The Studies for the Preparation of Platelet Lysate (PL) and the Effects of Platelet Lysate (PL) on the Proliferations for Microbial and the Human derived cells

After it became known that the platelets release growth factors, numerous research on the use of platelet rich plasma (PRP) for the purpose of wound healing and regeneration have been reported from various fields in the medical world. The research on PRP gels and platelet lysates (PL), which are derived from PRP, are continuously being reported. As PRPgels have an antibacterial effect and PL is considered as a possible substitute material for fetal bovine serum (FBS), these are added to the culture medium. Therefore, this study aimed at understanding the factors that effect on PRP production, proliferation of microorganisms, and proliferation of human derived cells. This study investigates the antibacterial effect of PRP gels by using the disc diffusion method on liquid PRP that has been activated by thrombin after freezing-thawing and treating with heparin. This is aimed at providing the conditions of freeze-thaw repetitions, platelet counts, and PL concentration for the production of PL; these conditions can be used instead of FBS when performing cell culture to produce PL by using HaCaT cell lines which are human derived cells. To observe the antibacterial effect, PRP and platelet poor plasma (PPP) from 20 volunteers were separated from the blood treated with CPDA-1 anti-coagulant. The PRP was concentrated to 8 times of the standard platelet count in the first stage and 11 times in the second stage. After performing the freeze-thaw on PRP and PPP, they were activated with thrombin, and heparin was added to prevent the gel formation. The antibacterial effects on S. aureus and P. aeruginosa were verified by using disc diffusion method, direct dropping method, and the experiments were repeated. The platelet concentration (PC) was set to 1 x 109/mL and 2 x 109/mL, and freeze-thaw was repeated 1 to 3 times to provide concentration conditions in PL production. HaCaT cell lines were cultured at 5% and 10% concentrations. The calculation of cell count was measured using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), and that of growth factors was measured using LuminexTM 200 system (Millipore Corporation, Austin, TX, U.S.A.) The inhibition zones of disc diffusion method and direct dropping method for S. aureus and P. aeruginosa were both 0mm. There was no statistically significant difference between the platelet counts according to the frequency of freeze-thaws. The effect of PL concentration and frequency of freeze-thaws on the growth of HaCaT cells was irrelevant to platelet count; 5% PL group significantly had higher cell counts than 1 x 109/mL 10% PL group. As for the growth factors, VEGF, PDGF-AB/BB, PDGF-AA, and EGF had significantly higher counts in the 2 x 109/mL PL group than in 1 x 109/mL PL, but VEGF and PDFG-AA did not show any correlation. In studying the proliferation of microorganisms, the antibacterial effect of PL against S.aureus and P. aeruginosa could not be observed. Also, the results of this study offers the conditions of freeze-thaw frequency, platelet count, and PL concentration for producing PL, which can be used in place of FBS during the cell culturing using HaCaT cell lines. The concentration of VEGF, PDGF-AA, and EGF showed tendencies to increase as platelet count and freeze-thaw frequency increased, but only VEGF and PDGF-AA showed positive correlation between their concentration and HaCaT cell counts. There was no significant difference in HaCaT cell proliferation of 1 x 109/mL PL and 2 x 109/mL PL. The 5% PL group showed higher rates than 10% PL group, and the rate was the highest in PL that underwent freeze-thaw twice. These conditions are considered as the most suitable for HaCaT culture.

Author dc:creator

• 백, 세연

Contributors dc:contributor

• 임, 영애
• 대학원 의학과
• 201024445

• 혈소판
• 혈소판풍부혈장
• 혈소판융해물
• 항균효과
• 냉해동
• 세포배양
• 성장인자
• 세포증식
• platelets
• platelet rich plasma
• platelet lysates
• antibacterial effect
• freeze-thaw
• cell culture
• growth factor
• cell proliferation

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000017309
000000017309

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/10944