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Bag-based rapid and safe seed-train expansion method for Trichoplusia nisuspension cells

Trichoplusia ni suspension cells (High Five™) used in conjunction with the baculovirus vector expression system (BEVS) are regarded as potential product system of new, recombinant virus-like particle (VLP) vaccines. In order to push vaccine development and production, biomanufacturers use single-use technology when- and wherever possible. This applies to upstream processing and in particular seed-train expansion ranging from cryopreserved vials via t-flasks, spinners (respectively shake flasks) to stirred stainless steel bioreactors. The stainless steel bioreactors deliver inoculum for seed bioreactors and have been increasingly replaced by wave-mixed single-use bag bioreactors during the last 5 years [1].

The approach presented for seed-train cell expansion of High Five suspension cells is based on the Biostat CultiBag RM50 optical (Sartorius Stedim Biotech). It was used for the production of cells for long-term storage and for the expansion of cells for subsequent production experiments. For long-term storage the cells were frozen at high cell concentrations (20 - 40 x 106 cells x mL-1) in 60 mL Cryobags and stored in nitrogen at -196 °C in vapour phase.

Initial experiments were aimed at the growth characterization of High Five suspension cells from a vial working cell bank (WCB). The High Five cells were grown in batch mode and in 250 mL single-use shake flasks (Corning and Sartorius Stedim Biotech) on a Certomat® CT Plus shaker (Sartorius Stedim Biotech) during six days (triplicates, 27 °C, 100 rpm, 25 mm shaking diameter). Afterwards a procedure was developed in which thawed cells from a Cryobag were directly transferred into and expanded in a Biostat CultiBag RM. Under optimal process conditions (500 mL starting volume, a starting cell density of 1 x 106 cells x mL-1, 27 °C, rocking angle of 6 °, 20 - 30 rpm, 0.2 vvm, DO set point 50%) growth rate (0.039 - 0.042 h-1), doubling time (18 - 20 h) and maximal cell density (7.8 - 8.9 x 106 cells x mL-1) showed good correlation with results arising from CultiBags which were inoculated with cells from shake flasks. This bag-based seed-train expansion allows time saving of about one week and reduces cross-contamination, both advantages being due to omitted intermediate cultivation steps in shake flasks.


  1. Eibl R, Löffelholz C, Eibl D: Single-Use Bioreactors-An Overview. Single-Use Technology in Biopharmaceutical Manufacture. Edited by: Eibl R, Eibl D. Wiley. 2010, 1: 33-51. 1

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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Bögli, N.C., Ries, C., Bauer, I. et al. Bag-based rapid and safe seed-train expansion method for Trichoplusia nisuspension cells. BMC Proc 5 (Suppl 8), P124 (2011).

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