High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor

2. Materials and Methods

2.1. Maintenance of Huh7.5 Cells

Huh7.5 cell cultures were maintained in cell culture T flasks in serum-containing medium (SCM): Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Paisley, UK) containing 4 mM GlutaMAX and supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma). In T175 flasks (Nunc, Roskilde, Denmark) and triple layer culture flasks (Nunc), Huh7.5 cells were passaged every 2–3 days. All cell cultures were maintained at 37 °C and 5% CO2.

2.2. HCV Virus Stocks

Two sequence-confirmed stocks of the genotype 5a recombinant SA13/JFH1core-NS5B [28], grown in monolayer Huh7.5 cell cultures in T flasks, were used in this study. A third passage virus stock with an infectivity titer of 5.8 log10 FFU/mL, grown under serum-containing (SC) conditions [39], was used to inoculate CelCradle™ cultures, shake flask cultures, and for virus characterization experiments. A fourth passage virus stock with an infectivity titer of 5.1 log10 FFU/mL [32], grown under SF conditions [39], was used for virus characterization experiments.

Source from: https://www.mdpi.com/2076-393X/10/2/249/htm 

评论

发表评论

此博客中的热门博文

Functional innovation of cell culture flasks

图片
Cell culture flasks   play an important role in medium-scale cell and tissue culture in the laboratory. Common specifications include 25cm2, 75cm2, 175cm2, and 225cm2, which can meet the needs of cell culture of different scales. Different from ordinary plastic bottles, the innovations in the design of cell culture flasks are reflected in the following aspects: 1. Bottleneck: The bottleneck of the cell culture flask is designed with torticollis, and the bottom is in the shape of an arc, which is convenient for the export of the culture solution. The bottleneck is relatively wide and there is no dead corner, so that the pipette and the cell scraper can go deep into every corner of the bottle, and the operation is more convenient. 2. Bottle cap: This cell consumable is equipped with two different bottle caps, which are suitable for different culture environments. The sealing cap is in a completely sealed state, and there are no vents on the cap. It is mainly used under carbon dioxide...
阅读全文

Application of cell culture flasks in the passage of mouse fibroblasts

图片
  Cell culture flasks   are a type of cell culture consumables that are widely used in the market, and are mainly suitable for adherent cell culture. Among them, this consumable is also used in the passage of mouse fibroblasts. Mouse fibroblasts are special cells used for non-medical purposes such as scientific research or industrial applications. Fibroblasts have vigorous functions and weak basophilic cytoplasm. They have obvious protein synthesis and secretion activities. Under certain conditions, they Can achieve mutual transformation with fiber cells. After receiving the fibroblasts, they need to be stabilized in the incubator for about 4 hours and then cultured or passaged according to the cell density, changing the medium. The passaging operation follows the following steps: 1. Aspirate the medium in the original cell culture flask, rinse the cells twice with PBS buffer, and add 1~2 ml of 0.25% EDTA for trypsin digestion (pay attention to the digestion time, usually cont...
阅读全文

Analysis of the causes of cell clumping in cell culture flasks

图片
  Cultivating cells is a very rigorous task. In addition to creating a good growth environment for cells, it must also promptly resolve various emergencies during the culture process. Cell clumping in   cell culture flasks   is a phenomenon often encountered. What is the reason for this situation? The clumping phenomenon of cells in the cell culture flasks can generally be analyzed from the following aspects: 1. The digestion process may be too rough, pipetting too hard, digestion for too long, too strong or toxic digestive juice, etc., will cause cell death. After the cell dies, it releases DNA, entangles other cells, and forms a sticky cell cluster. 2. Excessive cell centrifugation speed or too long time can also easily cause cell clumping. 3. When the digestion is not complete, the connection between the cells and the cell is not separated; when the digestion is over, the round cells tend to pile up and it is difficult to separate again. 4. Cells that are in poor condi...
阅读全文