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a University of Florida Scholars Program. We thank Austin Hinkle for helping with creating microfluidic devices as an intern at Micro-gRx."},{"name":"DOI","value":"10.26030/5asv-zv75"},{"name":"Data Source Accession","value":"GSE234465"},{"name":"Data Source Link","value":"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE234465"},{"name":"Experiment Platform","value":"Tissue Chips in Space"},{"name":"Flight Program","value":"International Space Station (ISS)"},{"name":"Funding","value":"We thank the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida NextGen Sequencing Core (RRID:SCR_019152) and Alberto Riva in the Bioinformatics Core (RRID:SCR_019120). The authors acknowledge that this study was supported by the National Institutes of Health National Center for Advancement of Translational Sciences (5UG3TR002598 to S.M.) and the Center for the Advancement of Science in Space (CASIS) user agreement #UA-2019-011 to the University of Florida. Space Tango, Micro-gRx and Micro Aerospace Solutions were funded under a National Aeronautics and Space Administration (NASA) task order award (to T.C. at Space Tango, L.K.M at Micro-gRx, and D.P. at Micro Aerospace Solutions)."},{"name":"Identifiers","value":""},{"name":"Managing NASA Center","value":"Kennedy Space Center (KSC)"},{"name":"Mission End","value":"01/13/2021"},{"name":"Mission Link","value":"https://osdr.nasa.gov/bio/repo/data/missions/SpaceX-21"},{"name":"Mission Name","value":"SpaceX-21"},{"name":"Mission Start","value":"12/05/2020"},{"name":"Project Identifier","value":"CUBELAB"},{"name":"Project Link","value":""},{"name":"Project Title","value":""},{"name":"Project Type","value":"Ground Study"},{"name":"Space Program","value":"NASA; NIH"},{"name":"Study Grant Number","value":""}],"description":"Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. 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All participants provided written informed consent, and the study protocol was reviewed and approved by the institutional review board at AdventHealth, Orlando (IRBNet  554559). Cells were received at the University of Florida through a signed Material Transfer Agreement. The active group engaged in structured exercise at least 3 times per week and the sedentary group exercised one or less times per week. Satellite cells from individual donors were isolated, pre-plated to remove fibroblasts and pooled in equal ratios to generate a mean cell stock of young, active cohort (YA; 21\u201340 years, male, n equals 5) and old, sedentary cohort (OS, 65\u201380 years, male, n\u2009 equals \u20095) prior to cryopreservation and shipment to the University of Florida through a Material Transfer Agreement and in the absence of any patient identifiable information. Muscle myoblasts were thawed and then cultured on T75 flasks coated with collagen I in Skeletal Muscle Growth medium (PromoCell, Heidelberg, Germany) to confluence. Immunopurification of mononuclear myoblasts was performed using mouse monoclonal 5.1H11 anti-CD56 antibody (DSHB Hybridoma Bank, Iowa City, IA). CD56+ myoblasts were confirmed by FACS as described.","name":"Growth protocol","parameters":[{"@id":"#139783235787504","parameterName":{"@id":"a501H74krDu15ta3jJQ0VvF7eOcWIKFAsKNna6Di","annotationValue":"Culture Media","comments":[],"termAccession":"http://purl.bioontology.org/ontology/MESH/D003470","termSource":"MESH"}},{"@id":"#139783235787024","parameterName":{"@id":"iv3lsRtDm6VbFzk3toRlZpLyZSCYkkM4CGEsrAVi","annotationValue":"Immunopurification Marker","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139783235786016","parameterName":{"@id":"b4VwZtHwmCSqXOaZDPDVDnK3W9DHrKgJEARk2D54","annotationValue":"Source Tissue Preservation Method","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}}],"protocolType":{"@id":"0gMEkcMNeDUnXJOXyMiO3HWs7cSThiG9RpoYliDD","annotationValue":"growth protocol","comments":[],"termAccession":"http://www.ebi.ac.uk/efo/EFO_0003789","termSource":"EFO"},"uri":"","version":""},{"@id":"#139783222261024","comments":[],"components":[],"description":"Pre-Flight Treatment:\nCustom microfluidic chips made from polydimethylsiloxane (PDMS) and containing two platinum 22-gauge electrode leads were obtained from Micro-gRx, INC, (Orlando, FL), sterilized in ethanol and autoclaved at 150\u2009C for 30\u2009min prior to use. Cell seeding was performed at NASA\u2019s Kennedy Space Station Processing Facility (SSPF) for flight and at the University of Florida laboratory for the ground test. Injectable hydrogel mixtures (3.3\u2009mg/mL rat tail collagen I, and 22% (v/v) Matrigel) were combined with the 5-donor pooled YA- or the 5-donor pooled OS-derived CD56+ enriched myoblasts. The cell-laden hydrogels were injected into the PDMS chips to a final cell density of 15 and 20 million cells/mL for pooled YA- and pooled OS-derived cells, respectively. Cells were allowed to polymerize at 37\u2009C for 60\u2009min and seeding ports were sealed using polylactic acid (PLA, Makerbot, Brooklyn, NY) plugs. Chips were perfused with degassed Skeletal Muscle Growth media supplemented with 0.1\u2009mg/ml Primocin (InvivoGen, San Diego, CA) at a constant flow rate of 1\u2009mL/h at 37\u2009C and 5% CO2 using programmable syringe pumps (Cole Palmer, Vernon Hills, IL). After 48\u2009h, the culturing medium was changed to degassed differentiation media I (MEM-a, 0.5% (v/v) ITS, 2%(v/v) B27,10\u2009uM DAPT, 1\u2009uM Dabrafenib, 20\u2009mM HEPES, pH 7.3 and, 0.1\u2009mg/mL Primocin) and delivered at an intermittent rate of 125 ul min\u22121 for 1\u2009min every 8\u2009h for 6 days before integrating the chips for flight or ground studies.