Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels

Abstract

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Syk inhibitors protect against microglia-mediated neuronal loss in culture

“…However, care should be taken when comparing this in vitro morphological data to in vivo data, where resting microglia are ramified and become more amoeboid with activation (e.g., Ennerfelt et al, 2022). In our in vitro model and as seen by others (Balion et al, 2022), resting microglia adopt a largely unramified morphology and become more adherent upon inflammatory stimulation, which in 2D cultures may resemble ramification, but may simply reflect greater adhesion to the coated plate (Wollmer et al, 2001). Irrespective of this difference of morphology in vitro and in vivo, Syk inhibition did not inhibit the morphological transition induce by LPS, and thus Syk inhibition does not block part of the inflammatory response of microglia to LPS.…”

Disease phenotypic screening in neuron-glia cocultures identifies blockers of inflammatory neurodegeneration

“…This may be a peculiarity of these cerebellar neuron-glia cultures: cerebellar microglia are particularly round and non-ramified in vivo , 132 and in vitro we and others find that they adopt a prominent and exposed position on the surface of cultures. 56 , 133 , 134 Elsewhere, microglia are more clearly ramified and adherent, which likely renders them resilient to wash steps and ICC. In our case, we therefore continued with a live-cell staining approach which also proved practical and effective.…”