Viability Of Cultures Research Articles

Evaluation of Yoghurt Fortified with Encapsulated Echium Oil Rich in Stearidonic Acid as a Low-Fat Dairy Food

Echium oil (EO) is oil rich in stearidonic acid (SDA) as well as linolenic acid, which have many health benefits, and protective roles in inflammation, cardiovascular disease (CVD) and cancer. The study aims to produce and evaluate yoghurt fortified with encapsulated echium oil (EEO) as a functional low-fat dairy food. The EEO was added to skimmed buffalo milk at rate of 0, 2, 4, and 6% (w/v) to create 4 treatments. Physiochemical, microbiological properties, and sensory acceptance were evaluated in the fresh samples and during the storage period at 5±2°C for 21 days. As the EEO increased, soluble nitrogen (WSN/TN ratio), acetaldehyde and viscosity increased while pH value decreased. In addition, the WSN/TN ratio, diacetyl, and viscosity gradually increased, but the pH value and acetaldehyde content decreased with increasing storage time. Yoghurt fortified with EEO had the lowest peroxide value compared to control sample. The addition of EEO had no effect on viability of starter culture during storage period. The best of sensory evaluation was achieved by addition 2% EEO.

Evidence for toxic cyanobacteria in sediments and the water-sediment interface of a tropical drinking water reservoir

Cyanobacterial blooms in drinking water reservoirs constitute a health risk for humans and animals, especially in tropical zones where climate conditions may favour their prevalence throughout the year. The presence, viability, and toxicity of cyanobacteria were determined in the euphotic zone, in the water-sediment interface, and in the upper sediment fraction of the Riogrande II reservoir (Antioquia, Colombia), which provides drinking water for 1.4 million people. Cyanobacterial identification and quantification were performed in environmental samples of the euphotic zone and of the sediment-water interface. In addition, the effects of environmental parameters on occurrences of cyanobacteria were assessed and two microcystin synthetase (mcy) genes were extracted from water, interface, and sediment samples and quantified using qPCR. The viability of the cells in cultures from these different matrices was determined using chlorophyll-a measurements. Chlorophyll-a concentrations representing the biomass of cyanobacteria were higher at the interface than in the euphotic zone at all sampling sites. The potentially toxic genera Dolichospermum and Microcystis predominated in the euphotic zone and the interface, respectively. Moreover, the copy numbers of mcy genes was higher in cyanobacteria sampled at the interface and the upper sediment layers than in cyanobacteria sampled in the euphotic zone, which were correlated with the order Chroococcales. No impact of variation in environmental conditions on cyanobacterial abundance was observed. Viability of cyanobacterial cultures sampled from the interface and the upper sediment fraction showed the ability of cyanobacteria from several orders to proliferate under lab conditions. These results show that the surface sediment layer from Riogrande II serves as a sink for potentially toxic cyanobacteria which have the potential to recolonize and bloom in the water column.

In vitro selection of birch for tolerance to salinity stress

In vitro modelling of stress is one of the promising avenues for plant breeding for tolerance to negative environmental factors. In this study we examined the effect of NaCl (0.5%) on callusogenesis and morphogenesis of stem explants of different birch genotypes: Betula pendula Roth, B. pendula Roth var. carelica (Mercklin) Hämet-Ahti, B. pendula f. ‘dalecarlica’ (L.f.) Schneid., B. pubescens Ehrh. In our experiments we used pre-selected microclones from our in vitro collection on NaCl (0.2-1.0%) selective media. The clones were contrasted by the degree of their sensitivity to salinity (so-called ‘stable’ and ‘sensitive’ microclones). With the use of stem callus cultures we identified informative, simple and reproducible indicators for the selection of salt-tolerant genotypes. Among these indicators were the frequency of callus formation and the viability of callus cultures, which were significantly higher in ‘stable’ group of microclones. Polyploid birch clones (2n=4x=56, 2n=3x=42) were more resistant to salination compared to diploid clones (2n=28). Our study has shown that the selection of salt-tolerant birch lines can be based on the plants’ genetic diversity presented in the collection (various species, varieties, hybrids, polyploids) and manifested in the process of in vitro cultivation, as well as in the cellular heterogeneity of callus cultures.

