Effective vitrification of human induced pluripotent stem cells using carboxylated e-poly-L-lysine q Kazuaki Matsumura 1 , Jung Yoon Bae 1 , Hak Hee Kim, Suong Hyu Hyon ⇑ Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan article info Article history: Received 24 December 2010 Accepted 13 May 2011 Available online 20 May 2011 Keywords: Human induced pluripotent stem cells Vitrification Cryopreservation Polyampholytes DMSO-free vitrification solutions 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 e-poly-L-lysine (PLL); this solution inhibited devitrification. Human iPS cells were vitrified in 200-lL vitrification solutions comprised 6.5 M 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: 2 M dimethyl sulfoxide, 1 M acetamide and 3 M 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 devit- rification 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 tera- toma 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. Ó 2011 Elsevier Inc. All rights reserved. Introduction Derivation of induced pluripotent stem (iPS) cell lines from hu- man somatic cells [23] offers profound potential for research and clinical use as tools for pharmaceutical applications and ultimately for use as cell sources for transplantation and tissue regeneration. In addition, iPS cells enable production of disease- and patient- specific pluripotent stem cells for cell therapy applications without using human oocytes or embryos [13,18]. It is also believed that these personalized stem cells would not be immunologically re- jected if reprogrammed. To avoid rejection, it is crucial for cells to be well preserved until just prior to their use. Future applica- tions of iPS cell-derived cells or tissues depend upon establishing efficient preservation methods. Cryopreservation is used for long-term preservation of biologi- cal materials containing cells. Two primary techniques of cryopres- ervation are slow freezing and vitrification, in which water transition directly to the glassy state without crystallization. Slow freezing methods, which utilize 10% dimethyl sulfoxide (DMSO) as a cryoprotectant, are effective for numerous cell lines, including mouse embryonic stem (ES) and iPS cells. However, human ES and iPS cells are quite sensitive to damage during freezing and thawing. In addition, the percent that survive after slow freezing cryopreservation is reported to be very low. Vitrification methods have been developed with the advent of preservation techniques for oocytes and embryos, primarily in the field of reproductive medicine. With further development in techniques, large-scale preservation would not be limited by any constraints. The volume of a cell suspension during vitrification is often in single- or a double-digit microliter value at most. Although human ES cells can be successfully cryopreserved by vit- rification [20], the protocol is impractical for large-scale use, as it requires open pulled straws and manual selection of stem colonies. Fujioka et al. [3] developed a simple, large-scale (200 lL) vitrifica- tion method for primate ES cells using a DAP213 solution contain- ing 2 M DMSO, 1 M acetamide and 3 M propylene glycol. However, human ES cells are not sufficiently maintained in an undifferenti- ated state on using DMSO as a cryoprotective agent [4]. A chemi- cally defined solution without DMSO has been identified to 0011-2240/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2011.05.003 q Statement of funding: This study was supported by the grant ‘‘The Project for realization of regenerative medicine’’ from the Ministry of Education, Science, Sports and Culture of Japan. ⇑ Corresponding author. Fax: +81 75 751 4141. E-mail address: biogen@frontier.kyoto-u.ac.jp (S.H. Hyon). 1 These authors contributed equally to this study. Cryobiology 63 (2011) 76–83 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo