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

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.

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.

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.

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.