Introduction Platelet-rich plasma (PRP) and platelet lysate (PL) are biologically active products used in regenerative medicine because of their high concentrations of growth factors, cytokines, and other bioactive molecules that promote tissue repair and modulate inflammation. Freeze–thaw cycles are commonly used to activate PRP and induce growth factor release. While the optimal number of freeze–thaw cycles has been evaluated in human PRP, this parameter has not been investigated in canine PRP products. The objective of this study was to evaluate platelet counts and concentrations of platelet-derived growth factor (PDGF), transforming growth factor–beta (TGF- β ), and tumor necrosis factor–alpha (TNF- α ) in a commercially available, pooled, freeze-dried canine PRP product subjected to multiple freeze–thaw cycles (1, 3, 4, 5, and 10). We hypothesized that (1) a significant proportion of platelets would be lysed by the fourth freeze–thaw cycle and (2) growth factor concentrations would decrease, whereas TNF- α concentrations would increase by the 10th freeze–thaw cycle. Methods Six vials of a commercially available canine PRP product from three separate manufacturing lots were evaluated. Each vial was reconstituted according to the manufacturer’s instructions and stored at −80 °C for 12–24 h. Samples were thawed in a dry bath at 37 °C. Additional freeze–thaw cycles were performed using liquid nitrogen, with thawing at 37 °C for samples undergoing 3, 4, 5, and 10 cycles. A complete blood count was performed before freezing and after each freeze–thaw cycle. Concentrations of PDGF, TGF- β , and TNF- α were quantified using enzyme-linked immunosorbent assays (ELISAs). Results Initial platelet and white blood cell counts were consistent with manufacturer-reported values. Platelet counts were significantly reduced following 4, 5, and 10 freeze–thaw cycles compared with initial PRP values. Concentrations of PDGF, TGF- β , and TNF- α were largely conserved across freeze–thaw cycles, although significant inter-sample variability was observed. Discussion Four to five freeze–thaw cycles effectively lysed platelets while preserving PDGF and TGF- β concentrations for at least 10 cycles. However, significant variability in growth factor and cytokine content was evident, even in a commercially available product. Further investigation is warranted to better define the effects of freeze–thaw cycles on activating PRP intended for clinical therapeutic use.
Sumner et al. (Wed,) studied this question.