Size and Shape Control of Ice Crystals by Amphiphilic Block Copolymers and Their Implication in the Cryoprotection of Mesenchymal Stem Cells

Abstract

Precise control over the size and shape of ice crystals is a key factor to consider in designing antifreezing and cryoprotecting molecules for cryopreservation of cells. Here, we report that a poly(ethylene glycol)−poly(L-alanine) (PEG−PA) block copolymer exhibits excellent cryoprotecting properties for stem cells and antifreezing properties for water. As the molecular weight of PA increased from 500, 760, and 1750 Da (P1, P2, and P3) at the same PEG molecular weight of 5000 Da, the β-sheet content decreased and α-helix content increased. Comparing P2 (PEG−PA; 5000−760) and P4 (PEG−PA: 1000−750), β-sheets increased as the PEG block length decreased. The critical micelle concentration of the PEG−PA block copolymers was in a range of 0.5−3.0 mg/mL and was proportional to the hydrophobicity of the PEG−PA block copolymers. The P1, P2, and P3 self-assembled into spherical micelles, whereas P4 formed micelles with cylindrical morphology. The difference in the block copolymer structure affected ice recrystallization inhibition (IRI) activity and cryopreservation of cells. IRI activity was assayed via mean largest grain size (MLGS), and interactions between polymers and ice crystal surfaces were studied by dynamic ice-shaping studies. The MLGS decreased to 58 → 53 → 45 → 35 → 23% of that of PBS, as the polymer (PEG−PA 5000−500) concentration increased from 0.0 (PBS; control) → 1.0 → 5.0 → 10 → 30 → 50 mg/mL. The MLGS of PEG 5k solutions (negative control) decreased to 74 → 71 → 64 → 44 → 37% of that of PBS in the same concentration range. P3 and P4 with a longer hydrophobic PA block developed elongated ice crystals at above 30 mg/mL. The dynamic ice-shaping study exhibited that ice crystals became needle-shaped, as the hydrophobicity of the polymer increased as in P2−P4. The cell recovery in the P1 system after cryopreservation at −196 °C for 7 days was 87% of that of the dimethyl sulfoxide (DMSO) 10% system (positive control). The cell recovery was 48% for the P2 system and drastically decreased to less than 30% of that of the DMSO 10% system in the P3, P4, PEG 5k, PEG 1k, PVA 80H, and PVA 100H systems. Current studies suggest that IRI activity, round ice crystal shaping, and membrane stabilization activity of P1 cooperatively provide excellent cell recovery among the candidate systems. Recovered stem cells exhibited excellent proliferation and multilineage differentiation into osteocytes, chondrocytes, and adipocytes. To conclude, the PEG−PA (5000−500) block copolymer is suggested to be a promising antifreezing cryoprotectant for stem cells.

Recent progress in cryoablation cancer therapy and nanoparticles mediated cryoablation

“…Ice crystals which are sharp and long may be advantageous to cryoablation if the intracellular ice crystals can pierce into organelles or the cell membrane. For instance, it was recently reported that aqueous dispersions of poly(ethylene glycol)-poly(l-alanine) (PEG-PA) could form round ice crystals while poly(vinyl alcohol) (PVA) could form needle-like crystals 197 . PEG-PA had high cryoprotection while PVA did not.…”

Hypothermic Stem Cell Storage Using a Polypeptide Thermogel

“…Cell recovery was assayed using trypan blue staining after cell plating in a 24-well culture plate. , All live and dead cells were resuspended in DMEM growth media (1 mL). The same number of live cells (5 × 10 4 live cells/well) was added to a 24-well culture plate.…”

“…In particular, DMSO used in the traditional cryopreservation system is toxic, and it damages mitochondria, interferes with intracellular cell signaling, and affects the differentiation of stem cells. Therefore, a very strict procedure for the postremoval of DMSO is required for in vivo as well as in vitro applications. − …”

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

“…In addition, self-assembling polymers have been reported as IRI-active compounds. The control of hydrophobic and hydrophilic balance is a key factor again in the design of these polymers because they need to adhere to the ice crystal surface rather than self-assembling. , Vitrification is another method to avoid ice crystallization, where a high concentration of cryoprotectants and a fast cooling procedure inhibit ice crystal formation. , …”