An Optimized Method for Single Cell Cloning of Human CAR-T Cells Based on FBS-Coated Plates

Area of Science:

• Immunotherapy
• Cell Biology
• Biotechnology

Background:

• Chimeric antigen receptor (CAR)-T cell therapy shows promise for hematologic malignancies and solid tumors.
• Current CAR-T cell production faces challenges in isolation and expansion.
• There is a need for simple, cost-effective methods for CAR-T cell isolation and expansion.

Purpose of the Study:

• To develop a simple and cost-effective method for isolating and expanding human CAR-T cells.
• To enhance the viability and functionality of CAR-T cells using coated culture plates.
• To improve T cell adherence for better expansion.

Main Methods:

• A two-dimensional (2D) culture technique using matrices pre-coated with glutaraldehyde and fetal bovine serum (FBS) was evaluated.
• Jurkat cells were transduced with an anti-prostate-specific membrane antigen (PSMA) CAR construct.
• FBS-coated and Matrigel-coated matrices were used for single-cell isolation and clonal expansion.
• Functional assays assessed CAR-T cell activation, proliferation, and IFN-γ release.

Main Results:

• Transfection efficiency reached 88.4% in Lenti-X 293T cells and 57.1% in Jurkat cells, with 34.1% showing surface CAR expression.
• Clonal expansion on FBS-coated matrices yielded 92.1% GFP-positive cells.
• CAR-T cells showed enhanced proliferation, activation (CD69, CD25), and IFN-γ release when co-cultured with LNCaP cells.

Conclusions:

• The novel FBS-coated plate method is efficient, economical, and scalable for CAR-T cell isolation and expansion.
• This approach improves T cell adherence, proliferation, and functional effectiveness.
• The method provides a foundation for advancing CAR-T cell therapies for solid tumors, with future research focusing on preclinical models.