Material below is adapted from the SfN Short Course Purification and Culture Methods for Astrocytes, by Shane Liddelow, PhD. Short Courses are day-long scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.
Astrocytes are star-shaped glial cells that can clear debris, form scars, and take a number of other actions in response to a variety of central nervous system (CNS) problems, such as brain tumors, stroke, and neurodegenerative disease. But alongside beneficial effects, reactive astrocytes may also have a negative impact on CNS health.
In order to understand the diversity of roles that these cells can play, researchers have recently devised methods for isolating and growing them in the lab. A comparison of these methods will reveal their potential in future research, as well as bring to light the ways in which these methods could be enhanced. Purifying a single cell type can be a powerful tool, but it is important to keep in mind whether the method you choose for purification will allow you to investigate the aspects of the cells that you need to study.
In 1980, Ken McCarthy and Jean De Vellis published the first astrocyte purification system, termed the MD-astrocyte model, in which astrocytes are isolated from cultures of neonatal rat brains. Though this model yields large numbers of cells and is inexpensive, it can take several weeks to purify the astrocytes, which are often contaminated with other cell types.
Another limitation of the model is that, once purified, cells must be grown in culture media containing serum, which results in transcriptional changes and changes in cell shape, meaning the cells are not always great models for mature astrocytes. Since 1980, several researchers have improved upon this model by growing the cells without serum or in three-dimensional conditions that preserve some of their astrocyte morphology. It is not known whether the cultured astrocyte-like cells could return to a more typical shape and transcriptional profile.
Another option for astrocyte purification is immunopanning (IP), developed by Ben Barres in 1988. This method involves passing neonatal CNS cells over antibody-coated dishes and, when done correctly, results in a pure population of astrocytes that retain in vivo gene expression profiles. Shortcomings of this method include its expense and the challenges of purifying adult astrocytes. The method takes much less time than the MD-astrocyte strategy, but it involves a lot of components.
For the most part, astrocytes isolated using IP do behave similarly to astrocytes in vivo. A modification of the IP method recently allowed researchers to study reactive astrocytes in cell culture, and then confirm that the cells secrete a neurotoxin in rodents that kills neurons. These findings highlight the importance of using a culture model that is consistent with in vivo astrocyte function.
It is also possible to purchase commercially available cell lines, though purchased astrocytes may be contaminated with other cell types or change over time. Commercially available astrocytes may also be activated in ways that do not parallel in vivo conditions. Astrocytes derived from induced pluripotent stem cells (iPSCs) are another choice that can yield large numbers of cells, and have the advantage of being human patient-derived. Astrocytes from iPSCs can be expensive to make because of the components and time — sometimes months — required.
Culture systems for microglia — another type of CNS cell that responds to inflammation — are also fraught with complications. And distinguishing the contributions of astrocytes and microglia has been challenging because in vivo, these cells often act together. Continuing to refine strategies for culturing both cell types will help researchers understand their roles and interactions, and inform our understanding of how they affect the brain during disease.
