Year: 2026
Type: article
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 CO 2 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 cm 2 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 C 2 C 12 myoblasts by high resolution respirometry. Results After 24 h, conventional T-25 flasks, full-filled to height of H 0 (2.25 cm) and with a hydrophobic-type gas exchange area of A 0 (0.2 cm 2 ) 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 O 2 s −1 10 −6 cells −1 ) remained unchanged between time zero and 24 h. At each medium depth ( 1 / 3 H 0 , 2 / 3 H 0 , and H 0 ) increasing the gas exchange area from (6A 0 , 12A 0 , and 18A 0 ) 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 / 3 H 0 ) with an enhanced hydrophilic gas exchange membrane of 18A 0 , 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 O 2 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 O 2 microenvironment.
Institution: University of Cagliari
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W7129045768