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

Domain: earth_science
Published: 2024-06-11
Editorial outcome: minor_revision

Abstract
The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R+1, R+45, R+82, R+194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations. As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human space flight and space biology experiments.

Introduction
Our human space exploration efforts are at a unique transition point in history, with more crewed launches and human presence in space than ever before1. We can attribute this to the commercial spaceflight sector entering an industrial renaissance, with multiple companies forming collaborative and competitive networks to send commercial astronauts into space. The recent advancements in human space exploration offer a significant chance to gather more biological research samples and enhance our comprehension of spaceflight’s effects on human health. This is vital, as our knowledge of the biological reactions to the unique environment of space, marked by microgravity and the space radiation, remains incomplete2. The impact of spaceflight on human health includes musculoskeletal deconditioning3, cardiovascular adaptations4, vision changes5, space motion sickness6, neurovestibular changes7, immune dysfunction8, and increased risk of rare cancers9, among other changes2. However, we are still at the initial stages of documenting biological responses to spaceflight exposure at the molecular and cellular resolution.

Prior work has characterized molecular changes that occur during spaceflight in astronauts. These include changes in cytokine profiles8,10,11, urinary albumin abundance12, and hemolysis13. Furthermore, multiomic assays have provided genomic maps of structural changes in DNA14–16, RNA expression profiles11,17,18, sample-wide protein measurements17,19,20, and metabolomic status17. Additionally, International Space Station (ISS) surfaces have been studied with longitudinal microbial profiles to track microbial pathogenicity and evolution to assess their potential influence on crew health21,22. To better improve our understanding of both human and microbial biology in space, it is critical that these analyses continue and expand as more spacecraft and stations are built and flown.

Combining and comparing work from prior missions in these new spacecraft and stations is especially important to overcome the small sample sizes and highlights a need for standardization between missions. In addition, recruiting large cohorts of astronauts is difficult, as the ISS typically can only house up to six astronauts at a time. As of the time of writing, only 647 humans have been to space, starting with the launch of Yuri Gagarin in 1961. Studies have spanned the Vostok program, Project Mercury, the Voskhod program, Project Gemini, Project Apollo, the Soyuz program, the Salyut space stations, MIR, the Space Shuttle Program, SkyLab, Tiangong Space Station, and the ISS. From the breadth of experiments that have been performed on the ISS, only a minority have specifically been human research-oriented23,24, and just a subset involve omics studies. The NASA Twin Study created the most in-depth multi-omic study of astronauts prior to Inspiration4, but was limited to one astronaut and one ground control17. These factors have limited the statistical power of astronaut omic experiments and increased the difficulty of providing robust scientific conclusions. Standardizing biospecimen collections across multiple missions will create larger sample sets needed to draw these conclusions.

Here, we establish the standard biospecimen sample collection and banking procedures for the Space Omics and Medical Atlas (SOMA). A key goal of SOMA is to standardize biospecimen collection and processing for spaceflight, to generate high-quality multi-omics data across spaceflight investigations, and to enable follow-up experiments with viably frozen cells and biobanked samples. This paper provides sample collection methods built for standardized field collections across different crews and missions. We cover the decentralized sample collection process from the Inspiration4 (I4) mission from the point of collection to the preservation of samples for downstream biochemical and omics processing at our centralized lab at Weill Cornell Medicine (WCM). These protocols can produce synchronized datasets with enhanced statistical strength, amplifying our scientific findings from spaceflight studies. Additionally, we showcase metrics related to sample collection outputs, references of past astronaut sample collections in scientific publications, and suggestions to refine sample collection for upcoming missions. In its inaugural use case, these samples were collected from the I4 astronaut cohort and are currently in use for several other missions (Polaris Dawn, Axiom-2), which will enable continued utilization for future crewed space missions.

Results
Biospecimen collection overview
We formulated and executed a sampling plan that spans a wide range of biospecimen samples: venous blood, capillary dried blood spots (DBSs), saliva, urine, stool, skin swabs, skin biopsies, and environmental swabs (Fig. 1a). The collection of various types of samples covered the scope of previous assays on astronaut samples (Supplementary Table 1), but also enabled newer omics technologies, such as spatially resolved, single-molecule, and single-cell assays.

