Rethinking viral vector quantification: a microfluidic approach to standardised functional titre assays

The quantification of lentiviral vector (LVV) potency via titration is a critical quality control step for cell and gene therapies. However, standard functional titre assays are fundamentally limited by an inability to detect multiple integration events, procedural variations, and, most critically, a mass transport limitation created by the fluid overlay in conventional well plates. These issues, compounded by a lack of standardisation, lead to significant inter-laboratory variability and a systematic underestimation of true vector potency. In this study, we employed a microfluidic approach to create a more precisely engineered assay environment. We systematically evaluated channel depth, incubation time, vector concentration, and multiplicity of infection (MOI) for their impact on assay linearity, sensitivity, limit of detection, and reproducibility. A 0.2 mm deep channel provided linearity and reproducibility comparable to 96-well plates yet offered shorter incubation periods, and enhanced sensitivity, detecting activity down to a MOI of 0.0625 (corroborated by qPCR analysis) - a level at which conventional well plates fail. This establishes our microfluidic platform as a device-based analytical standard that transforms functional titre quantification from a variable protocol into a more reliable engineering solution for quality testing in cell therapy manufacturing.