Separation of Activated T Cells Using Multidimensional Double Spiral (MDDS) Inertial Microfluidics for High-Efficiency CAR T Cell Manufacturing

Abstract

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.

Figure 1.. Overview of the modified CAR T cell manufacturing process utilizing MDDS sorting.

(a) schematics of the modified CAR T cell manufacturing process. (b) Channel configuration of the MDDS device (green: the first spiral channel with a smaller dimension, yellow: the second spiral channel with a larger dimension) and schematic diagram of the T cell sorting process.

Figure 2.. Proof-of-concept test of the separation of activated and unactivated T cells.

(a) Trajectories of the red-fluorescence-stained Dynabead+T cells (red) and the green-fluorescence-stained Dynabead−T cells (green) in the transition and outlet bifurcation regions of the MDDS device under various flow rate conditions. (b) Recoveries of Dynabead+T cells (red bar) and Dynabead−T cells (green bar) and purity of Dynabead+T cells (gray bar) in the IW and OW outputs under various flow rate conditions. (c) Analysis of activation level (assessed by measuring 4–1BB expression) of the IW and OW outputs under various flow rate conditions; red and green bars show the activation levels gated by ‘Dynabead+T cell’ and ‘Dynabead−T cell’, respectively, while gray bars represent the overall activation levels of all T cells without gating. (d) Fluorescent microscopic images of the IW and OW outputs obtained at the optimized flow rate condition, 3.5 mL/min; Dynabead+T cells (red-fluorescence-stained) and Dynabead−T cells (green-fluorescence-stained). MDDS device, multi-dimensional double spiral device; RFP, IW, inner wall; OW, outer wall.

Figure 3.. Improving CAR T cell manufacturing process by pre-transduction MDDS sorting.

(a) Timeline of the proposed workflow with descriptions of corresponding samples; solid arrows denote the application of treatment to the sample, while dashed arrows indicate the absence of treatment processing for the sample. (b) Analysis of activation level (assessed by measuring 4–1BB expression) across the unsorted control and outputs after MDDS sorting on Day 2; the red bar in the graph represents the proportion of 4–1BB+T cells out of the total live T cells in a given sample, indicated as “4–1BB+T cell (%)” while the blue bar indicates the average 4–1BB MFI of the total live T cells. (c) Viability analysis across the unsorted control and outputs on Day 2. (d) Analysis of CAR transduction (assessed by measuring GFP expression) across the unsorted control and outputs on Day 4; the red bar in the graph represents the proportion of GFP+T cells (meaning CAR+T cells) out of the total live T cells in a given sample, indicated as “GFP+T cell (%)” while the blue bar indicates the count of GFP+T cells (with respect to the unsorted control) from the same number of seeded/transduction-processed live cells (1×10⁶) on Day 2. (e) Cytotoxicity assay profiles across the unsorted control and outputs depending on various effector to target (E:T) ratios; GFP+ live T cells were employed as effectors, while NALM6 cells expressing a firefly luciferase reporter (FLuc) were utilized as targets. MDDS device, multi-dimensional double spiral device; IW, inner wall; OW, outer wall; E:T ratio, effector to target ratio; GFP, green fluorescent protein.

Figure 4.. Improving CAR T cell manufacturing by post-transduction MDDS sorting.

(a) Timeline of the modified workflow with descriptions of corresponding samples; solid arrows denote the application of treatment to the sample, while dashed arrows indicate the absence of treatment processing for the sample. (b) Analysis of activation level (assessed by measuring 4–1BB expression) across the unsorted control and outputs after MDDS sorting on Day 2; the red bar in the graph represents the proportion of 4–1BB+T cells out of the total live T cells in a given sample, indicated as “4–1BB+T cell (%)” while the blue bar indicates the 4–1BB MFI of the total live T cells. (c) Analysis of CAR transduction (assessed by measuring GFP expression) across the unsorted control and outputs after MDDS sorting on Day 2: proportion of the transduced cells; the term “GFP+T cell (%)” refers to the proportion of GFP+T cells (meaning CAR+T cells) out of the total live T cells in a given sample. (d) Analysis of separation efficiency: recovery rates of overall (gray bar), 4–1BB+ (red bar), 4–1BB− (green bar), GFP+ (orange bar) and GFP−T cells (purple bar); the expression of 4–1BB evaluates cell activation, whereas GFP expression evaluates CAR transduction. (e) Cytotoxicity assay profiles across the unsorted control and outputs depending on various effector-to-target (E:T) ratios; GFP+ live T cells were employed as effectors, while NALM6 cells expressing a firefly luciferase reporter (FLuc) were utilized as targets. MDDS device, multi-dimensional double spiral device; Dyna, Dynabead treatment; IW, inner wall; OW, outer wall; E:T ratio, effector to target ratio; GFP, green fluorescent protein.