Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

For several decades, cell lines have been widely used as a high throughput tool in preclinical studies and biological research. Cost-effectiveness, fast growth, and reduction of live animal use are some benefits of using these cells. However, genetic alterations and phenotypical changes accumulate after several passages in vitro¹. Misidentification of cell lines and genetic dissimilarity from primary cells can lead to irreproducible experiments and false conclusions^2,3,4,5. Therefore, in spite of some similarities to differentiated cells such as neurons (e.g., neurotransmitters, ion channels, receptors, and other neuron-specific proteins), neuronal cell lines cannot replicate the full phenotype of neurons. Using mature neurons is another option; however, these cells are non-dividing postmitotic cells that are difficult to propagate in culture. Moreover, re-entry into the cell cycle may precipitate apoptosis⁶.

Three-dimensional (3D) cell culture, organotypic slice cultures, and organoid cultures have been developed to provide an environment in which cells can arrange into a 3D form that mimics the in vivo setting. Thus, cell-to-cell communication, migration, invasion of tumor cells into surrounding tissues, and angiogenesis can be studied⁷. However, additional costs of using extra cellular matrix (ECM) proteins or synthetic hydrogels as a bedding, difficulty in imaging, and compatibility with high-throughput screening instruments are considerable drawbacks of 3D cell culturing. A major disadvantage of organotypic tissue slice culture is the use of a large number of animals and the adverse effects of axotomy, which leads to inaccessibility of targets and growth factors for axons, and consequently neuronal death⁸.

Therefore, an alternate approach, which avoids the problems with cell lines, the difficulty of growing mature cells, and complexity of tissues, is in vitro maturation of immature primary cells. Primary cells are derived directly from human or animal tissue and dissociated using enzymatic and/or mechanical methods⁹. The main principles of isolation, seeding, and maintenance in culture medium are similar regardless of the tissue source. However, the trophic factors necessary to promote proliferation and maturation are highly cell specific⁶.

Knowing the ‘birthdate’ of each cerebellar cell type is a prerequisite for designing a primary culture experiment. In general, Purkinje cells (PCs) and the neurons of the cerebellar nuclei (CN), are born before the smaller cells, including interneurons (e.g., basket, stellate cells) and granule cells. In mice, PCs emerge between embryonic day (E)10–E13, whereas CN neurons at approximately E9–E12¹⁰.

Other cerebellar neurons are born much later. For example, in mice, the Golgi subpopulation of interneurons are generated from VZ at (~E14−E18) and the remaining interneurons (basket cell and stellate cells) located in the molecular layer emerge from dividing progenitor cells in the white matter between early postnatal (P)0–P7¹¹. Granule cells are generated from the external germinal zone (EGZ), a secondary germinal zone that is derived from the rostral rhombic lip and goes through terminal division after birth. But before their precursors arise from the rhombic lip from E13–E16, the cells have already migrated rostrally along the pia surface to make a thin layer of cells on the dorsal surface of the cerebellum anlage. Nonneuronal macroglial cells such as astrocytes and oligodendrocytes, which originate from the ventricular neuroepithelium, are born at E13.5−P0 and P0−P7 respectively^{11,12,13,14,15}. Microglia are derived from yolk-sac primitive myeloid progenitor cells between E8–E10 and after invasion into the central nervous system can be detected in the mouse brain by E9¹⁶.

The method presented in this article is based on the one originally developed by Furuya et al. and Tabata et al.^17,18, which was optimized for primary culturing of Purkinje cells derived from Wistar rat cerebella. We have now adapted this method and carefully modified it to study the growth of mouse cerebellar neurons¹⁹. Unlike in our new protocol, cold dissection medium is the main washing buffer used during dissection and dissociation steps before adding seeding medium in Furuya’s protocol¹⁷. This buffer lacks the nutrition, growth factors, and hormones (all in Dulbecco’s modified Eagle medium:nutrient mixture F-12 [DMEM/F12]) that are necessary to support cell growth and survival during the aforementioned steps. In addition, based on our extensive experience with murine primary cerebellar cultures, we have used 500 μL of culture medium in each well (instead of 1 mL) and increased the tri-iodothyronine concentration to 0.5 ng/mL, which improves growth of neuronal cells, in particular those with a Purkinje cell phenotype, and promotes the outgrowth of dendritic branches in culture. The principal method featured in this article can be broadly applied to other small rodents (e.g., squirrels and hamsters) during embryonic development and can be used to study cerebellar neurogenesis and differentiation in the various embryonic stages, which differ between species.