Summary
This protocol describes the encapsulation of primary tumour-derived cells in LunaX CoreMatrix Low Stiffness ECM to establish patient-derived organoids. It provides starting conditions for reproducible 30 µL dome cultures in a defined, tuneable 0.5–1.5 kPa matrix.
What you’ll do
- Prepare LunaX ECM precursor and freshly reconstituted photoinitiator.
- Harvest expanded primary tumour cells and remove residual trypsin or protease.
- Resuspend cells at a recommended starting density of 1 × 10⁶ cells/mL.
- Dispense and photocrosslink 30 µL cell-laden ECM domes in a 24-well plate.
- Maintain PDO cultures for 3–14 days and optionally recover cells for downstream analysis.
Protocol at a glance
- Starting material: Primary tumour-derived cells isolated and expanded using LX-APR-001.
- Format: 30 µL domes in a clear, flat-bottom 24-well plate.
- Starting density: 1 × 10⁶ cells/mL, equivalent to 3 × 10⁴ cells per dome.
- Crosslinking: 10 mW/cm² for 60–90 seconds to achieve approximately 0.5–1.5 kPa.
- Culture period: 3–14 days, with medium replacement every 2–3 days.
Expected outcome
Rounded, often lobulated patient-derived organoids should form within the LunaX ECM dome and increase in size over the culture period. Formation and proliferation typically become apparent within 3–14 days, depending on tumour type and experimental endpoint.
Critical parameters
- Warm the LunaX ECM to 37 °C and protect the freshly prepared photoinitiator from light.
- Use photoinitiator solution only on the day it is prepared.
- Wash harvested cells twice to remove residual trypsin or protease, which can compromise hydrogel integrity.
- Remove all supernatant before resuspending cells to avoid diluting the ECM.
- Optimise cell density, stiffness, medium formulation and culture duration for each tumour type.
