June 24, 2013

Scale-up of adherent Vero cells grown on Cytodex microcarrierrs using ReadyToProcess equipment


Materials and methods
Cell line and cultivation media

Vero cells were obtained from ATCC (No. CCL81, ATCCLGC Standards, Teddington, Middlesex TW11 0LY, UK). The cultivation medium used was Dulbecco’s modified Eagle medium (DMEM)/Ham’s F12 (GE Healthcare Life Sciences) supplemented with glucose (1 g/L), glutamine (0.9 g/L), β-cyclodextrin (0.1 g/L) (Sigma-Aldrich Co., St. Louis, MO, USA), and soy peptone (2 g/L) (Kerry, Norwich, NY, USA). For bioreactor cultivations, Pluronic™ F-68 was added (2 g/L) (Sigma-Aldrich Co.). For cell propagation, the cultivation medium was supplemented with fetal bovine serum (50 g/L) (GE Healthcare Life Sciences). Inoculum was grown in Nunc™ T-flasks and Cell Factory™ systems (Thermo Fisher Scientific Inc., Waltham, MA, USA). For detachment, cells were washed with PBS-EDTA and incubated with Accutase™ (GE Healthcare Life Sciences). 

 
Microcarrier preparation
In bioreactor cultivation of the Vero cells, the microcarrier concentration was 3 g/L. Cytodex 1 microcarriers (GE Healthcare Life Sciences) were hydrated in PBS in a siliconized glass bottle (Sigmacote™, Sigma-Aldrich Co.) and washed three times with PBS prior to autoclaving for 15 min at 121°C. Before use, the microcarriers were washed with cultivation medium.

Bioreactor cultivation
Cultures at 10 L scale were grown in a WAVE Bioreactor 20/50 system (GE Healthcare Life Sciences). WAVEPOD II controller (GE Healthcare Life Sciences) was used to control temperature, pH, DO, and agitation. The culture parameters were maintained at 37°C, pH 7.1, DO 30%, and the agitation settings were 10 rpm/5°. Cultures at 50 L scale were run in a WAVE Bioreactor 200 system (GE Healthcare Life Sciences).
Cultivation conditions were the same as for the 10 L cultures except for the agitation setting, which were changed to 6 rpm/5°. 

 
Scale-up procedure and bead to bead transfer
Inoculum grown in cell factories was used to seed a 10 L microcarrier culture. The 10 L culture was subsequently used to inoculate a 50 L culture by bead to bead transfer. For cell transfer to the 50 L microcarrier culture, the heating and agitation were switched off and the microcarriers were allowed to settle. The cells were washed twice in PBSEDTA inside the Cellbag 50L bioreactor (GE Healthcare Life Sciences). For wash, the supernatant (90% of the volume) was removed and partly replaced with PBS-EDTA. After a second wash, the microcarrier suspension was transferred to a Sigmacote siliconized glass bottle. In a laminar flow hood, the residual buffer was removed and trypsin-containing buffer solution (0.2% trypsin and 0.02% EDTA in PBS w/o Ca2+, Mg2+), corresponding to 250% of the settled carrier volume, was added. The bottle was incubated at 37°C in a water bath and the suspension was mixed every 5 min. After 20 min, cell detachment was verified microscopically and the supernatant transferred to a glass bottle. The microcarriers were washed twice with cultivation medium and the supernatants were pooled. The cell suspension was used to inoculate a 50 L culture in a Cellbag 200L bioreactor (GE Healthcare Life Sciences).

Cell sampling and counting
During bioreactor cultivation, samples were taken daily to determine cell concentration and morphology as well as concentrations of selected metabolites. Microcarrier suspension was withdrawn via the sample port while the base unit was rocking continuously. A 1 mL sample was transferred to a tube. After settling of the microcarriers, 800 μL supernatant was removed and replaced with an equal volume of 0.1% crystal violet in 0.1 M citric acid and 1% Triton™ X-100. The suspension was vigorously mixed for 45 s and the released nuclei were counted using a hemocytometer. Cell morphology and attachment to microcarriers were determined using an inverted microscope with attached camera (Eclipse TS100, Nikon Instruments Inc., Melville, NY, USA).  The concentrations of glucose, lactate, glutamine, glutamate, and ammonium were measured in a Bioprofile Flex™ analyzer (Nova Biomedical Corporation, Waltham, MA, USA).

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Tags: vaccine; influenza; readytoprocess; single-use; cytodex; microcarriers; cell-culture; wave; bioreactor