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 mesen-chymal stem cells (MSCs). FTIR and circular dichroism spectraindicated that the content of the alpha-helical structure of PAKdecreased after conjugation with trehalose. Two protocols wereinvestigated in cryopreservation of MSCs to prove the significanceof the intracellularly delivered PAKT. In protocol I, MSCs werecryopreserved at-196 degrees C for 7 days by a slow-cooling procedurein the presence of both PAKTand free trehalose. In protocol II,MSCs were preincubated at 37 degrees C in a PAKTsolution, followed bycryopreservation at-196 degrees C in the presence of free trehalose for 7days 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 PAKTsolution. The recovered cellsfrom protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, andadipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Icerecrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly deliveredPEG-PAKTwas the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosisand caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT.1H NMR and FTIRspectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation