Ready-to-use cryopreservation of undifferentiated induced pluripotent stem cells (iPSCs) without detachment from culture plates using D-proline and a synthetic polymer

Abstract

Induced pluripotent stem cells (iPSCs) have been making a significant impact on the fields of regenerative medicine and cell biology. Several cell-freezing media for dispersed single-cell iPSCs are already commercially available. However, cryopreservation techniques for iPSCs cultured in 2D and 3D formats remain to be established. In this study, we developed a xeno-free cell-freezing medium containing D -proline and a synthetic block copolymer composed of 2-(dimethylamino)ethyl methacrylate (DEGMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC), designated PDEGMA- b -PMPC- b -PDEGMA, that enables the cryopreservation of iPSCs cultured in 2D on microplates without detachment of the cells. Prior to cryopreservation, 2D-cultured iPSCs were treated with TrypLE Select Enzyme to weaken their adhesion to the microplate surfaces. Subsequently, the cells were cryopreserved in the cell-freezing medium containing D -proline and PDEGMA- b -PMPC- b -PDEGMA at −80°C for 2 days. At 48 h after thawing, the cell recovery (cell viability) was at least 70 % relative to the cell viability before freezing, while the cell recovery with commercially available media was 1.2 % at most. The most effective composition of the cell-freezing medium was 10 vol% DMSO, 5 vol% Dulbecco’s modified Eagle’s medium, 85 vol% water, 1 % (w/v) PDEGMA- b -PMPC- b -PDEGMA, 2 % (w/v) D -proline, and 0.35 % (w/v) NaCl. The cell recovery value remained stable after 3 months of cryopreservation. Most importantly, the iPSCs maintained their pluripotency after cryopreservation in the newly developed cell-freezing medium.