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.

Comparative cultivation of single cell derived placental mesenchymal stromal cells in vitro / 上海交通大学学报（医学版）

Objective • To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (bFGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods • Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+bFGF, HPL and HPL+bFGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzed by inoculating P4 cells. Results • It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×107) cells/cm2 in FBS+bFGF group and (1.24×107) cells /cm2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+bFGF group were (5.58×106) cells /cm2 and (8.56×106) cells /cm2, respectively. For cell morphology, the P4 PMSCs of FBS+bFGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+bFGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+bFGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion • PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+bFGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+bFGF medium has no advantage on quality and quantity.