Poly(l-alanine-co-l-lysine)-g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells.

Abstract

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAKT) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAKT. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAKT and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAKT solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAKT solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAKT was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT. 1H NMR and FTIR spectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation.