JAXA Rodent Experiment Requirements Futoshi MATSUNAGA (JAXA IS) Splinter session; JAXA Rodent Experiment Requirements -Mouse Epigenetics- 21st July 2015 Futoshi MATSUNAGA (JAXA IS) Motoki TADA (JAMSS IS) Thank you for this opportunity to explain our Mouse Epigenentics experiment. In this session, I will explain especially on the standpoint of execution, requirements and constraints. Notice：This technical data is furnished on the condition that it　will be used by and disclosed to the receiving Cooperating agency and its contractors and sub contractors only for the purposes of fulfilling the Cooperating agency’s responsibilities under the Space Station Intergovernmental Agreement (IGA) and Memorandum of Understanding (MOU). It shall not be used for any other purpose, nor disclosed or retransferred to any other entity or government without prior written permission of the Japan Aerospace Exploration Agency (JAXA). 1 1

Significance of Mouse Epigenetics High priority payloads Mouse Epigenetics is the JAXA first rodent research in ISS. The success of this experiment is very important to expand the area of life science investigation of JAXA. Crew intensive payloads This experiment have important crew procedures and maintenance activities to keep live mice in good condition. Special significance payloads which may attract media attention This experiment is the first mission that keeps and returns living mammals from ISS for JAXA. At first, this slide show the rationale of significance of Mouse Epigenetics experiment. First, this experiment is one of JAXA’s highest priority payload because mouse epigenetics is… And this experiment is Crew intensive payload 2

Mouse Epigenetics Research team Univ. Tokyo Immune system Osaka Univ. AIST Reproductive system Endocrine system Univ. Tsukuba PI: Prof. Takahashi Musculoskeletal system Central nervous system Gifu Univ. Tokyo Med. Dent. Univ. Vestibular system Skeletal system This slide shows the research team of Mouse epigenetics. The PI is prof Takahashi from Univ. Tsukuba univ. covered musculoskeletal system and central nervous system. Collaboration and Cooperation NASA ARC (Ames Research Center) NASA KSC (Kennedy Space Center) 3

Schedule of Mouse Epigenetics Inc. 44 Inc. 45 Inc. 46 7 8 9 10 11 12 1 MHU installation and Function Checkout should be completed by 2 months before SpX-9 launch. Weekly maintenance (Food/Water supply, Cleaning) are performed. MHU Checkout Experiment <30days> More than 2 months (for mice procurement and T/S) Mouse Habitat Unit (MHU) Launched by HTV5 Live mice (12) Launched by SpX-9 Live mice (12) Returned by SpX-9 - Mouse Habitat Unit (MHU) will be launched by HTV-5, then installed into CBEF in SAIBO Rack. After the function checkout of MHU, Live mice will be launched by SpX-9. - Weekly maintenance (Food cartridge exchange, Water resupply, Waste collecting equipment exchange, etc.) will be performed once a week. - Live mice will be returned by SpX-9. - Returned mice will be dissected on the ground and each mouse organ will be analyzed by researchers. Next, the schedule of this experiment is shown in this slide. The hardware of this experiment, Mouse Habitat Unit and other instruments will be launched by HTV5 from Tanegashima, then they are installed into SAIBO Rack. Hardware installation and C/O should be completed by 2 months before SpX-9 launch for the plan of mice procurement and trouble shoot if that will be occurred in Checkout. After the functional C/O of MHU hardware is finished, 12 mice will be launched by SpX-9. Mice will be kept in mouse habitat cage unit during SpX-9 berthing, and weekly maintenance for food, water supply and cleaning of MHU is performed by crew task in once a week. 12 live mice are returned to the earth with SpX-9. Returned mice will be dissected on the ground as soon as possible, and each mouse organ will be analyzed by researchers. In addition the important point for JAXA is to launch Mouse Habitat Unit by HTV-5 and checkout will be performed by JAXA crew, Yui in Inc44/45. Launching mouse unit by JAXA vehicle and performed checkout by JAXA’s crew promote the public recognition and it is crucial for sustainable ISS project for us. 4

Operations flow of MHU Checkout CBEF JPM1F3 (Glove box) Laptop Setup (ELT2 or MSPR ELT) Glove Box assembling Power supply from JEM PS 120 Junction Box (or MSPR or MSPR2) μG/1G Interface Unit attachment μG/1G Interface Unit attachment Euthanasia Unit Setup Connect Euthanasia Unit and ISPR with CO2 Hose Mouse Habitat Cage Unit attachment (μG/1G) Transfer following items to Glove Box. Mouse Habitat Cage Unit Mouse Habitat Cage Unit Pwr Cable Sample Container for Euthanasia Mouse Habitat Cage Unit attachment Checkout from Ground Operations Checkout for Mouse Habitat Cage Unit Pwr Cable Euthanasia Unit Mouse Habitat Cage Unit detachment (μG/1G) :Crew operations :Ground operations μG/1G Interface Unit detachment Glove Box closeout 5

Overview of Mouse Habitat Unit (MHU) Six (6) mice in micro G < Micro-G > Six (6) mice in 1 G Circulated for 1G From here, I will shows the hardware overview of the Mouse Habitat Unit. CBEF of SAIBO Rack has micro G section and circulated artificial G section. 12 mice are individually kept in Mouse Habitat Cage Unit in CBEF on Saibo Rack. 6 mice in micro-G and 6 in artificial 1-G condition to identify micro gravity effects for mice. < 1-G > Twelve mice are individually kept in Mouse Habitat Cage Unit in CBEF on SAIBO Rack. Six mice in micro-G and 6 in 1-G condition. 6

Detailed operational flow (1G I/F Unit and Mouse Habitat Cage Unit attachment) 1G I/F Unit A/B This slide shows the detailed operational flow of assembling 1G unit mouse habitat cage unit adaptor. 1G cage unit adaptor is saparated 2 part, and each part will be installed into CBEF 1G part following the cage unit installation. 7

Hardware; Glovebox The JAXA Glove Box (GB) is a partially disposable soft bag. The GB is attached to the front panel of a rack inside Kibo before mice arrival on orbit. Maintenance and mice transfer tasks are performed by crew inside the GB. Power Panel Fan Frame Glove Sample Container Mouse Handling Forceps Soft bag 8

Detailed operational flow (Glove Box assembly) This slide shows detailed operational flow of assembling Glove Box. Grove Box is attached in front of JPM1F3 payload rack known as Kobairo Rack. The Glove Box Base A and B is attached to the seat track on the both side of Kobairo Rack. Next, Glove box panel is attached to the 9

Hardware: Transportation cage unit (TCU) Controller Cage Air Inlet Odor filter (Test model) Air Outlet Particle filter Feature 12 mice be contained in one single CTB powered locker. No cage refreshing requirement for 10 days No feed and water resupplying requirement for 10 days Feed Air outlet Air Inlet Water Waste collection area 10

Activity Overview Experiment <SpX-9 Berthing period (30days)> Live mice (12) Launched by SpX-9 Live mice (12) Returned by SpX-9 Dragon ■1G mice have to be kept in 1G condition at least 26 days. ▼Mice transfer from Transportation Cage Unit to Mouse Habitat Cage Unit prior to experiment start. Performed inside in Glove Box. ▼Weekly maintenance (Food cartridge exchange, Water resupply, Waste collecting equipment exchange, etc.) is planned to be performed once a week. 1st Food and Water supply should be performed between 1 week and 24 hrs before mouse arrival. ▼Waste collecting equipment exchange is performed inside Glove Box. ▼Mice transfer from Mouse Habitat Cage Unit to Transportation Cage Unit prior to mice return. Performed inside Glove Box. This slide shows more detailed crew task timing during experiment. Crew task activity overview include launch and return. Because 12 live mice is launched by SpX-9 and returned by SpX-9, they are breed during SpX-9 berthing. PD required that mouse should be on orbit at least 26 days. Red triangle indicated that food and water supply. Just before arriving mouse by SpX-9, Food and Water should be supplied to the mouse habitat cage unit. During keeping mouse in Mouse Habitat Cage Unit, food cartridge exchange and water supply is planned to be performed once a week. 1st food and water supply should be performed between 1 week and 24 hrs before mouse arrival. Soon after SpX-9 Dragon berthing, mice transfer from transportation cage unit to Mouse Habitat Cage Unit should be performed as indicated in yellow triangle. In addition to food and water supply, waste collecting equipment exchange will be performed once a week or two weeks inside Glove Box. I will explain about Glove Box later. At last, Mice transfer from Mouse Habitat Cage Unit to Transportation Cage Unit just before the mice return. These transfer task will be performed inside Glove Box. 11

