1. Rodent Habitat (RH) & Rodent Research (RR) Concept of Operations December 2013 ARC/Cecilia Wigley Rodent Research Operations Manager JSC/Brienne Shkedi Research Integration Manager JSC/Pete Hasbrook Program Science Office

2. Contents Rodent Research Concept of Operations – Project Overview – Hardware – Operations – Rodent Research – 1 Mission Overview (SpaceX-4) – Operations Requiring Two Crew – Procedures Suggested for RS Crew Member Participation RR-1 Data sharing opportunities

3. Project Overview Rodent Research (RR) includes of a suite of hardware developed by ARC – Rodent Habitat – provides on-orbit housing for rodents in an EXPRESS rack – Transporter – provides housing for rodents during ascent on Dragon – Animal Access Unit (AAU) – Interfaces with both the Habitat and Transporter for transfer of the animals between the units and access to the animals for science operations 3 – Hardware Support Kits (foodbars, clean up, and containment bags) – Experiment Unique Equipment (Specific to each flight based on science requirements) – Ex: Anesthesia Kit and Cardiac Puncture Kit being developed for RR-2 (on SpX-6)

4. Other Projects Supporting RR Operations and Science – Wetlab-2 (ARC) Provides modular, plug-and-play equipment that enables in situ sample collection, processing and analysis for space biology and biomedical research Flying with RR-1 on SpX-4: Basic Animal Processing System – Animal Health Kit, Anesthesia Kit, Basic Animal Dissection Kit – Bone Densitometer (Techshot) Provides bone density scanning capability on-orbit Needed for RR-2 on SpX-6 – Wetlab Kit (JSC Biostronautics Contract) Provides various generic lab equipment for sample processing and manipulation – Rodent Tissue Box (CASIS/Bioserve) Provides formalin and ethanol fixation of hind limbs Needed for RR-1 and RR-2 4 – MSG Life Science Ancillary Hardware (LSAH) (MSFC) Enhancements to support biological handling and clean up Air Filtration System, Exchangeable Glove System, and Decontamination System MSG Rear Wall Cover and Rodent Dissection Table also built by MSFC as a subset of this project. Project Overview

5. Based on existing AEM design Flown 27 times on Shuttle Modified to meet ISS needs Reduce acoustics Improve airflow Accommodate floor lighting Add cameras (4) Change power and data (STELLA) Single MLE unit houses 10 mice or 3 – 6 rats (2 on RR-1) Front breather Temperature and RH monitoring, no active thermal control Transfer animals to a clean Rodent Habitat with a full complement of food and water after 20 - 30 days to achieve longer duration missions Improved science and animal husbandry through video monitoring and in-flight access Adapted to operate at the ISS/EXPRESS Rack Launches and returns as passive stowage Powered only during duration of experiment operations Animals housed in two groups of five on either side of the Habitat Rodent Habitat with lid removed Rodent Habitat With new Access Unit mating access lid NASA Ames Research Center5 5 Rodent Habitat Hardware Overview November 14-15, 2012 Side A Side B

6. Rodents are launched in the Transporter – Single MDL – Current design is a front breather, but potentially switching to a rear breather – Power ~35 W for fans and lights – Each unit houses up to 20 mice. Multiple units may be flown depending on science requirements. Transporter Overview Front Breather Transporter Ducting used on SpaceX for airflow – Animals loaded in the Transporter at L-25 hrs – Or as late as possible. – Transporter certified for 10 days – Would support up to 2 launch attempts before change out with a new Transporter

7. Frozen Samples Refrigerated Stabilized Rodent Habitat w/o animals (passive transport) Transporter w/ animals (20 mice, 10 days support) SpX 3. Ascent ~ 3 days 4.ISS Dock and Animal Transfer NLT Dock + 48 hours, transfer animals from Transporters to Rodent Habitats (w/ AAU). Stow Transporter for return. 5. On-Orbit 7. Descent 8. Sample Recovery Generic RR Concept of Operations 7 Rodent Habitat w/ animals (10 mice, 30-45 days support) SpX EXPRESS Racks AEM 5. On-Orbit 6. Transfer Remaining Live Animals and samples Mouse Transfer Box MSG Tissue and Blood Samples DXA Bioprocessing and Analysis System Descent Activities may require MSG Cold Stowage Habitat On-Orbit ConOps: Animal Processing System Stabilized samples Mouse Transfer Box Dissections and DXA using the Animal Processing System and Bone Densitometer Animal Processing System Access Unit 1. Late Load Transporters 2. Launch (same or next) SpX Empty AEM-T (same or next) SpX Empty AEM-T