Electrochemical laser induced graphene-based oxygen sensor

• Highly sensitive DO biosensor based on LIG decorated with co-Pt. • Enriched layer of metallic Pt nanostructures on graphene composite detected by SEM. • Synergistic response to DO in H9C2 cell culture. • Extended linear dynamic range 30 µM to 400 µM and fast response time of 2 s. • Synergy of electrochemistry and SEM and XPS analyses to investigate surface modifications. A fully integrated dissolved oxygen (DO) sensor was fabricated using laser induced graphene (LIG). The LIG was patterned on polyimide (Kapton) film using CO 2 laser followed by the electrodeposition of colloidal platinum to form the electroactive sensing area. The sensing area was insulated in an oxygen gas permeable membrane for selective detection of DO. The fabricated sensor demonstrated excellent catalytic activity towards the reduction of oxygen with a sensitivity of 246.66 µA/mMcm 2 over a linear detection range of 30 µM to 400 µM. The sensor exhibited a limit of detection (LOD) of 2.4 µM DO and fast response time of 2 s. The LIG-based DO sensor also exhibited good selectivity and stability. The sensor was further evaluated in a cell culture system to monitor DO to maintain cell culture viability. The DO sensor showed good performance in detecting oxygen in fresh medium (82.2 µM), at 80% cell confluency (102.37 µM), and at 100% confluency (93.52 µM). The performance of the DO sensor offers great promise for detecting dissolve oxygen in cell culture systems for biomedical and environmental applications.

Разработка и влияние добавки из лимфоидной ткани птицы на жизнеспособность клеток в культуре

Introduction. Today, dietary supplements are an integral part of human diet. Some of them are made of hydrolysates of animal origin. Biologically active additives of immunomodulatory action can prevent various diseases. The research objective was to develop a dietary supplement from the bursa of Fabricius obtained from broiler chickens and evaluate its effect on cell viability in culture.
 Study objects and methods. The study featured biologically active supplement obtained by enzymatic hydrolysis of the bursa of Fabricius, immature stem cells, and adult differentiated cells of human dermal fibroblasts, HeLa and MCF-7 cancer cells, and extract of the bursa of Fabricius.
 Results and discussion. The research resulted in a new technology of dietary supplement production from the bursa of Fabricius of broiler chickens. It included washing, cutting, homogenization, proteolytic enzyme fermentation, and ultrafiltration. When introduced into the culture of mesenchymal stem cells, the dietary supplement caused a slight decrease in the cell viability at concentrations of 25 and 50%, which indicated a possible cytotoxic effect of the extract on mesenchymal cells. The extract did not affect the viability of human fibroblast culture and caused no cytotoxic effect. In MCF-7 culture, the extract had a dose-dependent cytotoxic effect, which lowered the relative cell viability. 
 Conclusion. The new dietary supplement based on the bursa of Fabricius of broiler chickens had a cytotoxic effect on stem cell cultures. However, it did not affect the cell viability and had no cytotoxic effect on human dermal fibroblasts. The effect depended on the cell culture. In the case of HeLa, the supplement stimulated proliferative activity, and in the case of MCF-7, it had a cytotoxic effect. Therefore, the new dietary supplement demonstrated some prospects as an active ingredient for various biologically active additives and immunomodulatory drugs.

Response of human cancer cells to simultaneous treatment with sorafenib and radiofrequency current.

Due to their alleged analgesic, anti-inflammatory and tissue regenerative effects, capacitive-resistive electrothermal therapy (CRET), which is based on non-invasive exposure to radiofrequency (RF) currents, is often applied to chemotherapeutically treated patients with cancer. Our previous studies have demonstrated that subthermal CRET currents can elicit a number of cell responses, including anti-proliferative effects, in the human liver cancer cell line HepG2. Such effects involve significant changes in the regulation of proteins involved in MAPK signaling pathways, which are also implicated in the cancer cell response to standard anticancer drugs such as sorafenib. This overlap in response pathways may lead to competitive, neutralizing or blocking interactions between the electrical and chemical treatments, thus raising questions on the advisability of CRET treatment for their analgesic, anti-inflammatory or other purposes in patients undergoing chemotherapy. The present study analyzed the effects of simultaneous treatment with sorafenib and 448-kHz, subthermal CRET current on the proliferation and viability of HepG2 cell cultures. Cell viability was assessed through Trypan blue or XTT assays, while flow cytometry was applied for cell cycle and apoptosis analysis. The expression of proteins involved in cell proliferation were assessed by immunoblotting and immunofluorescence. The results revealed no evidence to suggest that the electrical treatment counteracted or neutralized the cellular response to sorafenib at the different conditions evaluated. Furthermore, at the standard pharmacological sorafenib concentration, 5 µM, the combined treatment elicited an anti-proliferative response significantly stronger than that induced by each of the treatments when applied separately in HepG2 cells. These data do not support the hypothesis that CRET exposure may inhibit or diminish the effects of a chemotherapeutic drug used in cancer treatment, and highlights the requirement for further investigation into the cell response to the combined action of electrical and chemical treatments.