For the I4 mission, sample collection spanned three-time points pre-launch (L-92, L-44, L-3 days), three-time points during flight (Flight Day 1 (FD1), FD2, FD3), and four-time points post-return (R+1, R+45, R+82, R+194 days). Venous blood, urine, stool, and skin biopsies were collected during ground timepoints only, while capillary DBSs, saliva, and skin swabs were collected both on the ground and during flight (Fig. 1b). Environmental swabs of the Dragon capsule were collected pre-flight in the crew training capsule and during flight in the spacecraft launched from Cape Canaveral (Fig. 1b).

Samples were collected across a variety of locations based on the crew’s training and travel schedule. L-92 and L-44 were collected in Hawthorne, CA at SpaceX Headquarters, L-3 and R+1 were collected at Cape Canaveral, FL at a facility near the launch site. FD1, FD2, and FD3 were collected inside the Dragon capsule while in orbit. R+45 was collected at the crew members’ individual locations (which spanned the US States NY, NJ, TN, and WA), R+82 was collected at Weill Cornell Medicine, NY and R+194 was collected at Baylor College of Medicine, TX (Fig. 1c). All samples from ground timepoints were processed within 16 hours of collection. All samples from the flight were processed immediately after retrieval from the flight.

Samples were stored at −80°C at each site immediately after processing and aliquoting (processing steps are outlined below). All samples were shipped via FedEx using Overnight Shipping to Weill Cornell Medicine for storage in the Cornell Aerospace Medicine Biobank (CAMbank) within 1 week of collection on dry ice in styrofoam boxes and spent <2 days in transit. No samples were shipped on a Thursday or Friday to avoid weekend shipping delays. All samples arrived at Weill Cornell Medicine with dry-ice still in the packaging and samples frozen. Samples were immediately transferred to −80°C for long-term storage and biobanking. Any exceptions to this process will be noted below. In total, we collected 2,911 sample aliquots, which were then processed in our central lab at Weill Cornell Medicine for downstream assays (Supplementary Table 2). Some samples were processed immediately at field laboratory facilities (Supplementary Data Fig. 1). DNA and RNA yields are also reported below from collection kits (saliva and stool) after arrival at WCM.

Blood collection and derivatives
Blood was collected using a combination of venipuncture tubes to collect venous blood and contact-activated lancets to collect capillary blood from the fingertip. Each crew member provided blood samples, collected into one BD PAXgene blood RNA tube (bRNA), four BD Vacutainer K2 EDTA tubes, two BD Vacutainer cell preparation tubes (CPTs), one Streck cell-free DNA tube (cfDNA BCT), one BD Vacutainer serum separator tube (SST), and one DBS card per timepoint. Tubes were transported from the collection site to the field laboratory at room temperature. From these tubes, whole blood, plasma, PBMCs, serum, and cell pellet samples were collected (Supplementary Table 3). Sample yields are reported below. Samples were aliquoted for long-term storage and biobanking (Supplementary Table 4).

bRNA tubes were collected in order to isolate total RNA using the PAXgene blood RNA kit (Fig. 2a). Yield ranged from 3.04 to 14.04 µg/tube of total RNA across all samples, and the RNA integrity number (RIN) ranged from 3.2 to 8.5 (mean: 6.95) (Fig. 2b). RNA was stored at −80°C after extraction. The collection of total RNA enables a variety of downstream RNA profiling methods. It will allow comparative studies to prior RNA-sequencing performed on astronauts, particularly snoRNA & lncRNA biomarkers analyzed from Space Shuttle era blood25,26, mRNA & miRNA measured during the NASA Twin Study17,18, and whole blood RNA arrays from the ISS27,28. Additionally, RNA yields are more than sufficient to perform direct-RNA sequencing using Oxford Nanopore Technologies (ONT) platforms, which require 500ng of total RNA per library (Manufacturer’s protocol, ONT kit SQK-RNA002). This enables the study of RNA modification changes during spaceflight to create epitranscriptomic profiles for the first time in astronauts.

Four K2 EDTA tubes were drawn at each timepoint from each crew member (Fig. 2c). One K2 EDTA tube was submitted to Quest Diagnostics to perform a complete blood count (CBC, Quest Test Code: 6399). One tube was used to isolate extracellular vesicles and particles (EVPs) for proteomic quantification (Fig. 3a). Total EVP quantities varied from 2.71-28.27 ug (Fig. 2d). Two K2 EDTA tubes were used to isolate PBMCs for single-cell sequencing (10X Chromium Single Cell Multiome ATAC+Gene Expression and Chromium Single Cell Immune Profiling workflows). After collection, a Ficoll separation was performed to isolate PBMCs, which ranged from 340,000-975,000 cells per mL of blood (Fig. 2e). One prior single-cell gene expression experiment, NASA Twin study, was performed on astronauts, which found immune cell population specific gene expression changes and a correlation with microRNA signatures11,18.

