High-capacity in vitro models for the assessment of pain-related mechanisms

Abstract

Chronic pain affects one in five people. Yet, new efficient drugs with fewer adverse effects remain an unmet need for chronic pain. In vitro models that closer recapitulate relevant disease mechanisms are suggested as a way forward to support drug development and to decrease the failure rate of drug candidates during the transition from preclinical to clinical studies. While current more physiologically relevant in vitro models have shown the possibility to replicate certain aspects of chronic pain diseases, these models are often complex and do not allow the automated experiments at scale which are required in a drug screening context. Therefore, in this thesis methods to create scalable in vitro models that closer resemble in vivo pain transmission were developed and evaluated. Moreover, high-capacity in vitro models were applied for mechanism of action studies.

In study I, a high-capacity microchannel plate (MC-plate) was characterized for the application in neuroscience research. The MC-plate parallelizes 96 compartmentalized cell cultures in a conventional 384-well format and was demonstrated to be compatible with primary rodent dissociated neuronal cultures of the peripheral and central nervous system, as well as human induced pluripotent stem cell (iPSC)-derived sensory neurons. In addition, the plate design allowed selective axonal growth to adjacent compartments and the fluidic integrity supported localized treatment.

The MC-plate was further used to develop an in vitro co-culture model of dissociated dorsal root ganglia (DRG) neurons and spinal cord (SC) neurons (Study II). The cell populations were cultured in spatially separated compartments, to attempt to replicate the pain pathway. In this way, the axons of DRG neurons were growing through the microchannels, thus providing the possibility to model synaptic connections of the first pain transmission point in the nervous system. The functional synaptic connections between the two cell types were assessed with electric field stimulation (EFS). EFS was applied to DRG neurons and induced a transient calcium influx in SC neurons in a timed manner. These results were validated using antagonists targeting receptors of excitatory synaptic transmission. The timed signals in the SC neuronal compartments were fully or partially inhibited by the AMPA receptor antagonist NBQX and NMDA receptor antagonist MK801, respectively, confirming that synaptic mechanisms are involved in the observed signal.

In study III, a novel technique of cell adhesion-based bioprinting was evaluated and used as an alternative method to create spatially defined DRG cultures. Several cell lines were successfully patterned in 2D and 3D using the bioprinting method based on cell-surface interactions. Moreover, dissociated DRG neurons were printed and cultured for up to 9 days in 2D.

In study IV, the mechanisms contributing to pain relief, observed in patients with rheumatoid arthritis (RA) when treated with the JAK/STAT antagonist baricitinib, were studied using both in vitro and in vivo techniques. A previously established high-capacity in vitro DRG model, in a conventional 384-well format, was used to investigate the direct effect of baricitinib on neuronal excitability and morphology. In the in vitro experiments, baricitinib decreased excitability of DRG neurons. Baricitinib also altered the morphology of satellite glial cells (SGCs) which was quantified as a decrease in length of glial fibrillary acidic protein (GFAP) positive extensions of SGCs. Moreover, combining the in vitro results with in vivo experiments of a collagen antibody-induced arthritis model confirmed baricitinib targeting JAK/STAT and SGCs but it also indicated adaptor-associated protein kinase 1 (AAK1) as an additional target in DRGs that contributes to the observed analgesic effect.

In summary, this thesis evaluated methods to create and apply scalable in vitro methods to contribute to mechanistic studies and drug screening in chronic pain in a drug discovery context.