The increasing demand for platelet (PLT) transfusions requires a safe alternative to blood and PLT donation. In vitro production of platelets is currently explored, but achieving a commercially viable solution is hindered by the limitations of large-scale production and costs. This paper introduces a scalable solution that efficiently produces one platelet concentrate (PC) in under two hours, starting from mature megakaryocytes (MK).

Human CD34+ cells were seeded in a xeno-free medium containing thrombopoietin to differentiate into MK, and transferred into HemostOD’s modified rotating bed reactor – consisting of a pod containing microstructures – in a 200 mL bioreactor, for 2 hours at 900 RPM. Control PLTs were both from healthy donors (blood-PLT) or generated using a microfluidic chip (CHIP-PLT). PLT activation was done using ADP + PAR1 agonist peptide and analyzed by flow cytometry (FC). The pharmacokinetic study was done in immunodeficient clodronate-treated mice. Desialylation was measured using R. communis agglutinin. PLT (CD41+CD42b+) were counted by FC.

PLT generated in HemostOD’s bioreactor (HOD-PLT) were compared to CHIP-PLT and to blood-PLT. At resting state, 15.2+/-4.1% of CHIP-PLT were CD62P positive, while only 6.6+/-4.0% of HOD-PLTs were positive (mean+/-SD, n=3, P=0.057, 2-way ANOVA with Tukey multiple-comparisons test). Phosphatidylserine exposure was significantly reduced in HOD-PLTs (5.1+/-1.1%) compared to CHIP-PLTs (16.5+/-1.3%, P=0.01). Upon activation, PAC-1 binding and release of CD62P were increased in HOD-PLTs compared to CHIP-PLT (53.5+/-1.0% and 46.0+/-6.6% versus 24.0+/-4.1% and 32.0+/-1.1%, P<0.01). Sialylation (resting) and markers after activation of HOD-PLTs were similar to blood-PLT. After infusion in mice, survival of HOD-PLTs and blood-PLTs were also similar (half-life of 22+/-2 h and 17+/-2.0 h, respectively). Finally, 5,5*10^10 PLTs were produced using 4,5*10^9 MKs, reaching systematically a plateau between 90 and 120 minutes.

Functional platelets are generated in HemostOD’s bioreactor in 2 hours, sharing characteristics with blood-PLTs. HemostOD’s bioreactor in conjunction with HemostOD's proprietary human MK cell line thus has the potential to enable large-scale production of platelets for clinical use with an unlimited and stable source of megakaryocytes.