Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.

Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.

New treatment options of acute myeloid leukemia (AML) are rapidly emerging. Pre-clinical models such as ex vivo cultures are extensively used towards the development of novel drugs and to study synergistic drug combinations, as well as to discover biomarkers for both drug response and anti-cancer drug resistance. Although these approaches empower efficient investigation of multiple drugs in a multitude of primary AML samples, their translational value and reproducibility are hampered by the lack of standardized methodologies and by culture system-specific behavior of AML cells and chemotherapeutic drugs. Moreover, distinct research questions require specific methods which rely on specific technical knowledge and skills. To address these aspects, we herein review commonly used culture techniques in light of diverse research questions. In addition, culture-dependent effects on drug resistance towards commonly used drugs in the treatment of AML are summarized including several pitfalls that may arise because of culture technique artifacts. The primary aim of the current review is to provide practical guidelines for ex vivo primary AML culture experimental design.

Development of a selective and differential media for the isolation and enumeration of Bacillus cereus from food samples.

AIM: Identification and enumeration of foodborne pathogens in food stuffs are valuable concerns. In the present study, starch-blood-egg yolk-polymyxin B-trimethoprim-ceftazidime (SBYPTC) agar was established to isolate and specify the number of Bacillus cereus in food products. METHODS AND RESULTS: The effectiveness of the developed medium in selecting for B. cereus from pure cultures and food matrixes naturally contaminated by high levels of microbiota was estimated, and the results were compared with that of two commercially available MYPA and PMBA media. In pure cultures, there were no significant differences in the recoverability of B. cereus among the three media, however, SBYPTC agar showed a greater exclusivity. To examine SBYPTC performance in food, B. cereus were artificially inoculated into lettuce and potato samples with high background microbiota in two separated experiments. There were no significant differences between MYPA and PEMBA. However, SBYPTC manifested greater selectivity and exclusivity and made the differentiation easier by allowing growth of B. cereus in separated colonies and inhibiting competing microflora. CONCLUSION: Our results showed that SBYPTC has high selective properties in comparison with MYPA and PEMBA. Thus, it can be considered as a useful tool to monitor the existence and the number of B. cereus in foods especially those contaminated with high levels of microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: In the food industry, SBYPTC can be employed for food quality assurance to monitor B. cereus in food products contaminated with high levels of microbiota.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome.

BACKGROUND: In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. METHODS: Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.

Metabolic and proliferation evaluation of human adipose-derived mesenchymal stromal cells (ASC) in different culture medium volumes: standardization of static culture.

Adipose-derived mesenchymal stromal/stem cells (ASC) have acquired a prominent role in tissue engineering and regenerative medicine. However, the standardization of basic culture procedures in this cellular type is still not well established according to the main qualitative cellular attributes. We evaluate the cell growth profile of human ASC in a different culture medium volumes and their nutritional composition utilizing static cultivation. Culture medium volumes (5, 10 and 15 mL/25 cm2) in T-flasks were evaluated by kinetic parameters and the metabolic composition was determined by biochemical analysis and Fourier transform infrared (FT-IR) absorption spectroscopy. 50% renewal of culture medium volume every 48 h was adopted. Immunophenotypic characterization and cell differentiation were performed. There was no difference (p > 0.05) in the kinetic parameters of cell proliferation between the culture medium volumes or in FT-IR composition. However, the concentrations of glucose, glutamine, lactate, and glutamate varied significantly during the cultivation process as a function of the medium volume. ASC presented specific antigens and differentiation potential of mesenchymal stromal/stem cells. It was concluded that the minimal culture medium volume (5 mL/25 cm2 in static culture) was sufficient to maintain the stability, potency, and growth of ASC, representing an economic and safe standardization for this cell culture process.

A Decade of Development of Chromogenic Culture Media for Clinical Microbiology in an Era of Molecular Diagnostics.

In the last 25 years, chromogenic culture media have found widespread application in diagnostic clinical microbiology. In the last decade, the range of media available to clinical laboratories has expanded greatly, allowing specific detection of additional pathogens, including Pseudomonas aeruginosa, group B streptococci, Clostridium difficile, Campylobacter spp., and Yersinia enterocolitica. New media have also been developed to screen for pathogens with acquired antimicrobial resistance, including vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter spp., and Enterobacteriaceae with extended-spectrum ß-lactamases and carbapenemases. This review seeks to explore the utility of chromogenic media in clinical microbiology, with particular attention given to media that have been commercialized in the last decade. The impact of laboratory automation and complementary technologies such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is also assessed. Finally, the review also seeks to demarcate the role of chromogenic media in an era of molecular diagnostics.

pH stability of human preimplantation embryo culture media: effects of culture and batches.

