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) on vitronectin (Qk120) coated plates, without the need for daily feeding, enabling weekend-free maintenance and expansion.
Weekly schedule for routine maintenance of iPSCs
Monday
Passage iPSCs with 0.5 mM EDTA for detachment and culture in 5 µg/ml vitronectin (Qk120) 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), 40 ng/ml FGF2-G3 (Qk053) and 2 ng/ml TGF-β1 PLUS (Qk010))
Tuesday
Replace spent media with 5 ml fresh E8-like media
Wednesday
No media change
Thursday
Passage iPSC with 0.5 mM EDTA for detachment and culture in 5 µg/ml vitronectin coated 6-well plates using a 1:6 split ratio in E8-like media.
Friday
Replace spent media with 5 ml fresh E8-like media
Saturday/Sunday
No media change
Optional: 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.
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.
