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Microbial Cell Factories Nov 2008Here we describe a novel cultivation method, called EnBasetrade mark, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and...
BACKGROUND
Here we describe a novel cultivation method, called EnBasetrade mark, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.
RESULTS
High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel.Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.
CONCLUSION
The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.
PubMed: 19017379
DOI: 10.1186/1475-2859-7-31 -
ACS Biomaterials Science & Engineering Apr 2020Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling...
Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling hepatocytes to display improved levels of liver-specific functions. One common model is hepatocyte spheroids, which are spontaneously formed cell aggregates. Techniques for spheroid formation include the use of ultralow attachment plates and the hanging drop method, both of which are technically challenging and relatively low throughput. Here, we describe a simple-to-use platform that improves spheroid production and is compatible with genotoxicity testing by the comet assay. To achieve this, we created a chip containing a microwell array where dozens of spheroids are contained within a single well of a 96-well plate. The microwells are made from agarose, a nontoxic material suitable for cell growth and spheroid formation. HepG2 cells loaded into customizable microwells formed spheroids through agarose-assisted aggregation within one to two days. In addition, the agarose matrix allows the same platform to be used in DNA damage analysis. Specifically, the comet assay enables quantification of DNA breaks based on the increased migration of damaged DNA through agarose during electrophoresis. Here, we developed a modified comet assay and show that intact HepG2 spheroids cultured in microwells can be electrophoresed to reveal the extent of DNA damage following exposure to inflammatory chemicals (HO and SIN-1). With this SpheroidChip analysis method, we detected a dose-dependent increase in DNA damage and observed rapid repair of HO-induced DNA damage. In summary, we utilized an agarose microarray to condense what had required an entire 96-well plate into a single well, enabling analysis techniques that were cumbersome or impossible under conditions of a single spheroid per well of a 96-well plate.
Topics: Animals; Cell Culture Techniques; Hydrogen Peroxide; Mutagenicity Tests; Sepharose; Spheroids, Cellular
PubMed: 33145399
DOI: 10.1021/acsbiomaterials.9b01951 -
Frontiers in Microbiology 2018The objective of this study was to evaluate the capacity of 49 methicillin resistant (MRSA) from foods of animal origin (42 from dairy products and 7 from meat and meat...
The objective of this study was to evaluate the capacity of 49 methicillin resistant (MRSA) from foods of animal origin (42 from dairy products and 7 from meat and meat products) to form biofilms. Overall, a higher biofilm biomass was observed for those MRSA strains harboring SCC type IV, while 8 MRSA strains (5 from dairy products and 3 from meat and meat products) were classified as strong biofilm formers in standard Tryptic Soy Broth medium. When a prolonged incubation period (48 h) was applied for those 8 MRSA strains, an increased biofilm biomass accumulation was observed during the time course, whereas the number of viable cells within the biofilms decreased as the biomass increased. The capacity of biofilm production correlated pretty well between the experiments using polystyrene microtiter plates and stainless steel micro-well plates, and significant higher values were observed in stainless steel when glucose was added to TSB during the enrichment. Biofilms were further characterized by confocal laser scanning microscope (CLSM), confirming that proteins and α-polysaccharides were the predominant components inside the extracellular polymeric matrix of biofilms formed by MRSA strains. In conclusion, our results confirm that MRSA isolates from foods of animal origin have significant capacity for forming biofilms with a high protein content, which can play a key role for the successful dissemination of MRSA lineages food. Knowledge of the capacity of MRSA strains to produce biofilms, as well as characterization of the main MRSA biofilms matrix components, can help both to counteract the mechanisms involved in biofilm formation and resistance and to define more rational control strategies by using tailor-made cleaning agents.
PubMed: 30564226
DOI: 10.3389/fmicb.2018.03004 -
Journal of Gastrointestinal & Digestive... Dec 2011The diagnosis (endoscopy, and biopsy) and continued clinical management of Inflammatory Bowel Disease (IBD), remain highly invasive, expensive, and inconvenient for the...
