2.6 pH measurements
In addition to the online pH measurements from the STR, offline pH
readings were also taken daily from the microwell and STR cultures using
the RAPIDLab 348EX blood gas analyser (Siemens Healthineers, Erlangen,
Germany). For the STR cultures, the pH was recalibrated if the online
and offline pH value deviated by more than 0.05 pH units.
2.7 Quantification of
lentiviral vector infectious titre
To quantify the infectious titre in terms of transducing units per ml
(TU/ml), HEK293T cells were transduced with serial LV dilutions in the
presence of polybrene (8 µg/ml) (MilliporeSigma, Burlington,
Massachusetts, United States). Polybrene is a polycation agent which has
shown to enhance transduction by retroviral vectors (Davis, Morgan, &
Yarmush, 2002). The transduced cells were adherent HEK293T cells which
were derived from a GSK in-house research cell bank. Cell counts were
performed at the time of transduction. Following this, cells were
cultured for 3 days at 37°C in a 5% (v/v) CO2humidified incubator. The eGFP expression in the transduced cells was
measured by flow cytometry using the Accuri c6 Plus system (BD
Biosciences, Franklin Lakes, New Jersey, United States). The titre was
determined using the formula: Transducing Units/mL = [(% of GFP
positive cells) x (number of cells transduced) x (dilution
factor/transduction volume)].