Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Protein-Stabilizing Effect of Amphiphilic Block Copolymers with a Tertiary Sulfonium-Containing Zwitterionic Segment.

Tertiary sulfonium-containing zwitterionic block copolymers consisting of N-acryloyl-l-methionine methyl sulfonium salt (A-Met(S+)-OH) and n-butyl acrylate (BA) were newly synthesized to develop a novel protein stabilizer. The zwitterionic block copolymers were prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization of BA using a hydrophilic macro-chain-transfer agent (CTA) obtained from N-acryloyl-l-methionine (A-Met-OH) and subsequent postmodification. RAFT polymerization of A-Met-OH using poly(BA) macro-CTA, followed by postmodification, also afforded the target poly(A-Met(S+)-OH)-b-poly(BA). The block copolymers stabilized horseradish peroxidase (HRP) during storage at 37 °C for 5 days, and the protein-stabilizing effect was enhanced with increase in the A-Met(S+)-OH content. In particular, the block copolymer with ∼85% A-Met(S+)-OH content showed a significant protein-stabilizing effect at a temperature (37 °C) higher than the room temperature, which is highly desirable for practical and industrial applications. The addition of sucrose into the block copolymer-protein solution led to a considerable increase in the HRP activity under the same conditions. Excellent alkaline phosphatase stabilization at 37 °C for 12 days was also achieved using the block copolymers. The zwitterionic block copolymers with the optimal hydrophilic/hydrophobic balance were found to serve as efficient protein-stabilizing agents, in comparison with the corresponding homopolymer and random copolymers. Dynamic light scattering, zeta potential, transmission electron microscopy, and circular dichroism measurements revealed that the zwitterionic block copolymer stabilizes an enzyme by wrapping with a slight change in the size, whereas the secondary and ordered structures of the enzyme are maintained.

Synthesis of Zwitterionic Polymers Containing a Tertiary Sulfonium Group for Protein Stabilization.

The present study demonstrates the controlled synthesis and biological potential of poly( N-acryloyl-l-methionine methyl sulfonium salt)s (poly(A-Met(S+)-OH)s), which mimic dimethylsulfoniopropionate (DMSP), a compound produced by marine algae to protect their proteins. The novel sulfonium-containing zwitterionic polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of the amino acid-based monomer N-acryloyl-l-methionine (A-Met-OH) followed by a postmodification process in which the sulfide groups were reacted with iodomethane. The DMSP-mimic zwitterionic macromolecules were shown for the first time to exhibit low cytotoxicity and the ability to stabilize proteins. By adding the resulting poly(A-Met(S+)-OH)s to horse radish peroxidase (HRP) solution, the activity of HRP was maintained even after storage at 4 °C for several days. In addition, the protein activities were tested using peroxidase-labeled antibody to mouse immunoglobulin G (IgG-HRP) and alkaline phosphatase (ALP) after storage and for HRP after freeze-thaw cycles. Amphiphilic random copolymers, poly(A-Met(S+)-OH- co-BA)s, also exhibited excellent properties for protein stabilization.