Cell culture technologies are foundational to contemporary biomedical research and therapeutic development, providing controlled platforms to study cellular behavior, disease mechanisms, and drug responses. Among the physicochemical parameters governing in vitro systems, precise regulation of extracellular pH is essential for maintaining cellular viability, metabolic balance, and phenotypic stability. Carbon dioxide (CO₂) plays a central role in this process by modulating bicarbonate-based buffering systems that closely mimic physiological conditions. Beyond pH control, accumulating evidence indicates that CO₂ can directly influence cellular signaling, metabolism, gene expression, and stress responses. This review critically examines the role of CO₂ in mammalian cell culture systems, with emphasis on its interaction with buffering strategies, cellular development, and metabolic regulation. In addition, emerging medicinal and biotechnological applications of CO₂-regulated culture environments are discussed, highlighting their relevance to translational research, tissue engineering, and therapeutic manufacturing. By integrating fundamental principles with recent advances, this article provides a comprehensive perspective on CO₂ as both a regulatory and functional component of modern cell culture and biomedical applications.