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

Resumo

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.

Resumo

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.

Introdução

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), vascular endothelial growth factor (VEGF), and others^1,6,7. 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 cells^{10,11,12,13,14,15,16}. 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.

Protocolo

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.
      NOTE: The reference ranges for hemoglobin (Hb), red blood cell (RBC) count, and platelet (PL) concentration in healthy adults are described by Vajpayee¹⁹. In order to be suitable for blood donation, the donor’s blood analysis is required to be within these ranges.

2. Blood collection

1. Have the blood donors sit during the procedure.
2. Wear gloves. Fasten a tourniquet on the donor’s upper arm while the donor makes a fist.
3. Once a suitable vein is found, sterilize the skin twice with 70% alcohol and insert the needle.
4. Use a 21 G syringe to collect blood. Do not loosen the tourniquet during the blood draw.
   1. Use an 8.5 mL blood collection tube to collect the blood. Observe the state of the blood in the tube and change to a new tube as soon as the blood flow slows down.
   2. Collect a sufficient amount of blood to make the PRP. Collect four tubes of anticoagulated blood with a total volume of 34 mL. Invert the tubes 10 times to mix the blood with the anticoagulant.
   3. Use a 10 mL serum blood collection tube (see Table of Materials) without anticoagulant to collect blood for making the activator (see Step 4). Collect 10 mL of non-anticoagulant blood.

3. Double-spin method to make PRP

1. Take 40 μL of anticoagulated whole blood for the platelet count. Use filtered pipette tips to protect the pipette from blood contamination.
2. Centrifuge the anticoagulated whole blood for 7 min at 450 x g and room temperature. This is the first spin to obtain layered blood samples. The upper layer is the plasma fraction, the middle layer is the thin buffy boat, and the bottom-most layer consists of red blood cells.
3. Use a marker to make a line at 2 mm below the buffy coat.
4. Use a 20 mL syringe with a long cannula to collect the plasma just to the 2 mm mark below the buffy coat. The yellow plasma with buffy coat contains platelets, leukocytes, and some erythrocytes. Collect the plasma from two tubes into a serum blood collection tube. Combine the contents of 4 tubes into 2 tubes for a total volume of 16 mL of plasma collected.
5. Centrifuge the plasma with the buffy coat for 5 min at 1,600 x g and room temperature. This is the second spin. The supernatant of the layered blood samples is platelet-poor plasma (PPP).
6. Use a 20 mL syringe with a long cannula to transfer the PPP to a 50 mL serum blood collection tube containing 1 mL of liquid as a measure. Platelets that accumulated in the thrombocyte pellet in the remaining 1 mL plasma were used as the PRP. The total volume of PRP collected was 2 mL.
7. Vortex the PRP in each tube and then pool into one tube (total 2 mL).
8. Take 400 µL of the PRP for a platelet count.
9. Aliquot the PRP into several 1.5 mL tubes, containing approximately 400 µL in each tube. There is a total of four tubes. Approximately 500 µL PRP per tube is recommended.

4. Prepare the activator

1. Allow the 10 mL of blood without anticoagulant in a serum blood collection tube (step 2.3.3) to sit for 30 min at room temperature.
2. Centrifuge the blood sample without anticoagulant for 8 min at 2,015 x g and room temperature in a laboratory centrifuge.
3. Collect the supernatant as autologous thrombin.
4. Mix 0.5 M CaCl₂ and autologous thrombin in a 1:1 (v/v) ratio as an activator. For example, 500 µL of CaCl₂ was mixed with 500 µL of autologous thrombin. Make a total volume of 1 mL of the activator.

5. PRP activation

1. Mix PRP and the activator in a 10:1 (v/v) ratio. Mix 400 µL of PRP with 40 µL of the activator in each 1.5 mL tube, and then incubate for 10 min at room temperature. This is activated PRP in a coagulated form.

6. Storage of PRP

1. Centrifuge activated PRP at 9,000 x g for 10 min in a 4 °C laboratory centrifuge.
2. Collect the supernatant via pipette into a proper volume syringe.
3. Filter the supernatant through a 0.22 µm membrane.
4. Store the final PRP at −80 °C until use.

7. Measurement of platelet concentrations and growth factor levels

1. Count the platelet number in whole plasma and PRP using an automated hematology system (see Table of Materials).
2. Analyze concentrations of PDGF-BB, IGF, and EGF levels in whole plasma and activated PRP using commercially available ELISA kits (see Table of Materials).

8. Cell proliferation assay

1. Isolate hASCs using a previously described method¹⁸.
   1. Seed hASCs at a density of 1.0 x 10⁴ cells/well in 24-well culture plates and incubate in DMEM containing 10% FBS and antibiotics overnight at 37 °C. Use hASCs from passages 7-9 in experiments.
2. Replace cell media with serum-free DMEM. After 6 h, add PRP at the designated concentrations and incubate further for 48 h at 37 °C.
3. Incubate with WST-8 solution (see Table of Materials) for 1 h at 37 °C. Read absorbance at 450 nm with a multi-well plate reader.
4. Construct a standard curve: Seed hASCs at densities of 0, 6,250, 12,500, 25,000, 50,000, and 100,000 cells/well in 24-well plates in DMEM with 10% FBS for 3 h. Read the absorbance at 450 nm after incubation with WST-8 solution for 1 h at 37 °C
   1. Draw the standard curve by plotting the number of cells versus the A_{450 nm}.
5. Estimate the number of hASCs from the absorbance based on the standard curve.

Resultados

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 prolifera...

Discussão

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,...

Materiais

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