P04.02 Single-cell transcriptomic analysis reveals shifts in glioblastoma cell composition in different BMP4-treated primary tumor cultures

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. The well-known cellular heterogeneity of this cancer, which includes glioma tumor-initiating cells with stem cell characteristics (GSCs), (co)influences therapy resistance and tumor recurrence. Bone Morphogenetic Protein-4 (BMP4) promotes differentiation of GSCs towards astroglial lineage while suppressing oligodendrocyte maturation. Treatment with BMP4 is associated with increased survival in mice. BMPs exert effects in cell-type and context dependent fashion, but also generate subtle heterogeneity in transcriptional response among similar cells. We studied first the cell viability of BMP4-treated primary GBM cultures followed by single-cell RNA-sequencing (scRNA-seq) on two differently responding cultures, and found correlation between their responses and therapy sensitivity. MATERIAL AND METHODS Cell viability, proliferation and apoptosis were assessed in 17 patient-derived BMP4-treated GBM cell cultures. We selected one culture in which this treatment induced high in vitro therapeutic efficacy, and one in which the treatment was ineffective, for analysis by scRNA-seq and then compared the results on the initial panel of 17 cultures. RESULTS After 7 days of treatment with BMP4, cell viability ranged from 28% (referred to as highest in vitro therapeutic efficacy) to 132% compared to untreated cells. scRNA-seq of the previously mentioned cultures in passage 7 showed that all neural cell types that are usually found in freshly resected GBM, were also present in our cultures. In the culture where BMP4 induced high in vitro therapeutic efficacy, BMP4 induces the formation of a large new cell population displaying decreased cell proliferation, increased migration and cell death, while the pro-inflammatory cells were depleted. RNA-velocity analysis revealed that, the cycling of cells was greatly diminished in the culture where therapy with BMP4 was efficacious, whereas this was enhanced in the GBM culture with the lowest cell viability after treatment. Astroglial differentiation was induced in all BMP4-treated cultures, while neuronal differentiation was reduced most in the cultures in which BMP4 induced lower or no in vitro therapeutic efficacy. In the culture without therapeutic efficacy of BMP4 cell cycle arrest was not induced anymore. In addition, OLIG1/2 mRNA and protein levels seemed predictive for BMP4-therapy efficacy, while activation of translation-associated genes (RPL27A, RPS27) was a suitable, immediate post-therapeutic marker for this. CONCLUSION scRNA-seq of in vitro GBM cultures provides advanced insights into the mechanism underlying therapy efficacy of BMP4. Neural differentiation status is distinctive for therapeutic efficacy of BMP4 in vitro before and after therapy.

A comparative study between different preservation methods on the viability of some yeast cultures

The influence of chilling, freezing, and dry-ing preservation methods on morphological properties and viability of four yeast cultures, isolated Saccharomyces boulardii (SB1 and SB2), Saccharomyces cerevisiae (SC), and Kluyveromyces lactis (KL), was studied. chilling preservation had no effect on yeast colony morphology parameters till the end of the preservation period in contrast with freez-ing and drying preservation methods. Drying preservation resulted in the highest viability decrement % of yeast cultures ranged between 59.38 to 40.21% as compared with other preservation methods. There is a noticeable re-duction in D values of preserved yeast cultures by drying, which varied from (33 to 26 day) in comparison to chilling and freezing preserva-tion that recorded (119 to 83 and 180 to 57 day), respectively. The obtained results re-vealed that chilling preservation under proper conditions, as well as freezing, is more prefer-able for the preservation of studied yeast cul-tures than that preserved yeast by the drying method.

Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria.

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

Historical Development of the Puccinia triticina Population in South Africa.