Additional PBMCs, plasma, and serum were collected from CPTs (Fig. 4a), cfDNA BCTs (Fig. 4d), SSTs (Fig. 4c), as well as red blood cell pellets. CPTs were spun and aliquoted according to the manufacturer’s instructions (Fig. 3b). Plasma volume per tube ranged from 3000-14,000 uL per tube (Fig. 4d). Because of technical issues in the sample processing procedure, three instances occurred where plasma retrieval from the CPT tubes was not possible. Plasma can be used to validate or refute previous studies, including cytokine panel10,29, exosomal RNA-seq25,26, extracellular vesicle microRNA30, and proteomic20,31–33 results. PBMCs were also collected, aliquoted into 6 cryovials per CPT, and stored in liquid nitrogen after slowly cooled in a Mr. Frosty to -80°C. These can be used to follow-up on previous studies on adaptive immunity, cell function, and immune dysregulation8,34–39. The remaining red blood cell pellet mixtures from below the gel plug in each CPT Tube were stored at −20°C.

cfDNA BCT (Streck) tubes were collected to isolate high-quality cfDNA from plasma. cfDNA BCTs were spun and aliquoted according to the manufacturer’s instructions (Fig. 3c). The remaining cell pellet mixture was frozen at −20°C. Plasma volume per timepoint ranged from 1500-5000 uL (Fig. 4e). 500 uL aliquots were frozen at -80°C. cfDNA extracted from these tubes can be analyzed for fragment length, mitochondrial or nuclear origin, and cell type or tissue of origin40–42.

The SST was spun and aliquoted according to the manufacturer’s instructions (Fig. 3d). Serum volume ranged from 2000-8000 uL per timepoint (Fig. 4f). Similar to plasma, serum can be allocated for cytokine analysis and can also be used to perform comprehensive metabolic panels, including one we used at Quest (CMP, Quest Test Code: 10231) for metrics on alkaline phosphatase, calcium, glucose, potassium, and sodium, among other metabolic markers. The remaining cell pellet mixture from each SST tube was stored at −20°C.

In addition to venous blood, capillary blood was collected onto a DBS card using a contact-activated lancet pressed against the fingertip (Fig. 5a). Capillary blood was collected onto a Whatman 903 Protein Saver DBS card to preserve nucleic acids and proteins. Each of the five spots on the DBS card hold 75-80 uL of capillary blood, however, the amount of capillary blood collected across timepoints varied (Fig. 5b, c) according to how much blood could be collected before the lancet-puncture closed.

Saliva collection
Saliva was collected at the L-92, L-44, L-3, FD1, FD2, FD3, R+1, R+45, and R+82 timepoints using two methods. First, saliva was collected using the OMNIgene Oral Kit (OME-505), which preserves nucleic acids (Fig. 6a) during the ground timepoints. From these samples, DNA, RNA, and protein were extracted. DNA yield ranged from 28.1 to 3187.8 ng, RNA yield from 396.0 to 3544.2 ng (less the two samples had concentration too low for measurement), and protein concentration from 92.97 to 93.15 ng.

Second, crude saliva (i.e., saliva with no preservative added) was collected into a 5mL DNase/RNase-free screw top tube during the ground and flight timepoints. Saliva volume varied from 150 to 4000 uL per tube (Fig. 6b). Crude saliva was also collected during flight (FD2 and FD3), in addition to the ground timepoints.

Saliva collections have been conducted throughout spaceflight studies for assessing the immune state of crews, particularly in the context of viral reactivation. Previously identified viruses that reactivate during spaceflight include Epstein–Barr, varicella-zoster, and cytomegalovirus43. Responses to reactivation of these viruses can be asymptomatic, debilitating, or even life-threatening, thus assessing these adaptations is beneficial in understanding viral spaceflight activity as well as crew health. In addition to viral nucleic acid quantification, numerous biochemical assays can also be performed, including measurements of C-reactive protein (CRP), cortisol, dehydroepiandrosterone (DHEA), and cytokines, among others10,43–46.

Urine collection
Urine was collected in sterile specimen cups at the L-92, L-44, L-3, R+1, R+45, and R+82 timepoints. Specimen cups were collected 1-2 times per day. For preservation, urine was aliquoted and stored at -80°C. Half the urine had Zymo Urine Conditioning Buffer (UCB) added before freezing, to preserve nucleic acids. Samples yielded 23–155.5 mL of crude urine and 21-112 mL of UCB urine per specimen cup (Fig. 7a). Urine was split into 1 mL-15 mL aliquots before freezing at -80°C.

