Effect of lysed platelet count in platelet concentrates on various growth factor levels after freeze thaw cycles

Platelet concentrate (PC) has been used as substitute to the use of fetal bovine serum (FBS) in cell culture media. However, it’s use as additive in cell culture media showed inconsistent results on cell proliferation, and the inconsistent results may be due to variability of platelet counts or growth factor content. Standard protocols are lacking for the preparation of PC before it is applied as additive in cell culture media. The growth factor content of PC can be released by freeze-thaw cycles, which range from one to three cycles before use for cell culture media. This study aimed to measure base-line platelet counts and growth factor levels and compare to platelet counts and growth factor levels after one, two and three freeze-thaw cycles. In this study, we obtained PC from Indonesian Red Cross. The PCs were aliquoted and stored at -20oC and then subjected to 1 to 3 freeze-thaw cycles. The number of platelet before and after freeze-thaw cycles were measured using Sysmex XN 2000 (Pramita Lab). ELISA method was performed to measure the growth factors’ (TGF-β1, PDGF, EGF, IGF-1, VEGF) levels. Anova one-way, Spearman rank and a linear regression analysis were performed to analyze the data.

The average of platelet counts before and after 1 to 3 freeze-thaw cycle(s) are 582,833 ± 295,764 platelets/μL, 411,611 ± 329,078 platelets/μL, 273,417 ± 351,967 platelets/μL, and 179,167 ± 216,904 platelets/μL respectively. The platelet counts were decreased gradually and no significant difference among lysed platelet counts after 1 to 3 freeze-thaw cycle(s) (p > 0.05). Moreover, there were no correlation between platelet counts and growth factor levels (p > 0.05).

In conclusion, fresh and outdated PC from Indonesian Red Cross can be stored at -20oC followed by 1 to 3 freeze-thaw cycle(s) to release its growth factor contents to be used as FBS substitute in cell culture media.