Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA)

Human spaceflight is increasing due to the growth of commercial missions, offering an opportunity to collect more biological samples and understand spaceflight’s effects on human health. Despite known physiological impacts (e.g., musculoskeletal deconditioning, cardiovascular adaptations, neuro-vestibular changes, immune dysfunction, and elevated cancer risk), molecular and cellular responses remain incompletely characterized. Prior astronaut studies have been limited by small sample sizes, platform differences, and inconsistent protocols, which restrict statistical power and cross-mission inferences. To address these gaps, the study establishes standardized biospecimen collection and banking procedures for the Space Omics and Medical Atlas (SOMA), aiming to generate high-quality, multi-omics datasets across missions. The Inspiration4 mission serves as the inaugural use case, with a decentralized collection pipeline and centralized processing/biobanking at Weill Cornell Medicine, designed to enable synchronized datasets with enhanced statistical strength and support future missions.

The paper reviews prior spaceflight omics and biomedical work, including: changes in cytokine profiles, urinary albumin, and hemolysis; multi-omics spanning genomics (structural DNA changes), transcriptomics (mRNA, miRNA, lncRNA, snoRNA), proteomics, and metabolomics; longitudinal ISS surface microbiome profiling to assess pathogenicity and evolution; and the NASA Twins Study as the most detailed prior astronaut multi-omic investigation, albeit with only one astronaut and one ground control. It highlights human health studies across programs (Vostok, Mercury, Gemini, Apollo, Soyuz, Salyut, MIR, Shuttle, Skylab, Tiangong, ISS), noting that relatively few focused on human biology and even fewer on omics. The review underscores the need for standardized biospecimen protocols to enable cross-mission integration and larger effective sample sizes. It references numerous findings relevant to this work, such as spaceflight-associated immune changes, telomere dynamics, protein expression shifts, and microbial alterations on the ISS and in astronaut microbiomes.

