DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells

Figure 4

Cryopreservation of aderent human iPSCs in plates. Fluorescent microscopy of human induced pluripotent stem cells frozen in adherent state in 4-well plates. First row: Nanog (immune staining), second row: combined Nanog + Hoechst. The photos were taken on day 3 after thawing. In all cases, ethylene glycol (EG) and ROCK inhibitor Y-27632 were used.−80°C: uncontrolled freezing in a –80°C freezer in a Styrofoam box (~−1°C/min at the first stage of freezing). The yields of viable, and especially Nanog-positive cells, are very low (see Figure 3). −1°C/min: one-step freezing in a programmable freezer (Custom Biogenics, Inc, series 2100); Note 1: eroded edges of the colony and differentiation of the adjacent cells within the colony. 3St: three-step freezing protocol (described in Section 3.3) in a programmable freezer. 6St: six-step freezing protocol (described in Section 3.3) in a programmable freezer. Note 2: clear distinct edges of the colonies in BOTH cases and differentiated (Nanog-negative) cells (blue) outside the colony in the 3-Step protocol. QUANTA has confirmed substantially higher attachment efficiency and a larger 7AAD⁻/GFP⁺ pool of viable iPSCs in cases of multistep controlled freezing (Figure 3).