Cultivation strategies of a BA/F3 cell line for fundamental cell research

Background During the chronic myelogenous leukemia (CML) the Bcr-Abl oncoprotein is produced, which leads to unregulated cell proliferation. CML is treated with one of several targeting therapies such as imatinib, (formerly STI-571 [Glivec; Novartis, Switzerland]) a selective inhibitor that blocks tyrosin kinase activity of the Bcr-Abl oncoprotein. Apart from the second generation Bcr-Abl inhibitors, identifying novel direct or indirect downstream targets of Bcr-Abl could contribute significantly to the development of new synergistic treatment strategies against CML. The effects of imatinib on the protein expression of Bcr-Abl positive cells are being investigated [1]. A protein which is downregulated during treatment with imatinib (eukaryotic translation initiation factor eIF5A) was identified. This protein is a potentially promising target for single-agent and combined-treatment strategies for CML. For protein complex identification a high cell number is needed. This is difficult to be obtained reproducibly with flask cultures or roller bottles. The aim of this project was to develop and establish a reproducible bioreactor cultivation of murine suspension cell lines (BA/F3 p210), which yields a total cell number close to 1·10 cells required for analytics. Cells should be in exponential growth under constant culture conditions at the time of harvest. A small stirred tank bioreactor with a working volume of 150 mL was used to study and compare different operation modes: batch, fed-batch and continuous. Cell growth and glucose consumption were assessed as main culture parameters. Material and methods Cell lines: BA/F3 p210 and BA/F3 p210 eIF5A-2 (BA/ F3 = mouse pro B cells, p210 = Bcr-Abl oncoprotein (210kDa), eIF5A-2 = isoform of the eukaryotic translation initiation factor eIF5A). In a first step, a working cell bank was established and cell growth was characterized in T-flasks. Afterwards, different cultivation modes were tested in a stirred tank bioreactor (Vario1000, Medorex, Germany) as follows: batch: Cultivation volume Vstart = 350 mL, duration: 40 h fed-batch: Cultivation volume Vstart = 345 mL, duration: 64 h, Feeding took place every time Glucose concentration fell below 2 mM. Feed medium consisted on a mixture of batch medium and higher concentrations of glucose and glutamine. Continuous: Cultivation volume Vstart = 115 mL, dilution rate D = 0.049 h duration: 118.5 h. The scale-up experiment was performed in a 5 L stirred bench-top bioreactor (Biostat B, Sartorius Stedim Biotech GmbH) with pH and DO control.