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.

In Situ Synthesis of an Anticancer Peptide Amphiphile Using Tyrosine Kinase Overexpressed in Cancer Cells.

Cell-selective killing using molecular self-assemblies is an emerging concept for cancer therapy. Reported molecular self-assemblies are triggered by hydrolysis of well-designed molecules inside or outside cancer cells. This hydrolysis can occur in cancer and normal cells because of the abundance of water in living systems. Here, we report the in situ synthesis of a self-assembling molecule using a tyrosine kinase overexpressed in cancer cells. We designed a tyrosine-containing peptide amphiphile (C16-E4Y) that is transformed into a phosphorylated peptide amphiphile (C16-E4pY) by the overexpressed tyrosine kinase. Phosphorylation of C16-E4Y promoted self-assembly to form nanofibers in cancer cells. C16-E4Y exhibited selective cytotoxicity toward cancer cells overexpressing the tyrosine kinase. Self-assembled C16-E4pY induced endoplasmic reticulum stress that caused apoptotic cell death. Animal experiments revealed that C16-E4Y has antitumor activity. These results show that an enzyme overexpressed in cancer cells is available for intracellular synthesis of an antitumor self-assembling drug that is cell-selective.

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring.

We investigated d-amino acids as potential inhibitors targeting l-peptide toxins. Among the l- and d-amino acids tested, we found that d-tryptophan (d-Trp) acted as an inhibitor of melittin-induced hemolysis. We then evaluated various Trp derivatives and found that 5-chlorotryptamine (5CT) had the largest inhibitory effect on melittin. The indole ring, amino group, and steric hindrance of an inhibitor played important roles in the inhibition of melittin activity. Despite the small size and simple molecular structure of 5CT, its IC50 was approximately 13 µg/mL. Fluorescence quenching, circular dichroism measurements, and size-exclusion chromatography revealed that 5CT interacted with Trp19 in melittin and affected the formation of the melittin tetramer involved in hemolysis. Molecular dynamics simulation of melittin also indicated that the interaction of 5CT with Trp19 in melittin affected the formation of the tetramer.