OBJECTIVES
The diagnosis (endoscopy, and biopsy) and continued clinical management of Inflammatory Bowel Disease (IBD), remain highly invasive, expensive, and inconvenient for the pediatric patient. The objective of this study was to see if colonocytes obtained from stools of subjects with IBD and normal controls would demonstrate higher levels of inflammatory markers (Cox 2 in CD45+ and CD45- cells) and if the inflammatory process and treatment effects would be reflected in an altered cytokine expression in the subjects compared to controls.
SETTING
Outpatient hospital based pediatric gastroenterology clinic.
METHODS AND MAIN OUTCOME MEASURES
Stool samples (~ 1 gm), were obtained from 18 children between the ages of 4 and 18 diagnosed with IBD, and from a normal first degree relative. Colonocytes were isolated using the Somatic Cell Sampling Recovery (SCSR) system and assessed for the expression of COX-2, CD-45, IgA, IgG, IL6, IL18, TGF β, TNF, and IL16β using flow cytometry. In addition, levels of COX-2 and cytokeratin 19 transcripts were measured by microwell plate hybridization assay.
RESULTS
Expression of COX-2 and co-expression of IgA and IgG were significantly higher in the IBD cases compared to the controls. In ulcerative colitis, the expression of COX-2 and co-expression of COX-2 and CD45 were greater than that in patients with Crohn's disease. In contrast, cells expressing IgA and IgG were higher in Crohn's. Subjects on immunosuppressants and/or anti-inflammatory medications, expressed significantly lower levels of COX-2 and IL-18 compared to those who were not on treatment.
CONCLUSIONS
This study indicates that the use of disease markers on exfoliated colonic cells can be used for non-invasive assessment of disease status, for follow-up of response to treatment and for forecasting flare-up of disease before its symptomatic manifestations.
PubMed: 23519721
DOI: 10.4172/2161-069X.S8-001 -
Electrophoresis Jun 2017Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We...
Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We demonstrate a microchip that consists of electro-activated microwell arrays for positive dielectrophoresis assisted cell capture, DS measurements, and negative dielectrophoresis driven cell unloading; thus, providing a high-throughput cell analysis platform. To the best of our knowledge, this is the first microfluidic chip that combines electro-activated microwells and DS to analyze biological cells. Device performance is tested using Saccharomyces cerevisiae (yeast) cells. DEP response of yeast cells is determined by measuring their Clausius-Mossotti factor using biophysical models in parallel plate microelectrode geometry. This information is used to determine the excitation frequency to load and unload wells. Effect of yeast cells on the measured impedance spectrum was examined both experimentally and numerically. Good match between the numerical and experimental results establishes the potential use of the microchip device for extracting subcellular properties of biological cells in a rapid and nonexpensive manner.
Topics: Computer Simulation; Dielectric Spectroscopy; Electrophoresis, Microchip; Equipment Design; Gold; Lab-On-A-Chip Devices; Microelectrodes; Microfluidics; Models, Theoretical; Saccharomyces cerevisiae; Ultraviolet Rays
PubMed: 28256738
DOI: 10.1002/elps.201700020 -
Sensors (Basel, Switzerland) May 2014A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane...
A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane (PDMS) slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA) was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD) for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays.
Topics: Biosensing Techniques; Capillary Action; Equipment Design; Equipment Failure Analysis; Humans; Immunoassay; Immunoglobulin A; Microarray Analysis; Miniaturization; Reproducibility of Results; Saliva; Sensitivity and Specificity; Spectrometry, Fluorescence
PubMed: 24859022
DOI: 10.3390/s140509132 -
STAR Protocols Sep 2023The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time...
The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.
