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.
Method Article
Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells
In This Article
Summary
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 surgeries4,5. 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....
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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
- 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.
- Recommend donors to drink 500 mL of water 6 h before blood collection.
NOTE: The reference ranges for hemoglobin (Hb), red blood cell (RBC) count, and platelet (PL) concentration in healthy adults are described by....
- Recommend donors to drink 500 mL of water 6 h before blood collection.
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Results
Enriched concentrations of platelet and PDGF-BB in PRP
Concentrations of platelets and PDGF-BB in PRP increased 11.5-fold and 25.9-fold, respectively, as high as those in whole plasma. However, the concentrations of EGF in PRP were not changed and IGF was only 70% of that of whole plasma (Table 1). The experiments were replicated four times by the double-spin method.
Enhanced proliferation of hASCs by PRP stimulation
Cell proliferation.......
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Discussion
After PRP activation, several growth factors, such as PDGF, EGF, IGF, TGF-β, and VEGF1,6,7 "activate" cells and tissues of wounds promoting wound healing20,21. In the case of cosmetic reconstruction surgery, activated cells have been shown to induce healing and improve aesthetics22,23. Alternatively,.......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
Not applicable.
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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 |
References
- Eppley, B. L., Woodell, J. E., Higgins, J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plastic and Reconstructive Surgery. 114 (6), 1502-1508 (2004).
- Salcido, R. S.
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