Summary
This protocol describes how to isolate viable primary tumour cells from fresh biopsies or resected cancer tissue and expand them to approximately 80% confluence. The resulting cultures provide a physiologically relevant starting point for patient-derived organoids and other LunaX™ 3D models.
What you’ll do
- Prepare and mince fresh cancer tissue under aseptic conditions.
- Digest tissue with collagenase/hyaluronidase to release viable tumour-derived cells.
- Filter the suspension through 100 µm and 37 µm strainers before counting and viability assessment.
- Seed and expand isolated cells in supplemented organoid medium.
- Lift cultures for reseeding, analysis or downstream encapsulation in LunaX ECM.
Protocol at a glance
- Starting material: Fresh biopsy or resected cancer tissue transported in sterile medium on ice.
- Digestion: 1–2 hours at 37 °C and approximately 150 rpm, with gentle pipetting every 30 minutes.
- Initial seeding: Typically 1–3 × 10⁶ cells per T75 flask.
- Culture endpoint: Approximately 80% confluence, usually within 1–2 weeks.
- Next step: PDO generation in LunaX ECM using LX-APR-002.
Expected outcome
Viable tumour-derived cells should attach and progressively form a monolayer with minimal floating debris by the end of the first week. Expansion to approximately 80% confluence commonly takes 1–2 weeks, although success depends on tumour type, cellular composition and sample quality.
Critical parameters
- Process fresh tissue promptly and maintain appropriate biosafety controls for human-derived material.
- Mince tissue into approximately 0.5 × 0.5 × 0.5 mm fragments for consistent digestion.
- Confirm viability after filtration before committing cells to expansion.
- Refresh pre-warmed organoid medium twice weekly and monitor morphology throughout culture.
- Primary samples do not always establish proliferative cultures; successful expansion is reported for approximately 50% of breast cancer biopsy donors.