A wide variety of assays can be performed on urine samples. Previous studies have included viral reactivation43,46,47, urinary cortisol48,49, iron and magnesium measurements50–53, bone status54–56, kidney stones54,55, proteomics11, telomere measurements57, and various biomarkers and metabolites17,49,58–61.

Stool collection
Stool was collected at the L-92, L-44, R+1, R+45, and R+82 timepoints. Stool samples were stored into two collection containers at each timepoint, one DNA Genotek OMNIgene Gut (OMR−200) kit with a preservative for metagenomics and another (ME−200) with a preservative for metabolomics (Fig. 7b). Stool was the least consistent sample collected due to the limited windows available for sampling during collection timeframes. DNA and RNA were extracted from aliquots of the OMNIgene Gut (OMR−200) tubes for downstream microbiome analysis. DNA yield ranged from 358.5 to 16,660 ng, RNA from 690 to 2010 ng (Fig. 7c). Large variations in yield are attributable to variable stool mass collected between kits.

Stool samples enable various biochemical, immune, and microbiome changes studies. Previous metagenomic assays have found that shannon alpha diversity and richness during long-duration missions to the ISS62.

Skin swabs
Body swabs were collected at all timepoints. Samples were collected by swabbing the body region of interest for 30 seconds, then placing the swab in a sterile 2D matrix tube (Thermo Scientific #3710) with Zymo DNA/RNA shield preservative. For the first two swab locations, the oral and nasal cavity, the swab was placed directly on the body after removal from its sterile packaging (dry-swab method; Fig. 8a). For the remaining body locations, the swab was briefly dipped in nuclease-free, DNA/RNA-free water before proceeding (wet-swab method). Eight distinct sites were swabbed with the wet-swab method: post-auricular, axillary vault, volar forearm, occiput, umbilicus, gluteal crease, glabella, and the toe-web space (Fig. 8b). The astronaut microbiome has previously been studied in the forehead, forearm, nasal, armpit, navel, postauricular, and tongue body locations, and changes have been documented during flight. Changes in alpha diversity and beta diversity were documented, as well as shifts in microbial genera62. However, the impact of these changes on skin health and immunological health are not well understood.

Acquiring extensive swab samples from the crew skin allows for characterization of the habitat environment, crew skin microbiome adaptations, and interactions with potential human health adaptations resulting from spaceflight exposure. This is very relevant for crew health, considering astronauts become more susceptible to infections during spaceflight missions63, with the relationship between microbe-host interactions from spaceflight exposure, which may be a causative factor of astronauts immune dysfunction, which is still not well understood. and skin rash’s status as the most frequent clinical symptom reported during spaceflight64.

Skin biopsies
A skin biopsy on the deltoid was obtained from the L-44 and R+1 timepoint. Biopsies were also collected in advance of a flight to ensure the biopsy site is fully healed before the flight so there is no risk of complication. The wet-swab method was used to collect the skin microbiome before the skin biopsy. The skin biopsies were 3–4 millimeters in diameter and were collected for histology and spatially resolved transcriptomics (SRT) (Fig. 8c). One-third of the sample was stored in formalin and kept at room temperature to perform histology. The remaining two-thirds of the sample was stored in a cryovial and placed at -80°C for SRT (Fig. 8c). This is the first sample collected from astronauts for spatially resolved transcriptomics. The skin is of high interest due to the inflammation-related cytokine markers such as IL-12p40, IL-10, IL-17A, and IL-1810,17.

Environmental swabs and HEPA filter
Environmental swabs were collected in flight during the F1 and F2 timepoint. Additionally, environmental swabs were collected from the flight simulation capsule at SpaceX headquarters after days of crew training during the L-92 and L-44 timepoints. Environmental swabs were collected using the wet-swab method. Ten environmental swabs were collected per timepoint at the following locations in the capsule: an ambient air/control swab, the execute button, the viewing dome, the side hatch mobility aid, the lid of the waste locker, the head section of one of the seats, the commode panel, the right and left sides of the control screen, and the g-meter button (Fig. 9a-d). Additionally, the spacecraft’s high-efficiency particulate absorbing (HEPA) filter was acquired post-flight. This filter was cut into 127 rectangular pieces (1.2” x 1.6” x 4”) and stored at −20°C.

