iPSC-derived neurons and astrocytes in an OrganoPlate®

Screenable hiPSC neurons

Screenable hiPSC neurons

3D networks of neurons and glia in OrganoPlates®

3D networks of neurons (green) and astrocytes (magenta) differentiated from Axol neural stem cells

MIMETAS OrganoPlates® have proven to be an excellent platform to evaluate pharmaceutical compounds in functional 3D neuronal networks. OrganoPlates® contain 96 tissue chips that can be used to culture networks of brain cells, such as neurons and astrocytes, which can be subjected to parallel drug testing. Establishment of neural tissues in OrganoPlates® proceeds via two routes. As a first option, neuronal stem cells or neural progenitors are embedded in extracellular matrix (ECM) gel and differentiated over a period of 6-8 weeks to obtain mature cell types. Alternatively, mature iPSC-derived neurons and glia are embedded in ECM gel to establish defined neuronal populations that are electrophysiologically active within several days. Extensive series of experiments with neuronal cells, both in-house and by our clients, have shown that the OrganoPlate® supports 3D culture and differentiation of a wide spectrum of cerebral cell types, including glutamateric, GABAergic, and dopaminergic neurons, as well as astrocytes.

The assessment of neurotoxicity remains a major scientific challenge due to the complexity of the central nervous system. Current strategies to evaluate toxicity of chemicals and drug candidates are predominantly based on ex vivo or in vivo animal studies. These models have limited predictability for neurotoxicity in humans and are not amenable to high-throughput testing. Over the past few years, many research has focused on the genetically reprogramming of somatic cells into an embryonic-like state which results in induced pluripotent stem cells (iPSCs). These iPSCs offer a new tool for in vitro neurotoxicity screening. Previous experiment with iPSC-derived neurons have shown the compatibility of the OrganoPlate® with neuronal lineage differentiation. iPSCs were cultured and differentiated in the microfluidic channels for 6 to 12 weeks which resulted in a mixed population of neuronal cells.

In vitro brain models

Seeding neurons in ECM gels in OrganoPlates(r)
Seeding neurons in ECM gels in OrganoPlates(r)

Diseases of the central nervous system (CNS) represent one of the major problems in current health care. The brain is arguably the most complex organ in the human body and the disease mechanisms of many disorders remain to be fully understood. Although our knowledge on CNS disorders has tremendously increased over the last decades, this has not yet translated into effective treatments. This can in part be explained by the lack of physiologically relevant in vitro models.

Recently great advances have been made in the fields of induced pluripotent stem cell (iPSC) technology and developmental biology. Techniques have been developed that make use of cells of patients, for instance from skin, and reprogram them to a stem cell phase, from which these cells can be differentiated into a large myriad of cell types.

There is a need for a simple method that encompasses iPSC technology in a user-friendly platform to perform drug candidate screening on a large scale in labs across the world, while still mimicking the complexity of the human brain to the extent necessary to acquire physiologically relevant responses.

High-throughput drug candidate evaluation

Automated neuronal activity assays using calcium-sensitive dyes

Drug candidates and other compounds can easily be added to the 3D neuronal networks by means of a simple pipetting step. Various read-outs can be employed to evaluate the effects of the compounds. The viability of the cells can be assessed through fluorescent assays that evaluate plasma membrane integrity, or by assessing cell metabolism. We exposed co-cultures of mature human neurons and astrocytes to various concentrations of three different neurotoxic compounds and found concentration-dependent reductions in cell viability. Another, more sensitive, read-out for neurotoxicity was assessed by treating neuronal networks in the OrganoPlate® with various concentrations of methylmercury and evaluating neurite outgrowth. Lastly, compound effects on electrophysiology can be evaluated by employing calcium imaging techniques. Neuronal cells are loaded with a calcium sensitive fluorescent dye (Fluo 4-AM) and imaged over time to assess neuronal firing. We observed reduced firing after addition of GABA, the brain’s main inhibitory neurotransmitter, and TTX, a potent neurotoxin present in pufferfish, while addition of our medium control left the cells unaffected.

Outlook

The neuronal models in the OrganoPlate® are ideal for drug candidate evaluation, due to their user-friendless and compatibility with all standard laboratory equipment, such as pipettes, high-content imaging systems, and plate-readers. Moreover, the OrganoPlate® is the only system that incorporates microfluidic on-a-chip technology into a platform that allows high-throughput screening.

Personalized medicine represents the future of treating CNS disorders. With most disorders resulting from different causes with each patient, it is likely that tailor-made treatments are necessary to successfully target these diseases. Using iPSC technology, our method enables culture of 96 parallel neuronal-glial networks of one specific patient and allows testing of various compounds to evaluate the best treatment option, thereby increasing treatment efficacy while reducing adverse effects.

This work is part of an NC3Rs Crack-IT challenge and is performed with our partners IRAS and Cellular Dynamics International, in close collaboration with BASFSanofiGlaxoSmithKline and Abbvie.