Human space exploration is experiencing a surge in activity due to the rise of commercial spaceflight. This presents a valuable opportunity to expand our understanding of spaceflight's impact on human health, which currently remains incomplete at the molecular and cellular level. Previous research has identified various physiological changes in astronauts, including musculoskeletal deconditioning, cardiovascular adaptations, vision changes, immune dysfunction, and cancer risks. However, existing studies are limited by small sample sizes and a lack of standardization across different missions. The Inspiration4 mission, with its all-civilian crew, offered a chance to develop standardized biospecimen collection and processing procedures for the Space Omics and Medical Atlas (SOMA) initiative. SOMA aims to generate high-quality multi-omics data across spaceflight investigations and enable future research using viably frozen cells and biobanked samples. This paper describes the biospecimen collection process employed during the Inspiration4 mission, from sample collection to biobanking at Weill Cornell Medicine, providing a framework for future studies.

Prior research on astronauts has characterized molecular changes during spaceflight, including alterations in cytokine profiles, urinary albumin abundance, hemolysis, and genomic, transcriptomic, proteomic, and metabolomic changes. Studies of the International Space Station (ISS) surfaces have also investigated the longitudinal microbial profiles. However, these studies have been hampered by small sample sizes and inconsistencies in methodologies across missions. The NASA Twin Study, while providing in-depth multi-omic data, was limited to a single astronaut and a ground control. Standardizing biospecimen collection across missions is crucial for generating larger datasets to draw robust scientific conclusions.

A comprehensive sampling plan was designed to collect a diverse range of biospecimens from the four Inspiration4 crew members at multiple time points: before launch (L-92, L-44, L-3 days), during flight (FD1, FD2, FD3), and after return (R+1, R+45, R+82, R+194 days). The biospecimens included venous blood, capillary dried blood spots (DBSs), saliva, urine, stool, skin swabs, skin biopsies, and environmental swabs from the Dragon capsule. Venous blood was further processed to obtain serum, plasma, extracellular vesicles and particles (EVPs), and peripheral blood mononuclear cells (PBMCs). Samples were collected at various locations based on the crew's schedule, including SpaceX headquarters, Cape Canaveral, and the crew members' homes. Samples were processed within 16 hours of collection (ground samples) or immediately after retrieval (flight samples). All samples were stored at -80°C and shipped to Weill Cornell Medicine for long-term storage and biobanking. The paper details the specific protocols for each biospecimen type, including blood collection and processing (using PAXgene tubes, K2 EDTA tubes, CPTs, cfDNA BCTs, and SSTs), DBS collection, saliva collection (using OMNIgene Oral kits and crude saliva collection), urine collection (with and without Zymo Urine Conditioning Buffer), stool collection (using OMNIgene Gut and OMNImet-GUT kits), skin swab collection (using dry and wet swab methods), skin biopsies, and environmental swab and HEPA filter collection. Specific details on processing methods, including centrifugation protocols, RNA extraction, and EVP isolation are given. Yields for various biospecimens (RNA, EVPs, PBMCs, DNA, plasma, serum etc.) are reported.

A total of 2,911 sample aliquots were collected and processed. RNA yields from PAXgene blood RNA tubes ranged from 3.04 to 14.04 µg/tube, with RIN values ranging from 3.2 to 8.5. EVP yields ranged from 2.71 to 28.27 µg. PBMC yields ranged from 340,000 to 975,000 cells per mL of blood. DNA yields from OMNIgene Oral saliva kits ranged from 28.1 to 3187.8 ng, RNA yields from 396.0 to 3544.2 ng, and protein concentrations from 92.97 to 93.15 ng. Urine volumes ranged from 23 to 155.5 mL for crude urine and 21 to 112 mL for urine with UCB. Stool DNA yields ranged from 358.5 to 16,660 ng, and RNA yields from 690 to 2010 ng. The study provides detailed information on the quantity and quality of the collected samples. The data supports the feasibility of comprehensive multi-omics analysis from a range of biospecimens collected during spaceflight.

This study establishes a standardized biospecimen collection and processing protocol for commercial spaceflight missions. The comprehensive range of biospecimens and the rigorous approach to sample handling will facilitate high-quality multi-omic analysis. This will allow researchers to characterize human and microbial responses to spaceflight with greater resolution than previously possible, enhancing statistical power and reducing variability due to pre-analytical factors. The protocols developed here are robust and adaptable, providing a foundation for future spaceflight investigations, including long-duration missions and exploration-class missions to the Moon and beyond. The potential for precision medicine applications tailored to individual astronaut responses is also highlighted.

The Inspiration4 mission's biospecimen collection, using the SOMA protocols, represents a significant advance in aerospace medicine research. The standardized procedures and the extensive biobank will support future studies exploring the impact of spaceflight on human health, microbial adaptations, and the space environment. This framework is scalable and applicable to various spaceflight missions and providers, furthering our understanding of human physiology and informing future countermeasure development.

The study was limited by the lack of cold storage during the flight portion of the mission, which impacted the quality of saliva samples. Some inconsistencies in sample processing were unavoidable due to logistical constraints of the mission. The sample size, while larger than previous studies, is still relatively small, limiting the extent to which certain conclusions can be generalized.