Stem cells are invaluable tools in modern research, holding immense potential for regenerative medicine, disease modeling, and drug discovery. To harness their full potential, a carefully tailored environment is required.
At Creative Biolabs, we share a comprehensive protocol on feeder-dependent stem cell culture. This protocol outlines a dynamic method for culturing stem cells on feeder layers, enabling you to maintain their pluripotency, self-renewal, and overall robustness.
For MEFs or HFFs, culture cells in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin until reaching 70-80% confluence. Passage feeder cells at a ratio of 1:3 every 2-3 days. Prepare enough feeder cells for coating the plates for stem cell culture.
If starting from cryopreserved vials, thaw the vial quickly in a 37°C water bath, and transfer cells to a tube containing pre-warmed stem cell culture medium. Centrifuge the tube, then aspirate the supernatant. Resuspend the cell pellet in fresh stem cell culture medium supplemented with bFGF, and plate on prepared feeder cell-coated plates.
Incubate the plates at 37°C with 5% CO2 in a humidified incubator. Change the medium daily, gently aspirating the old medium and adding fresh stem cell culture medium with bFGF.
When colonies reach the appropriate size, wash the plates twice with PBS. Add an appropriate volume of Accutase or Trypsin-EDTA to the plate and incubate at 37°C until cells detach. Neutralize the enzymatic reaction by adding stem cell culture medium, and triturate to obtain a single-cell suspension. Passage the cells onto freshly prepared feeder cell-coated plates.
By following this protocol, we wish you were now equipped with the knowledge to cultivate stem cells in an environment that fosters their pluripotency and self-renewal. Contact us for more technical support.
References
For Research Use Only. Not For Clinical Use.
