture, suggesting that increased levels of CEL in C.elegans are attributable to ketone bodies.

Effects of consecutive culture of Penaeus vannamei on phosphorus transformation and microbial community in sediment.

Phosphorus (P) is highly related to water quality during shrimp culture. Recognizing P transformation in pond-based cultures is crucial for sustainable and healthy aquaculture. However, P transformation remains unclear in the sediment of Penaeus vannamei cultures, although commercial species have been pervasive worldwide. To determine P transformation, samples with different culture years were collected from Zhejiang province, China. Sequential chemical extraction was applied to reveal the composition of inorganic P, while phosphatase activity was used to evaluate the biomineralization of organic P. The results indicated that the consecutive culture of Penaeus vannamei promoted the dissolution potential of sedimentary P. This was attributed to anoxic iron reduction that increased the formation of loosely bound P and Fe (II)-P. However, this phenomenon was dominated by biomineralization, which transformed the organic P to inorganic P. The results suggested that consecutive culture changed the microbial community structure in the sediment as well as the gene functions. The Shannon Wiener index showed that increasing the culture duration significantly decreased the stability of the microbial community. Overall, this study suggests that long-term consecutive culture of Penaeus vannamei may increase the P release potential of the sediment, which increases the risk of pond eutrophication.

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.

Producing young, single and meaty oyster Crassostrea iredalei (Faustino, 1932) in grow‐out culture using pouches suspended from rafts

Growth and survival of the slipper oyster Crassostrea iredalei using traditional (tires and oyster shells) and new methods (pouches and trays) suspended from floating rafts were compared. After six months of grow-out culture, mean shell length (mm SL) and body weight (g BW) of oysters were significantly highest in those reared in pouches (89.32 ± 0.41 mm SL, 87.70 ± 0.70 g BW), followed by those in trays (83.16 ± 0.31 mm SL, 87.06 ± 0.72 g BW), then those harvested from oyster shells (76.42 ± 0.88 mm SL, 67.35 ± 1.03 g BW), and lastly from tires (72.30 ± 1.21 mm SL, 55.07 ± 1.49 g BW). Growth rates both for length and weight were almost comparable between oysters reared in pouches (6.05 ± 1.28 mm mo−1, 10.98 ± 2.05 g mo−1) and trays (5.01 ± 1.46 mm mo−1, 10.77 ± 2.58 g mo−1). Although 60.7% of the oysters harvested from pouches (n = 1071) were classified as large (85–100 mm SL) and jumbo (100–120 mm SL) and only 39.5% of those from trays (n = 1048), their meat yield did not significantly differ (pouches = 22.60 ± 0.86%; trays = 24.76 ± 0.93%). Survival in pouches (95.2 ± 0.77%) and trays (93.15 ± 2.10%) was almost parallel at harvest. Growing oysters using the new methods produced single, larger, meatier oysters of almost the same size at a shorter culture duration.

HepaRG Maturation in Silk Fibroin Scaffolds: Toward Developing a 3D In Vitro Liver Model.

In vitro liver models are necessary tools for the development of new therapeutics. HepaRG cells are a commonly used cell line to produce hepatic progenitor cells and hepatocytes. This study demonstrates for the first time the suitability of 3% silk scaffolds to support HepaRG growth and differentiation. The modulus and pore size of 3% silk scaffolds were shown to be within the desired range for liver cell growth. The optimal seeding density for HepaRG cells on silk scaffolds was determined. The growth and maturation of scaffolded HepaRG cells was evaluated for 28 days, where the first 14 days of culture were a proliferation period and the last 14 days of culture were a differentiation period using dimethyl sulfoxide (DMSO) treatment. After the first 14 days of culture, the scaffolded HepaRG cells exhibited increased metabolic activity and albumin secretion compared to monolayer cultured controls and preserved these attributes through the duration of culture. Additionally, after the first 14 days of culture, the scaffolded HepaRG cells displayed a significantly reduced expression of genes associated with hepatocyte maturation. This difference in expression was no longer apparent after 28 days of culture, suggesting that the cells underwent rapid differentiation within the scaffold. The functionalization of silk scaffolds with extracellular matrix (ECM) components (type I collagen and/or an arginylglycylaspartic acid (RGD)-containing peptide) was investigated to determine the impact on HepaRG cell attachment and maturation. The inclusion of ECM components had no noticeable impact on cell attachment but did significantly influence CYP3A4 expression and albumin secretion. Finally, the matrix support provided by the 3% silk scaffolds could prime the HepaRG cells for steatosis liver model applications, as evidenced by lipid droplet accumulation and expression of steatosis-related genes after 24 h of exposure to oleic acid. Overall, our work demonstrates the utility of silk scaffolds in providing a modifiable platform for liver cell growth.

Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells.

Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response.

Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture

Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.

Effect of glucose concentration and culture substrate on HUVECs viability in in vitro cultures: A literature review and own results

Some clinical studies reported that glucose variability increased the risk of developing diabetes-related late complications more than constant hyperglycemia, while others claimed that the evidence was not strong enough to support such a conclusion. A few in vitro studies investigated the effect of constantly high or variable glucose levels (VGLs) on endothelial cells (EC). The first aim of this work was to review these studies and demonstrate that most of them support the notion that viability and other metabolic parameters of EC deteriorate faster in cell cultures with VGLs than in cultures with stable normal or high glucose concentration. The second aim was to verify whether the effect of glucose concentration is the same regardless of other culture conditions such as the substrate on which the cells are grown. We cultured Human Umbilical Vein Endothelial Cells (HUVECs) for 7 or 14 days in constant (5 mM or 20 mM) or variable (switching between 5 mM and 20 mM once a day) glucose concentration in culture plates, which were either not-covered with any additional substrate or were covered with fibronectin or gelatin. We assessed the cell viability using a propidium iodide test. The ANOVA revealed that HUVECs viability was affected not only by glucose concentration and duration of the cell culturing but also by the type of substrate and interactions of these three factors. In conclusion, the effect of glucose level on EC viability should not be analyzed in isolation from other culture conditions that may amplify or attenuate this effect.

Monocyte-Derived Macrophages Contribute to Chitinase Dysregulation in Amyotrophic Lateral Sclerosis: A Pilot Study.

Neuroinflammation significantly contributes to Amyotrophic Lateral Sclerosis (ALS) pathology. In lieu of this, reports of elevated chitinase levels in ALS are interesting, as they are established surrogate markers of a chronic inflammatory response. While post-mortem studies have indicated glial expression, the cellular sources for these moieties remain to be fully understood. Therefore, the objective of this pilot study was to examine whether the peripheral immune system also contributes to chitinase dysregulation in ALS. The temporal expression of CHIT1, CHI3L1, and CHI3L2 in non-polarized monocyte-derived macrophages (MoMas) from ALS patients and healthy controls (HCs) was examined. We demonstrate that while CHIT1 and CHI3L1 display similar temporal expression dynamics in both groups, profound between-group differences were noted for these targets at later time-points i.e., when cells were fully differentiated. CHIT1 and CHI3L1 expression were significantly higher in MoMas from ALS patients at both the transcriptomic and protein level, with CHI3L1 levels also being influenced by age. Conversely, CHI3L2 expression was not influenced by disease state, culture duration, or age. Here, we demonstrate for the first time, that in ALS, circulating immune cells have an intrinsically augmented potential for chitinase production that may propagate chronic neuroinflammation, and how the ageing immune system itself contributes to neurodegeneration.

Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model.

Clinical experiments suggest that mesenchymal stem cells (MSCs) may be useful for tissue repair therapies or treatment of the autoimmune disorders. There is still lack of consensus concerning the age limit of MSC donors, majority of researchers suggest the autologous MSC therapies of patients not exceeding age limit of 55-60 yrs. The purpose of our study was to compare the selected parameters of MSCs from adipose tissue (adipose stem cell, ASC) collected from young and old rats of ages corresponding to patient's ages 25 yrs. and 80 yrs., respectively. The differences of parameters of ASCs from young and old animals were compared with the differences between ASCs from short-term (3 passage) and long-term (30 passage) in vitro culture. Cell morphology, surface marker expression, growth potential, metabolic activity, β-galactosidase activity, clonogenic potential, angiogenic potential, and differentiation ability of ASCs from young and aged animals and from in vitro cultures at 3rd and 30th passages were compared and analyzed. It may be concluded that ASCs may be applied for autologous transplantations in aged patients. Comparison of ASC aging dynamics depending on host aging or in vitro culture duration suggests that long-term in vitro culture may affect ASCs more than natural aging process of their host. We suggest that ASCs expanded in vitro prior to their clinical use must be carefully screened for the possible aging effects resulting not only from donor age, but from the duration of their in vitro culture.

