Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection...

Area of Science:

• Cellular therapy
• Biotechnology
• Quality Control

Background:

• Cellular therapy development requires robust quality control for therapeutic products.
• International Organization for Standardization (ISO) provides guidance on critical quality attributes (CQAs).
• Cell count and viability are crucial CQAs, but no standardized method exists for cell viability assay selection.

Purpose of the Study:

• To present strategies for characterizing and comparing acridine orange/propidium iodide (AO/PI) and acridine orange/4',6-diamidino-2-phenylindole (AO/DAPI) staining methods.
• To evaluate the comparability of cell viability measurements and identify causes of differences.
• To enable selection of a fit-for-purpose cell viability detection method for cellular therapeutic product development.

Main Methods:

• Comparison of AO/PI and AO/DAPI staining using heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models.
• Investigation of staining time effects and linearity analysis with varying live/dead cell mixtures.
• Validation of viability measurements using a long-term cell proliferation assay.
• Practical application on antibiotic-selected transduced Jurkat and THP-1 cells for functional genomics screening.

Main Results:

• AO/PI and AO/DAPI methods were characterized and compared under different cell death induction conditions.
• Staining time and cell mixture linearity were assessed for both methods.
• Viability measurements were validated against a long-term proliferation assay.
• A practical comparison demonstrated method selection for specific screening applications.

Conclusions:

• The proposed strategies allow for proper characterization, comparison, and selection of cell viability detection methods.
• This work addresses the lack of guidance on qualifying and selecting fit-for-purpose cell viability assays.
• Effective method selection is critical for reliable cellular therapeutic product development and manufacturing.