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An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level. We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination. This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain. Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells.
Surgical tools: sterilize all dissection scissors and forceps by autoclave
Note: Coat enough coverslips/plates for each dissection. PDL-coated coverslips can be stored for a few weeks at 4°C.
Dissection medium (DM): Use sterile ice cold Hank's Balanced Salt Solution (w/o Ca2+, Mg2+) (HBSS, Invitrogen 14175) supplemented with 10 mM HEPES (Invitrogen 15630).
Aggregate culture media: The DMEM/NBB27 medium contains DMEM/Neurobasal (1:1 vol:vol), 2% B27, 1 x Sato, 0.5 mM sodium pyruvate, 0.75mM GlutaMAX, 60μg/ml N-acetylcysteine, 5 μg/ml insulin, 10nM d-Biotin and 1% penicillin/streptomycin. To make 100 ml of the aggregate culture medium, mix 50 ml DMEM (w/o pyruvate/glutamine, Invitrogen 11960), 50 ml Neurobasal medium (Invitrogen 211034), 375 μl GlutaMax (100 x, Invitrogen 35050), 5.5 mg sodium pyruvate (Sigma P2256), 2 ml B27 (Invitrogen 17504), 6.3 mg N-acetylcysteine (Sigma A8199), 1 ml of Sato stock (100 x stock, see below), 25 μl of d-Biotin stock (4000 x stock, 40 μM in PBS stored as aliquots at -20°C. Sigma B4639), 100 μl of insulin (1000 x stock, 5 mg/ml in 0.01N HCl stored as aliquots at -20°C, Sigma 16634), and 1 ml penicillin/streptomycin (100 x stock, Invitrogen 15140), filter-sterilize and store at 4°C.
Sato 100 x stock solution: To make 40 ml of the stock: mix 40 ml Neurobasal with 400 mg apo-transferrin (Sigma T2252), 400 mg BSA (Sigma A9647), 10 μl of progesterone (25 mg/ml ethanol, stored as aliquots at -20°C. Sigma P8783), 64 mg putrescine (Sigma P7505), and 40 μl of sodium selenite (30 μM in PBS, stored as aliquots at -20°C, Sigma S5261). Filter sterilize the Sato stock solution, and store as aliquots at -20°C.
Neuron plating medium (PM ): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20°C, Invitrogen 25030), 25μM glutamic acid (100 stock, aliquots stored at -20°C) and 1% penicillin/streptomycin.
Neuron culture medium (CM ): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20°C) and 1% penicillin/streptomycin.
5-FdU stock: 100x stock, 1mM 5-fluoro-2'-deoxyuridine (Sigma F0503) and1mM uridine (Sigma U3003) in Neurobasal medium. Filter sterilized and stored as aliquots at -20°C.
Papain digestion solution (make fresh prior to dissection):
Trypsin inhibitor solution (make fresh prior to dissection):
Figure 1. Phase contrast images of cells forming aggregates at different days and aggregates after washing.
Figure 2. Phase contrast images of an aggregate culture 2 and 12 days after seeded on Matrigel-coated coverslips.
Early studies reported formation of synapses and mature myelin in rotation-mediated free floating aggregate cultures1. The CNS aggregate culture system described here combines serum-free growth of multipotent progenitor cells in three dimensional aggregates with the convenience of traditional 2D cultures to facilitate analyses of cell development, migration and differentiation in vitro. The system can be modified and used for neural precursor cell research and for investigation into neuron-glia interactions. For example, genetically modified cells such as oligodendrocyte progenitors2 may be added to the aggregate cultures. Effect of immune cells such as microglia and macrophages or various reagents on the development and differentiation of neurons and glia can be also studied. Reaggregates of purified retinal ganglion cells have recently been used to study CNS myelination in cocultures with oligodendrocytes in the absence of other cells3. In agreement with previous report4, Matrigel appears to be superior to PDL in that it promotes cell adhesion and accelerates neurite outgrowth and glia differentiation. Matrigel is therefore used as the substratum for cell aggregates in this protocol.
This study was funded in part by start-up funds from Texas A&M University