Optimization of initial culture stage of Vero and HEK293 cell lines

Introduction. Laboratory production of viral material in small quantities is performed, as a rule, using adherent cell lines and culture flasks of varying surface area. The need to increase product yield leads to either an increase in the number of flasks or a switch to other accumulation systems, such as roller bottles. One factor influencing the efficiency of cell adhesion and homogeneous monolayer formation is the rotation frequency of the roller bottle. There is a lack of available research data on the impact of rotation frequency on these parameters and determination of its optimal value, particularly based on cellular morphology.

Objective. To optimize the initial stage of roller cultivation for Vero and HEK293 cell lines, taking into account the effect of roller bottle rotation frequency on cell adhesion during seeding and monolayer formation.

Materials and methods. Experiments were conducted using two monolayer cell lines, Vero and HEK293. Seeding concentrations were taken from the cell line passports, amounting to 4×10⁴ cells/cm². Each cell line was seeded onto roller bottles and cultured according to the range of rotation frequencies (0.2, 0.3, 0.4, 0.5, and 0.6 rpm) using a Celrol Mid roller (Wiggens) in a RWD D180 CO₂ incubator. Following 1, 2, and 3 days of cultivation, the quality of cell adherence to the growth surface and monolayer formation was assessed by a TC5400 microscope (Meiji Techno).

Results. During cultivation of the Vero cell line, the rotation frequency up to 0.6 rpm did not significantly affect cell adhesion to the surface. The most homogenous cell distribution was observed at rotation frequencies of 0.4–0.5 rpm. The HEK293 cell culture is more sensitive to mechanical disturbances of the nutrient medium; as a result, at rotation frequencies above 0.2 rpm, abnormally rounded cell shapes and impaired adherence to the growth surface were observed. Furthermore, continued cultivation at this rotation frequency did not lead to the formation of a homogenous monolayer due to slow alternation between the respiration and nutrition phases. Consequently, after cell adherence to the surface, the rotation frequency of the roller bottle should be increased. 

Conclusions. For the Vero cell line, the optimal rotation frequency was established to be 0.4–0.5 rpm. For the HEK293 cell line, the rotation frequency should be at least 0.2 rpm during the first day followed by its increase to 0.5 rpm after 24 h. The tested cultivation conditions enable an efficient growth of these cell lines for the production of viral biomass.

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