Study design and timepoints: Four Inspiration4 crew members provided biospecimens at 10 longitudinal timepoints: pre-flight (L-92, L-44, L-3), in-flight (FD1, FD2, FD3), and post-flight (R+1, R+45, R+82, R+194). Sampling locations included SpaceX HQ (Hawthorne, CA), Cape Canaveral, FL, the Dragon capsule in low-Earth orbit, and post-flight clinical/lab sites across NY, NJ, TN, WA, and TX. Ground samples were processed within 16 hours; in-flight samples were processed immediately upon return. All samples were stored at -80°C after processing and shipped on dry ice to WCM within 1 week, with transit <2 days, and then biobanked in CAMbank. Biospecimen types: venous blood (processed into serum, plasma, PBMCs, extracellular vesicles/particles, and cell pellets), capillary dried blood spot (DBS) cards, saliva (preserved and crude), urine, stool (for metagenomics and metabolomics), skin swabs, skin biopsies, environmental swabs, and the Dragon HEPA filter. Venous blood collection: Per timepoint and crew member: 1 PAXgene blood RNA tube (bRNA), 4 K2 EDTA tubes, 2 CPT tubes, 1 Streck cfDNA BCT, 1 SST, and 1 DBS card. One K2 EDTA was sent to Quest for CBC; one K2 EDTA for EVP isolation; two K2 EDTA for PBMC isolation. Processing followed manufacturer instructions or specified protocols; aliquots were stored at -80°C; pellets at -20°C; PBMCs were viably frozen (Mr. Frosty to -80°C then liquid nitrogen). At R+194, CPT sodium citrate tubes were used. Tube processing details: SST: 1300×g, 10 min; serum aliquoted (500 µL) and stored at -80°C; pellet stored at -20°C. CPT: 1800×g, 30 min; plasma aliquoted to -80°C; PBMCs washed (2% FBS/PBS), pelleted (300×g, 15 min), resuspended in 10% DMSO in FBS, divided into 6 cryovials, slow-cooled in Mr. Frosty to -80°C; CPT pellet to -20°C. cfDNA BCT: 300×g, 20 min; plasma transferred and clarified at 5000×g, 10 min; 500 µL aliquots to -80°C; pellet to -20°C. bRNA: handled per PAXgene protocol; total RNA extracted using PAXgene kit and stored at -80°C. EVP isolation: From one 4 mL K2 EDTA tube shipped on ice to WCM. Plasma was prepared with sequential spins (500×g 10 min, then 3000×g 20 min) and stored at -80°C. EVPs were isolated by sequential ultracentrifugation: 12,000×g 20 min to remove microvesicles; 100,000×g 70 min to pellet EVPs; wash in PBS and repeat 100,000×g 70 min; final resuspension in PBS. DBS: Finger warmed and sterilized; lancet or 21G needle used; blood applied to Whatman 903 Protein Saver cards (5 spots per card). Cards stored at room temperature with desiccant. Saliva: Crude saliva collected into sterile tubes, aliquoted (500 µL) and frozen at -80°C. Preserved saliva collected using OMNIgene ORAL (OME-505), mixed with preservative and stored at -20°C; DNA/RNA/protein extracted using AllPrep; quantification by Qubit HS for DNA/RNA and Pierce Rapid Gold BCA for protein. Urine: Collected into sterile containers, kept at 4°C until aliquoting into 1–50 mL tubes; half stored neat at -80°C; half mixed with Zymo Urine Conditioning Buffer (UCB) before -80°C storage. Stool: Collected with DNA Genotek OM-AC1 aid, transferred into OMNIgene GUT (OMR-200) for microbiome and OMNImet-GUT (ME-200) for metabolome; stored at -80°C. For nucleic acids, 200 µL used for DNA (QIAGEN PowerFecal Pro) and 200 µL for RNA (QIAGEN PowerViral); remaining sample aliquoted and re-stored. Swabs: Sterile Isohelix swabs; for wet swabs, dipped in nuclease-free water (ground) or HFactor water (in-flight); oral and nasal collected dry. Each site swabbed 30 s using both sides. Swab tips placed into Matrix 2D Screw Tubes with 400 µL Zymo DNA/RNA Shield; stored at 4°C before -80°C transfer. Skin biopsies: Deltoid skin prepared with ChloraPrep and lidocaine with epinephrine; 3–4 mm punch biopsies obtained at L-44 and R+1; one-third in formalin (room temp) for histology; two-thirds at -80°C for spatially resolved transcriptomics; sutured with 5-0 or 4-0 nylon. Environmental sampling: Ten wet swabs per timepoint at defined capsule locations during L-92, L-44 (training capsule), and in-flight (F1, F2). Post-flight Dragon HEPA filter disassembled; activated carbon discarded; HEPA cut into 127 sections and stored at -20°C. Ethics: All subjects consented; WCM IRB Protocol 21-05023569. Logistics and storage: All ground samples processed within 16 h; flight samples stored at ambient during flight and processed post-landing; shipments via overnight courier on dry ice, avoiding weekend delays; all arrived frozen with dry ice present; long-term storage at -80°C or liquid nitrogen for cells.

- Scale and scope: 2,911 aliquots collected from four crew across 10 timepoints (L-92, L-44, L-3, FD1–FD3, R+1, R+45, R+82, R+194), spanning 289 days (2021–2022). Biospecimens included venous blood and derivatives (serum, plasma, PBMCs, EVPs), DBS cards, saliva (preserved and crude), urine, stool (microbiome and metabolome), skin swabs, skin biopsies, environmental swabs, and the capsule HEPA filter. - RNA from blood (PAXgene bRNA): Total RNA yield 3.04–14.04 µg per tube; RIN 3.2–8.5 (mean 6.95), sufficient for direct RNA sequencing (ONT requires ~500 ng per library) and comparative transcriptomics. - K2 EDTA/EVP pipeline: Plasma volumes for EVP isolation 0.6–1.7 mL; EVP yield 2.71–28.27 µg. PBMC isolation yielded 340,000–975,000 cells per mL of blood; single-cell multi-ome and immune profiling preserved. - CPT plasma and PBMCs: Plasma per CPT tube 3,000–14,000 µL; PBMCs aliquoted into 6 cryovials per tube and viably frozen. Three instances occurred where CPT plasma could not be retrieved due to technical issues. - cfDNA BCT: Plasma per timepoint 1,500–5,000 µL; 500 µL aliquots stored; suitable for cfDNA analysis (fragmentation, mtDNA/nuclear origin, tissue-of-origin). - SST: Serum per timepoint 2,000–8,000 µL; suitable for cytokines and clinical chemistry (e.g., Quest CMP Test Code 10231). One extra SST tube at R+45 for C004 increased yield. - DBS: Variable spot saturation across timepoints; five 75–80 µL spots per card when fully saturated. - Saliva: Preserved kit yields—DNA 28.1–3,187.8 ng; RNA 396.0–3,544.2 ng; protein 92.97–93.15 ng. Crude saliva volumes 150–4,000 µL per tube, including in-flight (FD2, FD3). - Urine: Per specimen cup, crude urine 23–155.5 mL; UCB-preserved urine 21–112 mL; aliquoted for diverse assays (e.g., viral reactivation, cortisol, electrolytes, bone/kidney markers, proteomics, metabolites). - Stool: Timepoints L-92, L-44, R+1, R+45, R+82; collected into OMNIgene-GUT (microbiome) and OMNImet-GUT (metabolome). DNA yield 358.5–16,660 ng; RNA 690–2,010 ng; variability due to sample mass. - Skin swabs and biopsies: Eight wet-swab sites plus oral/nasal dry swabs collected at all timepoints; first-ever astronaut skin biopsies for spatially resolved transcriptomics; samples split for histology and SRT. - Environmental sampling: Ten defined capsule surface swabs per timepoint pre-flight and in-flight; post-flight HEPA filter sectioned into 127 pieces and stored. - Logistics: All ground samples processed within 16 h; all shipments arrived on dry ice with samples frozen; centralized biobanking at -80°C (or LN2 for cells).

