Implementing CompacT SelecT Automated Cell Culture in Support of Alzheimer's Disease-Targeted Drug Discovery

Abstract

The CompacT SelecT is the latest generation automated cell culture system in the SelecT product line allowing incubation of up to 90 T-175 flasks and preparation of 210 assay-ready plates. We have successfully implemented the CompacT SelecT in support of a number of cell-based assays used in our Alzheimer's disease (AD) lead optimization programs. One of the distinguishing features of AD pathology is deposition of two neurotoxic forms of the beta-amyloid peptide (Aβ40 and Aβ42) in the brains of patients. It is thought that specifically lowering Aβ40 and Aβ42 in the brains of patients will halt the progression of the disease. The generation of Aβ requires sequential cleavage of the type-I integral membrane amyloid precursor protein (APP) by two proteases, β-secretase (BACE) then γ-secretase. In the specific examples presented here, we have transitioned two cell lines supporting drug discovery efforts for identifying β- and γ-secretase inhibitors (GSIs) from manual cell culture protocols to fully automation using CompacT SelecT. In Chinese hamster ovary (CHO) cells which over express wild-type APP (CHOAPP cells), robust secretion of Aβ40 was observed from cells cultured manually and with CompacT SelecT with signal:background ratios of 54–99 and 23–47, respectively. Despite the reduced signal:background observed with the CompacT SelecT cultured cells, the rank order of potency for a series of 18 BACE inhibitors in reducing Aβ40 secretion was identical when manually cultured cells were compared with CompacT SelecT cultured cells. The correlation coefficient when comparing the two sets of EC50 values was r2 = 0.99. Similarly, CHO cells that over express a reporter construct for the Swedish mutant APP that generates high levels of Aβ peptide (CHOAPP-NL cells) cultured with CompacT SelecT exhibited a reduced secretion of Aβ40 and Aβ42 when compared with manually cultured cells; signal:background ratios for secretion of Aβ40 from CHOAPP-NL cells were 57–71 and 16–31, and for Aβ42 secretion 8–18 and 4–11 comparing manual and automated cell culture, respectively. Similar to the observations in CHOAPP cells, an identical rank order of potency for a series of GSIs was observed when CHOAPP-NL cells cultured manually and in CompacT SelecT were compared; r2 = 0.99 and 0.98, respectively, for EC50 values for inhibition of Aβ40 and Aβ42 secretion. Taken together, the robust signal:background and identical pharmacological profile of inhibitors provide validation for the implementation of automated cell culture for CHOAPP and CHOAPP-NL cells in support of β- and γ-secretase-targeted drug discovery.