Human induced pluripotent stem cell (hiPSC) culture has become routine, yet pluripotent cell media costs, frequent media changes, and reproducibility of differentiation have remained restrictive, limiting the potential for large-scale projects. Here, we describe the formulation of a novel hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. B8 eliminates 97% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli -expressed, codon-optimized recombinant proteins: an engineered form of fibroblast growth factor 2 (FGF2) with improved thermostability (FGF2-G3); transforming growth factor β3 (TGFβ3) - a more potent TGFβ able to be expressed in E. coli ; and a derivative of neuregulin 1 (NRG1) containing the EGF-like domain. The B8 formula is specifically optimized for fast growth and robustness at low seeding densities. We demonstrated the derivation and culture of 34 hiPSC lines in B8 as well as maintenance of pluripotency long-term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing growth rate or capacity for differentiation. Thus, this simple, cost-effective, and open source B8 media, will enable large hiPSC disease modeling projects such as those being performed in pharmacogenomics and large-scale cell production required for regenerative medicine.

Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture