ISSN 1866-8836
Клеточная терапия и трансплантация

BS-06. Studying efficiency of T-cell transduction in microfluidic systems for creation of T-lymphocytes with chimeric antigen receptor

Vladislav V. Markelov1, Konstantin V. Arabuli2, Ivan N. Gaponenko1, Vladislav S. Sergeev1, Dina A. Senichkina1, Artem A. Potanin1, Valeriia O. Laushkina1, Mikhail V. Zyuzin2, Alena I. Shakirova1, Kirill V. Lepik1, Alexander D. Kulagin1

1 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia
2 School of Physics and Engineering, ITMO University, St. Petersburg, Russia


Contact: Dr. Vladislav V. Markelov, e-mail: marckelov.vladislav5@mail.ru

doi 10.18620/ctt-1866-8836-2023-12-3-1-176

Summary

Among the problems arising with T-lymphocytes carrrying chimeric antigen receptor (CAR-T) are those created by insufficient level of gene transduction to target cells. Modification of the geometric conditions for transduction may provide an appropriate tool. Using of microfluidic systems combining high surface area and low volume has the potential to improve transduction efficiency. The aim of this study is to investigate the efficiency of T-lymphocyte transduction in microfluidic systems to generate CAR-T cells.

Materials and methods

Peripheral blood mononuclear cells (PBMCs) were obtained from blood samples from healthy donors in the Ficoll density gradient using SepMate tubes. α/β T-lymphocytes of PBMCs were isolated by immunomagnetic separation. Particles labeled with anti-CD3/anti-CD28 antibodies were used for activation of α/β T cells. The structure of the anti-CD19 CAR included the domains: FMC63, 4-1BB, and CD3ζ. In the control group 1×106 activated α/β T-lymphocytes were transduced at MOI 5 according to the previously described protocol [Belovezhets et al., 2023]. Polymethylsiloxane microfluidic chips were chosen as the tested microfluidic system. They carried out the transduction of prototypes under static conditions for 6, 12, 18 and 24 hours. The chip channel height was 50, 100, 150, and 200 μm. The surface area of the bottom of the chips was 1 cm2. 0.2×106 activated α/β T lymphocytes were transduced onto the chips. Transduction in the control and experimental series was performed in X-VIVO-15 medium supplemented with 5% inactivated human serum, IL15 (10 ng/ml), IL7 (ng/ml) and protamine sulfate (10 µg/ml). After transduction, the cells were cultured in the same culture medium but in the presence of non-inactivated human serum. Transduction efficiency was assessed by flow cytometry at D+1 and D+ after transduction by staining cells with anti-FMC63 and anti-CD3 antibodies and vital dye 7-AAD.

Results

The level of transduction on D+1 varied slightly depending on the height of the chip. The stage of static transduction lasted for 6 h, the MOI rate was 30. The transduction rates for a chip with a height of 50 µm was 47.18%; for 100 µm, 41.58%; for 150 µm, 42.3%; for 200 µm, 43.13%. Cell viability after transduction in the chips of different heights was also similar being >99%. A tendency was found for the dependence of transduction efficiency (at MOI 5) on the duration of incubation in the chip: in control group, the level of transduction on D+1 was 41.72% ±21.74; in the 150-μm chip with 6-h incubation, it was 23.1%±14.13; at 12 h, 41.15%±18.1; at 18 h, 50.92%±15.69; at 24 h, 61.14%±12.36 (p=0.200 when compared to the control group). When using 100 µm-high chips cultured for 24 hours, the levels of 69.21% ±14.05 (p=0.133 compared to the control group) were obtained. At D7, there was a decrease of CAR-T cell numbers in the control group to 14.02%±9.83; in the 150-μm chip they showed following results: during 6 h-cultivation, 13.37% ±6.84; at 12 h, 13.18%±7.34; at 18 h, 20.71% ±1.64; at 24 h, 18.93% ±11.16; in the 100 µm chip during 24 h cultivation, 18.85% ±4.91.

Conclusion

Transduction in a microfluidic system provides a more efficient interaction of viral particles with T-lymphocytes in comparison with the control group, which is reflected in a high proportion of CAR-expressing cells on D+1. The level of transduction increases with increasing time of incubation of T cells in the chip. However, a small sample did not allow to determine statistically significant differences between the control and experimental groups. At D+7, the number of CAR-positive lymphocytes decreases, which is partly due to the pseudotransduction phenomenon and specific features of the lentivirus samples used. The height of a chip channel did not affect the transduction level and cell viability. Thus, microfluidic transduction in chips is promising and requires further study by the method of transduction of T-lymphocytes.

Keywords

Microchips, microfluidic transduction, CAR-T cells.


Supplement 12-3
09/30/2023

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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