Refined home-brew media for cost-effective, weekend-free hiPSC culture and genetic engineering

<ns3:p> Background Cost-effective, practical, and reproducible culture of human induced pluripotent stem cells (hiPSCs) is required for both basic and translational research. This is especially crucial for large-scale expansion of hiPSCs for cell therapy, which should be made accessible to many patients regardless of their socioeconomic background. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hiPSC culture. However, homebrewing of some recombinant growth factors for B8 can be a bottleneck towards both access and reproducibility of this technology. Moreover, we found the published B8 formulation to be suboptimal in normoxic hiPSC culture, which is widely used. Lastly, the suitability of B8 for applications such as genome editing or organoid differentiation remains to be assessed. Methods We formulated B8 with commercially available, animal-free growth factors, refined its composition to support normoxic culture of the widely-used WTC11 hiPSC line, and compared it to commercial Essential 8 (E8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle with flow cytometry, and investigated the transcriptional profiles by bulk RNA sequencing. We also assessed the efficiency of gene editing, single-cell sorting, and cardiac differentiation in both monolayer and organoids. Results hE8 performed similarly to commercial E8 in all the assays. Despite morphological changes, cells in B8+, our optimised variant of B8, expressed the pluripotency marker NANOG at the highest level. At the same time, cells grown in B8+ were primed towards a mesendodermal fate. B8+ outperformed other media with regard to genome editing <ns3:italic>via</ns3:italic> homology directed recombination, and was on par with other media in other assays. Conclusions Overall, optimised weekend-free media formulations promise to democratise the generation of engineered cells for a wide range of applications. </ns3:p>