Characterisation of Cultivation and Initial Proteome Analysis of the Novel Human Cell Line AGE1.hn

Abstract

The human cell line AGE1.hn (ProBioGen AG, Berlin, Germany) was originated from neural precursor tissue and has been adapted to serum-free conditions. During cultivation in shake flasks and bioreactors in 42-MAX-UB (serum-free, animal-component-free and chemically defined medium, Teutocell AG, Bielefeld, Germany), this cell line shows the tendency to aggregate, which leads to inexact cell counting and misleading lower viability. Methods for preventing agglomeration and the effects on protein expression are described here. For a first proteomic approach a protein-map out of 2DE-gels was generated. Almost 400 spots were isolated for tryptic digestion and conducted to MS-identification. Finally we identified 219 proteins with a significant score based on MASCOT-search. Identified protein-spots were labelled on a 2D-Protein map which can be used in further experiments with this cell line. To examine the influence of aggregates on protein expression, cultivation-experiments in shake flasks with increased calcium-concentration (fivefold) and three parallel bioreactors were performed, respectively. In this parallel approach according to the shake flask-cultivation, increased calcium-concentration and constant stirring speed in comparison to a standard cultivation with normal medium and adjusted stirring rate was used to induce aggregation.