3D tumour spheroids for drug testing

Who it is for

• Pharmaceutical and biotech oncology discovery groups testing small molecules or antibody-drug conjugates on 3D tumour models
• Contract research organisations that offer 3D spheroid drug screening
• Academic cancer biology and gravitational biology labs studying 3D growth, adhesion and signalling
• Space agencies and research funders with cancer and microgravity programmes

What earlier flights found

How LELP-1 runs it

Sample. Human cancer cell line chosen by the customer (for example FTC-133 follicular thyroid cancer cells, as flown before), about 1e5-5e5 cells per well. Optional fluorescent reporter line for in-flight imaging. Container. Large bay, 24-well cassette with gas-permeable lids, linked to the bay fluidics (medium, vehicle, drug, fixative and RNA-stabiliser reservoirs, waste bag). Well surface chosen by the customer: standard surfaces if the question is whether microgravity alone drives spheroid formation, or low-attachment surfaces for more even spheroids (these also form spheroids at 1 g). Launch. Cryopreserved at -80 C in the passive cryo cassette; thawed, washed free of cryoprotectant and seeded automatically in orbit. Light and gas. Dark except during imaging. No CO2: HEPES-buffered medium in the sealed bay with gas-permeable lids. No robotic-arm moves during the aggregation days if possible, to keep about 1e-6 g. A lower setpoint (CellBox-2 used 28 C) can be chosen to slow growth.

1. day 0

   Launch. Cells held at -80 C in the passive cryo cassette. Bay pre-conditioned at 37 C with medium reservoirs filled.

2. day 1

   Automatic thaw, dilution and wash to remove cryoprotectant; seed cell suspension into all 24 wells. Baseline brightfield image of every well.

3. day 2

   Free aggregation in microgravity. Brightfield imaging of each well every 12 h to count aggregates and measure their size.

4. day 4

   Imaging continues. Bay logs temperature, pressure, acceleration and dose so aggregation can be linked to any disturbance.

5. day 5

   Automatic partial medium exchange in all wells (Shenzhou-8 and CellBox-2 both exchanged medium 5 days into the flight).

6. day 6

   Dosing: 8 wells vehicle only, 8 wells low drug dose, 8 wells high drug dose, pumped from sealed drug reservoirs. Image all wells before and 2 h after dosing.

7. day 7

   Daily brightfield and fluorescence imaging (reporter or viability dye) to follow spheroid size and outline under drug. Optional: arm moves the bay to the central 40x microscope for close-up images.

8. day 10

   Add a viability dye to a subset of wells and image. Then fix: 12 wells with RNA stabiliser (for RNA and protein), 12 wells with buffered paraformaldehyde (for imaging and histology), 4 of each per dose group.

9. day 11

   Replace paraformaldehyde with buffer after about 24 h to avoid over-fixation. Set the bay to 4 C and hold until handover to the courier.

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. The catalog lists no stage for this microscope: it is drawn inside the microscope's mass.
• 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: 7 reagent bags in 4 layers (128 x 93 x 92 mm), every port on a front edge.
• The waste bag lies under the reagent bags and fills the room they free: they hold 712 mL, it needs 204 mL.
• The catalog gives 0.25 kg for structure and containment; as an aluminium box that is a 0.12 mm skin on 0.07 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

An identical large bay and cassette, with the same cell batch, medium, drug doses and fixatives, runs on the ground at 1 g. It replays the flight timeline and temperature log from telemetry, a few hours behind the flight. This follows CellBox-2, where ground controls used the same temperature and time profiles as the flight. A second ground set on a random positioning machine (simulated microgravity) is optional. Without an in-flight centrifuge, launch, re-entry and radiation effects cannot be fully separated from microgravity.

Coming home

Samples are fixed in orbit, so no live cells need to come back. On CellBox-2, fixed cells were stored at 4 C. Hold the bay at 4 C through re-entry, recovery and courier transport. Paraformaldehyde is replaced with buffer in orbit to prevent over-fixation. 96 h is our estimate; the customer should confirm it for their assays.

Limits of this flight

• Spheroid formation is not guaranteed. The CellBox-2 paper reports that no spheroids formed in the earlier CellBox-1 mission, where cells had become a confluent monolayer after launch delays.
• Lab time is 14 days at most. A standard ground spheroid screen (spheroid start, drug treatment, integrity and survival endpoints) takes about 170 h and fits. Measuring growth delay and regrowth after a drug needs 14 days or more of culture, which LELP-1 cannot offer after the thaw and aggregation days.
• Spheroids that form freely in microgravity vary in size and number. This makes drug-response data less uniform than plate-based ground screens.
• Large spheroids (millimetres across) develop gradients of oxygen and nutrients. This affects drug penetration, and the bay microscope cannot see inside them.
• No crew: wells cannot be checked by hand, individual spheroids cannot be picked, and doses cannot be changed except by pre-loaded reservoirs.
• Cells launch frozen and are thawed in orbit. Thaw stress and launch loads may affect early behaviour.
• No CO2 supply: HEPES-buffered medium only, and pH may drift over 10 days.
• Without an in-flight 1 g control, radiation, launch and re-entry effects cannot be fully separated from microgravity.
• Cells are fixed in orbit, so live spheroids cannot be brought back for regrowth tests or further drug treatment.
• The two heritage flights cited here used one thyroid cancer cell line (FTC-133). Results may not carry over to other tumour types.
• Neither heritage flight tested a drug on the spheroids. Drug dosing here is the LELP-1 proposal, not a flown result.