Collection of Biospecimens from the Inspiration4 Mission Establishes the Standards for the Space Omics and Medical Atlas (SOMA)

Abstract

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from the crew at different stages of the mission, including before (L-92, L-44, L-3 days), during (FD1, FD2, FD3), and after (R+1, R+45, R+82, R+194 days) spaceflight, creating a longitudinal sample set. The collection process included samples such as venous blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies, which were processed to obtain aliquots of serum, plasma, extracellular vesicles, and peripheral blood mononuclear cells. All samples were then processed in clinical and research laboratories for optimal isolation and testing of DNA, RNA, proteins, metabolites, and other biomolecules. This paper describes the complete set of collected biospecimens, their processing steps, and long-term biobanking methods, which enable future molecular assays and testing. 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 also aid future experiments in human spaceflight and space biology.

Figure 1:. Biospecimen Samples and Collection Locations.

(a) List of biospecimen samples collected over the course of the study. (b) Timepoints for each biospecimen sample collection. “L−” denotes the number of days prior to launch. “R+” denotes the number of days after return to Earth. “FD” denotes which day of the flight a sample was collected. (c) Location of each collection timepoint.

Figure 2:. bRNA and K2 EDTA Tubes.

(a) One 2.5mL bRNA tube was collected per crew member at each ground timepoint. (b) bRNA tube total RNA yields per sample (_μg) and RINs. (c) Four K2 EDTA tubes were collected per member at each ground timepoint. One tube was used for a CBC, one tube was used to isolate EVPs, and two tubes were used for isolation of PBMCs. (d) Plasma and EVP yields from the “[2] EVPS” tube on figure 2c. (e) PBMC yields per mL from the “[3] PBMCs” tubes on figure 2c.

Figure 3:. Tube Processing Steps.

Centrifuge (brown circles) and aliquoting (white and green boxes and circles) protocols for (a) K2 EDTA tubes designated for EVP isolation (b) CPTs (c) cfDNA BCTs and (d) SSTs.

Figure 4:. CPT, cfDNA BCT, and SST Yields.

(a) A spun CPT yields plasma, PBMCs, and a red blood cell pellet. PBMC from each tube were divided into 6 cryovials and viably frozen. Plasma was aliquoted and the pellet was frozen at −20C. (b) A spun cfDNA BCT yields plasma and a red blood cell pellet. Plasma was purified with an additional spin (see Fig 4a) then aliquoted. The pellet was frozen at −20C. (c) A spun SST yields serum and a red blood cell pellet. Serum was aliquoted and the pellet was frozen at −20C. (d) CPT plasma volumes per timepoint are reported. (e) cfDNA BCT plasma volumes per timepoint. (f) SST serum volumes per timepoint. An extra tube was drawn for C004 at R+45, resulting in a higher serum yield.

Figure 5:. DBS Collection Yields.

(a) Dried blood spot cards were collected preflight, during flight, and postflight. There were five spots for blood collection per card. (b) Blood collections varied in saturation level across blood spots and timepoints. These were classified as “full”, “partial”, and occasionally “empty”. (c) DBS card yields per blood spot, per timepoint, and per crew member.

Figure 6:. Saliva, Urine, and Stool Sample Collections.

(a) DNA, RNA, and protein yields from the OMNIgene Oral kits. (b) Volume of crude saliva collected per timepoint.

Figure 7:. Urine and Stool Sample Collections.

(a) Urine volumes per timepoint. Volumes are reported for both crude urine and urine preserved with Zymo urine conditioning buffer (UCB). (b) Timepoints that stool tubes were collected. “Gut” tubes are OMNIgene•GUT tubes for microbiome preservation. “Met” tubes are OMNImet•GUT tubes for metabolome preservation. (c) Stool “Gut” tube DNA and RNA extraction quantities.

Figure 8:. Skin Collection Locations and Sample Types.

(a) Dry swabs were collected from two body locations. (b) Wet swabs were collected from eight body locations. (c) Swabs were collected from the deltoid region. Immediately after, 3- or 4-mm skin biopsies were collected from the same area and divided for histology and spatially resolved transcriptomics.

Figure 9:. Capsule Swab Locations.

(a) Swab locations, descriptions, and label IDs. (b) Interior view of the SpaceX Dragon capsule. (c) View of the control panel located above the middle seats in the Dragon capsule. (d) View of the cupola (viewing dome) region from the outside. The rim of the dome was swabbed from the inside (ID 10).

Figure 10:. Dragon Capsule HEPA Filter.

(a) View of the un-cut HEPA filter. (b) HEPA filter during sectioning. (c) Cutting schema for the HEPA filter. The filter was split into 21 columns and 7 rows, creating a total of 147 preserved sections.