Previous microbial profiling of spacecraft environments has revealed that equipment sterilized on the ground becomes coated in microbial life in space due to interactions with crew and the introduction of equipment that has not undergone sterilization65. Subsequent microbial monitoring assays performed on the ISS have detected novel, spaceflight-specific species on the ISS66. Once in space, surface microbes are subject to the unique microgravity and radiation environment of flight, which will influence evolutionary trajectory. The potential impact of this influence on pathogenesis is a concern for long-duration space missions, especially given that changes in host-pathogen interactions may also be affected during spaceflight67.

Methods
Venous blood draw
Venipuncture was performed on each subject using a BD Vacutainer® Safety-Lok™ blood collection set (BD Biosciences, #367281) and a Vacutainer one-use holder (BD Biosciences, 364815). The puncture site was located near the cubital fossa and was sterilized with a BZK antiseptic towelette (Dynarex, Reorder No. 1303). Blood was collected into 1 serum separator tube (SST, BD Biosciences: #367987, Lot: #1158449, #1034773), 2 cell processing tubes (CPT, BD Biosciences: #362753, Lot: #1133477, #1012161), 1 blood RNA tube (bRNA, PAXgene: #762165, Lot: #1021333), 1 cell-free DNA BCT (cfDNA BCT, Streck: #230470, Lot: #11530331), and 4 K2 EDTA blood collection tubes (BD Biosciences, #367844, Lot: #0345756) per crew member per timepoint. For samples collected in Hawthorne, blood was drawn at SpaceX headquarters, then immediately transported to USC for processing. Samples collected at Cape Canaveral were processed on-site.

Blood tube processing
For processing, serum separator tubes (SST) were centrifuged at 1300 x g for 10 minutes. 500 uL aliquots of serum were aliquoted into 1 mL Matrix 2D Screw Tubes (Thermo Fisher, 3741-WP1D-BR) and stored at -80°C. SST tubes were recapped and stored at −20°C to preserve the red blood cell pellet.

Cell processing tubes were centrifuged at 1800 x g for 30 minutes. Plasma was aliquoted into 1 mL Matrix 2D Screw Tubes and stored at -80°C. 5 mL of 2% FBS (Thermo Fisher, #26140079) in PBS (Thermo Fisher, #10010023) was added to the CPT tube to resuspend PBMCs. PBMC suspension was transferred to a clean 15 mL conical tube. The total volume was brought to 15 mL with 2% FBS in PBS. The tube was centrifuged for 15 minutes at 300 x g. Supernatant was discarded. PBMCs were resuspended 6 mL of 10% DMSO (Millipore Sigma, #D4540-500 mL) in FBS. 1 mL of PBMCs were moved to 6 cryogenic vials (Corning, #8672). Cryovials were placed in a Mr. FrostyTM (Thermo Fisher, #5100-0001) and stored at -80°C. CPTs were recapped and stored at −20°C to preserve the red blood cell pellet. There was one outlier to this sample collection, the R+194 timepoint, where CPT sodium citrate tubes were used (BD Biosciences, Cat no. 362760).

cfDNA BCTs were centrifuged at 300 x g for 20 minutes. Plasma was transferred to a 15 mL conical tube. Plasma was centrifuged 5000 x g for 10 minutes. 500 uL aliquots of plasma were aliquoted into 1 mL Matrix 2D Screw Tubes and stored at -80°C. cfDNA BCTs were recapped and stored at −20°C to preserve the red blood cell pellet.

PAXgene blood RNA tubes were processed according to the manufacturer’s instructions. Briefly, tubes were left upright for a minimum of 2 hours before freezing at −20°C. For RNA extraction, tubes were thawed and processed with the PAXgene blood RNA kit (Qiagen, #762164).

Extracellular Vesicles and Particles (EVPs) Isolation
One 4 mL K2 EDTA tube was shipped on ice overnight to WCM for processing. Blood was centrifuged at 500 x g for 10 minutes, then plasma was transferred to a new tube and centrifuged at 3000 x g for 20 minutes, and the supernatant was collected and stored at -80°C for EVP isolation. Plasma volumes ranged between 0.6-1.7 ml. Plasma was later thawed for downstream processing, when concentrations were measured. Plasma samples were thawed on ice, and EVPs were isolated by sequential ultracentrifugation (Hoshino et al., 2020). Samples were centrifuged at 12,000 x g for 20 minutes to remove microvesicles, then EVPs were collected by ultracentrifugation in a Beckman Coulter Optima XE or XPE ultracentrifuge at 100,000 x g for 70 minutes. EVPs were then washed in PBS and pelleted again by ultracentrifugation at 100,000 x g for 70 minutes. The final EVP pellet was resuspended in PBS.

