Data from the NASA Rodent Research-1 (RR-1) mission showed that gene-expression levels in mouse livers are different depending on what tissue preservation protocol is used and that slow freezing is not an effective method for preserving signals in gene-expression data. In response to these and other observations the Rapid Freeze hardware was built for use on the International Space Station. The Rapid Freeze hardware freezes mouse tissues (Glovebox freezer) and whole carcasses (Cryochiller) at rates closely mimicking those attained with immersion in liquid nitrogen. Because this hardware will be used extensively on future rodent research missions it is crucial to understand whether or not it preserves signals in gene expression data in order to maximize the value of these rare and expensive spaceflight experiments. Therefore this study was designed with three goals: 1) To evaluate the temperature profile of the Cryochiller and Glovebox freezer cartridges (Rapid Freeze hardware) over time during mock on-orbit procedures; 2) To determine the freezing profiles of tissues and carcasses using Rapid Freeze hardware at both optimal and sub-optimal temperatures (to mimic on-orbit operations) compared with those frozen in liquid nitrogen (the laboratory gold standard) or frozen at -80 C (the current standard method); 3) To identify gene expression changes in a) tissues that were frozen via the Glovebox freezer and b) tissues dissected from whole or partial carcasses that were frozen via the Cryochiller versus tissues that were frozen via control methods (liquid nitrogen or -80C slow freeze) to assess how the Rapid Freeze hardware compares with laboratory gold standard practices and our current standard methods.

Data from the NASA Rodent Research-1 (RR-1) mission showed that gene-expression levels in mouse livers are different depending on what tissue preservation protocol is used and that slow freezing is not an effective method for preserving signals in gene-expression data. In response to these and other observations the Rapid Freeze hardware was built for use on the International Space Station. The Rapid Freeze hardware freezes mouse tissues (Glovebox freezer) and whole carcasses (Cryochiller) at rates closely mimicking those attained with immersion in liquid nitrogen. Because this hardware will be used extensively on future rodent research missions it is crucial to understand whether or not it preserves signals in gene expression data in order to maximize the value of these rare and expensive spaceflight experiments. Therefore this study was designed with three goals: 1) To evaluate the temperature profile of the Cryochiller and Glovebox freezer cartridges (Rapid Freeze hardware) over time during mock on-orbit procedures; 2) To determine the freezing profiles of tissues and carcasses using Rapid Freeze hardware at both optimal and sub-optimal temperatures (to mimic on-orbit operations) compared with those frozen in liquid nitrogen (the laboratory gold standard) or frozen at -80 C (the current standard method); 3) To identify gene expression changes in a) tissues that were frozen via the Glovebox freezer and b) tissues dissected from whole or partial carcasses that were frozen via the Cryochiller versus tissues that were frozen via control methods (liquid nitrogen or -80C slow freeze) to assess how the Rapid Freeze hardware compares with laboratory gold standard practices and our current standard methods.

The purpose of this study was to evaluate transcriptional changes in mouse spleens using a ground-based model for spaceflight. This model includes prolonged unloading and low-dose irradiation. Low-dose-rate gamma-radiation was delivered to 6-month old female C57BL/6J mice using 57Co plates (0.04 Gy) to simulate the radiation environment of spaceflight. Anti-orthostatic tail suspension was used to model the unloading fluid shift and physiological stress aspects of the microgravity component of spaceflight. Mice were hindlimb suspended and/or irradiated for 21 days. Mice were euthanized and spleens collected 7 days following treatment. RNA sequencing data was generated to assess transcriptional changes in these spleens.

This study compares standard laboratory protocols for tissue freezing and storage with a simulation of the delayed processing of liver specimens and long-term storage protocols used during the Rodent Research-1 (RR-1) payload. Liver samples from twelve-week old female C57/BL6 mice (snap frozen vs. 25 min. slow-frozen and stored for 3 days vs. 1 year) received from the RR-1 project were processed for extraction of RNA DNA and protein. RNA-seq whole genome bisulfite sequencing and mass spectrometry proteomics were performed. These multi-omics datasets will help distinguish the effects of freezing and storage time that could confound the interpretation of the Rodent Research-1 datasets.

