Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

Antifungal effects of synthetic human β-defensin 3-C15 peptide

OBJECTIVES: The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. MATERIALS AND METHODS: C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. RESULTS: C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05). CONCLUSIONS: Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

Importance of determining viability of Mycobacterium leprae inside macrophages—an in vitro method using uracil.

It has been demonstrated that Mycobacterium leprae, are capable of taking up uracil and incorporating it into trichloroacetic acid-insoluble materials, both as free suspension of bacteria, as well as when they are inside the macrophages, a host cell for their in vivo survival. Same amount of bacteria show better incorporation inside macrophages than as free bacterial suspension. Both types of incorporation are inhibited by rifampicin an antileprosy drug and an RNA synthesis inhibitor. Thus uracil uptake by Mycobacterium leprae inside macrophages has been used for standardising a rapid in vitro viability assay for the leprosy causing bacteria.