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Upstream processing for viral vaccines–General aspects
Published in Amine Kamen, Laura Cervera, Bioprocessing of Viral Vaccines, 2023
Lars Pelz, Sven Göbel, Karim Jaen, Udo Reichl, Yvonne Genzel
To allow a continuous growth/maintenance, passaging of cells is necessary due to depletion of nutrients, accumulation of toxic by-products, or limitation of the growth surface (adherent cells). During cell passaging, old medium is withdrawn and cells are seeded in fresh medium at a lower cell concentration. The passage number describes the number of these subcultivations. Although the growth of continuous cell lines is not limited per se, a regulatory limitation exists. For cells that pose a potential tumorigenicity hazard, including Vero and MDCK cells, the maximum allowed passage number for production is often limited to 20. In contrast, new designer cell lines (e.g., PER.C6) grown in suspension are often very well characterized and can be used for up to 100 passages [12]. The cell line, starting from the working bank, undergoes several passages with increasing volume during seed train expansion until inoculation at production scale. Furthermore, cells can be maintained at a small volume, e.g., laboratory scale experiments or vaccine development studies. Adherent cells are typically passaged once or twice a week, shortly before they reach confluence. For passaging, the attached cells have to be detached by proteases (e.g., trypsin). Use of trypsin (activity/concentration, incubation time, pH) and inactivation of protease activity by addition of either serum or medium needs to be optimized for the respective cell line, medium and the cultivation vessel (e.g., T-flask, roller bottle, cell factory, microcarrier culture). A typical split ratio for adherent cells is 1:4-1:10. For suspension cells, typical cell concentrations in batch mode reach 2E06-10E06 cells/mL with doubling times of 20−30 h. Suspension cells will be subcultured every 3 and 4 or 2 and 3 days at the late end of the exponential growth phase and are seeded usually at a cell concentration of 0.5-0.8E06 cells/mL, while a small fraction of the cell suspension is filled up with fresh medium (split ratio: 1:4-1:20). Preferably, no antibiotics are added to the cell culture medium to avoid masking of contaminations. Some of these possible contaminations only become evident when switching to active aeration and use of antibiotics can potentially create resistances. Therefore, when using antibiotics, a clear strategy of changing antibiotics in a regular mode needs to be followed.
Characterization of cytochrome P450s (CYP)-overexpressing HepG2 cells for assessing drug and chemical-induced liver toxicity
Published in Journal of Environmental Science and Health, Part C, 2021
Si Chen, Qiangen Wu, Xilin Li, Dongying Li, Nan Mei, Baitang Ning, Montserrat Puig, Zhen Ren, William H. Tolleson, Lei Guo
Collectively, our data demonstrated that the expression and function of CYPs were consistent among the cells at different passages encompassing 2-10. Next, we investigated whether the cell passage number could alter the cellular responses to stimuli by studying the responses to two drugs, amiodarone and dronedarone. Both amiodarone and dronedarone are used to treat cardiac arrhythmias and CYP3A4 is involved in the metabolism of both drugs. Earlier, we determined that the activation of CYP3A4 significantly increased the cytotoxicity of amiodarone, whereas CYP3A4 played the opposite role and counteracted the cytotoxicity of dronedarone.44,46 Here, we compared the CYP3A4 mediated toxicity between the cells at passage 2 and 10. Consistent with our previous studies, the cytotoxicity of amiodarone significantly increased in CYP3A4-overexpressing cells when compared to empty vector control cells. Conversely, the cytotoxicity of dronedarone significantly decreased in CYP3A4-overexpressing cells (Figure 4). These observations were consistent for cells at passage 2 and 10, demonstrating that the metabolic response to the insults was not affected by cell passaging.
Impact of collagen-alginate composition from microbead morphological properties to microencapsulated canine adipose tissue-derived mesenchymal stem cell activities
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Hyunkyu Lee, Heung-Myong Woo, Byung-Jae Kang
Canine ASCs were isolated from a 2-year-old male beagle using the collagenase perfusion technique according to methods described previously [16]. Canine ASCs were cultured under standard conditions in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS; Invitrogen) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific). The medium was changed at 48-h intervals until the cells reached 90% confluence, when they were trypsinized and stored in liquid nitrogen or subcultured. Cell density for the passaging of canine ASCs was approximately 2.5 × 104 cells/cm2. The cryopreserved canine ASCs were used up to passage five.
Proliferation of mouse embryonic stem cells on substrate coated with intact silkworm sericin
Published in The Journal of The Textile Institute, 2022
Chihiro Umehara, Ai Ai Lian, Yuichiro Funahashi, Keiko Takaki, Rina Maruta, Yuto Ohmaru, Yoko Okahisa, Takashi Aoki, Hajime Mori, Eiji Kotani
The cell line used to evaluate the efficacy of sericin coatings was mouse ES (EB5), which was derived from E14tg2a (American Type Culture Collection) by inserting the blasticidin S deaminase gene under the control of an Oct-4 gene promoter. A 6-well tissue culture plate made of polystyrene (AGC TECHNO GLASS Co., Ltd) was coated with autoclaved 0.1% gelatin type A (Millipore Sigma). Gelatin solution was added to each well, and the plate was incubated for 5min at room temperature. The solution was then removed, and the plate was left to air dry. EB5 cells were then maintained on gelatin-coated plates and cultivated with undifferentiation medium (GMEM (Invitrogen)) supplemented with 1% (v/v) fetal bovine serum (FBS) (CELLectTM, MP Biomedicals), 10% KnockOut Serum Replacement (KSR) (Invitrogen), 0.1mM nonessential amino acids (NEAA) (Invitrogen), 1mM sodium pyruvate (Invitrogen), 2mM L-glutamine (Wako Chemicals), 0.1mM 2-mercaptoethanol, blasticidin (500ng/mL) (Invitrogen), and 2,000U/mL (10ng/mL) recombinant human leukemia inhibitory factor (rhLIF) (Millipore Sigma) (Niwa et al., 2000; Otsuki et al., 2017). Undifferentiated EB5 cells harboring a blasticidin resistance gene were obtained through blasticidin selection. EB5 passaging was accomplished by washing the cells once with Dulbecco’s phosphate-buffered saline (D-PBS) (Thermo Fisher Scientific), followed by incubation with 0.05% trypsin-EDTA for 5min at 37 °C. Cells were seeded onto the gelatin-coated wells of the 6-well plate at a density of 1.0 × 104/well and cultured in 2mL of undifferentiation medium at 37 °C and 5% CO2 for 3days before the next passage.