The effect of short-term cryopreservation on the properties and functionality of platelet-rich plasma

Figures & data

Figure 1. Schematic layout of the methodological protocol. PRP was obtained by centrifugation at 580 x g for 8 minutes. The lower half of the plasma layer was transferred to a new tube to obtain PRP. One part was directly preserved at either −20ºC or −80ºC for 1 or 3 months. The other part was first activated by CaCl2 addition and once the clot was formed, the supernatant, also called platelet lysate (PL), was preserved under the same conditions. Finally, frozen PRP and PL were characterized and in vitro evaluated using fresh samples as a control.

Table I. Summary of PRP characteristics.

1. PRP Preparation
Initial blood volume	18 mL
Anticoagulant	Sodium citrate 3.8% (wt/V)
System	Close
Centrifugation	Yes
number	1
speed	580 x g − 8 min
Final PRP volume	4 mL per subject
2. PRP Characteristics
PRP Type	14-00-11 *
MPV	9.6 ± 0.6 fL
Red Blood Cells	<0.01 x 106/µL
White Blood Cells	<0.05 x 106/µL
Neutrophils	—
Lymphocytes	—
Monocytes	—
Eosinophils	—
Basophils	—
Activation	CaCl2 (10% wt/vol)
3. Application Characteristics
Dose	10%
Direct/Indirect	Direct
Cell line	Normal Human Dermal Fibroblast
4. Other remarkable PRP and study features
The product added to the cell cultures was the platelet lysate obtained after activation of PRP with calcium chloride (10%)

*Kon et al.¹⁶

Figure 2. Platelet concentration, size and activation after freezing preservation. Mean values of platelet concentration (A), size (B) and activation (C) in the PRP and PL according to different storage temperatures (−20/−80ºC) and time (Fresh, 1 month, 3 months). The platelet activation graph represents the CD62 and CD41 positive cells, relative to the fresh control. Error bars = standard deviation.

Figure 3. Platelet growth factors levels. Mean values of platelet growth factors concentration (A/D = PDGF, B/E = TGFB, C/F = VEGF) in the PRP according to activation time (A/B/C = before freezing, D/E/F = after thawing), storage temperatures (−20/–80ºC) and storage time (Fresh, 1 month, 3 months). Error bars = standard deviation. * p < .05; **** p < .0001.

Figure 4. Extra-platelet growth factors levels. Mean values of extra-platelet growth factors concentration (A / C = IGF-1, B / D = HGF) in the PRP according to activation time (A / B = before freezing, C / D = after thawing), storage temperatures (−20/−80ºC) and storage time (Fresh, 1 month, 3 months). Error bars = standard deviation. * p < .05; **** p < .0001.

Figure 5. Growth curves of dermal fibroblasts. Normal human dermal fibroblasts were cultured with a medium supplemented with either fresh or frozen platelet lysate in triplicate, and cell viability was measured every 24 hours. A = platelet lysate obtained before freezing; B = platelet lysate obtained after thawing. Error bars = standard deviation.

Figure 6. Fibrinogen levels and clot properties of the PRP after freezing. Mean values of fibrinogen levels (A), Young’s modulus (B) and dissipated energy (C) of the fibrin clot according to different storage temperatures (−20/−80ºC) and time (Fresh, 1 month, 3 months). Error bars = standard deviation.

Kon E, Di Matteo B, Delgado D, Cole BJ, Dorotei A, Dragoo JL, Filardo G, Fortier LA, Giuffrida A, Jo CH, et al. Platelet-rich plasma for the treatment of knee osteoarthritis: an expert opinion and proposal for a novel classification and coding system. Expert Opin Biol Ther. 2020;20(12):1–14. doi: 10.1080/14712598.2020.1798925.

 PubMed Web of Science ®Google Scholar