Stable bioreactor control reveals acidic pH-driven metabolic reprogramming and mitochondrial dysfunction in human lymphoblastoid cells

Human cells require precise pH regulation to maintain optimal physiological functions, yet the molecular impacts of acidic environments remain underexplored due to the lack of technologies capable of precisely controlling the cell culture environment. Here, we employed a gas-only bioreactor to stably control pH, dissolved oxygen, and temperature during the culture of human B lymphoblastoid cells. Integrated transcriptomic, epigenomic, and metabolomic analysis revealed that acidic pH (6.8) induces a metabolic shift towards glycolysis, suppresses energy-intensive processes such as cell proliferation, and triggers intracellular accumulation of lactate and oncometabolites and mitochondrial dysfunction. This environment also elevates reactive oxygen species (ROS) and upregulates inflammatory and immune pathways, leading to heteroplasmic shifts in a pathogenic mitochondrial mutation. Mechanistically, acidic pH caused marked depletion of intracellular NAD⁺ driven in part by PARP1 activation, and restoration of NAD⁺ levels via nicotinamide mononucleotide supplementation or PARP1 inhibition partially rescued cellular proliferation, mitochondrial genomic integrity, and stress-associated transcriptional programs. These findings highlight the critical role of pH homeostasis in cellular metabolism and immune response, offering insights into the cellular adaptations to acidic stress, which may inform therapeutic strategies for conditions like cancer and metabolic disorders.