BenchMasteryMolecular biology techniques & virtual lab

Co-culture

One cell type grows on a porous insert membrane and the other in the well beneath, so they communicate only through the shared medium; with an epithelium on the insert, TEER and transport across the layer report how the partner cells affect the barrier.

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Transwell inserts

One cell type grows on a porous insert membrane and the other in the well beneath, so they communicate only through the shared medium; with an epithelium on the insert, TEER and transport across the layer report how the partner cells affect the barrier.

How it differs from the standard method

  • Use 0.4 µm pores to keep cells apart; 3–8 µm pores let cells migrate.
  • Let an epithelium such as Caco-2 differentiate for about three weeks before co-culture.
  • Measure TEER at a stable temperature, with consistent electrode placement, subtracting a blank insert and multiplying by the area.
  • Each compartment can be sampled separately for cytokines or transported compounds.
In this lesson · about 2 min read
  1. Two cell types, one experiment
  2. Telling the effect apart from everything else
  3. How it goes wrong
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Two cell types, one experiment

In the body no cell lives alone. Epithelia sit on fibroblasts and are patrolled by immune cells, tumours are full of stroma and macrophages, neurons depend on glia.

0.4 µm

Transwell insert

Soluble factors cross, cells do not. Larger pores (3–8 µm) are for migration and allow cells through.

Ω·cm²

TEER

Electrical resistance across an epithelial layer, minus a blank insert, times the membrane area: a measure of how tight the barrier is.

E:T

Direct killing

Effector-to-target ratio; a titration of it gives a curve of killing against dose.

2Telling the effect apart from everything else

Co-culture results are easy to misread because there are more ways for something to change. Each cell type has to be grown in a medium that suits both.

3How it goes wrong

  • Large-pore inserts in an indirect experiment: cells migrate through and the compartments mix.
  • An epithelium tested before it has formed a barrier: there is little resistance to lose.
  • TEER reported in raw ohms, without a blank or area correction, or read as the plate cools.
  • No monoculture given the same stimulus: the effect cannot be credited to the partner cells.
  • Unlabelled populations in direct contact: nobody can say whose cells died.
  • No targets-alone wells: background death is counted as killing.
  • Effectors in poor condition (cytokine withdrawn) or cells kept apart in flat wells: little interaction.

Key terms

Practise these as flashcards →
Transwell insert

A cup with a porous membrane floor that hangs in a well, creating apical and basolateral compartments that share medium through the pores.

TEER

Transepithelial electrical resistance: (measured resistance − blank) × membrane area, in Ω·cm²; rises as tight junctions form.

Apical / basolateral

The top (lumen-facing) and bottom (tissue-facing) sides of a polarised epithelium, and the matching compartments of an insert.

Paracrine signalling

Signalling by secreted factors between nearby cells, the interaction an indirect co-culture isolates.

Effector : target ratio

The number of killer cells per target cell in a cytotoxicity assay.

Specific lysis

(Measured target death − spontaneous death) / (100% − spontaneous death): killing attributable to the effectors.

CFSE

A fluorescent dye that labels cells covalently, used to tell one population from another (and to follow division).

Conditioned medium

Medium taken from one cell type and given to another; a simpler, one-way alternative to co-culture.

When to use it

  • When the effect you study requires a second cell type, such as immune cells acting on an epithelium or tumour, or stroma supporting stem cells.
  • When you need to separate a contact-dependent effect from a secreted one, by comparing direct contact with a Transwell.
  • When monoculture results need to be tested in a setting closer to the tissue before moving to animals.

Compared with alternatives

Migration & invasion →

Uses the same inserts with 3–8 µm pores, but the read-out is the cells that cross the membrane rather than signals passing between compartments.

Organ-on-chip →

Adds flow, mechanical forces and separate channels for each cell type, at the cost of throughput.

3D culture & organoids →

Organoids and spheroids can be co-cultured with immune or stromal cells to add tissue structure to the interaction.

Flow cytometry & FACS →

The usual way to read out a direct co-culture, with each population identified by a label or marker.

Viability & cytotoxicity →

Plate-based release assays (LDH, calcein) measure killing without separating populations, which is simpler but less specific.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

One cell type grows on a porous insert membrane and the other in the well beneath, so they communicate only through the shared medium; with an epithelium on the insert, TEER and transport across the layer report how the partner cells affect the barrier.

  • Use 0.4 µm pores to keep cells apart; 3–8 µm pores let cells migrate.
  • Let an epithelium such as Caco-2 differentiate for about three weeks before co-culture.
  • Measure TEER at a stable temperature, with consistent electrode placement, subtracting a blank insert and multiplying by the area.
  • Each compartment can be sampled separately for cytokines or transported compounds.