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Objective: To investigate how delayed blood centrifugation affects the composition of the resultant platelet rich fibrin membrane (PRF, a concentrated growth factor preparation) and its biological effects towards gingival fibroblasts., Materials and Methods: Blood samples were collected from 18 healthy individuals and centrifuged immediately (T-0), or after a 1-6-minute delay (T-1-6, respectively), to generate PRF. Each PRF membrane was weighed. T-0 and T-6 membranes were incubated for 48 h in cell culture medium at 37 °C to create PRF "releasates" (soluble factors released from the PRF). Human gingival fibroblasts were incubated for 48 h with or without the releasates, followed by RNA isolation and real-time polymerase chain reaction to measure expression of select genes associated with granulation tissue formation, angiogenesis and wound contraction. Additional T-0 and T-6 membranes were used for visualization of leucocyte nuclei and platelets by immunostaining., Results: Immediate centrifugation (T-0) resulted in the largest membranes, T-6 membranes being on average 29% smaller. Leucocytes and platelets were significantly more abundant in T-0 than in T-6 samples. Majority of the fibroblast genes studied were consistently either upregulated or downregulated by the T-0 PRF releasates. However, centrifugation after a 6-minute delay significantly weakened the fibroblast responses., Conclusions: Delayed centrifugation resulted in smaller PRF membranes with fewer leucocytes and platelets and also significantly reduced on the expression of a set of healing-related gingival fibroblast genes., Clinical Relevance: The higher expression of wound healing-related genes in gingival fibroblasts by the immediately-centrifuged PRF membranes may increase their biological properties in clinical use., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)