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Patients suffering from acute or sustained thrombocytopenia require platelet transfusions, which are entirely donor‐based and limited by challenges related to storage and fluctuating supply. Developing cell‐culture technologies will enable ex vivo and donor‐independent platelet production. However, critical advancements are needed to improve scalability and increase megakaryocyte (Mk) culture productivity. To address these needs, we evaluated Mk production from mobilized peripheral blood CD34 +  cells cultured on a commercially available gas‐permeable silicone rubber membrane, which provides efficient gas exchange, and investigated the use of fed‐batch media dilution schemes. Starting with a cell‐surface density of 40 × 10 3  CD34 +  cells per cm 2  (G40D), culturing cells on the membrane for the first 5 days and employing media dilutions yielded 39 ± 19 CD41 + CD42b +  Mks per input CD34 +  cell by day 11—a 2.2‐fold increase compared with using standard culture surfaces and full media exchanges. By day 7, G40D conditions generated 1.5‐fold more CD34 +  cells and nearly doubled the numbers of Mk progenitors. The increased number of Mk progenitors coupled with media dilutions, potentially due to the retention of interleukin (IL)‐3, increased Mk production in G40D. Compared with controls, G40D had higher viability, yielded threefold more Mks per milliliter of media used and exhibited lower mean ploidy, but had higher numbers of high‐ploidy Mks. Finally, G40D‐Mks produced proplatelets and platelet‐like‐particles that activate and aggregate upon stimulation. These results highlight distinct improvements in Mk cell‐culture and demonstrate how new technologies and techniques are needed to enable clinically relevant production of Mks for platelet generation and cell‐based therapies.

Enabling Large‐Scale Ex Vivo Production of Megakaryocytes from CD34 + Cells Using Gas‐Permeable Surfaces