Human iPSC-derived excitatory neurons provide helpful physiologically relevant cell models to investigate neurodevelopmental and neurodegenerative disorders, although achieving healthy neuronal cultures can be challenging. bit.bio central nervous system (CNS) scientist Dr Kaiser Karim has studied the genomic mechanisms governing reprogramming of human iPSCs into glutamatergic neurons and contributed to research investigating the effects of energetic substrate availability on global neural network behaviour.
In this blog, Kaiser shares his top tips on glutamatergic cell culture to help you overcome some of the most common issues. By using careful handling to prevent cell detachment, promoting cell adherence, perfecting routine media changes, and using the right cell density, it’s possible to achieve regular and consistent cell culture success!
Go straight to the top tips!
Glutamatergic neurons are cells that produce the common excitatory neurotransmitter glutamate, ‘exciting’ other neurons. These cells can be found throughout the CNS, for example within the cortex, where they are abundant, as well as the spinal cord and hypothalamus. Glutamatergic neurons contribute to a number of CNS-related functions such as cognition, memory, learning, and sensory perception1. They have also been implicated in a number of neurodegenerative diseases, including Alzheimer’s disease2.
The human iPSC-derived ioGlutamatergic Neurons provided by bit.bio are reprogrammed iPSC-derived cells and provide a highly relevant and consistent in vitro human model3. They form networks of postsynaptic dendrites and presynaptic axon projections, recapitulating the type of human neuron-to-neuron communication networks found in the body4.
"With such complex and fragile structures, culturing glutamatergic neurons in vitro requires advanced planning and special attention", cautions Kaiser, and so here are some top tips for success.
Top tip #1: Careful handling can prevent cell detachment from culture surfaces
Cell detachment is a common problem scientists face when culturing glutamatergic neurons. ioGlutamatergic Neurons, for example, can form neurites after only two days in culture and are ready for experiments in as little as 11 days, although they can be fragile. “As neuronal cell bodies form connections across the culture plate via neurites, the disruption of one neuron can impact the entire culture,” says Kaiser. With careful handling, however, the development of these neuronal networks will increase and mature over time.
There are 4 key considerations surrounding medium changes that can reduce the risk of cell detachment:
Human iPSC-derived cell cultures generally benefit from ECM coatings on the cell culture plate surface. After much testing and optimisation, we’ve found that poly-d-lysine (PDL)-Geltrex works very successfully for iPSC-derived human neurons. PDL is a synthetic, non-ECM coating molecule that improves cell attachment to surfaces via electrostatic interactions. We resuspend PDL in borate buffer rather than water, which in our experience is a critical factor in achieving success. Geltrex is a soluble basement extracellular matrix-derived coating that contains a cocktail of laminin, collagen IV, heparin sulphate proteoglycans, and entactin/nidogen. The presence of such molecules plays a role in recapitulating the physiological microenvironment, promoting cell-cell interactions and network activity5,6. With this double layer surface coating, ioGlutamatergic Neurons can be cultured for over 2-3 weeks, to enable functional studies into neuronal maturation and activity, explains Kaiser.
Getting your media change protocol right throughout the entire length of a cell culture is essential, including timings. For ioGlutamatergic Neurons, Kaiser thaws the cells from cryopreservation and seeds them onto cell culture surfaces on a Monday. The cells then undergo a stabilisation phase in the presence of doxycycline (dox) for four days. During this time, 90% medium changes are necessary every 48 hours. So, for example, he carries out a 90% medium change on a Wednesday (i.e., day two post-thawing, with dox and DAPT), followed by the last 90% medium change on a Friday (day 4, without dox). Once the stabilisation phase is over, from day 6/7 onwards, Kaiser recommends replacing 50% of the medium every 48 hours (see diagram below for further steps).
“This protocol is also helpful for those times when ‘life happens’, and you can’t make it into the lab at the weekend to do a media change,” Kaiser adds.
Using the right cell seeding density of cryopreserved glutamatergic neurons generally makes it easier to isolate, stain, image and analyse individual neurons, dendrites, axons and synapses, says Kaiser. However, commonly recommended seeding densities of >100,000/cm2 often make it challenging to carry out these methods.
bit.bio’s ioGlutamatergic Neurons can be seeded out at a much lower density - a minimum of 30,000 cells/cm2, and are compatible with plates ranging from 6- to 384-well formats. “We are confident in the quality of our neurons and recommend cell seeding at 30,000 cells/cm2 for everyday experiments,” says Kaiser.
At this density, ioGlutamatergic Neurons can be used in routine experiments and have been pre-validated for immunocytochemistry, qPCR, and RNAseq. The cells have also been validated for morphology characterisation, including brightfield imaging and quantitative live-cell imaging and analysis (like neurite outgrowth). Furthermore, at this density, the cells are cost-effective without impacting quality.
“For complex experiments, such as analysing neuronal network activity using neuronal electrophysiology or calcium signalling, higher seeding densities may be necessary, including additional optimisation,” adds Kaiser.
Applying the tips above to prevent cell detachment and enhance cell adherence, combined with well-regulated and timed media changes and the right cell density, should help make your glutamatergic neuron culture successful from the start. For more tips and information on ioGlutamatergic Neurons cell culture, check out this helpful step-by-step video with Kaiser or our FAQs.
Do you have any cell culture tips to share? We’d love to hear from you!
About Kaiser Karim, PhD
Kaiser completed his PhD in Clinical Neurosciences at the University of Cambridge, under the supervision of Dr Mark Kotter (NIHR Clinician Scientist, Consultant in Neurosurgery, University of Cambridge and CEO bit.bio), where he studied the genomic mechanisms that govern reprogramming of human iPSCs into induced glutamatergic neurons. This work followed the pivotal 2017 paper published by Mark Kotter’s academic research group. He has also contributed to work looking into the effects of energetic substrate availability on global neural network behaviour. Kaiser’s research has provided a deeper understanding of bit.bio’s ioGlutamatergic Neurons and helped lay the foundations for its commercialisation.