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Culture of iPS Cells

Feeder-Free Culture

This is the method for culturing iPS cells without feeder cells. The iPS cells are originally cultured using mouse feeder cells. However, the feeder-free culture is gradually becoming popular because it is preferable for clinical use to eliminate xeno feeder cells or other animal-derived components for regenerative medicine.
Here, we introduce the method for conducting the feeder-free culture with the StemFit® AK02N medium and iMatrix-511.

1Passage

Passage (1) Preparation of the plate and medium

Passage (2) Passage

Instruments/Reagents to be Used

Point of Operation

[Plate coating]
Add iMatrix-511at the concentration of 0.3 – 0.5 μg/cm2.
After the addition of iMatrix-511, quickly mix the solution for uniform coating. to the plate is high. If there are many wells, the iMatrix-511/PBS solution can be prepared at one time with a 50-ml tube .Be careful of dryingdry.

[Preparation for passage]
Dispense the StemFit medium in the 50-ml tube and add Y-27632 at the the concentration of 10 μmol. To prevent missing the addition of Y-27632 or confusion of the medium, write a memo on the tube (such as, +Y-27632) after addition and tick off the related culture record.

[Passage]
Observe the cells for passage under microscopy and mark the differentiated cells (cells considered to be non-iPS cells) with a permanent marker. Attach a chip without a filter to the tip of the aspirator and remove the marked area by suction. If there are many areas to be aspirated, reverse a well during the aspiration of each area. Be careful not to dry the cells.

[Enzymatic treatment]
After 3 minutes of treatment with the 0.5 X TrypLE solution, observe under microscopy to confirm that the entire colony has turned white. Complete the treatment with the 0.5 X TrypLE solution within 4 minutes and then remove the cells by adding the Y-27632-supplemented medium after PBS washing,

[Medium change the next day]
It is recommended that the first medium change should be conducted between 22 hours and 26 hours because the medium contains Y-27632. An early medium change leads to a decreased survival rate after the medium change, and a late medium change leads to morphological change of iPS cells.

* This content is provided as reference information for your own experiments and research. This information is not a guarantee of results.


2Cryopreservation

Freezing (1) Preparation of freezing

Freezing (2) Cell freezing

Instruments/Reagents to be Used

Point of Operation

[Freezing]
Observe the cells to be frozen under microscopy and mark the differentiated cells (cells considered to be non-iPS cells) with a permanent marker. Attach a chip without a filter to the tip of the aspirator and remove the marked area by suction. If there are many areas to be aspirated, reverse a well during the aspiration of each area. Be careful of drying.

[Enzymatic treatment/removal]
After 3 minutes of treatment with the 0.5 X TrypLE solution, observe under microscopy to confirm that the entire colony has turned white. Complete the treatment with the 0.5 X TrypLE solution within 4 minutes and then remove the cells by adding the Y-27632-supplemented medium after PBS washing.

[Cell freezing]
After centrifuging, all cells are suspended in the frozen solution (STEM-CELLBANKER). Dispense them into the cryo tube and transfer to the container for freezing, and then store in the freezer at -80°C. The operation from suspension in the frozen solution to storage in the freezer at -80°C should be completed within 30 minutes.

* This content is provided as reference information for your own experiments and research. This information is not a guarantee of results.


On-Feeder Culture

This is the method to culture iPS cells with feeder cells (mouse embryo fibroblasts).

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