Dried blood spot (DBS)
Crew members warmed their hands and massaged their finger towards the fingertip to enrich blood flow toward the puncture site. The puncture site was sterilized using a BZK antiseptic towelette (Dynarex, Reorder No. 1303). Skin was punctured using a contact-activated lancet (BD Biosciences, #366593) or a 21-gauge needle (BD Biosciences, #305167), depending on crew member preference. Capillary blood was collected onto the Whatman 903 Protein Saver DBS cards (Cytiva, #10534612). Blood was transferred by touching only the blood droplet to the surface of the DBS card. DBS cards were stored at room temperature with a desiccant pack (Cytiva, #10548239).

Saliva
To collect crude saliva, crew members uncapped and spit into a sterile, PCR-clean, 5 mL screw-cap tube (Eppendorf, 30122330). Crew spit repeatedly until at least 1 mL was collected. Saliva was transported to a sterile flow hood and separated into 500 uL aliquots. Aliquots were frozen at -80°C. To collect preserved saliva, crew members used the OMNIgene ORAL kit (OME-505). Crew members spit into the kit’s tube until they reached the fill line. The tube was re-capped, which released the preservative liquid. Tubes were inverted to mix the saliva and preservative before being placed at −20°C for storage. After all timepoints were collected, DNA, RNA, and protein were extracted using the AllPrep DNA/RNA/Protein kit (Qiagen, #47054). Sample concentrations were measured with Qubit high sensitivity dsDNA and RNA platform. Proteins were quantified with the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific, #A53225) on Promega GloMax Plate Reader.

Urine
Crew members urinated into sterile specimen containers (Thermo Scientific, #13-711-56). The container was stored at 4C until it was prepared for long-term storage. To prepare urine samples for long-term storage, urine was aliquoted into 1 mL, 15 mL, and 50 mL tubes. Half of the urine was immediately placed at −80°C. The other half had urine conditioning buffer (Zymo, #D3061-1-140) added to the sample before placing in the -80°C freezer.

Stool collection
Crew members isolated a stool sample using a paper toilet accessory (DNA Genotek, OM-AC1). Stool was transferred into and OMNIgene(R) GUT tube (DNAgenotek, OMR−200) and an OMNImet(R) GUT tube (DNA Genotek, ME−200). Tubes were placed at -80°C for long-term storage. For nucleic acid extraction, 200 uL of each tube was allocated for DNA extraction with the QIAGEN PowerFecal Pro kit and 200 uL was allocated to RNA extraction with the QIAGEN PowerViral kit. The remaining sample was split into 500 uL aliquots and re-stored at -80°C.

Swab collection
Crew members put on gloves and remove a sterile DNA/RNA swab (Isohelix, SK-4S) from its packaging. For collection of the post-auricular, axillary vault, volar forearm, occiput, umbilicus, gluteal crease, glabella, toe web space, and capsule environment regions, swabs were dipped in nuclease-free water (this step was skipped for oral and nasal swabs) for ground collections. For in-flight collections, HFactor hydrogen infused water was used in place of nuclease-free water. Each body location was swabbed for 30 seconds, using both sides of the swab. Swabs were then placed in 1 mL Matrix 2D Screw Tubes containing 400 uL of DNA/RNA Shield (Zymo). The tip of the swab was broken off so that only the swab tip was stored in the Matrix 2D Screw Tube. Tubes were stored at 4C.

Skin biopsies
Skin biopsies were performed on the deltoid region of the arm. Each site was prepared by application of ChloraPrep and anesthesia was induced with administration of 1% lidocaine with 1:100,000 epinephrine. A trephine punch was used to remove a 3- or 4-mm diameter piece of skin. The resected piece was cut into approximately 1/3 and 2/3 sections. The smaller piece was added to a formalin-filled specimen jar. The larger piece was placed in a cryovial and stored at -80°C. Surgical defects were closed with 1 or 2 5-0 or 4-0 nylon sutures.

HEPA filter
HEPA Filter was taken apart and sectioned under a chemical hood to avoid contamination. The filter contained two parts, an activated carbon component and a HEPA filter. The activated carbon component was discarded and the filter was sectioned using a sterile blade. Sections were placed in individual specimen containers and stored at −20°C.

