Preparation of highly-concentrated autologous platelet-rich plasma for biomedical use

摘要

Cell therapy with platelets is a widely accepted approach for wound healing and tissue regeneration in medicine. However, with most available methods poorly concentrated platelet suspensions (up to 0.3?10sup6/sup?1/μl) or suspensions of mostly inactivated or lost platelets are obtained. In this study, we aimed to develop a simple and effective method for preparing a suspension of native and resting platelets with over 1?10sup6/sup 1/μl. Platelet-rich plasma (PRP) was obtained from fresh blood of healthy donors ( n = 5) collected using different amounts of heparin as the anticoagulant. Samples of PRP were spun down and re-suspended in autologous blood plasma. Count and vitality of platelets in each sample were determined by aggregation study on the Solar AP2110 aggregometer. Platelet shape and cytoplasmic granularity that indicate the nativity of platelets were monitored on the COULTER EPICS XL Flow Cytometer. This study of aggregation of platelets in PRP obtained using various amounts of heparin allowed us to reduce final concentrations to the amount that effectively prevented clotting and did not affect platelet reactivity (5 U/ml). PRP concentrated 5 times with a total concentration of cells of 1?10sup6/sup 1/μl was able to be activated by adenosine diphosphate (ADP) (aggregation rate 54 ± 7%). The amount of cells with altered shape and granularity in concentrated suspension was not higher than 20%. This finding means that the platelets would still be able to release a number of growth factors and other biologically active compounds after stimulation or injection into tissue during cell therapy. The decrease in heparin concentrations also minimizes haemorrhage in the injection site supporting biomedical use of the suspension. A simple and effective method for preparation of highly-concentrated PRP (1.2?106 1/μl) for biomedical use was developed. Aggregometry and flow cytometry proved that obtained platelets were resting and able to be activated. Being autologous, the preparation can be widely used for cell therapy without additional precautions.