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Expert tips on culturing human iPSC-Derived Sensory Neurons to aid your research

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Innovations in biotechnology and advances in stem cell biology are currently revolutionizing biomedical research and drug discovery.

One exciting breakthrough has been the ability to produce sensory neurons from human induced pluripotent stem cells (hiPSCs) and culture them in vitro on multi-electrode array (MEA) systems, to advance pain research and the discovery of effective pain therapies.

Although traditional in vitro cell-culture methods have used non-human mammalian neurons, it is now recognized that these animal-derived models lack physiological relevance and translatability to humans .

Here at Axol, our team of scientists have recently found that hiPSC-Derived Sensory Neuron Progenitors cultured on MEA systems can replicate the in vivo characteristics and firing responses of human sensory neurons and express the typical sensory markers involved in human nociception, offering a viable and more physiologically relevant model of human pain perception to replace animal-derived models.

Do you want to know how to culture hiPSC-Derived Sensory Neuron Progenitors in your lab? Then read on as our researchers offer you their top tips, helping you along the road to more reliable, translatable, and higher-impact research that could change the lives of patients around the world.

How to start culturing hiPSC-Derived Sensory Neuron Progenitors

In a recent study, researchers used our MEA chip cell-culture system to culture the hiPSC-Derived Sensory Neuron Progenitors, along with our Sensory Neuron Maintenance Medium and coating reagents. The study also used our step-by-step guideline to help with the set up and maintenance of the cell-culture itself, along with our MEA system guideline to assist with culturing the hiPSC-Derived Sensory Neuron Progenitors on an MEA system.

One key part of the protocol in particular, is to remove the non-neuronal population. This ensures a more homogeneous population of sensory neurons and is simple to do with the addition of mitomycin C to the Sensory Neuron Maintenance Medium after two days of culture. You should see the full effects of growth arrest after seven days.

The hiPSC-Derived Sensory Neuron Progenitors must then be maintained in the Sensory Neuron Maintenance Medium (containing growth factors Glial-Derived Neurotrophic Factor (GDNF), Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), and Neurotrophin-3 (NT-3)) for at least six weeks before performing any experiments; although you should start to see mature sensory neurons after just five weeks in culture.

Below our expert research partners share their five top tips to help your transition from conventional animal-derived cell-cultures to using more physiologically relevant hiPSC-Derived Sensory Neuron cell-cultures as easy as possible.

Five top tips for culturing hiPSC-Derived Sensory Neuron Progenitors

1. What types of cells will you see?

About 24 hours after thawing vials of the hiPSC-Derived Sensory Neuron Progenitors, you will see two types of cell under the microscope: fat, rounded neurons and darker, flatter cells.

Sometimes you will see more of the flat cells, because the hiPSC-Derived Sensory Neuron Progenitors are embedded in these, but don’t worry; the mitomycin C growth arrest treatment will get rid of these flat cells.

2. What happens after mitomycin C treatment?

It is normal to observe significant cell death after the mitomycin C treatment, which targets the flatter, darker, proliferating cells. This will make the population of hiPSC-Derived Sensory Neuron Progenitors more homogeneous (i.e. almost 80-90% pure sensory neurons).

From day five of cell culture, until around day ten, you’ll see the darker cells start to die and debris appear on the healthy cells, but don’t worry this all to be expected. After day 10, the sensory neurons will begin to become more prominent in the culture, with neurites becoming thicker from day 15 onwards. By this point, flat cells will be almost non-existent too.

3. How to ensure the best results from the mitomycin C treatment?

For each addition of mitomycin C to the Sensory Neuron Maintenance Medium, we advise you always make fresh mitomycin C to get the best results.

4. Don’t mistake hiPSC-Derived Sensory Neuron Progenitors for dying cells!

Under a phase contrast microscope, hiPSC-Derived Sensory Neuron Progenitors appear slightly rounder, have larger somas, and are lighter in color than other neuronal subtypes; be careful you do not mistake these for dying cells!

5. What will the sensory neurons look like after long periods in culture?

After longer culture periods (approximately five to six weeks in vitro ), neurites will become thicker and longer, and somas will become more spaced out.

Congratulations – you are now on the road to switching from conventional animal-derived in vitro cell-culture methods to using state-of-the-art hiPSC-Derived Sensory Neurons! Read our application note to find out how and why these cells can offer you a viable, more physiologically relevant in vitro model of human pain perception to make your research more reliable, translatable, and publishable.

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