RESEARCH QUESTION: How stable is the pH of human preimplantation embryo culture media during IVF culture and is there variation in pH between batches of culture media? DESIGN: To evaluate pH stability, three batches of three culture media were incubated in triplicate without embryos (sham culture) at CO2 levels recommended by the manufacturers (5% or 6%) for 4 days. To evaluate differences in pH between batches, the pH of three batches of five culture media was measured in triplicate during 1 day of sham culture. Linear mixed models were used for the analysis. RESULTS: An increase in pH during 4 days of culture was found in all three culture media, but the observed increased values were within the generally accepted range for clinical practice (pH 7.2-7.4). One medium was pH 7.1 in the first 2 days, but this was within the range provided by the manufacturer for that medium. Three out of five analysed media showed batch variation in pH that exceeded the generally accepted range for clinical practice. CONCLUSIONS: A relevant difference in pH was found between batches of human preimplantation embryo culture media. This suggests that the CO2 level of incubators may need to be adjusted for new batches of culture medium based on measured pH, to anticipate batch variability and safely accommodate limited pH increase over time. This study was unable to identify the cause of the differences in pH between batches, and further investigation on a larger number of batches and other media seems warranted.

Development of a versatile high-temperature short-time (HTST) pasteurization device for small-scale processing of cell culture medium formulations.

The compatibility of CHO cell culture medium formulations with all stages of the bioprocess must be evaluated through small-scale studies prior to scale-up for commercial manufacturing operations. Here, we describe the development of a bespoke small-scale device for assessing the compatibility of culture media with a widely implemented upstream viral clearance strategy, high-temperature short-time (HTST) treatment. The thermal stability of undefined medium formulations supplemented with soy hydrolysates was evaluated upon variations in critical HTST processing parameters, namely, holding times and temperatures. Prolonged holding times of 43 s at temperatures of 110 °C did not adversely impact medium quality while significant degradation was observed upon treatment at elevated temperatures (200 °C) for shorter time periods (11 s). The performance of the device was benchmarked against a commercially available mini-pilot HTST system upon treatment of identical formulations on both platforms. Processed medium samples were analyzed by untargeted LC-MS/MS for compositional profiling followed by chemometric evaluation, which confirmed the observed degradation effects caused by elevated holding temperatures but revealed comparable performance of our developed device with the commercial mini-pilot setup. The developed device can assist medium optimization activities by reducing volume requirements relative to commercially available mini-pilot instrumentation and by facilitating fast throughput evaluation of heat-induced effects on multiple medium lots.

Biotechnological production of itaconic acid-things you have to know.

Itaconic acid is one of the basic chemicals for the polymer industry, which can be produced on the basis of renewable raw materials. Since the middle of the twentieth century, itaconic acid has been produced industrially using the filamentous fungus Aspergillus terreus. But the demand for the organic acid is low due to the high production costs compared to alternative petrochemical manufactured raw materials. The high production costs are based on a low final titer, low productivities, and the usage of pure sugars, purified molasses, or starch hydrolysates, since the fungus reacts very sensitively to impurities in a culture medium. This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of itaconic acid. The technological achievements in the biotechnological production of itaconic acid are presented. Particular attention is paid to current achievements in terms of suitable alternative substrates and their applicability in fermentation processes. Also, the pathway of itaconic acid and especially the influences on the fermentation process, which must be known in order to achieve a high final titer of itaconic acid, a yield close to the theoretical yield, and high productivity.

Examining the sources of variability in cell culture media used for biopharmaceutical production.

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments.

BACKGROUND: Fungal laccase has profound applications in different fields of biotechnology due to its broad specificity and high redox potential. Any successful application of the enzyme requires large scale production. As laccase production is highly dependent on medium components and cultural conditions, optimization of the same is essential for efficient product production. RESULTS: Production of laccase by fungal strain Marasmiellus palmivorus LA1 under solid state fermentation was optimized by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L8) was designed using Qualitek-4 software to study the interactions and relative influence of the seven selected factors by one factor at a time approach. The optimum condition formulated was temperature (28 °C), pH (5), galactose (0.8%w/v), cupric sulphate (3 mM), inoculum concentration (number of mycelial agar pieces) (6Nos.) and substrate length (0.05 m). Overall yield increase of 17.6 fold was obtained after optimization. Statistical optimization leads to the elimination of an insignificant medium component ammonium dihydrogen phosphate from the process and contributes to a 1.06 fold increase in enzyme production. A final production of 667.4 ± 13 IU/mL laccase activity paves way for the application of this strain for industrial applications. CONCLUSION: Study optimized lignin degrading laccases from Marasmiellus palmivorus LA1. This laccases can thus be used for further applications in different scales of production after analyzing the properties of the enzyme. Study also confirmed the use of taguchi method for optimizations of product production.

Letter to the editor on 'The necessity of overhaul in perception of microbiological culture methods'.

