Application of xCELLigence real-time cell analysis to the microplate assay for pertussis toxin induced clustering in CHO cells

Abstract

The microplate assay with Chinese Hamster Ovary (CHO) cells is currently used as a safety test to monitor the residual pertussis toxin (PT) amount in acellular pertussis antigens prior to vaccine formulation. The assay is based on the findings that the exposure of CHO cells to PT results in a concentration-dependent clustering response which can be used to estimate the amount of PT in a sample preparation. A major challenge with the current CHO cell assay methodology is that scoring of PT-induced clustering is dependent on subjective operator visual assessment using light microscopy. In this work, we have explored the feasibility of replacing the microscopy readout for the CHO cell assay with the xCELLigence Real-Time Cell Analysis system (ACEA BioSciences, a part of Agilent). The xCELLigence equipment is designed to monitor cell adhesion and growth. The electrical impedance generated from cell attachment and proliferation is quantified via gold electrodes at the bottom of the cell culture plate wells, which is then translated into a unitless readout called cell index. Results showed significant decrease in the cell index readouts of CHO cells exposed to PT compared to the cell index of unexposed CHO cells. Similar endpoint concentrations were obtained when the PT reference standard was titrated with either xCELLigence or microscopy. Testing genetically detoxified pertussis samples unspiked or spiked with PT further supported the sensitivity and reproducibility of the xCELLigence assay in comparison with the conventional microscopy assay. In conclusion, the xCELLigence RTCA system offers an alternative automated and higher throughput method for evaluating PT-induced clustering in CHO cells.

Fig 1. Cell index in real time after treatment of CHO cells with variable concentrations of pertussis toxin.

(A) Treatment with standard pertussis toxin. (B) Treatment with in-house pertussis toxin. Control cells were incubated with culture medium only. Results of one representative experiment are shown [n = 1 for all pertussis treatment and n = 6 for the cell controls (Average of cell control represented)]. Error bars represent standard deviations.

Fig 2. Cell index in real time after treatment of CHO cells with pertussis toxin alone or preincubated with anti-pertussis toxin or irrelevant non-PT antibodies.

(A) Standard pertussis toxin (1 IU/mL = 6.7 ng/mL), average of 2 or more replicates are shown for each condition. (B) In-house pertussis toxin (6.8 ng/mL), average of 3 or more replicates are shown for each condition. Antibodies were serially diluted to PT to antibody molar ratios of 1 to 30000 (285.2 μg/mL), 1 to 6000 (57.0 μg/mL) and 1 to 1000 (11.4 μg/mL). Error bars represent standard deviations.

Fig 3. Mean cell index of pertussis toxin treatments versus cell control.

(A) Standard pertussis toxin results with at least n = 15 for each toxin treatment and n = 302 for cell control (C.C.). (B) In-house pertussis toxin with at least n = 7 for each toxin treatment and n = 60 for C.C. condition. Statistical analysis was performed by Kruskal-Wallis test followed by Dunn’s multiple comparison test (GraphPad Prism v. 8.0) (**** p <0.0001, *** p < 0.001). Error bars represent standard deviations.

Fig 4. Modifications to the cell layer by microscopy method 48 h after treatment with standard PT or gdPT.

(A) Control cells versus cells treated with 1 IU/mL to 0.125 IU/mL of PT (corresponding to Table 3, experiment 3, replicate 2) and the scoring of each well by microscopy (positive: ≥ 80% cell clustering per well; negative: < 80% cell clustering). (B) CHO cells exposed to concentrations of gdPT from 400 μg/mL to 50 μg/mL. Cytotoxicity +/- indicates low visible level of cell death.