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Background: Platelets play a key role in the treatment of thrombocytopenia. Nowadays, platelets (PLTs) are only obtained through blood donation. However, due to the limitations in their preparation and storage, they are produced in laboratories, especially through bioreactors that convert megakaryocytes from stem cells into large-scale injectable PLTs. Materials and Methods: In this study, the CD34 cells isolated from cord blood were differentiated into megakaryocytes. A 6-chamber bioreactor with a two-layer collagen scaffold, several ECM factors, and human cryoprecipitate were used to simulate the structure of the bone marrow. After the addition of megakaryocytes to the scaffold, PLTs were produced due to the flow pressure and the interaction between the scaffold structure and the ECM factors. Results: CD41 + cells were expanded 100 times as much as CD34 + cord blood stem cells. The mean PLT release from one megakaryocyte in the pure collagen scaffold was 17.42 PLTs. Once fibrin, fibronectin, hyaluronic acid, and cryoprecipitates were added to collagen, the mean PLT release was 21.4, 22.4, 23.9, and 27.37, respectively. With the simultaneous addition of three factors to collagen (CFFH) and then four factors (CFFHC), the number of PLTs reached 30.52 and then 34. Conclusion: Functional PLTs can be produced on a large scale with a multi-chamber bioreactor using a combination of ECM and cryoprecipitate with collagen scaffolding. [ABSTRACT FROM AUTHOR]