Recently, we proposed a Good Manufacturing Practice (GMP)‐compliant production process for freeze‐dried mesenchymal stem cell (MSC)‐secretome (lyo‐secretome): after serum starva-tion, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze‐dried, obtaining a standardized ready‐to‐use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo‐secre-tome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL‐E) and the new one (HPL‐S). From the same pool of platelets, two batches of HPL were ob-tained: HPL‐E (by repeated freezing and thawing cycles) and HPL‐S (by adding Ca‐gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti‐elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distri-bution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.
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