# Upregulation of the TCA Cycle and Oxidative Phosphorylation Enhances the Fitness of CD99 CAR-T Cells Under Dynamic Cultivation

## TL;DR

Dynamic cultivation using a wave bioreactor improves CAR-T cell function by enhancing their metabolism and memory-like properties.

## Contribution

This study reveals that dynamic cultivation promotes mitochondrial metabolic reprogramming in CD99 CAR-T cells.

## Key findings

- Wave bioreactor-cultured CAR-T cells show faster proliferation and stronger cytotoxicity.
- These cells retain enhanced function and display reduced immune checkpoint molecule expression.
- Metabolomic profiling shows increased TCA cycle activity and oxidative phosphorylation.

## Abstract

The manufacturing process contributes significantly to the proliferation, metabolic state, and functional persistence of chimeric antigen receptor (CAR)-T cells. However, how different culture systems regulate CAR-T cell metabolism and thereby influence their long-term antitumor activity remains poorly understood. In this study, we compared dynamic cultivation using a wave bioreactor with static expansion systems (gas-permeable and conventional T-flasks) for the production of CD99-specific CAR-T cells. CAR-T cells expanded by the wave bioreactor exhibited faster proliferation and stronger cytotoxicity during culture. Upon repeated antigen stimulation, they retained these enhanced functional properties and showed the reduced expression of immune checkpoint molecules, preferentially preserved memory-like subsets, and displayed transcriptional features consistent with memory maintenance and exhaustion resistance. Targeted metabolomic profiling revealed enhanced Tricarboxylic Acid (TCA) cycle activity and features consistent with sustained oxidative phosphorylation, supporting mitochondrial-centered metabolic reprogramming. In a Ewing sarcoma xenograft model, wave bioreactor-cultured CAR-T cells showed a greater percentage of memory-like tumor-infiltrating lymphocytes. Collectively, these results indicate that wave bioreactor-based dynamic cultivation promotes mitochondrial metabolic reprogramming, which is characterized by an enhanced TCA cycle and sustained oxidative phosphorylation, thereby sustaining CAR-T cell functionality and providing a robust platform for the manufacturing of potent and durable cellular therapeutics.