Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Summary

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Abstract

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

Introduction

Activated platelet-rich plasma (PRP) is prepared by centrifugation of whole blood and is shown to contain platelets well above baseline levels¹. Autologous RPP has been widely used in surgical treatment, including wound healing², bone injury³, and aesthetic surgeries^4,5. After activation of platelets in PRP, the α-granules present in platelets release several growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factors (IGFs), transforming growth factor beta (TGF-β), va....

Protocol

The study was approved by the Ethics Review Board of Kansai Medical University in accordance with the ethical guidelines of the Helsinki Declaration of 1975. All specimens were collected and used with informed consent from the donors.

1. Preparation

1. After obtaining informed consent, take blood from healthy adult donors. Here, blood was collected from four male blood donors between the ages of 28-38.
   1. Recommend donors to drink 500 mL of water 6 h before blood collection.<.......

Discussion

After PRP activation, several growth factors, such as PDGF, EGF, IGF, TGF-β, and VEGF^1,6,7 "activate" cells and tissues of wounds promoting wound healing^20,21. In the case of cosmetic reconstruction surgery, activated cells have been shown to induce healing and improve aesthetics^22,23. Alternatively,.......

Materials

Name	Company	Catalog Number	Comments
20 mL Syringe, Terumo syringe lock type	Terumo, Tokyo, Japan.	SS-20LZP
50 mL Tube, Polypropylene Conical Tube	Corning, NY, USA.	352070
Automated Hematology System	Sysmex Corp., Tokyo, Japan	XE-2100
Blood Collection Needle, SafeTouch PSV set with luer adapter, 21 G x 3/4”	Nipro, Osaka, Japan.	32-384
Blood Collection Tube, ACD Solution A Blood Collection Tube, 8.5 mL	BD Vacutainer, NJ, USA.	364606
Blood Collection Tube, Serum Blood Collection Tube/monovette, 10 mL	BD Vacutainer, NJ, USA.	366430
Calcium Chloride, 1 mEq/ mL,	Otsuka Pharmaceutical Factory, Tokushima, Japan.	3215400A1061
Cannula, BS non-bevel needle, 18 G (1.2 mm) x 75 mm	BS Medical, Tokyo, Japan.	BS-81007	Not for sale
Cell Counting Kit-8	Dojindo Molecular Technologies, Kumamoto, Japan	CK04
Centrifuge	Kokusan, Tokyo, Japan.	H-19F
Centrifuge	Eppendorf, Hamburg, Germany.	5415R
EnSpire 2300 Multilabel Reader	PerkinElmer, Inc., Waltham, MA, USA
Filter Unit	Merck Millipore, Co. Cork, Ireland.	SLGP033RS
Human EGF Quantikine ELISA Kit	R&D Systems, Minneapolis, MN, USA	DEG00
Human IGF-I Quantikine ELISA Kit	R&D Systems, Minneapolis, MN, USA	DG100
Human PDGF-BB Quantikine ELISA Kit	R&D Systems, Minneapolis, MN, USA	DBB00
Pipette Tip, ART 1000 Reach Barrier Tip	Thermo Scientific, MA, USA.	2079-05-HR
Pipette, Nichipet EXII 100-1000 μL	Nichiryo, Saitama, Japan.	00-NPX2-1000
Sterling Nitrile Power-Free Exam Gloves	Kimberly-Clark	50707
Yamazen Alcohol for Disinfection	Yamazen Pharmaceutical, Osaka, Japan.	A7L07