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The large-scale culture of human induced pluripotent stem cells (hiPSCs) is essential for developing new pharmaceuticals and regenerative therapy methods. While for the development of cultured meat products, mass production of animal myoblasts is necessary. Both hiPSCs and animal myoblasts consume d-glucose as their energy source and produce l-lactate, which accumulates in cell culture media and inhibits cell proliferation. To make large-scale cell culture economically feasible, l-lactate removal and subsequent reuse of media are of high importance. The adsorption technique is attractive for l-lactate removal due to its low cost, ease of operation, and scalability. The current study is dedicated to 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) intercalated Mg–Al layered double hydroxide (LDH), which acts as a biocompatible anion-exchanger in media. HEPES‧Mg–Al LDH was able to remove l-lactate from hiPS cells and myoblast-relevant media selectively while mostly retaining d-glucose. Adsorbent exhibited dose-dependent cytotoxicity to hiPSCs and C2C12 cells, mainly related to elevated osmolarity, HEPES, and Mg levels and adsorption of media micro components. By employing alternatively prepared sol-gel derived HEPES‧Mg–Al LDH, the required adsorbent dose for efficient l-lactate removal was reduced to a safe level. The current study thoroughly evaluates Mg–Al layered double hydroxides as suitable adsorbents for cell culture media regeneration and discusses the limitations of Mg–Al LDHs in systems relevant to hiPS cells and C2C12 cells. This work promotes the cost-effective large-scale production of cells and gives insight into the limitations of Mg–Al LDHs applied to systems of biological origin.