Single Use Bioreactors Successful Cell–Cultivation in Single Use Bioreactors

Author / Editor: ANIL KUMAR RATHOD / Dominik Stephan

Single use technology is one of the leading technolgies in the biotech sector. But can this promising cultivation method really live up to the high expectations? In a case study, Sartorius explains how it successfully cultivated Sf9 cells using single use vessels...

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Cultivation of Sf9 cells in the single use bioreactor Biostat RM 20/50 optical system.
Cultivation of Sf9 cells in the single use bioreactor Biostat RM 20/50 optical system.
(Picture: Sartorius Stedim Biotech, Bengaluru)

Insect cells like mammalian cells are able to produce eukaryotic proteins (i.e. posses the ability of post-translational modifications, protein folding etc). The most commonly used insect cell line is Sf9 (Spodoptera frugiperda ovary cells). These cells can be growth in suspension culture or adherent as monolayer. A variety of recombinant proteins can be obtained by utilising the baculovirus expression system.

The Biostat RM optical (figure 1) is the most advanced single use bioreactor using rocking motion technology. The pillow shaped cultivation chamber is rocked back and forth, creating waves which provide mixing with low shear.

The liquid surface is constantly renewed, thereby enabling efficient mass transfer between head space and media. The cultivation chamber itself is a single use bag composed of a multilayer film with pharmaceutical grade Ethyl Vinyl Acetate (EVA) as the contact layer. This single use bags are easy to use, reduce validation costs, and remove the need for cleaning, sterilising, and provide stress free convenient culturing. In this application notes, we tried to demonstrate the use of single use Cultibag RM system in cultivation of Sf9 cell line.

Single Use Bioreactors: Preferred in Various Applications

Spodoptera frugiperda (Sf9) cell line was procured from Invitrogen and cultivated in Serum free sf-900-II SFM media (GIBCO, 10902). The Cultibag RM 2 L Optical bag (DBO002L) was filled with 800 ml of media under aseptic condition and the bag was inflated with air.

Thereafter, DO and pH sensors were inserted in respective ports and equilibrated for two hrs at eight rocks per minutes. DO and pH sensors were calibrated as per manufacturer instructions. The pre culture was seeded in the Cultibag at seeding density of 0.7 x 10^6 cells/ml. Temperature was set to 28°C, DO to 60 per cent, pH to 6.2. Rocking was varied from 18 to 25 rocks/minutes and an angle of 6°.

A 5ml of sample was collected every day to determine viable cell density by trypan blue exclusion method and glucose and lactate estimation in the spent media. (Enzyme based assay kit from Chema diagnostic kit for Glucose assay and Radiant kit for Lactate assay was used).