In the field of cell therapy, the efficiency and stability of T cell culture directly affect the success or failure of clinical applications. The operational differences between 96 well and 6-well plates, as two common culture carriers, are not only reflected in their scale, but also involve key details of cellular microenvironment regulation.

1. Uniformity of cell inoculation

The high-throughput characteristics of the 96 well plate require more precise cell suspension distribution. It is recommended to use a multi-channel pipette combined with low-speed centrifugation (200 × g, 5 minutes) to promote cell sedimentation and avoid the "edge effect" caused by liquid surface tension in the edge pores. The single well volume of 6-well plates is relatively large (2-4mL), and manual mixing using the "cross shaking plate method" is required to ensure uniform distribution of cells.

2. Medium replacement strategy

The 96 well plate is prone to disturbing adherent cells during half volume liquid exchange, and the supernatant can be slowly aspirated and discarded at a 45 ° angle; If using serum-free culture medium, it is necessary to pre add matrix gel (such as Corning) ® Matrigel enhances adhesion. A 6-well plate can be replaced with 50% of the culture medium first using the "gradient exchange method", and then the full amount can be replaced 24 hours later to reduce cell stress.

3. Gas exchange optimization

When stacking 96 well plates for cultivation, it is recommended to use plate covers with breathable membranes (such as Greiner Bio One CELLSTAR) ®）， And control the stacking to not exceed 3 layers to avoid oxygen deficiency in the central hole. The 6-well plate can be directly placed in a wet mixed incubator containing 5% CO ₂, but attention should be paid to the distance between the edge of the plate and the box fan to prevent local excessive evaporation.

4. Phenotypic monitoring techniques

It is recommended to use a high-resolution imaging system (such as ImageXpress Micro) for multi-point scanning of 96 well plates, which automatically eliminates bubble interference data through AI algorithms. 6-well plates are more suitable for pre amplification before flow separation. It is recommended to use the "diagonal puncture method" to continuously puncture 3 times along the diagonal with a 1mL gun head during sampling to ensure sample representativeness.

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