Cell propagation Adherent cells

Adherent insect cell release from solid surfaces generally does not require trypsinization, unlike anchorage-dependent mammalian cells. Insect cells, like mammalian cells, need rigorous aseptic manipulation during cell transfer, inoculation, and propagation in bioreactors. A minimal inoculum density is required for both cell types. Typically, insect and mammalian cell cultures are initiated with inocula of 1-2 X 105 cells per milliliter of liquid medium (Agathos, 1991). Table 2.3 summarizes the major differences and similarities between these cells. 

Figures 9.2 and 9.3 show schemes that illustrate inoculum development from the cryotubes to production scale for suspension and adherent cells, respectively. In these hypothetical process schemes, the expression production bioreactor is used arbitrarily for any of the types of bioreactor presented in the next section of this chapter. In general, different flasks and several intermediate bioreactors are used for cell propagation to reach the quantity of cells necessary to inoculate the production bioreactor. The number of propagation steps is a function of the final scale of the production bioreactor.

Hypothetical example of the propagation of adherent cells, using hollow-fiber modules as the production bioreactor in the final stage. The amount of cells usually obtained at the end of each step is also indicated. 

Adherent cells are propagated in T25, T75, or T182 tissue culture flasks. Flasks can be used from various venders (e.g., Greiner). 

To understand the force generation and its propagation through the actin cytoskeleton here we have proposed a bio-chemo-mechanical framework. In this approach, the characteristic behavior of various types of cell types can be modeled and quantified. The properties of the model ingredients were based upon observations and mechanical measurements from both living cells and individual cytoskeletal filaments. It turns out that actin is one of the major players that determines the elastic properties of adherent cells. Individual actin filaments are treated as linear elastic cables due to their observed tensile response and a detailed analysis of their mechanical properties. Two regular types of cells were modeled and forces at the focal adhesions were measured. While the circular cell shows a characteristic... 

Adherence to Principles of GMP in a Prechnical Developmental Process Efficient Standardized MSC Propagation Using Low Cell Seeding Density Superior MSC Proliferation Resulting from HPL-Driven as Compared to FBS-Driven Cultures... 

The BHK-21 (baby hamster kidney) cell line consists of adherent fibroblasts, that can also be adapted to suspension culture, and was isolated from five 1-day-old hamsters (McPherson and Stoker, 1962). These cells are commonly used for virus propagation (polio, rabies, and foot-and-mouth disease) for production of veterinary vaccines. 

Most El-deficient recombinant adenoviruses are propagated in the HEK-293 and PER.C6 cell lines (and derivatives thereof) as discussed in Section 10.1.2.3. The generation of RCAs is minimal in PER.C6 but remains a concern for HEK-293. Both cell lines have been documented for GMP manufacturing [110]. The production of these cells can be accomplished by a variety of methods, which depend on whether adherent or suspension cell lines are being used. 

Once prepared, feeders can be kept in culture for up to 2 wk prior to use, but must remain as an intact monolayer. Most researchers prepare feeders on gelatin-coated plates. Although this is not essential for the propagation of the fibroblasts themselves, it promotes tight adherence to the substratum, and is also essential for the adherent growth of ES cells to any regions devoid of fibroblasts. 

Fig. 9 Intercellular interaction via ECM. (a) A number of cells adhered to the elastic ECM generate contractile strain, which propagate through the ECM and send mechanical signals to neighboring cells, (b) Cell spends energy W ocAF to contract and deform the matrix by pulling through the adhesion points.

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