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 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), vascular endothelial growth factor (VEGF), and others1,6,7. These growth factors play an important role in cell proliferation8, migration9, and differentiation9.
To date, several studies have reported the proliferation-stimulating effect of PRP in different kinds of cells10,11,12,13,14,15,16. One of these cell types is human adipose-derived stem cells (hASCs); hASCs exist in human adipose tissue and can be easily collected in large numbers. The regenerative effect of hASCs further suggests a potential use in clinical applications8. Our previous studies reported that compared to non-activated PRP, activated PRP had a marked proliferative effect on hASCs and human dermal fibroblasts (hDFs)8. In addition, we reported that PRP promotes the proliferation of hDFs through an ERK1/2 signaling pathway17. Recently, we also reported that PRP promotes the proliferation of hASCs through the ERK1/2, JNK and Akt signaling pathways18. In hASCs, PRP plays an important role as a supplement that promotes proliferation. Knowledge about the effect of PRP on hASCs will help the development of large-scale culture methods and enable further studies on the mechanism of proliferation in hASCs.
In this report, we introduce and describe a method for preparing PRP from whole blood using centrifugation. This method uses the double spin method to easily prepare a stable sample of PRP. To assess the biological function of PRP, we measured concentrated platelet counts and the concentration of several proliferation factors. We also confirmed the growth stimulatory effect by using the prepared PRP for culturing hASCs.
Protocol
Representative Results
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,…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Not applicable.
Materials
| 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 |
Riferimenti
- 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. Autologous platelet-rich plasma in chronic wounds. Advances in Skin & Wound. 26 (6), 248 (2013).
- Marx, R. E., et al. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 85 (6), 638-646 (1998).
- Bhanot, S., Alex, J. C. Current applications of platelet gels in facial plastic surgery. Facial Plastic Surgery. 18 (1), 27-33 (2002).
- Man, D., Plosker, H., Winland-Brown, J. E. The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin glue) in cosmetic surgery. Plastic and Reconstructive Surgery. 107 (1), 238 (2001).
- Eppley, B. L., Pietrzak, W. S., Blanton, M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plastic and Reconstructive Surgery. 118 (6), 147-159 (2006).
- Lubkowska, A., Dolegowska, B., Banfi, G. Growth factor content in PRP and their applicability in medicine. Journal of biological regulators and homeostatic agents. 26, 3-22 (2012).
- Kakudo, N., et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plastic and Reconstructive Surgery. 122 (5), 1352-1360 (2008).
- Kakudo, N., Morimoto, N., Kushida, S., Ogawa, T., Kusumoto, K. Platelet-rich plasma releasate promotes angiogenesis in vitro and in vivo. Medical Molecular Morphology. 47 (2), 83-89 (2014).
- Liu, Y., Kalen, A., Risto, O., Wahlstrom, O. Fibroblast proliferation due to exposure to a platelet concentrate in vitro is pH dependent. Wound Repair and Regeneration. 10 (5), 336-340 (2002).
- Lucarelli, E., et al. Platelet-derived growth factors enhance proliferation of human stromal stem cells. Biomaterials. 24 (18), 3095-3100 (2003).
- Kilian, O., et al. Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro. European Journal of Medical Research. 9 (7), 337-344 (2004).
- Kanno, T., Takahashi, T., Tsujisawa, T., Ariyoshi, W., Nishihara, T. Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation. Journal of Oral and Maxillofacial Surgery. 63 (3), 362-369 (2005).
- Gruber, R., et al. Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Platelets. 15 (1), 29-35 (2004).
- Koellensperger, E., von Heimburg, D., Markowicz, M., Pallua, N. Human serum from platelet-poor plasma for the culture of primary human preadipocytes. Stem Cells. 24 (5), 1218-1225 (2006).
- Kocaoemer, A., Kern, S., Kluter, H., Bieback, K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 25 (5), 1270-1278 (2007).
- Hara, T., et al. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway. Journal of Artificial Organs. 19 (4), 372-377 (2016).
