Transferring fresh or frozen cultures to the DEF-CS system
- Coating of cell culture vessels
- Dilute the required volume of DEF-CS COAT-1 in D-PBS +/+ before use. Make a 1:10 dilution.
- Mix the diluted DEF-CS COAT-1 solution gently and thoroughly by pipetting up and down.
- Add an appropriate volume of diluted DEF-CS COAT-1 solution to a cell culture vessel (use 0.1 ml/cm2), making sure that the entire surface is covered.
- Incubate the cell culture vessel at 37°C ± 1°C, 5% CO2, and >90% humidity for a minimum of 20 min, or at room temperature (15–25°C) for 0.5–3 hr.
- Aspirate the DEF-CS COAT-1 solution from the cell culture vessel immediately before use.
- Preparing supplemented DEF-CS medium
- Prepare the appropriate volume of supplemented DEF-CS medium by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to DEF-CS Basal Medium.
- Prepare fresh medium on the day of intended use and warm it to 37°C ± 1°C immediately before use. Discard any leftover warm medium.
- Warm all other necessary reagents to room temperature (15–25°C) before use.
Transferring hiPS cells from a fresh MEF feeder culture
Fresh cultures should be transferred on days when they would normally be passaged, using a consistent culture area (e.g., cells cultured on MEF feeder cells in a 35-mm dish should be transferred to a 35-mm dish for culturing with the DEF-CS system).
- Check MEF-cultured colonies under a microscope; photo document as necessary.
- Aspirate the medium from the cell culture vessel and wash the cell layer once with D-PBS –/–.
- Add 20 μl/cm2 of TrypLE Select Enzyme (1X) to the cell culture vessel and incubate for 5 min, or until the cell layer has detached. Detachment can be aided by swirling the cell culture vessel or by tapping the side of the cell culture vessel firmly but gently.
NOTE: Any inactivated MEFs that are detached and passaged to the DEF-CS system can be neglected, as they will NOT affect the result.
- Resuspend the cells in pre-warmed, supplemented DEF-CS medium (40 μl/cm2) and pipet up and down several times to ensure a single-cell suspension (The cells will aggregate if left too long in TrypLE Select Enzyme).
- Centrifuge the cells at 200g for 2–5 min.
- Discard the supernatant carefully and resuspend the cell pellet in the appropriate volume of prewarmed, supplemented DEF-CS medium. Add the cell suspension to the newly coated cell culture vessel.
- Tilt the vessel backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface, then place in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.
Transferring cryopreserved, MEF-cultured hiPS cells
Thaw the cells onto the same culture area you would use for thawing the cells onto an MEF feeder layer.
- Thaw the cells according to your preferred protocol.
- Transfer the cells to a newly coated cell culture vessel with prewarmed, supplemented DEF-CS medium.
- Tilt the vessel backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface, then gently place in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.
The single-cell passaging method employed by the DEF-CS culture system causes iPS cells to initially assume a distinct morphology and sparser distribution relative to cells cultured using colony-based passaging methods. However, as the cells proliferate and form denser populations, morphologies commonly associated with undifferentiated stem cells (e.g., high nucleus-to-cytoplasm ratio, clearly defined borders, and prominent nucleoli) emerge.
- It may take 2–5 passages to adapt a cell line to the DEF-CS culture system. Newly transferred cells might initially grow at a slightly slower rate. A suitable passage interval might, therefore, be between 3 and 7 days for the first few passages.
- Use a 1:10 dilution of DEF-CS COAT-1:D-PBS +/+ for the first few passages to provide extra support during the adaptation process.
- To prevent cell loss during scale-up, we recommend not counting cells at passage when the total number of cells is quite low.
- If the hiPS cells were sparsely seeded or thawed in aggregates, they will grow as colonies on COAT-1. As a general rule, when passaging hiPS cells that are growing as colonies, the area covered by the cells at passage should not be less than 20% of the area of the destination vessel.
- For passages involving cells growing in a homogeneous monolayer (normal DEF-CS culture system characteristics), cells are ready for passage when they have acquired the morphology displayed in Figures 3 and 4 in the Cellartis DEF-CS 500 Culture System User Manual. However, if cells remain sparsely distributed after seven days in culture, a passage is still recommended. The area of the destination vessel should be 3–6 times the area of the current vessel.
- Once cells have been scaled up to a T-25 flask, they should be cultured according to the Cellartis DEF-CS 500 Culture System User Manual.