When cells proliferate in a culture medium, metabolites such as lactic acid gradually accumulate and eventually hinder cell growth. Herein, we considered various layered double hydroxides (LDH) and layered double oxides (LDO) for the selective removal of lactate in aqueous solution and a culture medium by anion exchange. NO3 center dot Mg-Al LDH showed high lactate adsorption, low adsorption of glucose (a nutrient in culture medium), appropriate pH stability, and low cytotoxicity within a limited contact time. In contrast, NO3 center dot Ni-Al LDH, NO3 center dot Ca-Al LDH, Mg-Al LDO, and Cl center dot Mg-Al LDH had a lower performance than NO3 center dot Mg-Al LDH. To further improve the performance, the anion of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, a common pH buffer in culture media) was tested as the intercalated anion in Mg-Al LDH. HEPES center dot Mg-Al LDH (Mg/Al = 2) was found to be the most promising adsorbent for culture medium regeneration because its adsorption behavior was similar to that of NO3 center dot Mg-Al LDH, while it had a much lower cytotoxicity. The traditional methods for regenerating a culture medium by removing accumulated lactate to increase the cell density in large-scale cell culture and lower the costs, including electrolysis, cell modification, and modification of the culture medium's composition, are complicated to operate and expensive. The adsorption method using HEPES center dot Mg-Al LDH presented in this study, instead, can be easily operated and has low costs. Thus, it is expected to be applied in actual cases for regenerating culture media.