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

Abstract

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 EGFlike 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 weekendfree 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.

Differentiation of Human induced Pluripotent Stem Cells to Authentic Macrophages using Fully Defined, Serum Free, Open Source Media

“…In order to culture iPSC, researchers are generally reliant on commercial media that are not only proprietary, but also very expensive, limiting the ability of many laboratories to undertake iPSC-based research. There has been a recent push to lower this cost (Kuo et al, 2020). The alternative that we describe here is also cheaper than the commercial alternatives.…”

Design of chemically defined synthetic substrate surfaces for the in vitro maintenance of human pluripotent stem cells: A review

“…In this study, bFGF were replaced to be thermostable variant of bFGF‐G3 capable to induce pERK in FGF‐starved cells, even after the medium had previously been stored for extended periods at 37°C. Besides, more potent TGFβ3 was applied to replace TGFβ1 40 . Above medium were summarized in Table 2.…”

Harnessing pluripotent stem cells as models to decipher human evolution

“…A single organoid differentiation experiment can cost thousands of dollars – before sequencing. Recent advances that promise, for example, minimal cost media for iPSC maintenance [164] are welcome steps, but differentiation protocols often require highly specialised media as well as multiple recombinant proteins or synthetic small molecules. And even as prices continue to drop, other limitations, such as the need to culture 10s or 100s of organoids at a time, are becoming more relevant.…”