Feasibility studies of oncornavirus production in microcarrier cultures

Abstract

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

Microcarriers for Upscaling Cultured Meat Production

“…Proteases are generally used in combination with chelating agents for Ca 2+ that reduce the ionic strength required for cell binding. The specifics of this protocol are highly dependent on the microcarriers, cells and enzymes used (116,117). To achieve an animal-free production process, animal-derived proteases can be replaced FIGURE 4 | Process requirements and variables for MC based bioprocesses in three scenarios: (1) MCs are serving as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible.…”

Cell growth on electrospun nanofiber mats from polyacrylonitrile (PAN) blends

“…New microcarriers can also be added to confluent microcarrier cultures during periods of virus production, and Manousos et al (98) used this technique to cause a new wave of cell proliferation and production of oncornavirus. It is also possible to scale up microcarrier cultures of human fibroblasts by allowing confluent microcarriers to settle with new microcarriers.…”

High‐yield retroviral production using a temperature‐modulated two‐stage operation

“…If other types of highly efficient reactors, for example, roller bottles (Faff et al, 1993), microcarrier reactors (Manousos et al, 1980), packed-bed reactor (Kang et al, 2000), hollow fiber reactors (Boorsma et al, 2002), or other various bioreactors recently reviewed (Merten, 2004), are combined with this circulating strategy with cold reservoir, mass production of high-titer retroviral supernatant is foreseeable. Certainly, such an operating strategy can be extended to clinically acceptable retroviral production systems (e.g., amphotropic retroviruses produced from HEK293-based producer cells) with higher yielding reactors under optimal culture conditions for selected producer cells and viral vectors.…”