Unarguably, clinical microbiology has got a boost from NGS technology, but in the process of this transition it has suffered a huge setback. Computational biology can find the microbial genomic variations and can link it to drug resistance, but it has so far underestimated the crucial role of microbial culture medium. The constituents and growth conditions of the medium have been documented to shuffle genomic, epigenetic and metabolic aspects of the bacterial pathogens. Ignoring these in vitro-driven evolutions and attributing the variations as normal bacterial features, responsible for drug resistance is a huge mistake. Unfortunately, it has been the trend since the inception of NGS in microbiology arena. No wonder, drug resistance is spreading unrestrained like wildfire and drug discovery is lagging behind. The urgent need to standardize culture medium by simulating it to human physiological conditions can salvage the situation and result in correct interpretations.

A consensus introduction to serum replacements and serum-free media for cellular therapies.

The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.

Comparative analyses of industrial-scale human platelet lysate preparations.

BACKGROUND: Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. STUDY DESIGN AND METHODS: Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. RESULTS: Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. CONCLUSION: Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures.

Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality.

Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448-1458. © 2017 Wiley Periodicals, Inc.

Novel Method Enabling the Use of Cryopreserved Primary Acute Myeloid Leukemia Cells in Functional Drug Screens.

The ability to assess antileukemic drug activity on primary patient samples is a powerful tool in determining potential drug targets and selection of therapeutic agents with biological and functional rationale. We previously established small molecule inhibitor screens for use on freshly isolated leukemia cells for this purpose. Here we describe a method that produces functional small molecule inhibitor screening results using cryopreserved primary acute myeloid leukemia cells. This method was established to take advantage of biorepositories containing archival material, such as those established by the Children's Oncology Group, and to enable validation of potential pathway dependencies uncovered by genomic analysis. Various conditions used to thaw and culture cryopreserved specimens were assessed for effect on viability, differentiation, and the ability to recapitulate sensitivity results obtained on fresh samples. The most reproducible results were obtained by quick-thawing and culturing samples in cytokine rich media before performing drug screens. Our data suggest that cytokine-enriched media aids in maintaining the viability and numbers required to perform functional analysis on cryopreserved leukemia cells. This method can aid in producing informative data on therapeutic targeting and precision medicine efforts in leukemia by making use of biorepositories and bio banks.

A high-throughput template for optimizing Drosophila organ culture with response-surface methods.

The Drosophila wing imaginal disc is a key model organ for molecular developmental genetics. Wing disc studies are generally restricted to end-point analyses of fixed tissues. Recently several studies have relied on limited data from discs cultured in uncharacterized conditions. Systematic efforts towards developing Drosophila organ culture techniques are becoming crucial for further progress. Here, we have designed a multi-tiered, high-throughput pipeline that employs design-of-experiment methods to design a culture medium for wing discs. The resulting formula sustains high levels of proliferation for more than 12 hours. This approach results in a statistical model of proliferation as a function of extrinsic growth supplements and identifies synergies that improve insulin-stimulated growth. A more dynamic view of organogenesis emerges from the optimized culture system that highlights important facets of growth: spatiotemporal clustering of cell divisions and cell junction rearrangements. The same approach could be used to improve culture conditions for other organ systems.

Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

BACKGROUND: Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5+ T lymphocytes to assess their T cell stimulatory capacity. RESULTS: The microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum were found to be superior in their functional T lymphocyte priming capacity and to elicit significantly less non-specific proliferation. CONCLUSIONS: EqMoDC generated with horse serum-supplemented medium showed improved morphological characteristics, higher cell viability and exhibited a more robust performance in the functional T cell assays. Therefore, horse serum was found to be superior to FBS for generating equine monocyte-derived dendritic cells.

Optimizing culture conditions for free-living stages of the nematode parasite Strongyloides ratti.

The rat parasitic nematode Strongyloides ratti (S. ratti) has recently emerged as a model system for various aspects of parasite biology and evolution. In addition to parasitic parthenogenetic females, this species can also form facultative free-living generations of sexually reproducing adults. These free-living worms are bacteriovorous and grow very well when cultured in the feces of their host. However, in fecal cultures the worms are rather difficult to find for observation and experimental manipulation. Therefore, it has also been attempted to raise S. ratti on Nematode Growth Media (NGM) plates with Escherichia coli OP50 as food, exactly as described for the model nematode Caenorhabditis elegans. Whilst worms did grow on these plates, their longevity and reproductive output compared to fecal cultures were dramatically reduced. In order to improve the culture success we tested other plates occasionally used for C. elegans and, starting from the best performing one, systematically varied the plate composition, the temperature and the food in order to further optimize the conditions. Here we present a plate culturing protocol for free-living stages of S. ratti with strongly improved reproductive success and longevity.