In contrast to many other countries, the virulence and genetic diversity of the South African Puccinia triticina population before 1980 is unknown, because of the absence of regular and systematic race analysis data and viable rust cultures. Herbarium specimens housed at the National Collection of Fungi, Biosystematics Unit, Plant Health and Protection, Agricultural Research Council, Pretoria, South Africa (SA), provided the opportunity to investigate the genetic development of the population using isolates collected between 1906 and 2010. Five subpopulations that survived between 21 and 82 years in the field were found. While three of these could represent the original races that entered SA during European settlement, two appear to be recent exotic introductions into SA, most probably from other African countries. The demise of the three oldest subpopulations might be from the release of resistant wheat cultivars. The population is clonal, where new virulence develops through single step mutations and selection for virulence. Although a possible case of somatic hybridization was found, sexual reproduction appears to be absent in SA. This study confirmed the importance of annual surveys in SA and its neighboring countries for the timely detection of new virulent races that could threaten wheat production in SA.

Measuring 3D Cell Culture Viability in Multiple 3D Printed Scaffolds Within a Single Miniature Electrical Impedance Tomography Sensor

The use of 3D cell culture for tissue engineering and regenerative medicine applications often challenges conventional biochemical and optical assays. Impedance‐based cellular assays have shown their potential to retrieve dielectric parameters pertaining to cell behavior such as viability, proliferation, and differentiation for 2D adherent cell culture. Herein, simultaneous 3D impedance imaging and viability measurements of multiple large (>2 mm) 3D cell cultures embedded in collagen gels are demonstrated. The method is facilitated by low‐resistance 3D printed scaffolds that can hold a 3D cell–gel system throughout cell culture while being transparent to impedance imaging. It is shown in silico and in vitro that frequency‐difference electrical impedance tomography (fd‐EIT) can nondestructively and in a label‐free way differentiate a variety of cell concentrations with a single miniature sensor in real time. This study paves the way toward the development of EIT imaging for the quantitative and noninvasive evaluation of tissue engineering products.

Ciprofloxacin reduces tenocyte viability and proteoglycan synthesis in short-term explant cultures of equine tendon.

Fluoroquinolones are an effective, broad-spectrum antibiotic used to treat an array of bacterial infections. However, they are associated with an increased risk of tendinopathy and tendon rupture even after discontinuation of treatment. This condition is known as fluoroquinolone-associated tendinopathy, the underlying mechanisms of which are poorly understood. While many factors may be involved in the pathophysiology of tendinopathies in general, changes in tenocyte metabolism and viability, as well as alteration of proteoglycan metabolism are prominent findings in the scientific literature. This study investigated the effects of ciprofloxacin, a common fluoroquinolone, on cell viability, proteoglycan synthesis, and proteoglycan mRNA expression in equine superficial digital flexor tendon explants after 96 h treatment with between 1–300 µg/mL ciprofloxacin, and again after 8 days discontinuation of treatment. Ciprofloxacin caused significant reductions in cell viability by between 25–33% at all dosages except 10 µg/mL, and viability decreased further after 8 days discontinuation of treatment. Proteoglycan synthesis significantly decreased by approximately 50% in explants treated with 100 µg/mL and 300 µg/mL, however this effect reversed after 8 days in the absence of treatment. No significant mRNA expression changes were observed after the treatment period with the exception of versican which was down-regulated at the highest concentration of ciprofloxacin. After the recovery period, aggrecan, biglycan and versican genes were all significantly downregulated in explants initially treated with 1–100 µg/mL. Results from this study corroborate previously reported findings of reduced cell viability and proteoglycan synthesis in a whole tissue explant model and provide further insight into the mechanisms underlying fluoroquinolone-associated tendinopathy and rupture. This study further demonstrates that certain ciprofloxacin induced cellular changes are not rapidly reversed upon cessation of treatment which is a novel finding in the literature.

Design and optimization of 3D-bioprinted scaffold framework based on a new natural polymeric bioink.

