Getting Started with Organoids

ESSENTIAL EQUIPMENT

Successful organoid culture requires a CO2 incubator (37°C, 5% CO2), laminar flow biosafety cabinet, inverted microscope with phase contrast, centrifuge (300-500g capability), and low-attachment culture plates. Cold storage (-80°C freezer, liquid nitrogen) is essential for Matrigel handling and organoid banking.

KEY REAGENTS

• Matrigel/BME: Basement membrane matrix (Corning, Cultrex)—keep on ice
• Growth Factors: R-spondin, Noggin, EGF, Wnt3a (tissue-specific combinations)
• Media: Advanced DMEM/F12 with supplements (STEMCELL IntestiCult, Hepaticult)
• ROCK Inhibitor: Y-27632 for survival during passaging and thawing
• Dissociation: TrypLE, Dispase, or mechanical dissociation

BASIC PROTOCOL

• Day 0: Isolate tissue/cells, embed in Matrigel domes, allow to solidify 15-30 min
• Days 1-7: Add growth medium, change every 2-3 days, observe formation
• Day 7-14: First passage when organoids reach 200-400μm diameter
• Passaging: Mechanical or enzymatic dissociation, re-embed in fresh Matrigel
• Banking: Cryopreserve in 10% DMSO with ROCK inhibitor

COMMON PITFALLS

Warm Matrigel causes premature polymerization—always keep on ice. Over-confluent organoids become necrotic centrally; passage before exceeding 500μm. Mycoplasma contamination is common—test regularly. Poor growth often indicates spent growth factors; ensure R-spondin and Wnt activity are validated.