Weekend-free protocol for iPSC maintenance

Weekend-free culture of human induced Pluripotent Stem Cells (iPSCs)

Feeder-free human induced pluripotent stem cell (iPSC) culture is highly repetitive and costly due to the requirement for daily media changes. iPSCs need Fibroblast growth factor 2 (FGF-2) and transforming growth factor b1 (TGF-b1) present at precise concentrations to maintain pluripotency. Achieving this in vitro can be challenging due to low thermostability of these growth factors.

This protocol update describes a method for maintaining the pluripotency of iPSCs using Qkine FGF2-G3 (Qk053) and TGF-β1 PLUS (Qk010) without the need for daily feeding, enabling weekend-free maintenance and expansion.

Weekly schedule for routine maintenance of iPSCs

Weekly schedule for routine maintenance of iPSC


Passage iPSCs with 0.5mM EDTA for detachment and culture in 5 µg/ml vitronectin coated 6-well plates using a 1:6 split ratio in E8-like media (DMEM/F12 with 1.1 mg/ml sodium bicarbonate, 2 mg/ml sodium chloride, 0.13 mg/ml L-ascobic acid, 1x Insulin-Transferrin-Selenium (ITS-G) (Thermo Fisher Scientific, 41400045), 40ng/ml FGF2-G3 (Qk053) and 2ng/ml TGF-β1 PLUS (Qk010))


Replace spent media with 5ml fresh E8-like media


No media change


Passage iPSC with 0.5mM EDTA for detachment and culture in 5µg/ml vitronectin coated 6-well plates using a 1:6 split ratio in E8-like media.


Replace spent media with 5ml fresh E8-like media


No media change

Optional: Validate pluripotency using immunocytochemistry

Validate pluripotency using immunocytochemistry

Validation of pluripotency markers in iPSC grown in E8-like media.

iPSC cultures were fixed and stained with the pluripotency markers OCT-4, NANOG and SOX2 and secondary antibodies Donkey anti-Mouse AlexaFluor™ 647 or Donkey anti-Goat AlexaFluor™ 488 and Hoechst 33258. Brightfield image (A), NANOG expression (green, B), OCT-4 expression (red, C), combined NANOG, OCT-4 and Hoechst 33258 (D). Brightfield image of stained colony (E), SOX2 expression (green, F) combined SOX2 and Hoechst 33258 (G, blue) scale bar = 150µm.

Featured resources

FGF2-G3 brochure

Animal-free and xeno-free growth factor diagram

Nine-point Quality Commitment

Animal origin-free proteins manufacturing

TGF beta family technote

the first quality- matched animal-free TGF-ß1, 2 and 3 proteins for improved cell culture media development.

Contact our expert team

Our dedicated team of stem cell specialists is available to answer any queries and to give expert support when required.

Weekend-free culture of human iPSCs

Qkine growth factors are manufactured to the highest quality standards and are free from animal-derived contaminants, delivering low endotoxicity and high purity. At Qkine, we are committed to raising the standards of growth factors, cytokines and related proteins to better support long-term and complex neural stem cell culture. We are a science-led team, please reach out with any questions or requests.