Human platelet lysate processing

Buffy coats were obtained from the IdISBa Biobank with approval from the Ethics Committee of the Balearic Islands (CEI-IB) (IB 1995/12 BIO), following ethical approval of the project by the CEI-IB (IB 3656118 PI). From these buffy coats, platelet concentrates were prepared following the conventional procedure used in blood banks with minor modifications. Briefly, six fresh buffy coats containing 25% to 40% residual plasma were pooled without any consideration of blood group, sex, and/or age. When possible, buffy coats with platelet concentration above 200 × 10⁹ platelets/l were used. Buffy coats from donors who had taken non-steroidal anti-inflammatory drugs (NSAIDs) were excluded.

Selected buffy coat bags were washed with 0.9% NaCl and centrifuged at 650 ×g for ten minutes. Then, leucocytes were filtrated to finally obtain a platelet concentrate. Platelet concentration was determined and adjusted at 1,200 to 1,800 × 10⁹ platelets/l. To obtain PL, ten bags of six buffy coats were pooled to obtain a macro-pool, and three freeze/thaw cycles (-80°C/37°C) were performed to lyse more than 80% of platelets. Then, a centrifugation at 5,050 ×g for 20 minutes at room temperature discarded cell debris and supernatant was filtered by 40 µm pore size membrane (GVS Filter Technology, Italy). Then, microbiological control assays were performed by using the BACT/ALERT system (bioMérieux, France) for aerobic and anaerobic growth. Finally, the whole batch was aliquoted in 50 ml tubes and stored at -20°C until use.

Then, to eliminate small cell debris, PL was centrifuged at 1,500 ×g for 15 minutes at 4°C and supernatant was filtered through 0.8 µm pore size membrane (Sartorius, Germany). Afterwards, PL that was to be used for EV isolation by size exclusion chromatography (SEC) was aliquoted in 5 ml aliquots and stored at -20°C until use.