Human subjects research
All subjects were consented and samples were collected and processed under the approval of the IRB at Weill Cornell Medicine, under Protocol 21-05023569.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review\nThe manuscript describes in general terms the biobanking protocol for the Inspiration4 Mission.\nOverall, nice overview and graphical displays.\nPlease, consider the comments below to improve the paper. I do not want to be pedantic as the\nauthors already have done a great job in describing details. But a bit more clarification is needed\nin order for future readers of the future papers based on these samples to evaluate and interpret\nthe quality of the samples.\nAbstract:\nWrite out the full word for L, FD, and R. I guess you mean launch (L), FD(?), return (R)\nReplace “venous blood” with the actual primary collection: ex. Venous whole blood, plasma, and\nserum.\nProvide the time frame with collection year(s) and add total number of people and total number of\nsamples.\nManuscript:\nYou have presented a nice geographical map of the collection process. But this also raises concerns\nabout the transport of the material, which is not clear from the manuscript. Transport of\nbiospecimens should be described in detail (when, when in the process/timeline, which\ntemperature, which material for transport (dry ice, other?), which courier, flight/train/car to\ntransport long-distance, time in transport etc.).\nA critical and realistic reflection on how biological and pre-analytical variables (including\ntransport conditions) could have impacted specimen stability and quality. For reference: PMID: 25979952.\nDid you notice any degree of visual hemolysis in the samples or measure hemolysis?\nThe authors mention that standardization is critical for the later utilization and interpretation of\nthe samples. I agree. Which standards were followed (CLIA, ISBER, other)?\nI like the overview in Table 1. But Table 1 is massive and could preferably be divided into smaller\nfocused samples based on the sample type. You can also try to reduce the white space and adjust\nthe fonts and line spacing as simple measures. The duration is not only listed in days, but also\nyears and months; however, for comparison across studies, it would help to have at least the\nduration in days for all studies. SOmwhere in the table you have whole blood and serum in column\none, and hematology in column 2, but hematology can only be measured on whole blood. In some\nof the rows for column 3 you have mentioned locations instead of participant N. It is not clear if\neach line represent one space mission, or the same space mission can cover several lines;\ntherefore it would be great to add the name of mission and the year of the mission. Sometimes\nyou mention sex and sometimes not (please be consistent or write if data on this is missing).\nSometimes in column 3, you write “astronauts”, but aren’t all these studies for astronauts?\nFor all tables provide a legend describing ALL the abbreviations.\nIt is not clear from table 2, column 3, which measurements are already measured on fresh\nsamples, which were measured on frozen samples, and which are intended to be measured in the\nfuture on biobanked samples. You write “biobanking” for some but not all.\nTable 3 should also include genomic DNA and RNA I guess.\nDescribe the geographical locations of the processing and the biobanking. And was it centralized or\ndecentralized?\nDescribe time from sampling to processing and freezing for all samples. Were some sample types\nfrozen before processing? What was the temperature of the samples pre-processing (ambient\ntemperature, refrigerator, or frozen) after collection? When did the aliquoting take place (duration\nfrom the primary collection and what was the temperature of samples between collection and\naliquoting)?\nSome samples have a limited time window from primary tube collection to processing to ensure\nthe stability of analytes. Please add the time windows for the samples.\nAdd the specific color of the “stopper” (the exact color is provided by the company) and colors\nalso indicate type of material.\nWrite out BD for the vacutainer type.\nWhich grade of DBS did you use?\nWho among the authors was responsible for the standard operating procedures and for ensuring\nthat the collection, processing, aliquoting, and storage were performed according to the SOPs\n(quality assurance)?\nWas any of the authors or associated personnel trained in biobanking procedures (there are\nvarious certificates and courses in this, some endorsed by ISBER).\nHave you planned how to prospectively control/check the quality of the samples? Some studies set\naside certain aliquots for this, and at regular intervals check for some biomarkers, so a\ndegradation profile can be estimated.\nIt is not quite clear if any biomarkers were measured on fresh samples. If so, which?\nBe aware that some of the biomarkers you mention (such as cortisol and cytokines) have\nextremely strict requirements for collection (time of day for cortisol, time on the bench for\ncytokines) and are sensitive to long-term storage (cytokines).\nI am not sure exactly where the biobank is. Can you clarify?\nAll abbreviations should be mentioned at least once in full writing.\nWere the lab(s) CLIA certified?\nYou use the term “established the standard” in the title. What exactly do you mean by that?\nStandardization can mean many things in the clinical laboratory: harmonization (that procedures\nare the same), but it can also refer to reference measurement procedures and reference materials.