Induction Protocol of Human-Induced Pluripotent Stem Cells
GUIDELINE
The generation of human iPS cells has had a tremendous impact on the treatment of degenerative diseases. Because iPS cells have similar functions to ES cells, but bypass many of the ethical and legal bottlenecks that embryonic stem cell research has been facing. So, they have a very promising application in the medical field and have become a hot spot for stem cell research.
METHODS
- Pre-coat one T25 bottle with 0.1% gelatin for approximately 10 min.
- When the cells are well grown, they are digested with 0.25% trypsin, collected into a 15 mL centrifuge tube, and counted with a platelet counting plate. Afterward, remove the number of cells needed for the infection experiment (e.g. 1×105).
- If 1×105 cells are infected, the required amount of virus is calculated at an infection ratio of 10:1. The required amount of each virus is added to a 15mL centrifuge tube, mixed, and filtered through a 0.45 μm filter to remove impurities such as cell debris from it to avoid any effect on subsequent experiments.
- Then 1×105 cells are added to the filtered virus solution and the final volume of the solution is made up to 5mL. 5μL of polybrene (10ug/ml) is added and the whole system is mixed and transferred to a precoated T25 bottle, shaken, and placed in a 37°C incubator overnight.
- After 24 hours of virus infection (day 1), the cells infected yesterday are digested and spread onto the pre-prepared MEF. Just spread 5×104 of cells per T75 bottle, and spread 2 T75 bottles.
- On day 2, change the DMEM culture medium to the hESC culture medium and continue the culture. After that, change the solution every 2 days.
- Observed day by day, when the cells grow to day 5-7, small cell aggregates are visible, and the cell morphology has changed, from shuttle-shaped to round, growing together.
- Until day 12, the culture medium is changed to a 1:1 mixture of CM+HES (hESC normal culture medium) to provide sufficient nutrients to the cells because the MEFs have been used for too long.
- When the cells grow to about day 20, the ES-like clone can be picked onto the new MEFs and continue to be cultured and amplified according to the hESC culture method.
Creative Bioarray Relevant Recommendations
- Induced pluripotent stem cells (also known as iPS cells or iPSC) are a type of pluripotent stem cell that can be generated directly from adult cells. We offer human iPSC lines derived from various primary cells by different inducing methods.
NOTES
- From the pre-experiment, the ratio of lentivirus infection of MRC-5 cells is 10:1 (i.e., all cells can be infected when 10 virus particles correspond to one cell).
- Virus infection can be divided into suspension infection and wall infection, and both methods are feasible and do not affect the efficiency of infection.
- Please prepare the trophoblast cells before starting the virus infection experiment.
- The induction of hiPSCs is a long-time process, and after the quality of the feeder layer decreases, the use of a conditioned culture medium is an option. The conditioned culture medium contains various factors secreted by feeder layer cells that are important for the growth of ESCs or iPSCs.
