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.

A polymethoxyflavone mixture extracted from orange peels, mainly containing nobiletin, 3,3',4',5,6,7,8-heptamethoxyflavone and tangeretin, suppresses melanogenesis through the acidification of cell organelles, including melanosomes.

BACKGROUND: Skin color is determined by melanin contents and its distribution. Melanin is synthesized in melanosomes of melanocytes, catalyzed by tyrosinase, melanogenic enzymes. Regarding the process of melanin synthesis, melanosomal pH is considered to play an important role, because it has been reported to differ between Caucasian and Black melanocytes. OBJECTIVE: Although polymethoxyflavone (PMF) has many beneficial effects, it has not been reported which PMF suppresses melanogenesis. In this study, we identified the mechanism underlying the effect of PMF on melanogenesis METHODS: We determined the effects of a PMF mixture extracted from orange peels on melanogenesis, on tyrosinase expression, on the localization of tyrosinase and on the acidification of organelles, including melanosomes, in HM3KO human melanoma cells. RESULTS TREATMENT: with the PMF mixture elicited the suppression of melanogenesis, the degradation of tyrosinase in lysosomes and the mislocalization of tyrosinase associated with the acidification of intracellular organelles, including melanosomes. The neutralization of cell organelle pH by ammonium chloride restored melanogenesis and the correct localization of tyrosinase to melanosomes, which had been suppressed by the PMF mixture. CONCLUSION: These results suggest that the PMF mixture suppresses the localization of tyrosinase to melanosomes and consequently inhibits melanogenesis due to the acidification of cell organelles, including melanosomes.

A polymethoxyflavone mixture, extracted from orange peels, suppresses the UVB-induced expression of MMP-1.

Ultraviolet (UV) B is the main cause of skin photoageing, which has characteristic features such as deep wrinkles. UVB increases the expression of matrix metalloproteinases (MMPs) in the skin and can cause wrinkles by disrupting components of the extracellular matrix, such as collagen fibres. We now report that a polymethoxyflavone (PMF) mixture, extracted from orange peels, suppresses the UVB-induced expression of MMP-1 that involves the inhibition of c-jun N-terminal kinase (JNK) activity. Furthermore, the PMF mixture also inhibits the UVB-induced phosphorylation of JNK. Therefore, the results suggest that the PMF mixture suppresses the UVB-induced expression of MMP-1 through the inhibition of JNK phosphorylation and should be useful as an antiphotoageing agent.

Orange peel extract, containing high levels of polymethoxyflavonoid, suppressed UVB-induced COX-2 expression and PGE2 production in HaCaT cells through PPAR-γ activation.

Ultraviolet light (UV) induces an inflammatory response in the skin by cyclooxygenase (COX)-2 expression and prostaglandin (PG) E2 production. Citrus peel has been used as a natural medicine. It contains polymethoxyflavonoids (PMFs) as a major ingredient, which have anti-inflammatory activity. We obtained orange peel extract containing high levels of PMFs. The extract suppressed UVB-induced COX-2 expression and PGE2 production in HaCaT cells. Furthermore, it was found that this extract acted as a peroxisome proliferator-activated receptor (PPAR)-γ agonist. The suppression of UVB-induced COX-2 expression by this extract was inhibited by GW 9662 and T0070907, which are both PPAR-γ antagonists. It is therefore suggested that orange peel extract, containing high levels of PMFs, suppresses UVB-induced COX-2 expression and PGE2 production through PPAR-γ. Hence, these extracts could provide useful protection against or alleviation of UV damage.