3D culture & organoids
Cells, usually a tumour or stromal line, are seeded into ultra-low-attachment round-bottom wells, hanging drops or agarose, where they aggregate into one compact sphere per well that develops oxygen and proliferation gradients as it grows.
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Spheroids
Cells, usually a tumour or stromal line, are seeded into ultra-low-attachment round-bottom wells, hanging drops or agarose, where they aggregate into one compact sphere per well that develops oxygen and proliferation gradients as it grows.
How it differs from the standard method
- Seeding density and culture time set the size; plan for the size range your assay needs.
- Round-bottomed wells give one spheroid per well; flat wells give several aggregates.
- Above roughly 400–500 µm diameter a necrotic core forms in most dense lines.
- Change half the medium gently; spheroids are not attached.
1Why grow cells in three dimensions
On flat plastic every cell sits in the same environment: the same stiffness underneath, full access to oxygen and nutrients, no neighbours above or below. Real tissue is nothing like that.
The oxygen limit
In a dense spheroid oxygen is used up within about 150–250 µm of the surface; beyond that cells stop dividing and then die.
Spheroids
Cells that cannot attach to the plate gather and compact into one sphere per round-bottomed well.
Organoids
EGF, Noggin and R-spondin in a Matrigel dome let intestinal stem cells rebuild crypts.
2Size, gradients and the necrotic core
A spheroid grows only from its surface. Cells in the outer rim divide, those a little deeper become quiescent as oxygen falls, and once the radius exceeds the distance oxygen can diffuse before it is consumed, the centre becomes anoxic and dies.
3How it goes wrong
- Too many cells per well or too long in culture: oversized spheroids with large dead cores.
- Flat-bottomed low-attachment wells: several irregular aggregates per well.
- A tissue-culture-treated plate: cells attach and no spheroid forms.
- Unmixed suspension while plating: sizes drift across the plate.
- Full medium aspiration: spheroids lost or torn.
- Warm Matrigel or medium added before the dome sets: flat domes and 2D growth.
- Missing R-spondin: organoids die within a few days.
- Too many fragments per dome: fused, dark organoids.
Key terms
Practise these as flashcards →A roughly spherical aggregate of cells grown without attachment to a surface; widely used as a simple 3D tumour model.
A self-organising 3D structure grown from stem cells that reproduces some of the architecture and cell types of an organ.
The dead centre of a large spheroid, where oxygen and nutrients do not reach.
Plasticware with a hydrophilic, protein-repellent coating that prevents cells from attaching.
A basement-membrane extract from a mouse sarcoma, rich in laminin and collagen IV, that gels at 37 °C and supports organoid growth.
A drop of matrix and cells set on a plate and covered with medium; the usual format for organoid culture.
A secreted protein that binds LGR4/5 receptors and enhances Wnt signalling, required to maintain intestinal stem cells in culture.
The outgrowth of crypt-like domains from an intestinal organoid, a sign of active stem cells.
When to use it
- When the response you study depends on tissue structure, cell–cell contact or gradients, such as drug penetration, hypoxia or invasion.
- When you need a renewable, patient-derived model of an epithelium (gut, liver, airway, tumour) that keeps its cell types.
- When a monolayer result needs testing in a more tissue-like setting before moving to an animal model.
Compared with alternatives
Monolayer culture is simpler, cheaper and easier to image and quantify; use it unless the third dimension matters for the question.
Many organoids start from pluripotent stem cells, whose maintenance and differentiation come first.
Chips add flow, mechanical forces and several tissue compartments, at the cost of throughput and complexity.
Scaffolds impose a designed geometry and stiffness instead of letting cells self-organise.
Whole spheroids and organoids are too thick for widefield imaging; light-sheet or confocal imaging resolves their interior.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cells, usually a tumour or stromal line, are seeded into ultra-low-attachment round-bottom wells, hanging drops or agarose, where they aggregate into one compact sphere per well that develops oxygen and proliferation gradients as it grows.
- Seeding density and culture time set the size; plan for the size range your assay needs.
- Round-bottomed wells give one spheroid per well; flat wells give several aggregates.
- Above roughly 400–500 µm diameter a necrotic core forms in most dense lines.
- Change half the medium gently; spheroids are not attached.