This aimed at the design and production of engineered 3D scaffold prototypes using a natural polymeric bioink made of chitosan and poly-γ-glutamic acid with a specific focus on 3D-bioprinting process and on 3D framework geometry. Prototypes were produced using a 3D bioprinter exploiting layer-by-layer deposition technology. The 3D scaffold prototypes were fully characterized concerning pore size and size distribution, stability in different experimental conditions, swelling capability, and human dermal fibroblasts viability. Hexagonal framework combined with biopaper allowed stabilizing the 3-layers structure during process manufacturing and during incubation in cell culture conditions. The stability of 3-layers structure was well preserved for 48 h. Crosslinking percentages of 2-layers and 3-layers prototype were 88.2 and 68.39, respectively. The swelling study showed a controlled swelling capability for 2-layers and 3-layers prototype, ∼5%. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results showed good biocompatibility of 3-layers prototype and their suitability for preserving 48 h cell viability in 3D cultures. Moreover, a significant increment of absorbance value was measured after 48 h, demonstrating cell growth. Bioink obtained combining chitosan and poly-γ-glutamic acid represents a good option for 3D bioprinting. A stable 3D structure was realized by layer-by-layer deposition technology; compared with other papers, the present study succeeded in using medical healthcare-grade polymers, no-toxic crosslinker, and solvents according to ICH Topic Q3C (R4).

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Highly self-healable and injectable cellulose hydrogels via rapid hydrazone linkage for drug delivery and 3D cell culture

To meet the rising demand of injectable hydrogels with self-healing, robustness and biocompatibility for biomedical engineering, the reversible ketoester-type acylhydrazone linkages was used for the fabrication of novel cellulose-based hydrogel. The ketoester-type acylhydrazone bond exchanged rapidly, endowing the hydrogels with highly efficient self-healing performance without any external stimuli under physiological environment, which was hardly achieved with the widely used arylhydrozone bond. The dynamic hydrogels exhibited tunable mechanical property, pH responsiveness, injectability and biocompatibility, demonstrating immense applications prospect for various biomedicines, such as drug and cell delivery. The pH-responsive controlled release of model drug doxorubicin (DOX) loaded in the hydrogel was demonstrated. In addition, benefitting from the excellent biocompatibility and the reversible ketoester-type acylhydrazone bonds, cells were encapsulated in the hydrogels as 3D carrier. The covalent adaptable network intensified injectability of cell-laden hydrogels and improved the long-lasting viability for cell culture, showing great potential in the biomedical field.

Humanized Biomimetic Nanovesicles for Neuron Targeting.

Nanovesicles (NVs) are emerging as innovative, theranostic tools for cargo delivery. Recently, surface engineering of NVs with membrane proteins from specific cell types has been shown to improve the biocompatibility of NVs and enable the integration of functional attributes. However, this type of biomimetic approach has not yet been explored using human neural cells for applications within the nervous system. Here, this paper optimizes and validates the scalable and reproducible production of two types of neuron‐targeting NVs, each with a distinct lipid formulation backbone suited to potential therapeutic cargo, by integrating membrane proteins that are unbiasedly sourced from human pluripotent stem‐cell‐derived neurons. The results establish that both endogenous and genetically engineered cell‐derived proteins effectively transfer to NVs without disruption of their physicochemical properties. NVs with neuron‐derived membrane proteins exhibit enhanced neuronal association and uptake compared to bare NVs. Viability of 3D neural sphere cultures is not disrupted by treatment, which verifies the utility of organoid‐based approaches as NV testing platforms. Finally, these results confirm cellular association and uptake of the biomimetic humanized NVs to neurons within rodent cranial nerves. In summary, the customizable NVs reported here enable next‐generation functionalized theranostics aimed to promote neuroregeneration.

Use of encapsulated lactic acid bacteria as bioprotective cultures in fresh Brazilian cheese.

There is great interest for biopreservation of food products, and encapsulation may be a good strategy to extend the viability of protective cultures. In this study, Lactobacillus paraplantarum FT-259 and Lactococcus lactis QMF 11 were separately encapsulated in casein/pectin (C/P) microparticles, which were tested for antilisterial and anti-staphylococcal activity in fresh Minas cheese (FMC) stored at 8°C. The encapsulation efficiency for both lactic acid bacteria (LAB) was 82.5%, with viability over 6.2 log CFU/g after storage of C/P microparticles for 90days under refrigeration. Interestingly, free Lb. paraplantarum and free Lc. lactis grew significantly in refrigerated FMC, both in the presence and absence of pathogens, but only the first significatively grew when encapsulated. Encapsulation increased the antilisterial activity of Lb. paraplantarum in FMC. Moreover, Lc. lactis significantly inhibited listerial growth in FMC in both its free and encapsulated forms, whereas Staphylococcus aureus counts were only significantly reduced in the presence of free Lc. lactis. In conclusion, these results indicate that C/P microparticles are effective carriers of LAB in FMC, which can contribute for the assurance of the safety of this product.

