In vitro human cord blood platelet lysate characterisation with potential application in wound healing

Abstract

Platelets contain abundant growth factors and cytokines that have a positive influence on the migration and proliferation of different cell types by modulating its physiopathological processes. As it is known that human umbilical cord blood platelet lysate (UCB-PL) contains a supraphysiological concentration of growth factors, in the present study, we investigated its effectiveness in wound-healing processes. Human UCB-PL was obtained by the freeze/thaw of platelet concentrate (1.1 × 10⁹ platelets/L), and its effect was evaluated on human or mouse endothelial cells, monocytes, fibroblasts, and keratinocytes in different concentrations. Human UCB-PL was observed to have high levels of pro-angiogenic growth factor than peripheral blood platelet-rich plasma. Among the cell lines, different concentrations of human UCB-PL were necessary to influence their viability and proliferation. For L929 cells, 5% of total volume was necessary, while for human umbilical vein endothelial cell, it was 10%. Cell migration on monocytes was increased with respect to the positive control, and scratch closure on keratinocytes was increased with respect to serum-free medium with only 10% of human UCB-PL. We concluded that the human UCB-PL may be useful to produce a large amount of standard platelet concentrates sufficient for several clinical-scale expansions avoiding inter-individual variability, which can also be used as a functional tool for clinical regenerative application for wound healing.

Keywords: human umbilical cord blood; in vitro characterisation; platelet lysate; wound healing.

Figure 1

Umbilical cord blood platelet lysate (UCB‐PL) effect on viability and proliferation of L929 cells and human umbilical vein endothelial cells (HUVECs). Viability (A and C) and proliferation (B and D) were assessed by MTT assay and BrdU incorporation assay, respectively, following 72 and 48 hours of incubation with different UCB‐PL concentrations (1%, 3%, 5%, 10%, and 20%). The percentage of cell viability and proliferation were calculated vs the complete medium (assumed as 100%). Data are means ± SD of values obtained from three independent experiments with four replicates each. *p < .05 vs both serum‐free medium and PL 10%; ^Δ p < .05 vs both serum‐free medium and PL 10%; ^§ p < .05 vs serum‐free medium, PL 1% and PL 20%; °p < .05% vs serum‐free medium, PL 1%, PL 20%

Figure 2

The chemotactic effect of umbilical cord blood platelet lysate (PL) on THP‐1 cell line was assessed following 2 hours of cell migration towards different PL concentrations (5%, 10%, and 20%). Positive and negative controls are represented by complete culture medium and serum‐free medium, respectively. Data (n = 3) are means ± SD of four replicates. ^§ p < .05 vs both serum‐free medium; *p < .05 vs serum‐free medium, 1% PL, 5% PL, and complete culture medium

Figure 3

Phase‐contrast micrographs of scratch test; effect of different concentrations of UCB‐PL on HaCat cells migration. The scratch wounds closure was evaluated after 10, 20, and 30 hours of exposure to culture medium added to 10% of FBS and 1%, 5%, and 10% of UCB‐PL. All data are representative of three independent experiments with n = 3 by group and are means ± SD