A recent publication in the journal Stem Cell International briefly discussed the freezing of induced pluripotent stem cells derived from adult equine fibroblasts. While assessing the effectiveness of the induced pluripotent stem cell freezing method was not the focus of the study (rather, the generation of the iPS cells was the focus) it does provide an at least workable protocol.
Of course, induced pluripotent stem cells can differ based on the cells that they are derived from and the method used to induce pluipotency, so this is by no means an endorsement of the cell freezing protocol for all types of iPS cells. Nor is it necessarily an optimized cell freezing protocol. It may, however, serve as a good starting point, and we at least know the protocol works for iPS cells derived from adult equine fibroblasts.
The researchers used an isopropanol-based cell freezing container equilibrated to 4°C. The iPS cells were dissociated into clumps of approximately 100-200 cells, collected in 15 ml tubes and washed with iPS cell medium. The medium is described as a “conventional medium containing α-minimum essential medium (α-MEM) with deoxyribonucleosides and ribonucleoside (Invitrogen, Mulgrave, Australia), supplemented with 2 mmol/mL glutamax (Gibco, Invitrogen, Mulgrave, Australia), 0.1% (v/v) Mercaptoethanol (Gibco), 1% (v/v) nonessential amino acid (NEAA) (Gibco), 1% (v/v) ITS (10 μg/mL insulin, 5.5 μg/mL 125 transferrin, 6.7 ng/mL selenium; Gibco), 5 ng/mL human LIF (Millipore, North Ryde, Australia), 10 ng/mL βFGF (Millipore), 10 ng/mL EGF (Invitrogen), 0.5% (v/v) penicillin-streptomycin (Gibco), and 20% (v/v) FBS.” The cells were then centrifuged at 400g for 3 minutes, the supernatant was discarded, and cells were resuspended in iPS cell medium. 500μL of cell suspension was added to cryovials and freezing medium was then added, which consisted of 80% FBS and 20% DMSO. The vials were frozen to −80°C overnight and then transferred to a LN2 tank at minus 196°C for long-term storage. To thaw the cells, the cryovials were placed in a water bath at 37°C for 1 minute. Cells were then transferred to a 15 ml tube and 10 mL of iPS cell culture medium was slowly added. The cells were centrifuged at 400g for 3 minutes, the supernatant was discarded, and the cells were resuspended in iPS medium for use.
All in all it’s a pretty standard cell freezing protocol, aside from the cell-specific medium. One thing that we found that was noteworthy was their use of an alcohol-based cell freezing container. The team could have saved themselves the expense and hassle of using alcohol if they opted for a BioCision CoolCell to freeze their induced pluripotent stem cells. The CoolCell’s design ensures reproducible freezing without ever needing alcohol, making cell freezing cheaper and easier than ever.
The research article that we discuss, “Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC” can be found here.