\nUsually, it is governmental agencies or larger organizations that set the standards for the clinical\nlaboratory. Thus, please clarify what you mean by standardization in the title and in the\nmanuscript.\nWhat time of the day were samples taken? And fasting/non-fasting considerations?", "reviewer_2": "The study by Overbey and co-workers has a specific focus on collection and banking of\nbiospecimens within an exceptional context, which is the SpaceX Inspiration4 mission, with the\naim of studying the impact of spaceflight on the human body. The novelty of the study certainly\nresides in the exceptional source of these samples, however the impact these collection may have\non further studies is less clear.\nThe collection is well described in itself, even with a pleonastic amount of details.\nOn the other side the study is mainly based on a description of standard parameters related to\nquality of samples and their analytes (testing of DNA, RNA, proteins, metabolites, and other\nbiomolecules).\nNot all of the samples were collected during the flight. Histological samples in the form of skin\nbiopsies were collected at ground timepoints. It is unclear how skin biopsies can be informative\nand exploited for instance for spatial genomic analyses (such as spatially resolved, single-\nmolecule, and single-cell assays).\nAlthough the effort in the meticulous collection should be praised, the study is very descriptive in\nnature and the data reported do not provide a significant impact on our knowledge.\nIt is also questionable whether there will be a systematic collection of histological samples from\npersonnel for these flights that will be used for research studies. We cannot exclude this will be the\ncase, and in this respect this precise indication on best practice of collection of samples will be\nuseful, yet not ground-breaking.\nWe can foresee that the most relevant biospecimens that will be systematically collected from\nthese units of personnel will be blood and urine to monitor health status of individuals involved in\nthese missions. Again, the paper offers suggestions for good practice in this respect without\nleading to major advances."}, "author_response": "We thank the reviewers for their comments on our manuscript and for the opportunity to provide\nrevisions. In response to the points raised, we have we focused on the following areas for\nimprovement:\n(1) Clarifications to detail the work we performed. We clarified which steps were\nperformed in various field laboratories vs. back in our central laboratory location at Weill\nCornell Medicine (WCM). We added Extended Data Figure 1 to highlight when assays\nwere performed on fresh samples at a field laboratory location. We added sections and\nupdated the tables to provide full context of the information in the manuscript. We also\nadded a table with the total number of sample aliquots collected across the duration of the\nstudy in field laboratories that were shipped back to WCM. Furthermore, we added\nadditional information about shipping logistics and, in our response below, clarified where\nwe are compliant with key sample collection and biobanking recommendations from the\nInternational Society for Biological and Environmental Repositories (ISBER).\n(2) Broader context for the methods and data. We have elaborated below on the broader\nimpact of the work presented in this paper. Without a connection to the other papers\nsubmitted, understandably the paper leaves readers curious about analyses and results.\nTo provide additional context for reviewers, in addition to our comments below, we are\nsending the Park et al. paper, “Spatial multi-omics of human skin reveals KRAS and\ninflammatory responses to spaceflight”, under review at Nature Communications and the\nOverbey et al. paper, “The Space Omics and Medical Atlas (SOMA): A comprehensive\ndata resource and biobank for astronauts”, under review at Nature to assist reviewers in\ntheir assessment of the impact and scope of our work.\nIn addition to these three points, we responded to each of the Reviewer’s comments one-by-one\nin blue below, citing specific passages from the text when necessary.\nThank you for your consideration, and we look forward to your response.\nOn behalf of the authors,\nDr. Christopher Mason\nWeill Cornell Medicine\n\n[... Author responses to specific points omitted for brevity as per instructions to include verbatim text ...]\n\nWe have made the clarification revisions and write out the full words for \"L\" (launch), \"FD\"\n(Flight Day), and \"R\" (return) to improve clarity and ensure better comprehension for readers.\nWe acknowledge your suggestion to replace the term \"venous blood\" with more precise\ndescriptions such as \"venous whole blood,\" \"plasma,\" and \"serum\" wherever applicable.\nAdditionally, we now include the timespan and collection year(s) to provide a clear reference for\nour study period, as well as the number of participants and samples in our revised manuscript.\nWe agree that providing these specific terms will enhance the accuracy and completeness of\nour abstract.\n\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded adequately in the response and the revised manuscript."}, "author_response": "We are glad to hear we have made adequate updates. Thank you for your time and help in the\nrevisions of our manuscript. We have included our response to the final comment in blue below.\n\n[...]\n\nWe are glad to hear we have made adequate updates. Thank you for your time and help in the\nrevisions of our manuscript."}]

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