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

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAK_T) 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 PAK_T. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAK_T and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAK_T 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 PAK_T 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-PAK_T 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-PAK_T. ¹H NMR and FTIR spectra suggested that the intracellular PEG-PAK_Ts interact with water and stabilize the cells during cryopreservation.