To understand the molecular mechanisms affected by spaceflight it is essential to achieve high quality sample preservation on-orbit for downstream gene expression analysis. However sample preservation protocols must also be compatible with available equipment and crew time. NASA s Rodent Research (RR) missions have used various methods for euthanasia carcass preservation and tissue preservation. This study extends the sample preservation study performed by GeneLab in GLDS-49 which examined conditions used for the RR-1 mission to include conditions used for multiple RR missions and is designed to help determine factors which may confound data analysis. To determine whether these various factors affect changes in gene expression this ground-based study generated gene expression profiles measured by RNAseq from the livers of 20-21 week-old female C57BL/6J mice. Multiple interacting factors were investigated: 1) To understand how euthanasia protocols affect gene expression when mouse carcasses are slow frozen mice were euthanized by either euthasol injection ketamine/xylazine injection or CO2 inhalation and carcasses slow frozen on dry-ice mimicking carcass preservation in the MELFI on the ISS. Carcasses were thawed and RNA extracted from livers; 2) To understand how carcass preservation protocols affect gene expression mice were euthanized with euthasol and carcasses preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater following three-way segmentation. Carcasses were thawed and RNA extracted from livers; 3) To understand how tissue preservation protocols affect gene expression mice were euthanized with euthasol and livers dissected and processed immediately or preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater. Liver samples that were processed immediately were homogenized in RLT buffer and then either immediately further processed for RNA extraction or were stored for 70 days at -80C post-homogenization in sample RLT buffer prior to RNA extraction.

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes single cell transcriptional profiling data from the spleens from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

RR-1 is a validation flight to evaluate the hardware operational and science capabilities of the Rodent Research Project on the ISS. RNA DNA and protein were purified from liver tissues from RR-1 mice (female C57BL/6J 16wk old at time of launch). From each group two liver samples were collected and frozen immediately after euthanasia. The rest of the liver samples from each group were collected from frozen carcasses dissected post-flight. RNA-Seq whole genome and RNA BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed. RNA extracted from these tissues was re-sequenced; these data are available as part of GLDS-168 (https://genelab-data.ndc.nasa.gov/genelab/accession/GLDS-168).

The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight, particularly visual impairment, and joint tissue degradation. To this end, a flight group (FLT) of 10-week-old male C57BL/6J mice were launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean, the animals were transported to Loma Linda University (LLU) for testing, euthanasia, and dissection on 9/18/2018. Ground Control (GC) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However, all the GC mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018. Because this resulted in a different cohort of mice being used for the GC controls as compared to the flight (FLT) groups, two cohort controls were included in the study. The first, Cohort Control 1 (CC_C1), was from the same cohort as the FLT animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second, Cohort Control 2 (CC_C2), was from the same cohort as the GC, and was sacrificed and dissected 2-8 days after the GC, (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. Upon dissection, spleen tissues were preserved in liquid nitrogen and stored at 80 C before RNA was extracted. Only the flight (FLT, n of 10) and Ground Control (GC, n of 10) samples were processed and analyzed in this study. Libraries were generated using a 3’ Tag-seq approach and sequenced at a targeted depth of 40 M clusters (SE 93 bp).

RR-1 is a validation flight to evaluate the hardware operational and science capabilities of the Rodent Research Project on the ISS. RNA DNA and protein were purified from liver tissues from RR-1 mice (female C57Bl6/J 16wk old at time of launch) including eight from the Flight group and eight from the Ground Control group. From each group two liver samples were collected and frozen immediately after euthanasia (Flight mice dissected on-orbit after total 37 days after launch Samples FLT-M21 M22 and corresponding Ground Control samples GC-M31,M32). An additional six samples from each group were collected from frozen carcasses dissected post-flight (Samples FLT-M25,M26,M27,M28,M29,M30 and corresponding Ground Control samples GC-M35,M36,M37,M38,M39,M40). RNA-Seq whole genome and RNA BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed.

The objective of the Rodent Research-23 missions (RR-23) was to better understand the effects of spaceflight on the eyes, specifically on the structure and function of the arteries, veins, and lymphatic vessels that are needed to maintain vision. To this end, twenty male, C57BL/6J, 16-17 weeks old mice were delivered to the ISS on SpaceX-21 in a single transporter, transferred to two rodent habitats, and maintained in microgravity for 38 days. Flight mice were then returned to Earth alive (Jan 13th, 2021). After splashdown in the Atlantic Ocean, mice were transported to Kennedy Space Center via helicopter. The 20 Flight, 20 Habitat Ground Control (HGC), and 20 Vivarium Ground Control (VGC) mice were removed from Rodent Transporters (Flight and HGC) or vivarium cages (VGC), placed into shipping containers, and flown to Texas A and M University. There, mice underwent post flight procedures, before euthanasia and tissue collection. Flight, HGC and VGC animals were euthanized and dissected on Jan 14th, 17th or 20th of 2021, respectively. Spleens were preserved by immersion in RNAlater and stored at -80C until RNA was extracted, and libraries generated and sequenced (target 60 M clusters per sample, PE 150 bp). This dataset features 9 samples from the Flight group, 10 samples from the HGC group, and 9 samples from the VGC group.