CNS Toxicity | Mimetas

CNS Toxicity

CNS Toxicity

3D CNS model for neurotoxicity testing

The rapid progress in the development of human induced pluripotent stem cells (iPSCs) allow us to grown human brain in a manner that recapitulate the inherent physiological characteristics of the neurological system. Here we describe the development of a human in vitro 3D central nervous system (CNS) model from iPSC derived neurons and astrocytes in the OrganoPlate®. These models are used for screening of neurotoxicity as well as for modelling of diseases such as Epilepsy, Parkinson’s disease and Alzheimer’s disease.

Benefits

  1. Formation of 96 3D human iPSCs derived CNS model in the OrganoPlate®
  2. Complex neuronal network formation applicable for high throughput neurotoxicity screening
  3. Precise imaging of neuronal outgrowth and histochemical stainings
  4. Individual tracing of electrophysiology through calcium imaging
  5. Reduce the use of animals as the gold-standard in neurotoxicity testing

Figure 1

Method

Figure 2b
Figure 2a

Figure 2

A MIMETAS OrganoPlate® 2-lane was used that harbours 96 individually perfused chips. To obtain 3D neuronal network structures, neurons and astrocytes derived from human iPSCs were mixed with Matrigel® and seeded in the OrganoPlate®. Perfusion was induced by placing the OrganoPlate® on the OrganoFlow® to enhance the diffusion of growth factors from the medium compartment towards the cells in the adjacent compartment. Complex networks of neurons and astrocytes were formed after 24 hours of culture as shown by expression of neuronal marker β-III-tubulin and astrocyte marker glial fibrillary acidic protein (GFAP) (figure 2). 

Toxicity testing

Figure 3c
Figure 3a/b

Figure 3

Exposure to Methylmercury shows a dose dependent effect on the viability of the co-culture. The effect of the neurotoxicant was shown with 1) immunofluorescent staining to assess network integrity using the β-III-tubulin and GFAP marker (figure 3a), 2) TMRM assay to assess the mitochondrial membrane potential (figure 3b) and 3) WST-8 assay to assess the cell viability. The neurons and astrocytes both were affected by the neurotoxicants as shown by the disrupted network of the neurons and changed morphology of the astrocytes.

Calcium imaging

Electrophysiological activity of the 3D neuronal network can be visualized by imaging of calcium efflux (see figure 4). Each pulls corresponds to a train of action potentials. Image analysis allow to trace each individual neuron through time, allowing to extract information on pulse duration, frequency and intensity. This data can for example be used to test for seizurogenicity of compounds (i.e. causing epilepsy).

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Figure 4

This work was supported by NC3Rs Innovation Platform through the CRACK IT Neuratect Challenge; NeuroScreen-3D: microfluidic microtiter plates for in vitro neurotoxicity screening.