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Analytics and virus production processes
Published in Amine Kamen, Laura Cervera, Bioprocessing of Viral Vaccines, 2023
TCID50- Tissue Culture Infectious Dose 50 uses a similar principle of applying a suspension of the virus at different concentrations onto a plated tissue culture. In this case, the diffusion of the newborn viruses is not limited to inducing several cycles or replications on the same plated culture. Ultimately the whole culture should be infected and lysed. Therefore, the read-out of such assays is evaluating the cytopathic effect of a virus suspension. Such an assay lasts between 4 to 7 days [4]. The test is commonly performed on 96-well plates with replicates to evaluate the percent of tissue cultures that have been infected after several potential replication cycles. Thus, the wells that are infected are counted, among the wells where the tissue culture is healthy. A statistical analysis is performed to determine the number of viral particles in this assay by targeting the conditions where 50% of the wells are infected. A conversion factor based on the Poisson law distribution (TCDI50 = 0.69 × PFU) has been proposed to compare the results obtained for the two infectivity assays.
Industrial Biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Cell culture is the process by which cells are grown under controlled conditions. In practice, the term “cell culture” has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Animal cell culture became a common laboratory technique in the mid-1900s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the nineteenth century. Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible, but this cannot always be accomplished.
Microbiological Considerations in the Selection and Validation of Filter Sterilization
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
If we leave the realm of true bacteria when can find another class of highly pleomorphic prokaryotic organisms-mycoplasma. These organisms lack the true cell wall characteristic of true bacteria and typically range in size from 0.2 to 0.8 mm, although smaller examples among the family mycoplasmataceae have been reported. In the last 10 years, the filtration of mycoplasmas has become an issue, particularly with respect to the manufacturing of biotechnology and biological products. Largely, this is because mycoplasma can be endemic in some of the ingredients used in the preparation of cell culture and tissue culture media. Studies published by Sundaram et al. clearly indicate that if a challenge level of 107 cfu was used in conjunction with many commercial 0.2-mm filters complete retention will not occur. This led to the suggestion that perhaps filters in the range of 0.1-mm pore size rating should be considered sterilizing grade filters rather than 0.2 or 0.22 mm pore size rating filters.
Mussel-inspired self-healing hydrogel based on gelatin and oxidized tannic acid for pH-responsive controlled drug release
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Yangyan Tang, Ximeng Sun, Jiangchuan Ma, Qishe Yan
The effects of all hydrogel samples on the proliferation and viability of 293 T cells were determined by CCK-8 assay [33, 34]. Firstly, 293 T cells were cultured in 293 T medium (with 5% inactivated fetal bovine serum) in an atmosphere of 95% air and 5% CO2 at 37 °C. Secondly, before 293 T cells seeding, the hydrogel samples were entirely purified in the PBS solution (pH 7.4), sterilized using 75% ethanol for 12 h and then placed in a 24-well plate with cell medium to swell to the swelling equilibrium state. The 293 T cells were cultured in 293 T medium on hydrogel samples (about 6 mm in diameter and 2 mm in thickness) in a 24-well tissue culture plate at a density of 2 × 104. Cells incubated in tissue culture plate containing the same cell culture media were set as a negative control. After 12 and 36 h of incubation, the medium was extracted, and then 500 μL fresh medium containing 10% (V/V) CCK-8 solution was added back to the 24-well plate. After culturing for 2 h, the cell medium was transferred to a 96-well plate, and the absorbance of the resulting medium in test (A1) and control (A0) samples were measured at the λmax of 450 nm. The viability of the control group was set for 100%. Therefore, relevant cell viability was defined as the following equation (7):
Repurposing pharmaceutical excipients as an antiviral agent against SARS-CoV-2
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Manisha Malani, Prerana Salunke, Shraddha Kulkarni, Gaurav K. Jain, Afsana Sheikh, Prashant Kesharwani, Jayabalan Nirmal
A study demonstrated that the SARS-CoV enters in Vero-E6 cells via lipid rafts. Ninety percent of infectivity of pseudotyped SARS-CoV was inhibited by MβCD-treatment [123]. Further a study showed that methyl beta cyclodextrin has the potential to reduce the infection of SARS-CoV on pretreated Vero-E6 epithelial cells [124]. Moreover, a study proposed that cyclodextrins can mimic as heparan sulfates when modified with mercaptoundecane sulfonic acid and can act as nontoxic virucidal agent. They are effective antivirals against Respiratory syncytial virus in respiratory tissue culture models and Herpes simplex virus-2 in vaginal tissue culture models [125]. Moreover, they are also effective in mice when administered prior to intra-vaginal Herpes simplex virus-2 inoculation [125].
Magnetic stimulation of the angiogenic potential of mesenchymal stromal cells in vascular tissue engineering
Published in Science and Technology of Advanced Materials, 2021
Ana C. Manjua, Joaquim M. S. Cabral, Carla A. M. Portugal, Frederico Castelo Ferreira
Cell culture on monolayer (2D) was performed first on standard polystyrene tissue culture plates (TCP), in the absence and in the presence of the magnetic field. This study allows us to investigate the impact of the magnetic field on the MSCs without effects driven by the interactions between cell and scaffolds. MSCs proliferation and immunophenotype profile were characterized. The number of MSCs increased steeply on the first days, reaching confluency after 48 h, with a total amount of 50,000 cells/mL. Cells number started then to slowly decrease until the end of the experiment (96 hours). Notably the magnetic field does not seem to impact on the proliferation curves, which show the same behavior over time in the presence and absence of the magnetic field at a value of 0.08 T (Figure 2(a)).