NS21: Re-defined and modified supplement B27 for neuronal cultures

https://doi.org/10.1016/j.jneumeth.2008.03.013Get rights and content

Abstract

In vitro culturing of primary neurons is a mainstay of neurobiological research. Many of these culture paradigms have taken advantage of defined culture media rather than serum additives that contain undefined survival factors to facilitate experimental manipulations and interpretation of the results. To culture neurons in the absence of serum, defined supplements such as B27 are now widely used. However, commercially available supplements exhibit large variability in their capabilities to support neurons in culture. We re-optimized and modified earlier published formulations of B27 using 21 different ingredients (NS21). NS21 supports neuronal cultures of high quality as manifested by their morphological characteristics, formation of synapses, and postsynaptic responses. Much of the variability in the quality of B27/NS21 was due to variability in the quality of different sources of bovine serum albumin. Furthermore, we found that holo-transferrin used in NS21 is preferable over apo-transferrin used in B27 for the quality of neuronal cultures.

Introduction

Neuronal cultures are widely used to study neuronal development including neurite and synapse formation, neurotransmitter release, subcellular distribution and trafficking of neuronal proteins such as neurotransmitter receptors, and homeostasis of electrical signaling. Initially those cultures depended on the use of sera for factors that are critical for cell survival and growth. Media supplements such as B27 were developed with defined components that eliminate the need for supplementation with serum (Bottenstein and Sato, 1979, Romijn, 1988, Romijn et al., 1984). Such supplements were widely welcomed. Particularly B27 is used by many investigators for a number of different neuronal culture systems (Christopherson et al., 2005, Colledge et al., 2003, Craven et al., 1999, Deisseroth et al., 1996, El-Husseini et al., 2000, Mi et al., 2004, Passafaro et al., 2003, Pratt et al., 2003, Roche et al., 2001, Sans et al., 2005, Schluter et al., 2006, Stellwagen and Malenka, 2006, Tai et al., 2007, Thiagarajan et al., 2002, Tomita et al., 2004, Tsui and Malenka, 2006, Ullian et al., 2001).

In theory the use of defined supplements reduces the variability of the culture conditions. It thereby limits the potential for detrimental effects of components that could affect the health of cultures. However, a number of laboratories have experienced large differences in their neuronal cultures over the last 4–5 years when using commercially available supplements (see below and, e.g., Schluter et al., 2006, Tsui and Malenka, 2006). Commercial supplements available earlier including B27 supported neuronal cultures of excellent quality including neurons derived from hippocampus, retinal ganglia (RGCs), and dorsal root ganglia (DRG) cells. However, more recently supplements available in the United States have largely failed to reliably promote healthy neuronal cultures.

The reason for this variability is likely due to the fact that several components such as bovine serum albumin and transferrin are isolated from biological sources. As sources and isolation procedures differ to some degree they introduce variability in those biological components. We re-evaluated published formulations (Brewer and Cotman, 1989, Brewer et al., 1993, Romijn, 1988, Romijn et al., 1984). We found that the exact source and vendor for some components are critical. Although based on the original B27 formulation, our formulation varies from commercially available B27 by using components of explicitly defined origin (vendor, precise product) and by the use of holo- rather than apo-transferrin. To ensure that the formulation is not confused with the currently widely used commercially available B27 we call this formulation NS21 (Neuronal Supplement 21). We chose this name because we optimized and evaluated this supplement for neuronal cultures and because it has 21 ingredients. The precise composition of NS21 is given in Table 1.

Section snippets

Primary cultures of rat hippocampal neurons

Low-density cultures of dissociated hippocampal neurons were prepared as given earlier (Lim et al., 2003). In brief, hippocampi were dissected from E18 embryonic rats (Sprague–Dawley, Harlan), treated in Hank's balanced salt solution (HBSS; Invitrogen) with trypsin (0.03%) for 15 min at 37 °C, washed three times with HBSS, and triturated with a fire-polished Pasteur pipette. After pieces of tissue that were not dissociated settled, cells in the supernatant were collected by centrifugation (1100 

Primary hippocampal cultures

Under optimal conditions pyramidal neurons in primary hippocampal cultures develop multiple, well arborized dendrites. This morphology is illustrated by immunofluorescence microscopy following antibody staining for the Ca2+- and calmodulin-dependent protein kinase II (CaMKII) (Fig. 1B, C, F and G). CaMKII is one of the most prevalent proteins in neurons. It is localized throughout dendrites and in neurons with fully mature synapses at dendritic spines, the postsynaptic sites of glutamatergic

Discussion

Our work identifies the growth medium component B27 as a major source of variability that has become detrimental to several types of primary neuronal cultures. Our comparisons of the effects of B27 batches obtain before and after 2004 and of NS21 on the quality of hippocampal cultures are largely based on consecutive rather than parallel experiments as we did not have available earlier B27 after problems became obvious. However, the striking difference between earlier and more recent B27 became

Acknowledgments

The authors wish to thank Dr. D.-X. Yang and L. Sowers for excellent technical assistance with cell cultures and immunostainings and Dr. Nicola Allen for help with RGC recordings. This work was supported by NIH Grants NS035563, NS046450, and AG017502 to JWH, AG013730 and NS039358 to JM, and DA15043 to BAB and a Larry H. Hillblom Fellowship (BS).

References (28)

Cited by (233)

  • In vivo nanoscopic landscape of neurexin ligands underlying anterograde synapse specification

    2022, Neuron
    Citation Excerpt :

    Tissues were washed with HBSS containing 10% FBS and dissociated by passing through plastic pipette tips in the HBSS containing 0.05% DNase and 12 mM MgSO4. 1.5 × 105 Cells were plated onto 13-mm cover glasses coated with poly-l-lysine (P2636, Sigma-Aldrich) and incubated in Neurobasal medium (21103049, Gibco/Thermo Fischer Scientific) containing NS21 supplement (Chen et al., 2008), 50 U/ml Penicillin, 50 mg/ml Streptomycin (Invitrogen), 2 mM l-Glutamine and 2% FBS in 5% CO2 at 37 °C. Thirty minutes to one hour after initial incubation, the culture medium was changed to a fresh medium without FBS.

View all citing articles on Scopus
View full text