Ann Lab Med.  2014 Jan;34(1):43-50. 10.3343/alm.2014.34.1.43.

Effects of Platelet Lysate Preparations on the Proliferation of HaCaT Cells

• Baik SY
• Lim YA
• Kang SJ
• Ahn SH
• Lee WG
• Kim CH

• KMID: 1781359
• DOI: http://doi.org/10.3343/alm.2014.34.1.43

Abstract

BACKGROUND
Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures.
METHODS
Cell density in three pooled platelet concentrates (PC) were adjusted to 1x10(12)/L and 2x10(12)/L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA).
RESULTS
Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2x10(12)/L than 1x10(12)/L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts.
CONCLUSIONS
The 5% PL from PC with a cell density of 1x10(12)/L prepared by two FT cycles treatment was the most effective condition that supported steady HaCaT cell proliferation. Our finding may be useful for preparing PL-supplemented cell culture media.

Keyword

Platelet lysate; Cell culture; Freeze-thaw; Growth factor; Cell proliferation

MeSH Terms

Blood Platelets/chemistry/*metabolism
Cell Line
Cell Proliferation/drug effects
Culture Media/pharmacology
Epidermal Growth Factor/chemistry/pharmacology
Humans
Platelet-Derived Growth Factor/chemistry/pharmacology
Vascular Endothelial Growth Factor A/chemistry/pharmacology
Culture Media
Epidermal Growth Factor
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Standard curves of Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) for HaCaT cell proliferation according to the frequency of passage in the 5% PL and 5% FBS groups. Optical density (OD) means the amount of formazan dye generated by the activity of dehydrogenases in living cells.Abbreviation: FBS, fetal bovine serum.

• 

  Fig. 2 HaCaT cell morphology according to the frequency of passage. There were no obvious morphological differences between HaCaT cells cultured in 5% fetal bovine serum (FBS) and 5% platelet lysate (PL) or between cells from different passages; (A) The first passage of 5% FBS. (B) The first passage of 5% PL with 1×109/mL. (C) The third passage of 5% FBS. (D) The third passage of 5% PL with 1×109/mL.

• 

  Fig. 3 Pearson's correlations between HaCaT cell count and vascular endothelial growth factor (VEGF) or platelet-derive growth factor-AA (PDGF-AA).

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