Overview of Mouse Habitat Unit Cage Main Function: Feeding automatically Watering automatically Two redundant water nozzle Keeping quality of life well (Air/Odor filter, Fans, Waste collecting equipment) Observation with camera & LED light Day & Night cycle 12 h day & 12 h night cycle Day 7:00- 19:00 (GMT) Camera/Light Watering unit Temp sensor Automatic Feeder Cage Cage Lid Automatic Feeder Waste collecting equipment Food cartridge This slide shows overview of Mouse Habitat Unit cage. It can keep one mouse inside it. The Cage unit have automatic feeder and watering unit. Watering unit has two water nozzle for redundancy. Also, it has odor filter, fans, waste collecting equipment for keeping mouse environment quality well. We can observe mouse from ground by CCD camera and LED light that can be controlled and operated from ground commanding,. Day and night cycle is planned to be 12h day and 12h night on 7:00-19:00 on GMT. Mouse waste is collected in Waster collecting equipment by air flow made by two funs attached on side of cage unit. Odor filter unit Fans Fig. Mouse Habitat Cage Unit 12

Hardware: Mouse Habitat Cage Unit (HCU) -Observation by video camera- Camera Features All 12 mice can be observed during real-time operations (with selected channel of view). All video images are recorded on external devices. An electrically controlled wiper with water tank is equipped to clean the window. Behavioral analysis can be conducted. Nighttime monitoring is possible with infrared rays (IR). Typical food (CRF-1) Special food bar (CRF-1) 13

Operations flow of Mouse Epigenetics Experiment start CBEF JPM1F3 (Glove box) μG/1G Interface Unit attachment (might have already been done) Glove Box assembling μG/1G Interface Unit attachment Transportation Cage Unit transfer from launch vehicle SpX-9 launch Mouse transfer from Transportation Cage Unit to Mouse Habitat Cage Unit Mouse Habitat Cage Unit attachment (μG/1G) Mouse Habitat Cage Unit attachment Maintenance operations: Once or twice per week Food cartridge exchange Water supply Odor filter exchange Waste collecting equipment exchange Off-nominal operations: Water gel insertion Mouse Habitat Cage Unit exchange Euthanizing Mouse Habitat Cage Unit transfer from Glove Box to CBEF Centrifuge rotation start Glove Box disassembling :Crew operations :Ground operations A Mouse care period (30 days) 14

Operations flow of Mouse return preparation CBEF/ELT2 JPM1F3 (Glove box) A Glove Box assembling Transportation Cage Unit watering and setup under Glove Box Mouse Habitat Cage Unit detachment (μG/1G) Mouse Habitat Cage Unit transfer from CBEF to Glove Box Mouse transfer from Mouse Habitat Cage Unit to Sample Container Mouse transfer from Mouse Habitat Cage Unit to Transportation Cage Unit Transportation Cage Unit transfer to berthed vehicle SpX-9 return :Crew operations :Ground operations μG/1G Interface Unit detachment Glove Box disassembling 15

Video Recording requirement during experiment This table shows the video recording requirement during experiment. Light/Dark cycle is changed at 19:00 and PD requested 2hrs recording everyday for nominal observation for mouse status check. It require real time downlink during AOS. In addition, additional 2 hrs recording is required for 4 days after experiment start. Also, weekly maintenance task is followed by 2 hrs video recording to check the food and water apparatus status. These recording require real time down link during AOS and video recording during LOS are followed by downlink as long as resource is available. 16

