In Vitro Model to Study Pain

A multicellular system of dorsal root ganglions created by sound


Highlights

  • Dorsal root ganglions can be aggregated into multicellular systems

  • The degree of cell condensation can be controlled by cell density and patterning conditions

  • Enhanced neuronal communication and synchronised discharge was observed in patterned cells

The culture of dorsal root ganglion (DRG) cells is a useful model to study pain-associated mechanism. Classical, 2 dimensional mono layer cell cultures fail to recapitulate the in vivo situation and the multicellular structure is lost. The spatial organisation of DRGs is paramount for inter neuronal communication and can only be achieved in 3-dimensional constructs of accurate cell density and organisation. With sound, DRGs were aggregated into a multicellular system in collagen. With this, the first step towards an in vitro model to study pain was achieved. By tuning various parameters, such as frequency and acceleration, cell density and culture conditions, the degree of condensation can be tuned and its effect on a hypoxic or necrotic core was investigated. Simultaneously, the models allows to study the effect of the multi cellular structure on calcium signal synchronisation. (1)

Figure 1 A) DRG (cell line ND7/23) were aggregated into concentric rings using sound. B) The gentle, contactless method can also be used to pattern primary bovine DRG. In A and B DRGs were stained using Calcein-AM/Ethidium homodimer-1 (live dead staining). C) Aggregated DRG neurons developed neurite outgrowth after 2 days. Immunofluorescent labeling of neurofilament 200 (NF200 in green)and calcitonin gene-related peptide (CGRP in purple) was used to characterize the morphology of neurite outgrowth.


 

Figure 2 A) and A’) The neuronal aggregation is associated with a higher degree of calcium signaling synchronisation. Pairs of neurons were randomly sampled in either sound aggregated (patterned) or randomly distributed (control) DRG cells. B) and B’)Sound-induced aggregation caused a higher frequency of synchronized calcium events as compared to the control group. Synchronisation events are represented by horizontal lines.The neuronal discharge was captured using calcium imaging (Fluo4). (2)


 

Figure 3 The ratio of synchronised calcium events divided by non-synchronised calcium events was corelated with the degree of cell aggregation represented by the cell number imaged per field. These data provide evidence that a higher degree of aggregation correlates with a higher degree of synrchonisation events. The linear regression is displayed in red.

 

Experimental Conditions

Biomaterial: CollagenType 1, rat tail.

Cell Type: Cell line ND7/23 or Bovine Dorsal Root Ganglions Labware: Custom Made Labware, AO Workshop


References

(1) Results presented at eCM Congress 2021. https://www.ecmconferences.org/abstracts/2021/Collection3/poster.pdf

(2)Ma et al. Direct and Intervertebral DiscMediated Sensitization of Dorsal Root Ganglion Neurons by Hypoxia and Low pH, Neurospine 2020.