- Lai, F., et al. Platelet-rich plasma enhances the proliferation of human adipose stem cells through multiple signaling pathways. Stem Cell Research and Therapy. 9 (1), 107 (2018).
- Vajpayee, N., Graham, S. S., Bem, S. Basic examination of blood and bone marrow. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22, 509-535 (2011).
- Cervelli, V., et al. Use of platelet-rich plasma and hyaluronic acid in the loss of substance with bone exposure. Advances in Skin & Wound. 24 (4), 176-181 (2011).
- Nicoli, F., et al. Severe hidradenitis suppurativa treatment using platelet-rich plasma gel and Hyalomatrix. International Wound Journal. 12 (3), 338-343 (2015).
- Cervelli, V., et al. platelet rich lipotransfert: our experience and current state of art in the combined use of fat and PRP. BioMed Research International. 2013, 434191 (2013).
- Gentile, P., et al. Platelet-Rich Plasma and Micrografts Enriched with Autologous Human Follicle Mesenchymal Stem Cells Improve Hair Re-Growth in Androgenetic Alopecia. Biomolecular Pathway Analysis and Clinical Evaluation. Biomedicines. 7 (2), (2019).
- Gentile, P., Scioli, M. G., Orlandi, A., Cervelli, V. Breast Reconstruction with Enhanced Stromal Vascular Fraction Fat Grafting: What Is the Best Method. Plastic and Reconstructive Surgery. Global Open. 3 (6), 406 (2015).
- Gentile, P., Casella, D., Palma, E., Calabrese, C. Engineered Fat Graft Enhanced with Adipose-Derived Stromal Vascular Fraction Cells for Regenerative Medicine: Clinical, Histological and Instrumental Evaluation in Breast Reconstruction. Journal of Clinical Medicine. 8 (4), (2019).
- Gentile, P., Piccinno, M. S., Calabrese, C. Characteristics and Potentiality of Human Adipose-Derived Stem Cells (hASCs) Obtained from Enzymatic Digestion of Fat Graft. Cells. 8 (3), (2019).
- Scioli, M. G., Bielli, A., Gentile, P., Cervelli, V., Orlandi, A. Combined treatment with platelet-rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose-derived stem cells in three-dimensional collagen scaffolds. Journal of Tissue Engineering and Regenerative Medicine. 11 (8), 2398-2410 (2017).
- Marx, R. E., Garg, A. K. . Dental and craniofacial applications of platelet-rich plasma. , (2005).
- Kakudo, N., Kushida, S., Kusumoto, K. Platelet-rich plasma: the importance of platelet separation and concentration. Plastic and Reconstructive Surgery. 123 (3), 1135-1136 (2009).
- Kakudo, N., Kushida, S., Minakata, T., Suzuki, K., Kusumoto, K. Platelet-rich plasma promotes epithelialization and angiogenesis in a splitthickness skin graft donor site. Medical Molecular Morphology. 44 (4), 233-236 (2011).
- Kushida, S., et al. Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems. Journal of Artificial Organs. 17 (2), 186-192 (2014).
- Morimoto, N., et al. Exploratory clinical trial of combination wound therapy with a gelatin sheet and platelet-rich plasma in patients with chronic skin ulcers: study protocol. British Medical Journal Open. 5 (5), 007733 (2015).
- Kushida, S., Kakudo, N., Morimoto, N., Mori, Y., Kusumoto, K. Utilization of Platelet-Rich Plasma for a Fistula With Subcutaneous Cavity Following Septic Bursitis: A Case Report. Eplasty. 15, 31 (2015).
- Eby, B. W. Platelet-rich plasma: harvesting with a single-spin centrifuge. Journal of Oral Implantology. 28 (6), 297-301 (2002).
- Castillo, T. N., Pouliot, M. A., Kim, H. J., Dragoo, J. L. Comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems. The American Journal of Sports Medicine. 39 (2), 266-271 (2011).
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