Topics: Humans; Organoids; Brain; Head
PubMed: 37715950
DOI: 10.1016/j.xpro.2023.102502 -
International Journal of Neonatal... Oct 2020Mucopolysaccharidosis Type II (MPS II), also known as Hunter syndrome, is a lysosomal storage disorder (LSD) caused by a deficiency of the lysosomal enzyme...
Mucopolysaccharidosis Type II (MPS II), also known as Hunter syndrome, is a lysosomal storage disorder (LSD) caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). MPS II satisfies all criteria defined by the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) for inclusion in the Recommended Uniform Screening Panel (RUSP) for newborn screening, apart from the fact that only minimal prospective population screening data are available. This report details the analytical validation, clinical validation, and implementation of a fluorometric assay for measurement of IDS activity in newborn dried blood spot (DBS) specimens at the Missouri State Public Health Laboratory (MSPHL). The assay is performed in a microwell plate format requiring approximately 15 min of hands-on time per plate and an incubation time of two hours. The analytical validation of this assay included linearity, analytical sensitivity, precision, and carry-over testing. Clinical validation was completed using more than 5000 deidentified presumptive normal newborn DBS specimens as well as seven specimens from patients known to be affected with MPS II. Following validation, MSPHL began prospective screening using the IDS assay on 1 November 2018. In the first 18 months of screening (to 30 June 2020), 146,954 specimens were prospectively screened using the method. Two newborns were identified with severe Hunter syndrome and the assay had a presumptive positive rate of 0.022%.
PubMed: 33124617
DOI: 10.3390/ijns6040079 -
Asian Pacific Journal of Cancer... 2001Hepatitis B virus (HBV), distributed throughout the world, is classified into seven geographically separated genotypes designated A to G. Since the prevalence of HBV...
Hepatitis B virus (HBV), distributed throughout the world, is classified into seven geographically separated genotypes designated A to G. Since the prevalence of HBV infection in isolated ethnic Tibetan populations in China, and the HBV genotypes involved have been hither to remained unclear, we collected 262 blood samples from four isolated villages in the east and west regions of Tibet. The prevalence of HBV infection was estimated by EIA for HBV Ag and HBV Ab. The HBV genotypes were determined by a PCR-microwell plate hybridization method using plasma DNA. The prevalence of HBV Ag and HBV Ab positives was 19.1% (50/262 cases) and 29.0% (76/262 cases), respectively. We detected only the C genotype (20/20 cases), this being known as a predominant type of HBV among Mongoloid populations in Asia. The results revealed, for the first time, that Tibetan villagers have a high rate of infection with HBV of C genotype, in line with the available data for chronic hepatitis and liver cancer.
PubMed: 12718622
DOI: No ID Found -
SLAS Technology Dec 2017Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial...
Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial susceptibility testing (AST) of patient bacterial isolates. However, increasingly, pathogens are developing resistance to a broad range of antimicrobials, requiring AST of alternative agents for which no commercially available testing methods are available. Therefore, there exists a significant AST testing gap in which current methodologies cannot adequately address the need for rapid results in the face of unpredictable susceptibility profiles. To address this gap, we developed a multicomponent, microscopy-based AST (MAST) platform capable of AST determinations after only a 2 h incubation. MAST consists of a solid-phase microwell growth surface in a 384-well plate format, inkjet printing-based application of both antimicrobials and bacteria at any desired concentrations, automated microscopic imaging of bacterial replication, and a deep learning approach for automated image classification and determination of antimicrobial minimal inhibitory concentrations (MICs). In evaluating a susceptible strain set, 95.8% were within ±1 and 99.4% were within ±2, twofold dilutions, respectively, of reference broth microdilution MIC values. Most (98.3%) of the results were in categorical agreement. We conclude that MAST offers promise for rapid, accurate, and flexible AST to help address the antimicrobial testing gap.
Topics: Anti-Infective Agents; Bacteria; Humans; Microbial Sensitivity Tests; Microscopy; Time Factors
PubMed: 28837780
DOI: 10.1177/2472630317727721