If the requester already has the somatic cells to be reprogrammed in their laboratory, these can be sent frozen on dry ice or alive in a 25cm2 flask. It is strongly recommended to have a minimum of 500,000 cells per cryovial. Additionally, the cells should have a low passage number and be capable of proliferation, as senescent cultures are not easily reprogrammable (except for PBMCs). No cryovial of cells will be kept in our laboratory. Unused cells will be returned to the requester along with the iPSC clones.
This service spans approximately three months and includes:
- Amplification of somatic cells
- Sampling of cells for parental genotype
- Seeding for transduction and freezing of remaining cells
- Mycoplasma testing before infection
- Transduction with Sendai virus
- Seeding on feeder cell layer (3 different densities)
- Daily observation and medium change
- Selection of approximately 15 clones per lineage directly on Matrigel
- Culture of iPSCs on Matrigel and mTSeR plus without antibiotics
- Amplification of the 5 best clones morphologically
- Freezing of 5 cryovials per clone (Passages 3 and 4)