Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.
The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.
Part 1: Setting up (1-2 days before dissection) (Not shown on video)
Part 2: Preparing for Dissection (day of dissection) – (Demonstrated in video)
All of the following procedures are performed in a tissue culture hood unless noted.
Part 3: Dissection and Meninges Removal
Part 4: Cell suspension
Part 5: Percoll Gradient Separation
Part 6: Pre-plating and Plating
This protocol is based on modifications of procedures that have been described in the past3,7,11. There are several important points to note as discussed below.
The granule neurons and progenitor cells adhere within 2 hours of plating. Healthy cells have a round morphology under the phase-contrast microscope4. Within 24 hours after plating, healthy cells will spread evenly around the coverslips or the plastic well and will form processes. At the time of the first medium change, after 24 hours, there will be a few dead floating cells. This is normal, as a few cells will die or become unhealthy during the dissociation process. However, if after 48 hours the cells are clumped together with varicosities on their processes, either the cells are unhealthy or the poly-D-lysine substrate is toxic. As with most substrates, the efficacy of poly-D-lysine is lot dependent and each new lot must be tested. Images of healthy cultures can be found in references 4,11, and 20. Healthy cells can be maintained in culture for up to two weeks8.
Granule neuron progenitors begin to differentiate upon plating8,12,13,14. Once the optimal plating density is established for your studies, it should be maintained, because proliferation is promoted by factors such as Notch signaling15, and cells will proliferate more at higher densities. The proliferation of granule neuron progenitors can be substantially prolonged by adding sonic hedgehog (Shh) to the medium12,13,14. Laminin can also be added as a substrate in addition to poly-D-lysine to promote neurite outgrowth16,17. These features of the granule cell culture offer a system for studying either the biology of granule neurons or the regulation of differentiation of progenitors cells into neurons6,18,19.
Isolated cerebellar cells from postnatal mice are initially comprised of a mixture of granule neurons and granule neuron progenitors at different stages of the cell cycle8. There are also astroglia and interneurons present in the isolation/culture and further purification of granule neurons and granule progenitors (95%-99%) is attained by the Percoll gradient separation4. Enriched granule cells obtained without the use of the Percoll gradient separation step have been used to study the regulation of proliferation and differentiation of granule neuron progenitors, 18,19,20. In corroboration of this, we find that cells in the population isolated by bypassing the Percoll gradient separation step respond robustly to Shh by remaining in the cell cycle for several days, and that without Shh addition, there is very little proliferation in the cultures as indicated by staining with the cell proliferation maker, Ki67. However, if the cultures are to be used beyond several days in vitro, it is recommended that the Percoll gradient step be included to decrease contamination of the granule neurons by proliferating non-neuronal cells.
We thank Barbara Carletti and Anna Marie Kenney for invaluable suggestions. HYL is supported by the Training Grant “Hormones: Biochemistry and Molecular Biology”, DK07328. Supported in part by NIH grant 5R01 NS16951 (CAM).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Cell strainer with 70μm mesh pore | BD Biosciences | 352350 | ||
Spinal Needle | BD | 405182 | 20G x 3-1/2 inches | |
Poly-D-Lysine mol wt>300,000 | Sigma | P1024 | Each new batch of Poly-D-Lysine must be tested. | |
Percoll | Sigma | P-1644 | ||
Penicillin-Streptomycin (100X) | Sigma | P4333 | ||
HBSS | Invitrogen | 14175-103 | Must be calcium and magnesium free. | |
Neurobasal A-Medium | Invitrogen | 10888-022 | ||
Glutamax I supplement | Invitrogen | 35050-061 | ||
B-27 Serum Free Supplement (50x) | Invitrogen | 17504-044 | ||
12-mm circle coverslips | Carolina Biological Supply | 63-3029 | These are made in Germany by Deckgluder. | |
25-mm circle coverslips | Carolina Biological Supply | 63-3037 | These are made in Germany by Deckgluder. | |
Papain Dissociation System | Worthington Biochemical Corporation | LK 003150 | Kit is good for five isolations. | |
Permaset scissors | Roboz | RS6782 | ||
Dumont #5 (Dumostar) | Roboz | RS4978 | ||
4-well culture dish | Nunc | 176740 | ||
6-well culture dish | Nunc | 140685 |