C. elegans muscle and ageing

Who it is for

• University C. elegans and muscle biology groups studying spaceflight muscle loss and ageing
• Space agency life-science and astronaut countermeasure programmes
• Pharma and biotech teams screening drugs against muscle wasting, sarcopenia or muscular dystrophy in a whole animal
• Companies that make C. elegans phenotyping tools (for example microfluidic strength or imaging assays) and want flight data

What earlier flights found

How LELP-1 runs it

Sample. C. elegans hermaphrodites: wild-type N2 plus one muscle-relevant strain chosen by the customer, for example dys-1 (BZ33), a body-wall muscle GFP reporter, or the Q35::YFP polyglutamine line. Each well holds about 50-100 age-synchronised L1 larvae. Container. 24-well gas-permeable culture cassette (about 0.5 mL liquid per well, thin gas-permeable film lid) inside the sealed bay. Two identical large modules fly: Module A (young-adult time point) and Module B (aged time point). Each bay block holds only one temperature, so the two time points need separate bays. Launch. Live, starved L1 larvae arrested in buffer (M9 with cholesterol) without food, held at 12 C. CERISE used a similar approach: L1 larvae held in stasis in buffer, then fed on orbit to start the experiment. Light and gas. Dark, except for short LED pulses during imaging. Air headspace through the gas-permeable lid in the sealed bay; no CO2 needed. Worm number and liquid volume are kept low because MME ground tests found gas exchange limits growth in sealed bags.

1. day -2

   Ground: bleach gravid adults, hatch eggs overnight in buffer to get synchronised L1 larvae, load each well, seal the cassettes, close the triple containment. Load reservoirs: freeze-dried E. coli OP50 food, FUdR, paraformaldehyde fixative, RNA preservation solution. Hold at 12 C.

2. day 0

   Launch. Both modules hold 12 C; larvae stay arrested without food.

3. day 1

   After orbit checkout and once the arm is parked (quasi-steady microgravity), raise both modules to 20 C. Pump bacterial food into every well to start development. Take baseline brightfield images of each well.

4. day 2

   Daily, both modules: 30-60 s brightfield video per well (swimming frequency, body length, stage); fluorescence images if a reporter strain flies. Downlink a compressed subset.

5. day 3

   Worms near L4: add FUdR to both modules (NIS used 75 uM) to stop new offspring before egg laying starts. Top up food.

6. day 5

   Module A (about day 1 of adulthood): final video and fluorescence imaging. Then fix 12 wells with paraformaldehyde (for muscle staining and body length) and fill 12 wells with RNA preservation solution (for gene expression). Drop Module A to 4 C and hold it there until the courier hands it over.

7. day 7

   Module B: top up food and FUdR. Continue daily imaging. Repeat every 2-3 days.

8. day 13

   Module B (about day 9 of adulthood): final video and fluorescence imaging. Fix 12 wells and preserve 12 wells for RNA, as in Module A. Drop Module B to 4 C.

9. day 14

   Deorbit and splashdown with both modules at 4 C. Recovery ship hands the sealed modules to the customer's courier (about 20 min after splashdown); cold transport at 4 C to the lab (about 1-2 days).

The payload module, in CAD

Every item in the module sheet, drawn and packed into the large bay deck by deck. The back face mates with the bay: blind-mate connector, and a cold wall against the bay's thermoelectric block. The front face carries Dexter-L's grapple fixture, and the fluid lines run down a channel just behind it. What you load before launch is marked blue in the configurator's payload guide.

CAD generated by cad/build_modules.py (CadQuery, OpenCascade kernel) from the catalog's module items: concept geometry, not a released design. Standard parts keep their real formats; the rest are sized from their catalog mass. The front panel and grapple fixture are left off in the 3-D view and are in the STEP.

• The XY stage travels 96.5 x 57.9 mm, so every one of the 24 wells reaches the objective; with the stage the imaging stack is 232 x 151 mm, which sets the module's depth budget.
• Reagent volumes the catalog does not state are split evenly from 75 % of the reservoir item's mass. The waste bag holds the spent medium and washes; fixatives and stabilisers stay in the wells and come home.
• Fluid pack: 4 reagent bags in 2 layers (164 x 120 x 46 mm), every port on a front edge.
• The waste bag lies under the reagent bags and fills the room they free: they hold 675 mL, it needs 169 mL.
• The catalog gives 0.80 kg for structure and containment; as an aluminium box that is a 0.38 mm skin on 0.24 kg of posts and shelves. Thin: the bay rails have to carry the launch loads, or the module needs more structure mass.
• The grapple fixture stands 26 mm proud of the front panel, into the bay opening; everything else is inside the bay envelope.

Ground control

Two identical ground modules with the same worm batches, food, reservoirs and cassette lots, run in a flight-like bay. Each step follows the flight timeline, with the bay's recorded temperature profile replayed about 1-2 days behind the flight (micro-16 replicated the flight temperature profile on a two-day delay). Ground modules are fixed, preserved, chilled and shipped the same way. This control cannot separate microgravity from space radiation or launch and re-entry loads. That needs an in-flight 1 G centrifuge control, as flown on CERISE and NIS.

Coming home

Fixed and RNA-preserved worms return sealed at 4 C. Module A samples are about 9 days past preservation at splashdown and about 10-11 days by the time they reach the lab. The 336 h (14 day) window is OUR ESTIMATE, and the customer must check it on the ground for both the fixative and the RNA solution. Heritage flights froze worms for gene and protein work: liquid nitrogen within 2 h of landing on ICE-First, and -80 C on orbit on CERISE, MME and micro-16. Only the NIS morphology samples were PFA-fixed and held at 4 C. If live worms are wanted for after-flight motility or lifespan tests, an extra well set must return unfixed at 15-20 C and reach the lab within about 48 h; this is also our estimate.

Limits of this flight

• 14 days of lab time covers development and early ageing (to about day 9 of adulthood) only; the worms' full lifespan cannot be followed in orbit
• No on-orbit freezing to -80 C. All the heritage gene and protein datasets we checked came from frozen worms, so chemical preservation (RNA solution, paraformaldehyde) at 4 C is a change that must be validated on the ground
• No crew, so the micro-16 strength assay (crew loaded worms into micropillar devices) cannot be repeated as flown; strength would need a new automated chip
• Without an in-flight 1 G centrifuge, the experiment cannot separate microgravity from radiation and launch or re-entry effects; CERISE and NIS had this control
• Sealed wells limit oxygen and food, so worm number per well must stay low; MME ground tests found gas exchange limited growth in sealed bags, and a lid-only well has less gas-exchange area per mL than the heritage bags
• The bay microscope is low magnification; sarcomere and mitochondrial detail needs the 40x central microscope or post-flight staining
• Arm moves raise acceleration to about 1e-4 to 1e-3 g, which may disturb swimming measurements; imaging must avoid those periods
• About 2 h to hand-over and 1-2 days to the lab means live worms partly readapt to 1 g before any post-flight motility test
• Worm muscle has no satellite stem cells and differs from human muscle; results point to mechanisms and need follow-up in mammalian models
• Two L bays per run use a large share of the 16 large bays