Extended Data Fig. 1: Details of CAR T cell process on the microbioreactor and gas-permeable well plate.
From: A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells
a, Left: Image of the microbioreactor system, comprised of a base station controller and a CO2 controller supporting up to four ‘pods’, which allows highly parallelized synchronous or asynchronous running of up to four separate cultures per instrument (W: 41 cm × D: 43 cm × H: 36 cm). Right: Image of the sterile, single-use consumable, consisting of a microfluidic chip or ‘cassette’ (W: 5 cm × D: 8 cm × H: 1 cm) connected to a bottle rack assembly (W: 10 cm × D: 13 cm × H: 18 cm). b, In an optimization experiment, T cells were isolated and activated on Day 0, transduced on either Day 0 (concurrent with activation) or Day 2, and expanded up to Day 14 in the microbioreactor, with either a step-wise ramping up of perfusion flow rate from 1 v.v.d. to 4 v.v.d. (starting at 1 v.v.d. approximately 48 h after transduction, ramping up by 1 v.v.d. every other day) or a constant high perfusion flow rate of 4 v.v.d. (starting from approximately 48 h after transduction). Cells were sampled on Day 14, the percentage of total live cells expressing CAR were quantified by flow cytometry, the viable cell count (VCC) in number of cells were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter, and the percentage of total live cells that were CCR7+ CD45RA+ (naïve and stem cell memory, Tn/Tscm), CCR7+ CD45RA– (central memory, Tcm), CCR7– CD45RA– (effector memory, Tem), and CCR7– CD45RA+ (terminally differentiated effector, Temra) were quantified by flow cytometry. c, In an optimization experiment, T cells were isolated and activated on Day 0, transduced on either Day 0 (concurrent with activation) or Day 1, and expanded up to Day 14 in the microbioreactor or gas-permeable well, with a constant high perfusion flow rate of 4 v.v.d. (starting from Day 2). Cells were sampled on Day 14, the percentage of total live cells expressing CAR were quantified by flow cytometry, the viable cell count (VCC) in number of cells were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter, and the percentage of total live cells that were CCR7+ CD45RA+ (naïve and stem cell memory, Tn/Tscm), CCR7+ CD45RA– (central memory, Tcm), CCR7– CD45RA– (effector memory, Tem), and CCR7− CD45RA+ (terminally differentiated effector, Temra) were quantified by flow cytometry. d, Image of the gas-permeable well plate. Created with BioRender.come, Details of activation, transduction, and expansion culture conditions on the microbioreactor and gas-permeable wells. Starting cell numbers were 2 million T cells in 2 mL culture volumes for both culture vessels. f, Culture volumes (dashed lines) and total volumes of medium used (solid lines) over time for the microbioreactor and gas-permeable wells. g, The microbioreactor dissolved O2 setpoint was 80%. When dissolved O2 levels fall below 80%, O2 would be injected into the headspace of the cassette to maintain dissolved O2 levels at a minimum of 80%. The microbioreactor pH setpoint was 7.40 ± 0.05. When pH is above 7.45, more CO2 (above the minimum of 5%) would be injected into the headspace of the cassette to lower the pH, and when pH is below 7.35, a basic carbonate/bicarbonate solution would be injected into the growth chamber to raise the pH. Dissolved O2 and pH control were activated 2 h after transduction.
