This research aims to develop and optimise an in vitro system for use as a high-throughput screening method to identify novel therapeutic agents which enhance (re)myelination, reducing the number of animals used in the study of neurodegeneration and de-myelinating diseases.
Treatments which enhance, maintain, or restore myelin, are a promising therapeutics for preventing further neurological decline in disorders of the myelin sheath, such as multiple sclerosis.
A number of potential molecular targets have been identified but further progress has been limited as identifying specific treatments is time-consuming, expensive, labour intensive and requires large numbers of animals for research and testing. An in vitro model for screening molecular libraries and identifying potential treatments which enhance myelination could potentially overcome these bottlenecks, and reduce animal use.
Research details and methods
This research will build upon an existing cell culture model of myelination using embryonic mouse spinal cord in order to develop a high throughput screening method.
The cell culture system will be adapted for use in multi-well formats, 96 or 384 well plates. A robotic system will be established and validated for liquid handling of parameters such as plating and feeding of cell cultures to optimise reproducibility.
To eliminate potential bias in collecting results, an objective automated analysis system will be developed using a high content screening microscope, and template analysis protocols will be developed to quantify effects of myelination.
The aim is to develop a high throughput screen, which uses a minimal number of animals, to identify therapeutic agents that will enhance remyelination in the context of a complex cellular environment mimicking that of the CNS in vivo.
The objectives are:
(i) to adapt an established myelinating cell culture system, from embryonic murine spinal cord, to a multi-well (96 or 384) format, that will minimise animal usage by a factor of at least ten.
(ii) automate handling and analysis for high throughput
(iii) automate data acquisition and analysis for high throughput and to eliminate user bias
(iv) validate the methodology, using factors known to modulate myelination in vitro, to confirm it provides the sensitivity and specificity required for high throughput screen
(v) to achieve i-iv using minimum numbers of experimental animals
(i) adapt existing myelinating cell culture techniques into a multi-well format to minimise animal usage
(ii) establish and validate the use of a robotic integrated liquid handling platform to automate all dispensing, feeding and processing steps
(iii) accelerate objective data acquisition using an IN CELL Analyzer 2000 high content screening microscope (GE Healthcare)
(iv) develop template analysis protocols using currint IN CELL level 3 software to quantify effects on myelination
Scientific and medical opportunities
The proposed project will provide ultimately a high throughput screening protocol that will have diverse scientific/medical applications. In the first instance it will provide, for the first time, the opportunity to screen molecular libraries for compounds that stimulate myelination by endogenous progenitor cells; an approach predicted to provide substantial clinical benefits in multiple sclerosis and other de- or dysmyelinating disorders of the central nervous system. It will also facilitate studies of autoantibody mediated effects in neurological diseases.
Bijland S et al. (2019). An in vitro model for studying CNS white matter: functional properties and experimental approaches. F1000Research 8:117. doi: 10.12688/f1000research.16802.1
Cumberworth SL et al. (2017). Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected. Acta Neuropathologica Communications 5(1):50. doi: 10.1186/s40478-017-0450-8v