Rapid indicators for monitoring the health of Chlamydomonas nivalis biomass during preservation

Microalgae are photosynthetic microorganisms that have increasingly attracted interest in the last decade due to their potential for industrial applications. One crucial aspect for the industrial-scale valorisation of algae biomass concerns the quality control during its preservation before being treated to obtain the end-product. Monitoring biomass quality is essential and can be potentially accomplished with the aid of specific biochemical indicators called biomarkers. In this context, the main aim of this work was to identify potential indicators of microalgae biomass viability that could be used as markers of its quality during storage/preservation for commercial operations. The health status of a suspension of Chlamydomonas nivalis was assessed at 4 ºC and 25 ºC during 10 days of storage. The use of the pulse-amplitude-modulation technique, based on chlorophyll fluorescence, was a valuable indicator of the culture viability. The measurement of DNA and of chlorophyll in the supernatant, indicative of cell lysis, also provided satisfactory results; in the case of the DNA, the limit of detection was 3.9 µg DNA·mL−1. The effect of different concentrations of a well-known and cheap preservative, acetic acid, was also evaluated at 4 ºC. This work identified three suitable biomarkers to be used as rapid indicators of the quality of the microalgal suspension prior to its reception for biorefinery activities.

Development and Validation of a Long-Term 3D Glioblastoma Cell Culture in Alginate Microfibers as a Novel Bio-Mimicking Model System for Preclinical Drug Testing.

Background: Various three-dimensional (3D) glioblastoma cell culture models have a limited duration of viability. Our aim was to develop a long-term 3D glioblastoma model, which is necessary for reliable drug response studies. Methods: Human U87 glioblastoma cells were cultured in alginate microfibers for 28 days. Cell growth, viability, morphology, and aggregation in 3D culture were monitored by fluorescent and confocal microscopy upon calcein-AM/propidium iodide (CAM/PI) staining every seven days. The glioblastoma 3D model was validated using temozolomide (TMZ) treatments 3 days in a row with a recovery period. Cell viability by MTT and resistance-related gene expression (MGMT and ABCB1) by qPCR were assessed after 28 days. The same TMZ treatment schedule was applied in 2D U87 cell culture for comparison purposes. Results: Within a long-term 3D model system in alginate fibers, U87 cells remained viable for up to 28 days. On day 7, cells formed visible aggregates oriented to the microfiber periphery. TMZ treatment reduced cell growth but increased drug resistance-related gene expression. The latter effect was more pronounced in 3D compared to 2D cell culture. Conclusion: Herein, we described a long-term glioblastoma 3D model system that could be particularly helpful for drug testing and treatment optimization.

Severe Acute Respiratory Syndrome Coronavirus 2 Normalized Viral Loads and Subgenomic RNA Detection as Tools for Improving Clinical Decision Making and Work Reincorporation.

BackgroundSARS-CoV-2 RT-PCR provides a highly variable cycle-threshold (Ct) value that cannot distinguish viral infectivity. Subgenomic RNA (sgRNA) has been used to monitor active replication. Given the importance of long RT-PCR positivity and the need for work reincorporation and discontinuing isolation, we studied the functionality of normalized viral loads (NVL) for patient monitoring and sgRNA for viral infectivity detection.MethodsNVL measured through the Nucleocapsid and RNA-dependent-RNA-polymerase genes and sgRNA RT-PCRs were performed in 2 consecutive swabs from 84 health-care workers.ResultsNVL provided similar and accurate quantities of both genes of SARS-CoV-2 at two different time-points of infection, overcoming Ct-value and swab collection variability. Among SARS-CoV-2-positive samples, 51.19% were sgRNA-positive in the 1 stRT-PCR and 5.95% in the 2 ndRT-PCR. All sgRNA-positive samples had >4log10RNAcopies/1000cells, while samples with ≤1log10 NVL were sgRNA-negative. Although NVL were positive until 29 days after symptom onset, 84.1% of sgRNA-positive samples were from the first 7 days, which correlated with viral culture viability. Multivariate analyses showed that sgRNA, NVL and days of symptoms were significantly associated (p<0.001)ConclusionsNVL and sgRNA are two rapid accessible techniques that could be easily implemented in routine hospital practice providing a useful proxy for viral infectivity and COVID-19 patient follow-up.