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.

The unique ability of Vδ1 ⁺ T cells to recognize targets in a major histocompatibility complex (MHC)-unrestricted manner makes them promising candidates for immunotherapy. This review explores the biological underpinnings of their advantages, particularly their ability to penetrate and migrate through solid tumors, their antigenic promiscuity, and their functional plasticity in inflammatory tumor microenvironments. Based on this, we analyze the therapeutic pipeline derived from this biology, including targeted antibodies, bispecific molecules and a new generation of cellular products, among which chimeric antigen receptor (CAR)-engineered constructs have shown encouraging clinical responses. Nonetheless, translation into the clinic can often present challenges; we tackle the unresolved issues of product standardization, subset complexity, and microenvironmental suppression, offering practical ways forward. This review aims to serve as a reference to deepen the understanding of the anti-tumor value of Vδ1 ⁺ T cells and facilitate their translational development.