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

Abstract

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.

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use

“…This, together with the efficient gas exchange and ready availability of abundant nutrients in culture medium, may explain the much more rapid and efficient expansion in G-Rex compared with T-flasks. G-Rex has been used by other groups for the optimal expansion of several types of blood cells, including tumor-infiltrating lymphocytes (TILs) [19,20], antigen-specific T cells [8,12,17,[33][34][35], gd T cells [23], Tregs [21,22], NKs [15], CIKs [25], CAR-Ts [16,24], megakaryocytes [36] and red blood cells [37] (reviewed in [9]). In most of these published cases, medium addition or exchange, as well as culture splitting, was performed with cell counting at various phases of the culture to define optimal dilution and collection times.…”

Beyond the thrombus: Platelet‐inspired nanomedicine approaches in inflammation, immune response, and cancer

“…PEVs are produced as a by‐product of platelet activation. They are natural carriers of proteins, organelles, and nucleic acids and hold tremendous promise as direct cell messengers for novel therapeutics because of their natural targeting to bone marrow stem cells, amenability to large‐scale/commercial manufacture, and potential low immunogenicity 124,125 . In addition, there is compelling evidence that PEVs interact with other cells to alter their fate and functionality.…”

The Blood Circulating Rare Cell Population. What Is It and What Is It Good for?

“…General interest in MKC for therapeutic applications may lay in thrombopoiesis as to replace platelet transfusion therapy [47]. Hereby, a prospective application is the re-infusion of ex vivo/in-vitro generated autologous MKC as to prevent or reduce chemotherapy-induced thrombocytopenia or treatments in any condition of reduced platelet production [46,48,49]. So far, the in-vivo generation consisted of derivations from HSC [50] and fibroblasts [48] thus requiring lengthy, complicated and low efficiency protocols.…”