Neuronal Cells

Neuroscience is a rapidly evolving field that encompasses a variety of cell types, including neurons and neuronal stem cells.

In vitro culture of these diverse cell types requires appropriate culture surfaces for attachment and proliferation/ differentiation, as detailed in the examples below. NG-108 rat glioma/mouse neuroblastoma cells and PC-12 cells, two neuronal cell lines, require different surfaces for attachment. NG-108 cells attach loosely to TC-treated cultureware, but when they are cultured on BD BioCoat™ laminin cultureware they exhibit a more typical neuronal morphology (Figure 1). PC-12 cells, derived from a transplantable rat pheochromocytoma, develop neurites in response to NGF when they are cultured on collagen I (Figure 2).

Other surfaces, including BD BioCoat Poly-D-Lysine cultureware1 and BD BioCoat Poly-D-Lysine/Laminin2, can also be used to culture PC-12 cells. Primary neuronal cells utilize different attachment surfaces depending on their origin and the composition of the media used during culture. Primary mouse cortical neurons and primary mouse basal forebrain cholinergic neurons have been cultured on BD BioCoat Poly-L-Lysine Cultureware3 and BD BioCoat Poly-D-Lysine/Laminin Cultureware4, respectively. Primary human neural stem cells have been grown under serum-containing conditions in tissue culture-treated BD Falcon cell culture flasks5. Using serum-free conditions, Thonhoff, et al., showed that neuronal stem cells maintain their capacity to differentiate into both Tuj1+ neuronal cells and GFAP+ astroglial cells on BD™ PuraMatrix™ peptide hydrogel while differentiation of neuronal stem cells grown on BD Matrigel™ Matrix was skewed toward GFAP+ astroglial cells6. Both BD PuraMatrix6-8 and BD Primaria™9 are defined, xeno-free surfaces for 3D and 2D culture, respectively, which are compatible with neuronal cells. BD Primaria cultureware enhances neuronal cell attachment as compared to tissue culture-treated cellware, as shown with chick embryo spinal cord neurons (Figure 3). These examples* illustrate the need for an appropriate growth surface which is determined by the cell type and whether a xeno-free surface with defined media is required by the experimental model.

*Other examples available in references 10-12.

Figure 1. Effects of BD Biocoat Laminin Cultureware on NG-108 Neuroblastoma Cells

Figure 1. Effects of BD Biocoat Laminin Cultureware on NG-108 Neuroblastoma Cells

NG-108 rat glioma/mouse neuroblastoma cell morphology is surface dependent. Cells cultured on tissue culture plastic are loosely adhered and remain rounded (A). Cells cultured on BD BioCoat Laminin cultureware exhibit a spindle-shaped morphology and dendritic processes (B).

Figure 2. PC12 Neurite Outgrowth, Cultured on BD BioCoat Collagen I Cultureware

Figure 2. PC12 Neurite Outgrowth, Cultured on BD BioCoat Collagen I Cultureware

PC12 cells were maintained in DMEM with 10% FBS, 5% horse serum and 1% penicillin/streptomycin. For neurite generation, approximately 15,000 cells/well were plated in BD Falcon™ 96-well imaging plates that were coated with BD™ collagen I, rat tail using 1.8 μg collagen per well. After 24 hours, the medium was replaced with differentiation medium (DMEM with 0.1% FBS, 0.05% horse serum, 100 ng/ml NGF). The medium was replenished every third day for 10 days. For imaging, cells were fixed with 3.7% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton-X-100 for 5 minutes. Neurites were stained with a primary mouse anti-β-tubulin antibody (Cat. No. 556321) using 0.125 μg antibody/well followed by AlexaFluor® 488 goat anti-mouse IgM at a concentration of 0.25 μg/well. Hoechst 33342 was used at 0.1 μg/well to stain the nuclei. To prevent the dissociation and fracture of fragile neuronal networks, the number of washes in the fixation and processing steps were minimized and extra care was taken in aspirating and dispensing liquids in wells. Images were acquired on a BD Pathway™ bioimager as a 4x4 montage using a 20x objective (0.75 NA).


 
Figure 3. Chick Embryo Spinal Cord Neurons cultured on BD Primaria Cultureware

Figure 3. Chick Embryo Spinal Cord Neurons cultured on BD Primaria Cultureware

When chick embryo spinal cord neurons are cultured on BD Primaria™ cultureware, growth is enhanced and extensive neurite development occurs. In this experiment, cells clumped and detached from traditional TC plates after 20 days in culture (A) but remained viable and differentiated on BD Primaria cultureware (B).

References

  1. Willard MD, Willard FS, Li X, Cappell SD, Snider WD, Siderovski DP. (2007) Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation. EMBO J. 26:2029.

  2. Rosario M, Franke R, Bednarski C, Birchmeier W. (2007) The neurite outgrowth multiadaptor RhoGAP, NOMA-GAP, regulates neurite extension through SHP2 and Cdc42. J Cell Biol. 178(3):503.

  3. Wetzel M, Li L, Harms KM, Roitbak T, Ventura PB, Rosenberg GA, Khokha R, Cunningham LA. (2008) Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia. Cell Death Diff. 15:143–151

  4. Schnitzler AC, Lopez-Coviella I, Blusztajn JK. (2008) Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons. Nat Protocols. 3(1):34.

  5. Redmond Jr. DE, Bjugstad KB, Teng YD, Ourednik V, Ourednik J, Wakeman DR, Parsons XH, Gonzalez R, Blanchard BC, Kim SU, Gu Z, Lipton SA, Markakis EA, Roth RH, Elsworth JD, Sladek Jr JR, Sidman RL, Snyder EY. (2007) Behavioral improvement in a primate Parkinson’s model is associated with multiple homeostatic effects of human neural stem cells. Proc Natl Acad Sci. 104(29):12175.

  6. Thonhoff JR, Lou DI, Jordan PM, Zhao X, Wu P. (2008) Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res. 1187:42.

  7. Gelain F, Bottai D, Vescovi A, Zhang S. (2006) Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3- dimensional cultures. PLoS ONE. 1(1):e119.

  8. Aguirre A, Rizvi TA, Ratner N, Gallo V. (2005) Overexpression of the epidermal growth factor receptor confers migratory properties to nonmigratory postnatal neural progenitors. J Neurosci. 25(48):11092.

  9. Holgado-Madruga M, Moscatello DK, Emlet DR, Dieterich R, Wong AJ. (1997) Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc Natl Acad Sci. 94:12419.

  10. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 321:1218.

  11. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa SI, Muguruma K, Sasai Y. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 25:681.

  12. Flanagan LA, Rebaza LM, Derzic S, Schwartz PH, Monuki ES. (2006) Regulation of human neural precursor cells by laminin and integrins. J Neurosci Res. 83(5):845.

AlexaFluor is a trademark of Invitrogen Corporation.
CytoFlour is a trademark of Applied Biosystems.
MetaMorph is a registered trademark of Universal Imaging Corporation (UIC).
mTeSR1 and WiCell is the property of WiCell Research Institute.
PuraMatrix is a registered trademark of 3DM, Inc.
Victor2 is a trademark of PerkinElmer, Inc.


For Research Use Only. Not for use in diagnostic or therapeutic procedures.