Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Summary

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Abstract

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12–18.

Introduction

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.

Protocol

All animal procedures were performed in accordance with institutional regulations and the Guide to the Care and Use of Experimental Animals from the Canadian Council for Animal Care and has been approved by local authorities (“the Bannatyne Campus Animal Care Committee”). All efforts were made to minimize the number and suffering of animals used. Adequate depth of anesthesia was confirmed by observing that there was no change in respiratory rate associated with manipulation and toe pinch or corneal reflex.</p…

Representative Results

Based on the different birthdates of neuronal subtypes in the cerebellum, cultures from E12−E18 mouse embryos yielded different cell types. Large projection neurons, such as CN neurons (E9−E12) and PCs (E10−E13), emerged early during cerebellar development. In mice, granule and Golgi cells arose between ~E13–E18 and underwent terminal divisions up to postnatal week 4. Replacing old medium I with fresh culture medium II at days in vitro (DIV) 7 will eventually prevent gl…

Discussion

The use of primary cultures is a well-known method applicable for all types of neurons^17,18,19. In the presented protocol, we explain how to isolate cerebellar neurons and maintain their viability with optimum survival in vitro for a maximum of 3 weeks. Primary culture of cerebellar cells, which were isolated at E15−E18, confirms the collection of three classes of large neurons: PCs, Golgi cells, and CNs. The cell bodies a…

Materials

Adobe Photoshop CS5 Version 12	Adobe Inc
Anti-Sodium Channel (SCN)PN4 (Nav1.6)	Sigma	S0438	3 μg/mL
Bovine Serum Albumin	Millipore Sigma	A3608
CALB1	Swant Swiss Antibodies (Polyclonal)	CB38	1/5000 dilution
CALB1	Swant Swiss Antibodies (Monoclonal)	300	1/1000 dilution
Cytosine β-D-arabinofuranoside or Cytosine Arabinoside (Ara-C)	Millipore Sigma	C1768
DNase I from bovine pancreas	Roche	11284932001
Dressing Forceps	Delasco	DF-45
Dulbecco’s Modified Eagle’s Medium (DMEM-F12)	Lonza	12-719F
Fisherbrand Cover Glasses: Circles	fisher scientific	12-545-81
Gentamicin	Gibco	15710-064
Hanks’ Balanced Salt Solution (HBSS)	Gibco	14185-052
Insulin from bovine pancreas	Millipore sigma	I5500, I6634, I1882, and I4011
Large Scissor	Stoelting	52134-38
L-glutamine	Gibco	25030-081
Metallized Hemacytometer	Hausser Bright-Line	3100
Microdissection Forceps	Merlan	624734
Pattern 5 Tweezer	Dixon	291-9454
Phosphate Buffered Saline (PBS)	Fisher BioReagents	BP399-26
Poly-L-Ornithine	Millipore Sigma	P4638
Progesteron (P4)	Millipore sigma	P8783
PVALB	Swant Swiss Antibodies	235	1/1500 dilution
Samll Scissor	WPI Swiss Scissors, 9cm	504519
Sodium Selenite	Millipore Sigma	S9133
Transferrin	Millipore Sigma	T8158
Tri-iodothyronine (T3)	Millipore Sigma	T2877
Trypsin	Gibco	15090-046
Zeiss Fluorescence microscope	Zeiss	Z2 Imager