The study addresses critical barriers in astronaut biomedical research—small sample sizes, inconsistent protocols, and mission heterogeneity—by implementing standardized, scalable biospecimen collection and banking within SOMA. The comprehensive, longitudinal sampling across pre-, in-, and post-flight enables detection of spaceflight-associated changes while controlling for inter-individual variability. Centralized processing and rigorous documentation reduce pre-analytical noise that can confound high-dimensional, multi-omic analyses, helping to distinguish true biological signals from batch or handling artifacts. The protocol suite is adaptable across commercial and agency missions, fostering cross-mission comparisons and meta-analyses to increase statistical power. In-flight constraints (e.g., lack of cold stowage) were managed by prioritizing specimens stable at ambient temperature and annotating deviations for downstream analyses. The archived aliquots and viable cells support immediate targeted clinical assays (CBC, CMP) and future multi-omic investigations, including novel modalities (e.g., direct RNA sequencing, spatial transcriptomics of skin). Together, these efforts create a foundation for precision medicine approaches in spaceflight, particularly as civilian crews introduce greater phenotypic heterogeneity.

This work establishes the SOMA standard operating procedures and a robust logistical framework for collecting, processing, and biobanking diverse human, microbial, and environmental biospecimens in spaceflight studies. The Inspiration4 mission demonstrates feasibility across decentralized sites and in-flight conditions, yielding a large, well-annotated repository suitable for multi-omic profiling, validation of prior findings, and discovery of new biological responses to spaceflight. The protocols are generalizable to future commercial and governmental missions and are already informing studies (e.g., Polaris Dawn, Axiom-2). The resulting biobank and methodological standards will accelerate cross-mission integration, improve statistical power, and support the development of personalized countermeasures. Future work should continue refining in-flight stabilization options, optimize yields under mission-specific constraints (e.g., EVA), and expand longitudinal cohorts to further enhance generalizability and mechanistic insight.

- In-flight sample handling constraints: No cold stowage during flight led to ambient storage of in-flight samples until return, potentially affecting analyte stability. Of the in-flight sample types, only DBS and swabs were preserved at room temperature (DBS cards; DNA/RNA Shield for swabs); crude saliva lacked preservative in-flight and will have a distinct profile versus ground timepoints. - Pre-analytical variability: Despite standardization, decentralized field processing introduced unavoidable inconsistencies (e.g., site-specific equipment and timing differences). There were three instances where CPT plasma could not be retrieved due to technical issues. - Sampling variability: Stool collection was least consistent due to limited collection windows, contributing to variable nucleic acid yields. DBS spot saturation varied by timepoint and subject. - Cohort size and generalizability: The cohort consisted of four civilian astronauts; while longitudinal sampling improves power, broader cohorts are needed to generalize findings across diverse populations and mission profiles.