092 Biophysical parameters of adherent human induced pluripotent stem sells for the rational design of cryoprotectant addition and removal procedures

Abstract

Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 (2012) 144–151) To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287–298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4 °C, 21 °C, and 37 °C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlue®. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4 °C, 21 °C, and 37 °C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37 °C than 4 °C or 21 °C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs. Source of funding: This project was funded by a Collaborative Research Contract from Life Technologies with Oregon State University. Conflict of interest: None declared. adam.higgins@oregonstate.edu