8. 20 mice launched in one Transporter, 10 per side – 10 NASA Mice: 16-week-old C57Bl/6 female – 10 CASIS Mice: Ten 16-week-old female mice (5 MuRF-1 knockout and 5 wild type, C57Bl/6 background Transferred to 2 Rodent Habitats on-orbit – NASA RH: 5 per side – CASIS RH: Split on each side 5 knockout/5 wild type Ops for CASIS Mice (SpX-4 Return) – 0g exposure for minimum 21 days, but as long as possible – Dissection of all 10 mice as follows, to occur over a max 3 day period Alternate wild type/knockout mice, 1st hindlimb dissected and frozen, 2nd hindlimb be dissected and formalin-fixed. Transfer to ethanol 48 +6 hours, Liver and spleen dissected from 5 wild type mice. Preserve per NASA protocol below. Remainder of each mouse carcass frozen – Frozen samples returned at -32C or colder Ops for NASA Mice (SpX-5 Return) – Housed in RH for 30 days (± TBD, pending biocompatibility test end Sept) – Dissect liver and spleen from 2 animals Fix spleens in RNA Later. Freeze livers in a cryovial. Remainder of each mouse carcass frozen – Remaining 8 animals euthanized and frozen for body weight measurements – Frozen samples are returned at -80C RR-1 Mission Design (SpaceX-4) 8

9. Freeze/pack CASIS samples for return RR-1 Timeline & Ops Constraints 9 NLT 48 hr after berthing; based on 10 day Transporter Lifetime Requires unpacking of Habitat and associated kits, configuring Habitat for ops in EXPRESS First health check ASAP after hatch open CASIS min 21 days in 0 g NASA min 30 days in RH CASIS dissection w/in 3 days max AHC: Animal Health Check Transfer: Transporter to Habitats WLC: Water Level Check T stow: Bag and stow Transporter X Stow: Bag and stow Habitat Dissect: Euthanize & Dissect Euth: Euthanize remaining animals TT: Tissue Transfer CBI: Cold Block & Ice Brick Insert CB: Crew Brief (15 min)

10. Operations Requiring Two Crew Certain Rodent Research procedures will require 2 crew members – Confirmed during operations validation test (OVT) in August – Recommended by MDS crew during debrief Most important during initial ops and dissections until prime crew member is comfortable with the activity. ALL procedures performed handling the animals are done with both hands inside the AAU or MSG. Second crew member needed as an assistant. – Animal Transfer from Transporter to Habitat Holding the flashlight Record keeping of animal number, type, condition Potential Simultaneous Ops: second crew can also assist with prepping the Habitats while the primary crew member is configuring the Transporter and AAU – Dissection and Tissue Transfer Operations Reading lengthy/multi-page procedures Pointing out key steps (such as matching proper labels for animals and their tissues) Record keeping as needed Retrieving any missing items as needed (i.e. primary needs more gloves or gauze or other lab supplies) Potential Simultaneous Ops: second crew member could also do CASIS tissue transfers in the glove bag (day 22/23 on the timeline in my charts) while the primary crew finishes the CASIS dissections. – Some experiment specific training may be required for second crew member depending on the level of participation. 10

11. 11 Transfer of animals from Transporter. Required helper holding flashlight and recording number/type of animals pulled from the Transporter. Limited space inside the MSG with RR equipment set up. No room to bring procedures inside. Operator with hands inside the MSG relied on helper to read procedures and point out key steps during the long dissection operations.

12. Procedures Suggested for RS Crew Member Participation 1.Procedure reader for all activities except the initial transfer operations 2.Time keeper for dissections 3.LSAH set-up in the MSG 4.All cold stowage activities – assumes they have received systems training on MELFI and Glacier – Coldbag configuration – MELFI inserts – Glacier inserts 5.Formalin to Ethanol tissue transfer – assumes CASIS is providing OBT for this activity 6.Cleaning and stowing the AAU after animal access operations 7.Stowing kits at the end of sessions 8.Water level assessments 9.Configuring and bagging the Transporter and Habitats for stowage and return in Dragon 10.Final cleanup of MSG Estimated RR-1 Russian crew time: 25-30 hours; – See 39/40 OOS for additional details

13. RR-1 Data Sharing Opportunities RR-1/SpaceX-4 Data to be provided to Russian Scientists – Data from 10 NASA mice can be provided to Russian scientists All mice will be euthanized Up to two will be dissected on-orbit (removal of liver and spleens). Cadavers and specimens will be returned on SpaceX-5 Data can be provided after NASA analysis of returned samples – via geneLAB? Tissue sharing is not available on RR-1 – CASIS data is proprietary and will not be shared with Russian scientists Possibilities for broader collaboration on future NASA Rodent Research missions – requires more discussion – Tissue sharing – Russian-defined rodent specimens – Expanded crew time sharing