\n\nFor spaceflight tissues, the sterile CubeLab fluidic system was flushed with sterile Phosphate-Buffered Saline (PBS) followed by priming with degassed differentiation media II (MEM-a, 0.5% (v/v) ITS, 2%(v/v) B27, 20\u2009mM HEPES, pH 7.3 and, 0.1\u2009mg\u2009ml-1 Primocin). PBS effluent was collected, plated, and incubated at 37\u2009C for 5 days followed by RT (20\u201325\u2009C) for 5 days (sterile PBS was plated as control). No colonies were present confirming sterility of the fluidic system. 48\u2009h prior to launch (L-2), tissue chips were exchanged with 1\u2009ml media, disconnected from the syringe pump, inspected under a 10x microscope and 16 chips were integrated into a custom designed 9U CubeLab (Space Tango, Lexington KY). Of the 16 chips, there were eight chip replicates of the YA mean cohort and eight chip replicates of the OS mean cohort (8x YA and 8x OS). On L-3, flight chips were secured PDMS-side down onto fluid connection ports which allowed for fluid-tight mating to the manifold chip posts via press fit connections. A low pulsatile annular gear pump and Takasago-modified valves, controlled fluid to each chip. Two thermoelectric coolers located on the opposite side of the chips ensured consistent temperature (37\u201339\u2009C). The stimulation circuit was connected to eight chip\u2019s stimulation leads via blue wire and connectors custom to the stimulation board mounted on one side of the manifold. 200\u2009ml of chilled, degassed media II was added to the source bag and placed into the cold flask (4\u20138\u2009C). Chilled RNALater (20\u2009ml) was added to a second source bag and placed into the cold flask (4\u20138\u2009C). Fluid from each chip (200\u2009ml) was routed to general waste. The payload was sealed, purged with 5% CO2 using an air gas canister and the thermal protocol was initiated to bring the manifold to 37\u2009C. Prior to handover, a leak test was performed to verify pressure integrity and the CubeLab was integrated into the powered accent utility locker (PAUL). Once in the PAUL, the camera system was initiated and calibrated for each chip position and the Media Exchange-Waste sequence I (outlined below) was initiated prior to handover to NASA at L-1.\n\nFlight Treatment:\nOn-orbit payload operation  After docking (L\u2009+\u20092), the CubeLab was installed by crew members and operated from within the PAUL. After install, the following protocols were initiated: (1) The Media Exchange-Waste sequence 2. During this event, media was flowed to each chip every 8\u2009h at 125\u2009ul per \u2009min for 1\u2009min and output volume was sent to general waste. (2) The Z-Stack event. During this event, the microscope (10x magnification) was moved to chip N and captured six phase contrast slices at 12\u2009um increments at each chip\u2019s pre-defined position immediately before and after each Media-Exchange-Waste event. (3) The Stimulation and Video event was initiated on L\u2009+\u20099. During this event, 8 chips (4x YA and 4x OS) were electrically stimulated. The remaining 8 chips (4x YA and 4x OS) were non-stimulated. The intended program involved moving the microscope to chip N\u2019s ideal position and recording video at 25\u2009fps for 40\u2009s (pre-stimulation phase), then at the 41\u2009s mark after video initiation, a stimulation waveform (3\u2009V, 2\u2009Hz, 2msec) on chip N was elicited and video was to be recorded for an additional 40\u2009s (stimulation phase). Finally, at the 82\u2009s mark after video initiation, the stimulation waveform ceased, and video was to be recorded for 40\u2009s (recovery phase) for total of 120\u2009s of video for each of the 8 chips. The program was to be repeated every 24\u2009h for 5 days. The executed program included stimulation of the 8 chips for 30\u2009min on one day prior to initiation of fixation.  Asynchronous ground experiment  The ground experiment was performed at the University of Florida approximately 30 days following the return and de-integration of the flight study. Aliquots from the same lot of pooled YA and pooled OS cells (8x YA and 8x OS) used for flight were used for the ground study and the 16 tissue chips were seeded and differentiated as described in the Preparation of Bioengineered Myobundles (Flight and Ground) section. On the equivalent of day \u201cL-3\u201d, ground chips remained connected to the syringe pumps in the cell incubator (37\u2009C and 5% CO2) and differentiation media I in 3\u2009ml syringes were changed to degassed differentiation media II and delivered to the ground chips every 8\u2009h at 125\u2009ul per \u2009min for 1\u2009min to mimic the integration of the flight chips into the CubeLab and the switch from media I to II. On the equivalent day as \u201cL\u2009+\u20099\u201d, 8 chips (4x YA and 4x OS) were electrically stimulated with wave form 3\u2009V, 2\u2009Hz, 2msec for 30\u2009min using a custom microcontroller and circuit board as described1. No video was collected as we did not have the capability to record inside the incubator. The remaining 8 chips (4x YA and 4x OS) were non-stimulated.  On-orbit payload operation  The Fixation event. During this event, RNALater was initiated to each chip at 125 \u2009ul per \u2009min for 5\u2009min at termination of the experiment. Crew members removed the CubeLab from the PAUL and transferred to the ICEBERG cold stowage asset, at \u221235\u2009C. Continuous telemetry from the CubeLab environment was recorded in the customer dashboard portal. Z-stacked images of each chip per day and the stimulation video were downlinked to the portal system. 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