Co-cultures and multi-cell cultures

Fig 1. Measuring network activity in microglia-neuronal co-culture. (A) Representative calcium activity traces of ioGlutamatergic Neurons expressing Incucyte® Neuroburst Orange Lentivirus in monoculture or co-culture with ioMicroglia at day 15 post-addition. Monitored using the Incucyte® Neuronal Activity Analysis software. (B) Quantification shows increased mean network correlation in co-culture, with comparable mean burst duration (mean ± SEM, n = 3–12). Data courtesy of Sartorius.

Co-culturing ioMicroglia with ioGlutamatergic Neurons increases synchronised firing without altering burst duration. This demonstrates how defined cell co-culture systems can reveal microglial influence on neuronal activity, supporting studies of neuroinflammation and neuro-microglial network dynamics.

Fig 2. Live-cell imaging of GFP ioMicroglia in co-culture with ioGlutamatergic Neurons. ioGlutamatergic Neurons were cultured to day 10 post-thaw. GFP ioMicroglia, cultured to day 10 post-thaw, were added directly to day 11 ioGlutamatergic Neurons. Co-cultures were maintained for a further 3 days before live-cell imaging on a Leica DMi8.

Constitutive GFP expression allows for the tracking of ioMicroglia within a neuronal network without antibody staining. This supports live-cell imaging of microglial morphology, motility and activation state, and simplifies cell sorting in multi-cell cultures.

Fig 3. Confocal imaging of motor neuron–skeletal muscle co-culture. ioMotor Neurons were co-cultured with ioSkeletal Myocytes and analysed by confocal microscopy at day 30. Alpha-bungarotoxin (yellow) staining highlights acetylcholine receptor expression on co-cultured ioSkeletal Myocytes. Meanwhile, desmin (cyan) and microtubule-associated protein 2 (red) identify ioSkeletal Myocytes and ioMotor Neurons, respectively. (40X magnification.)

In co-cultures of ioMotor Neurons and ioSkeletal Myocytes, acetylcholine receptor expression indicates early neuromuscular junction-like organisation. This co-culture method provides a foundation for studying neuromuscular junction formation and related cellular processes.

Fig 4. MEA analysis of glutamatergic, GABAergic, and astrocyte tri-cultures. ioGlutamatergic Neurons and human iPSC-derived astrocytes were cultured alone or with increasing numbers of ioGABAergic Neurons and analysed on a 48-well Axion Maestro Pro MEA platform. (A) Network burst frequency increases in excitatory cultures and decreases with the addition of inhibitory neurons. (B) Active electrode counts show sustained spontaneous activity across conditions. Data courtesy of Charles River Laboratories.

ioGABAergic Neurons integrate into excitatory co-cultures to form functional networks and modulate overall network activity. This tri-culture system facilitates the modelling of excitatory–inhibitory interplay in vitro, enabling further study of circuit dynamics relevant to epilepsy and neurodegeneration.

Fig 5. Immunofluorescent characterisation of a quad-culture model. Representative immunostaining shows ioOligodendrocyte-like cells expressing MBP (green), ioGlutamatergic Neurons expressing NF-200 (red) and TUBB3 (red), ioMicroglia expressing IBA1 (yellow), human iPSC-derived astrocytes expressing S100B (yellow), and DAPI counterstain (blue). Cultures were analysed on day 14.

By combining neurons, oligodendrocyte-like cells, microglia, and astrocytes, this quad-culture demonstrates successful integration of multiple human CNS cell types into a single in vitro system, offering a platform to study neurodegenerative processes relevant to Alzheimer’s disease and multiple sclerosis.

Fig 6. Automated generation of human neuronal microtissues. ioGlutamatergic Neurons and iPSC-derived astrocytes were bioprinted on the Inventia RASTRUM™ platform to form 3D co-cultures in a CNS-mimetic hydrogel, supporting high-throughput screening in 96- and 384-well formats. Data courtesy of Inventia Life Science.

Consistent inputs matter even more as model architecture grows. ioCells have been bioprinted into reproducible 3D neuronal microtissues and integrated into BrainZell brain organoids, where ioMicroglia add measurable inflammatory signalling, cytokine release and pharmacological modulation.

Start from one of our verified ioCells combinations, from neuron–glia co-cultures to neuromuscular and tri-cultures, then extend it with our optimisation guide. Each protocol gives step-by-step instructions, media recipes and recommended readouts.

Working with a combination we have not verified yet? The multi-cell culture protocols and optimisation guide covers coating, plating strategy, seeding densities, media transitions and validation of functional crosstalk.