Effective vitrification of human induced pluripotent stem cells using carboxylated ε-poly-l-lysine

Abstract

Derivation of human induced pluripotent stem (iPS) cells could enable their widespread application in future. Establishment of highly efficient and reliable methods for their preservation is a prerequisite for these applications. In this study, we developed a vitrification solution comprising ethylene glycol (EG) and sucrose as well as carboxylated ε-poly-l-lysine (PLL); this solution inhibited devitrification. Human iPS cells were vitrified in 200-μL vitrification solutions comprised 6.5M EG, 0.75 M sucrose and 0 or 10%w/v carboxylated PLL with 65 mol% of the amino groups converted to carboxyl groups [PLL (0.65)] in a cryovial by directly immersing in liquid nitrogen. After warming, attached colony and recovery rates of human iPS cells vitrified by adding PLL (0.65) were significantly higher than those for cells without PLL (0.65) and vitrification solution (DAP213: 2M dimethyl sulfoxide, 1M acetamide and 3M propylene glycol). Furthermore, even after warming at room temperature, attached colony and recovery rates of iPS cells vitrified with PLL (0.65) were reduced to a lesser extent than those vitrified with either DAP213 or EG and sucrose without PLL (0.65). This could be attributed to inhibition of devitrification by PLL (0.65), as differential scanning calorimetry indicated less damage after vitrification with PLL (0.65). In addition, human iPS cells vitrified in the solution with PLL (0.65) had normal karyotypes and maintained undifferentiated states and pluripotency as determined by immunohistochemistry and teratoma formation. Addition of PLL (0.65) successfully vitrified human iPS cells with high efficiency. We believe that this method could aid future applications and increase utility of human iPS cells.