Neuroscience

Multiple culture systems – one counting method

In neuroscience, a variety of cell-based models are being used to study the central and peripheral nervous systems, such as the brain and local neurons of a tissue, respectively.

The key questions that are being addressed are those of neural development, neural degradation and regeneration, and what signals affect the neural system not only locally, but also from the perspective of a whole organism, especially with the rise of microbiome research.

These research areas contribute to understanding the physical workings of the nervous system and how they affect the organism.

Neural cell sources and culture systems

Blue cells floating around in a blue substance
Work on neural cells in culture has long been done by obtaining a sample of mammalian brain tissue and culturing it in a physiologically relevant medium in a dish. Whether working with mature neuronal cells from the brain or neural stem cells (NSCs) obtained from the subventricular zone (SVZ) of the forebrain, the true advantage is that you can study actual neurons that have recently been assisting brain function in a whole organism. However, it is difficult to grow such cultures for a long time, as the neuronal niche is difficult to re-create, and neurons are often damaged during dissection and placement into the culture. Additionally, they could get infected with contaminants over time, as some culture systems run for several months to mature and test the brain-like structures in vitro. Many scientists prefer to work with human cells instead of animal cells, significantly limiting access to the working material.
An alternative source of cells is the neural progenitor cells (NPCs), obtained from neural differentiation of either embryonic or induced pluripotent stem cells (ESCs and iPSCs, respectively). Often grown in sphere cultures, called neurospheres1, these cultures can be maintained by regular passaging, where the spheres are dissociated, diluted and put back into culture. Further differentiation into mature neural cells offers a limitless material for studying the function of neural cells and identifies factors that can modify their responses to stimuli.

Differentiation into a more refined in vivo-relevant neural system has been tremendously elevated by the development of cerebral organoids, or mini-brains, by the Lancaster-Knoblich duo2. These complex neuronal structures contain cells of several different brain regions and are unique as a representative model of the brain.

There are many different culture systems within neuroscience, beginning with simple 2D cultures of adherent neurons, and increasing in complexity to the full 3D cultures of cerebral organoids. However, it is not a simple switch from 2D to 3D; rather it is a step-by-step evolution of culture systems from neurospheres and neural aggregates over neural rosettes to cortical spheroids and cerebral organoids. Each system has its benefits and challenges and is being used for exploring different types of scientific questions.

Neural disease studies

Neural tissues of the central nervous system (CNS) were traditionally thought to not be able to repair or regenerate upon injury3. During the past few decades, since the discovery of NSCs and NPCs, it is evident that there is significant plasticity within these systems, along with research into using them for a wealth of different studies, including4,5:

  1. Neurotoxicity testing
  2. Drug target validation and testing
  3. Brain development and injury modeling
  4. Disease modeling
  5. Cellular therapies to treat CNS conditions
  6. Neural tissue engineering (gene therapy) and repair
  7. Personalized medicine

Researchers are looking into cell therapies for conditions such as Parkinson’s disease, spinal cord injuries, Alzheimer’s disease and multiple sclerosis. In addition, they are trying to understand disease etiology and progression of new diseases such as microcephaly in newborns induced by the Zika-virus. They are turning to dendritic vaccines and other cell-based immunotherapies to combat glioblastoma, as well as exploring possibilities with personalized medicines, for example combining iPSC generation from patient samples with gene editing and using the differentiated cells to treat genetic diseases.

Solutions for controlling your neural cell cultures

In the Aggregated Cell Count Assay, you lyse and count the cells for the total cell count. For the dead cell count you stain the sample with DAPI before counting.

One of the initial and most basic challenges when culturing neural cells is maintaining tight control of the cell number both during culture system setup, maintenance, differentiation and testing. Especially in complex spheroid or organoid systems that must mature over prolonged periods, you are highly dependent on controlling the process along the way, so there is an optimal cell material to perform your testing protocols.

Since neural cells often grow in aggregates or more complex 3D cultures, it can be very challenging to dissociate them for accurate counting.

The NucleoCounter® NC-202™ automated cell counter robustly counts mammalian cells from complex samples and determines their viability. Even if the sample contains debris, the NC-202™ will give you unequaled reproducible data across samples, cell types, users, and instruments.

The automated cell counter features counting applications specifically for cells grown in suspension or adhesion, on microcarriers, and organoid cultures. Also, our de-clumping reagents ensure fast and consistent procedures to separate cells in aggregates and scaffolds.

With the robust NC-View™ software algorithm and unique Via2-Cassette™ sampling device, which eliminates human interference, you can focus on your experimental data collection and focus because you can rely on your initial cell culture setup.

References

  1. V Tropepe, S Hitoshi, C Sirard, et al.: Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron. 2001 Apr;30(1):65-78.
  2. MA Lancaster, M Renner, C-A Martin, et al.: Cerebral organoids model human brain development and microcephaly. Nature. 2013 Sep 19;501(7467):373-9.
  3. BA Reynolds, RL Rietze: Neural stem cells and neurospheres—re-evaluating the relationship. Nature Methods. 2005; 2, 333–336.
  4. CG Gross: Neurogenesis in the adult brain: death of a dogma. Nature Reviews Neuroscience. 2000; 1, 67–73.
  5. L Ottoboni, B von Wunster, G Martino: Therapeutic Plasticity of Neural Stem Cells. Front. Neurol. 2020; 11, 148.