Diagnosis of knee prosthetic joint infection; aspiration and biopsy

BackgroundProsthetic joint infection (PJI) is a significant cause of morbidity and mortality following knee replacement surgery. The diagnosis can be challenging and is based on a combination of clinical suspicion, radiographic findings and also biochemical/ microbiological investigations. Our Aim was to review the role of aspiration and biopsy in the diagnosis of PJI in Total Knee Arthroplasty (TKA). Method/resultsAspirated synovial fluid should be analysed by direct culture, via blood culture bottles, EDTA bottles for cell count and ‘point of care’ testing such as leucocyte esterase or alpha defensin. Synovial WCC and PMN cell percentage are important steps in diagnosis of both acute and chronic PJI. A minimum of 5 deep samples using a 5 clean instrument technique should be obtained and sent for tissue culture done either blind or arthroscopic. Formal fluoroscopic guided interface biopsy has also been described with excellent results. In a recent series of 86 TKRs preoperative arthroscopic biopsy group had a sensitivity of 100%, specificity of 94.7%, positive predictive value of 87.4% and a negative predictive value of 100%. ConclusionIn the presence of clinical suspicion with raised biomarkers, it is recommended that aspiration +/- biopsy with synovial fluid testing is performed. Direct culture and cell count are recommended. ‘Point of care tests’ such as Leucocyte Esterase testing should be considered. Duration of culture, including pathogen and host factors, should be discussed with a local microbiology/ID department in the context of a formal multi-disciplinary team.

The Culture Medium Optimization of Serratia plymuthica UBCF_13 To Produce Antifungal Compounds

Bacteria can produce secondary metabolites compounds that act as antifungal agents. But in natural conditions, the production of secondary metabolites is low and requires certain growth conditions. One of the methods to increase secondary metabolites production is the modification of the culture medium. In this study, four environmental factors combination was used to optimize the optimal culture medium for S. plymuthica UBCF_13 to produce antifungal compounds. The environmental factors were pH 8, 2 % peptone, 1 mM MnSO4, and 8 hours culture duration. In inhibitory activity test to C. gloeosporioides fungi, cell-free supernatant and cell culture of S. plymuthica UBCF_13 were applied. The results showed that the highest inhibitory activity of cell-free supernatant application (17.80%) was obtained from a medium that adjusted with pH 8, 1 mM MnSO4, and 8 hours duration. While the highest inhibitory activity of cell culture application (44.53%) was obtained from the medium with a combination of pH 8, 2% peptone, 1 mM MnSO4, and 8 hours culture duration. In the SDS-PAGE visualization, 4 protein bands appeared which were thought to be closely related to the inhibitory activity of antifungal compounds of S. plymuthica UBCF_13. The optimum medium was found to be a medium with a combination of pH 8, 2% peptone, 1mM MnSO4, and 8 hours of culture duration.

Antifungal abilities of Serratia plymuthica UBCF_13 produced in different types of media and culture time

Serratia plymuthica strain UBCF_13 is a potential biocontrol agent to inhibit pathogen causing anthracnose disease in plants, Colletotrichum gloesporioedes. The production of bacterial antifungal compounds is affected by nutrition and the growth phase of bacteria. This study is aimed to determine the optimal medium and duration of culture for the production of antifungal compounds. This study used three types of liquid media culture for UBCF_13 [King’s B, Potato Dextrose, and tryptic Soy] and six variations in duration culture [8, 16, 24, 32, 40, and 48 hours]. Extracellular that has been produced was used for antifungal activity assay against C. gloeosporioides. The results showed that the extracellular produced in the PDB with 48 hours culture duration could inhibit 18%. C. gloeosporioides growth. Different nutritional compositions for UBCF_13 cultures show affect the metabolism and compound produced as well. This was indicated by differences in protein profiles of the intracellular and extracellular. Further studies are necessary to be carried out, particularly to see the mechanism of the nutrient medium that could affect the metabolism of UBCF_13 in the synthesis of antifungal compounds.