Propagating Human iPS Cells with the DEF-CS™ Culture System
What are the characteristics of human iPS cells cultured with the DEF-CS system?
Human iPS cells cultured with the DEF-CS system are typically 98% Oct-4 positive, as determined by flow cytometry. They also express common pluripotency markers, including the transcription factors Oct-4 and NANOG and the surface markers SSEA-4, TRA-1-60, and TRA-1-81. The cells do not express the differentiation marker SSEA-1. Cells grown in the DEF-CS system have been shown to differentiate in vitro and in vivo into all three germ layers.
What is included in the system?
All reagents for human iPS cell culture are supplied, including three media additives and a matrix solution used to coat culture vessels for cell attachment. Enzymes and buffers for passaging are not included, but we recommend using TrypLE Select Enzymes (Cat. No. 12563029, Life Technologies). (Other enzymes can be used but require optimization.)
What is the composition of the medium?
DEF-CS Basal Medium is serum free and is not feeder conditioned, however its composition is proprietary. The kit also includes three media additives and a matrix solution; the compositions of these are also proprietary.
What enzyme is used to passage cells?
The DEF-CS system employs enzymatic single-cell passaging and seeding. The system is optimized for use with TrypLE Select Enzyme (Cat. No. 12563029, Life Technologies). Other digestive enzymes may be used with optimization.
Does the basal medium contain antibiotics?
Yes, it contains penicillin and streptomycin.
Why doesn't the provided volume of DEF-CS GF-3 correspond to the provided volume of DEF-CS Basal Medium?
DEF-CS GF-1 and GF-2 are added to the basal medium when changing the media and during cell passaging. DEF-CS GF-3 is added to the basal medium only when passaging cells, not during each media change, therefore less volume is needed.
How many cells can be grown with the DEF-CS system?
One DEF-CS 500 kit (containing 500 ml basal medium) is sufficient to culture cells on approximately 600 cm2 at a harvesting density of 1.5–3.0 x 105 cells/cm2. This corresponds to 24 T25 culture flasks, eight T75 culture flasks, or four T150 flasks.
What is the expected growth rate of the cells?
The typical generation time—the time needed for the cell population to double—of human iPS cells cultured with the DEF-CS system is 36 ± 5 hours.
How often should I change the medium?
We recommend changing the medium every day; however, when the cell density is sparse, you can change the medium every other day. It is important to change the medium the day before and the day after passaging.
How frequently should I passage cells?
Most human iPS cell lines can be passaged every 3–4 days. We suggest the following weekly schedule: passage on Monday and Thursday, using a slightly higher seeding density on Monday, then change the media on Tuesday, Wednesday, Friday, and Sunday. We recommend seeding 4.0–5.0 x 104 cells/cm2 and passaging at a density of 1.5–3.0 x 105 cells/cm2.
How many passages can be performed on human iPS cells cultured with the DEF-CS system?
Cells maintain pluripotency and a normal karyotype for at least 25 passages or approximately 65 population doublings.
Does the culture system allow automation?
Yes, the system supports single-cell suspension seeding, allowing control over seeding and harvesting densities and promoting a homogeneous stem cell population. These features, plus no need for cell selection, make the DEF-CS system ideal for mass producing cells with automation.