What if If a mouse looks unhealthy JPM1F3 (Glove box) Glove Box assembling CO2 Vlv Unit regulate CO2 concentration and supply CO2 gas via Glove Box. Power supply from JEM PS 120 Junction Box (or MSPR or MSPR2) Euthanasia Unit Setup Connect Euthanasia Unit and ISPR with CO2 Hose via Glove Box Transfer mouse with Mouse Habitat Cage Unit into Glove Box. Sample Container setup for euthanasia in Glove Box Euthanasia with CO2 gas supply (Gas supply for 16 min) This slide shows the procedure of euthanasia of mice. At first, crew assembles Glove Box because euthanasia is performed in Glove Box. Next, after connecting the power cable for Glove Box, Euthanasia unit is setup. CO2 valve unit regulate CO2 concentration and supply CO2 gas via Glove Box. CO2 concentration is regulated under the AVMA guidelines. After transfer the mouse into mouse sample container for euthanasia, CO2 gas is supplied for 16 min for euthanasia. After confirming the euthanasia, mouse is store into MELFI with triplicated ziplock bag. This procedure is approved by JAXA IACUC. Transfer Euthanized sample into MELFI with Ziplock bag Glove Box closeout Sample Container This procedure is approved by JAXA IACUC (Institutional Animal Care and Use Committee) 17

Rodent research in space NASA with AEM (Animal Enclosure Module) Rodent experiments on the ISS began in 2014 using AEM, which had been used for several Space Shuttle missions. Rodent experiments to be conducted twice a year are planned from now on. Experiments using a centrifuge are not planned. For the first ISS rodent experiment in 2014, 20 female mice were launched and no live return was planned. Onboard dissection was successfully completed. Italian Space Agency with MDS (Mice Drawer System) A 90-day mice experiment was conducted on the ISS/Kibo module used for NASA in 2009. Six mice were launched, but three died. No further experiment onboard the ISS has been planned. Russia with unmanned satellite named “Bion” Rodent experiments were conducted from 1970 to 1980. An artificial gravity facility was equipped for the Bion-4 experiment in 1977. In 2013, the Bion-M1 satellite was launched with 45 mice onboard, and returned 30 days later. Only 16 mice were alive due to system failure. (Source: NASA website) (Source: ASI website) (Source: IBMP website) 18

What if If a mouse looks unhealthy JAXA veterinarian for Mouse Epigenetics experiment makes a decision of euthanasia. Crew task (2 hrs (TBD) has to be planned to euthanize unhealthy mouse within 24 h after decision of euthanasia. MELFI and Video restricted resource will be required if euthanasia is planned. Euthanasia is performed with Euthanasia Unit in Glove Box. Euthanized mice is stowed in MELFI (-95 deg. C) and returned for ground analysis (8cm x 5cm x 13cm / 1 sample)(max 4). At last, I will explain the off nominal procedures of mouse epigenetics experiment. In the case of mouse looks unhealthy, JAXA veterinarian for Mouse Epigenetics experiment makes a decision of euthanasia. Within 24 hrs after decision of euthanasia, about 2hrs crew task has to be planned to euthanize unhealthy mouse. Up to 4 euthanized mice are planned to be returned to the earth, it require MELFI location and video restricted resource will be required if euthanasia is planned. Euthanasia is performed with Euthanasia unit in Glove box. The size of each euchanized mouse is 8 by 5 by 13 centimeters because it will be stored in triplicate ziplock bag. Euthanized sample is wrapped with triplicate Ziplock bag 19

Significance of Mouse Epigenetics High priority payloads Mouse Epigenetics is the JAXA first rodent research in ISS. The success of this experiment is very important to expand the area of life science investigation of JAXA. Crew intensive payloads This experiment have important crew procedures and maintenance activities to keep live mice in good condition. Special significance payloads which may attract media attention This experiment is the first mission that keeps and returns living mammals from ISS for JAXA. 20