A novel culture flask for clinostat-based simulation of extraterrestrial gravities

Perra, Giovanni

Co-prime

;Fais, Giacomo

Co-prime

;Dessì, Debora;Concas, Alessandro;Follesa, Paolo;Cao, Giacomo;Lai, Nicola

2026-01-01

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Abstract

Introduction: Conventional T-flasks (T-25) filled to capacity are frequently employed to minimize shear stress arising from fluid motion during ground-based microgravity simulations using a clinostat or random positioning machine (RPM). However, this approach can introduce confounding factors, such as hypoxia and CO2 accumulation that affect cell metabolism and function. Therefore, in vitro platform simulating microgravity is crucial to distinguish true gravity-dependent responses from culture artifacts. Here, we proposed an innovative engineered culture system (F-25) with a growth area of 25 cm2 primarily designed for full-filled and clinostat experiments. Methods: We assessed the effects of static and rotational (i.e., microgravity) full-filled cultures including conventional T-25 and eighteen customized F-25 with different medium depths, gas exchange areas and membrane types on mitochondrial function of intact C2C12 myoblasts by high resolution respirometry. Results: After 24 h, conventional T-25 flasks, full-filled to height of H0 (2.25 cm) and with a hydrophobic-type gas exchange area of A0 (0.2 cm2) showed intact cellular respiration (ICR) and maximal uncoupled respiration (ET) rates that were more than twice those measured in partially-filled controls, whose values (40 ± 3 for ICR and 60 ± 4 for ET pmol O2 s−1 10−6 cells−1) remained unchanged between time zero and 24 h. At each medium depth (1/3H0, 2/3H0, and H0) increasing the gas exchange area from (6A0, 12A0, and 18A0) led to a progressive decrease in ICR and ET rates reaching control values. The best optimized F-25 flask configuration, combining reduced medium depth (1/3H0) with an enhanced hydrophilic gas exchange membrane of 18A0, maintained ICR and ET rates similar to partially-filled controls. The F-25 flask was further tested to assess mitochondrial function under simulated Mars, Moon, and space gravity conditions following 24 h of exposure. Under different extraterrestrial gravity conditions, ICR and ET rates were again twice than those of partially-filled controls but remained unchanged in optimized F-25 flask. Discussion: The latter one provides a reproducible and relevant baseline, avoiding confounding factors related to O2 delivery for clinostat-based simulations. The F-25 flask setup, which allows controlled oxygenation and minimized hydrostatic artifacts, offers a versatile platform not only for space biology, but also for hypoxia studies, 3D culture systems, and tissue engineering applications requiring a defined O2 microenvironment.