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 quadriceps 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 quadriceps; 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 quadriceps; 3) To understand how tissue preservation protocols affect gene expression mice were euthanized with euthasol and quadriceps immediately dissected and preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater.

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 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. A Basal Control (BSL) was housed in standard cages at Kennedy Space Center (KSC) and euthanized one day after launch of the FLT animals (8/15/2017). Ground Control (GC) and Vivarium Control (VIV) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However all the GC and VIV mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC and VIV studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018 while the VIV was euthanized and dissected 6/22/2018 - 6/23/2018. Because this resulted in a different cohort of mice being used for the GC and VIV controls as compared to the flight (FLT) and basal (BSL) groups two cohort controls were included in the study. The first Cohort Control 1 (CC_C1) was from the same cohort as the FLT and BSL 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 VIV animals and was sacrificed and dissected 2-8 days after the GC and VIV groups (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. To clarify the connections between treatment groups and animal cohorts the following group abbreviations are used in the sample metadata: Flight (FLT_C1); Basal (BSL_C1); Ground Control (GC_C2); Vivarium Control (VIV_C2) Cohort Control 1 (CC_C1); Cohort Control 2 (CC_C2). Upon dissection livers were preserved in liquid nitrogen and stored at -80 C before RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated. or implanted with vehicle or treatment-filled nDS and launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of dorsal skin from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=9) ISS Terminal (n=9); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=6). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

Female C57BL/6CR mice were flown onboard STS-135 for 13 days and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Liver tissue samples that were used for microarray analysis for GLDS-25 were provided to GeneLab. GeneLab extracted RNA added ERCC control spike-in to the samples and performed RNA-Seq analysis.

In the NASA Rodent Research-1 (RR-1) validation study, ten 16-week-old female C57BL/6J mice were flown to the ISS for 37 days before euthanasia and subsequent dissection (Flight group). Due to crew time constraint, only two (out of ten) mice were dissected immediately after euthanasia to recover spleen and liver tissues on the ISS. The remaining eight animals were euthanized, then intact carcasses were placed in a pre-chilled cold stowage container and stored in the MELFI. There were respective cohorts of age-matched basal animals which were euthanized one day after launch as a baseline control (Basal control group) as well as age-matched ground control animals kept in an ISS Environmental Simulator at Kennedy Space Center (KSC) on a 4-day delay to mimic spaceflight conditions (Ground control group). In addition, the NASA Validation study also had a cohort of age-matched vivarium control animals that were housed in the vivarium cages and followed the same experimental timeline and process as the spaceflight animals (Vivarium control group). This dataset was generated only from Flight (n of 4) and Ground control (n of 8) animals that were euthanized and preserved intact on-orbit for subsequent dissection on the ground. Libraries were generated using a 3’ Tag-seq approach and sequenced at a depth of 40 M clusters (SE 93 bp).

In the Rodent Research Reference Mission (RRRM-1), forty female BALB/cAnNTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 10-12 week-old and twenty 32 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 22-23 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 40 days and allowed to recover for 2 days (Live Animal Return, LAR) before sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice housed in flight hardware in matched environmental conditions), basal controls (10 mice sacrificed 1 day after launch), and vivarium controls (10 mice housed within standard vivarium habitats). Thus RRRM-1 included a total of 160 mice. This datasets features ribodepleted total RNA-seq data from livers dissected from all groups in RRRM-1. Data from 7-10 livers per group are included. All samples include either Mix 1 or Mix 2 of the ERCC spike-in control.

Female C57BL/6CR mice were flown onboard STS-135 for 13 days and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Liver tissue samples that were used for microarray analysis for GLDS-25 were provided to GeneLab. GeneLab extracted RNA, added ERCC control spike-in to the samples, and performed RNA-Seq analysis.

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. A Basal Control (BSL) was housed in standard cages at Kennedy Space Center (KSC) and euthanized one day after launch of the FLT animals (8/15/2017). Ground Control (GC) and Vivarium Control (VIV) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However, all the GC and VIV mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC and VIV studies were therefore rescheduled and begun in May, 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018, while the VIV was euthanized and dissected 6/22/2018 - 6/23/2018. Because this resulted in a different cohort of mice being used for the GC and VIV controls as compared to the flight (FLT) and basal (BSL) groups, two cohort controls were included in the study. The first, Cohort Control 1 (CC_C1), was from the same cohort as the FLT and BSL 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 VIV animals, and was sacrificed and dissected 2-8 days after the GC and VIV groups, (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. To clarify the connections between treatment groups and animal cohorts, the following group abbreviations are used in the sample metadata: Flight (FLT_C1); Basal (BSL_C1); Ground Control (GC_C2); Vivarium Control (VIV_C2), Cohort Control 1 (CC_C1); Cohort Control 2 (CC_C2). Upon dissection, livers were preserved in liquid nitrogen and stored at -80 C before RNA was extracted, libraries generated (stranded, ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).