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Go-CART: an animal-free system for the assessment of CAR T cell function
  1. Pradip Bajgain1,
  2. Usanarat Anurathapan1,
  3. Ayumi Watanabe1,
  4. John Wilson2,
  5. Sujita Sukumaran1,
  6. Norihiro Watanabe1,
  7. Helen Heslop1,
  8. Cliona Rooney1,
  9. Malcolm Brenner1,
  10. Ann Leen1 and
  11. Juan Vera1
  1. Aff1 grid.39382.33000000012160926XBaylor College of Medicine Houston TX USA
  2. Aff2 Wilson Wolf Corporation New Brighton MN USA

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Meeting abstracts

The preclinical development of chimeric antigen receptor (CAR) T cell therapy has been hindered by the inadequacy of current in vitro methods to predict T cell performance (i.e. migration, proliferation and anti-tumor activity), which do not recapitulate in vivo conditions. For example, most T cell functional assays are performed over a relatively short timeframe (6-18 hrs), and platforms to assess T cell migration and prolonged T cell-tumor cell interactions are very restricted. To overcome these deficits, we developed the Go-CART – an in vitro, compartmentalized culture system that allows long-term assessment of multiple biological parameters simultaneously. The Go-CART is a six chambered device (C1-C6) connected by 2mm channels that form an “S” shaped, 11.5cm path, allowing the generation of a chemokine gradient (Figure 1). Thus, T cells and tumor cells can be physically separated, allowing for simultaneous in vitro assessment of T cell migration, anti-tumor effects, and persistence. Furthermore, the gas-permeable base of the Go-CART allows for prolonged T cell-tumor cell interaction (>2 weeks) without medium/nutrient replenishment.


To explore whether we could establish a chemokine gradient in the Go-CART, we added 24µg of MCP1 to compartment 1 (C1) and evaluated the chemokine levels in all compartments. After 72 hours, we observed the formation of a gradient that could drive T cell migration (196.79, 78.52, 56.80, 9.79, 2.52 and 0.64ng/ml MCP1, C1-C6, respectively). Next, to assess whether T cell migration could be induced by chemokine-producing tumor cells, we established a 3D tumor model (AlgiMatrix seeded with 1.00E+06 CAPAN1 tumor cells) in C1 and three days later, added 2.00E+07 CAR-PSCA-FFluc-GFP T cells to C6. We monitored T cell migration by periodic bioluminescence imaging and after 5 days, observed an accumulation of T cells at the tumor site (C1) (7.69E+06 and 3.19E+08 p/s/cm2/sr, days 1 and 5, respectively). In contrast, T cells failed to localize in the absence of tumor cells. Finally, to assess the utility of the Go-CART in evaluating anti-tumor effects, we established an AlgiMatrix-3D tumor model in C1 with 1.00E+06 CAPAN1-FFLuc-GFP tumor cells and three days later, added 2.00E+07 CAR-PSCA T cells to C6. Under these conditions, we observed a progressive decrease in the tumor signal [3.52E+08 (day 0) and 3.31E+07 (day 15) p/s/cm2/sr]. In contrast, in the absence of T cells, the tumor signal progressively increased [2.69E+08 (day 0) to 2.47E+09 (day 15) p/s/cm2/sr]. Thus, our results demonstrate that the Go-CART can be used to dynamically assess multiple parameters required for CAR T cell function.