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

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.

Figure 1. Morphology of neurons in primary hippocampal cultures

Primary hippocampal cultures were grown in NS21 containing holo-transferrin (B,F; labeled “NS21”), NS21 in which apo-transferrin was substituted for holo-transferrin (A,E; labeled “NS21 ApoT”), an earlier B27 lot (C, G), and a recent B27 lot (D, H) at different times. Cultures were fixed after 11 (A-D) or 18 DIV (E-H), permeabilized, and stained for CaMKIIα. CaMKII can be detected throughout dendrites including filopodia-like protrusions at 11DIV (arrows in B,C) and spines at 18 DIV (arrows in F,G). The density of filopodia-like protrusions and spines of dendritic segments of 11 and 18 DIV cultures, respectively, was quantified by counting those in 50 μm long segments of proximal dendritic shafts in a blind fashion (I,J; given are mean±SEM from 15 randomly chosen fields in 2 independent experiments). Only earlier B27 or NS21 containing holo-transferrin fully supported the formation of filopodia-like structure at 11DIV and spines at 18DIV, with NS21 in which holo-transferrin had been replaced with apo-transferrin being less effective and recent B27 only marginally supporting formation of spines at 18DIV and not at all filopodia at 11DIV. Scale bar is 10 μm.

Figure 2. AMPA receptor mEPSCs in pyramidal neurons cultured with NS21

Recordings were performed from 11 DIV neurons clamped to -70 mV with picrotoxin, MK801, and tetrodotoxin present. Insert at lower right: The average AMPA receptor mEPSC was assembled from multiple events in several recordings.

Figure 3. Comparison of RGC cultures grown with NS21 and commercial lots of B27

Shown are phase contrast micrographs of live retinal ganglion cells (RGCs) grown for 5 days in commercial lots of B27 (Invitrogen) from 2006 (A) and 2004 (B) or in NS21 (C). No astrocyte feeder layers were present in these cultures. NS21 supported the growth and survival of purified RGCs (C) and was comparable to commercial B27 lot from 2004 (B). Note the significant clumping of RGCs grown commercial B27 from 2006 (A), which is typical of cultured supplemented with recent B27 lots. Scale Bar is 30 μm.

Figure 4. Astrocytes promote formation of functional synapses in RGC cultures grown with NS21

A-C. Immunostaining for the presynaptic marker synaptotagmin (A, red) and postsynaptic marker PSD-95 (B, green) reveals numerous colocalized synaptic puncta (yellow puncta in C) in RGC cultures grown in the presence of a feeder layer of astrocytes. Scale Bar is 30 μm. D. Whole cell patch recording of a mixture of spontaneous EPSCs and mEPSCs as observed in the absence of tetrodotoxin from RGCs grown with NS21 with astrocytes present. As expected, astrocytes increased spontaneous EPSCs (large downward deflections) and mEPSCs (small downward deflections) above control (not shown) in RGC cultures supplemented with NS21 to a level that was comparable to that observed earlier with optimal B27 lots (Ullian et al., 2001).

Figure 5. Images of axons in mouse DRG cultures at 7, 10 and 14 DIV

Dissociated DRG cells were cultured with the indicated supplement. Shown are phase contrast images taken from live cultures at 7, 10, and 14 DIV (top) and quantification of the number of axons that displayed beading as a sign of their disintegration (bottom; given are mean±SEM from 4 randomly chosen fields in 2 independent experiments). Neurons grown with recent B27 media showed axonal beading at 10 DIV and complete axonal degeneration at 14 DIV. Neurons grown with NS21 containing apo-transferrin (“NS21 ApoT”) instead of holo-transferrin also exhibited signs of axonal degeneration in form of little blebs although blebbing was less pronounced. Neuron cultured with NS21 with holo-transferrin or optimal earlier B27 lots showed little evidence of axonal degeneration. Scale Bar is 50 μm.

Figure 6. Images of somata of mouse DRG cultures at 14 DIV

Dissociated DRG cells were grown using the indicated supplement. Shown are phase contrast images taken from live cultures at 14 DIV (top) and quantification of the number of somata that displayed irregular shapes as sign of their disintegration (bottom; given are mean±SEM from 4 randomly chosen fields in 2 independent experiments). Neurons in suboptimal recent B27 media showed massive degeneration of cell bodies at 14 DIV indicated by loss of clearly visible boundaries to neighboring cells and more amorphous overall appearance. Neurons in NS21 containing apo-transferrin also exhibited to a large degree signs of somatic deteriorating. However, neurons grown with NS21 containing holo-transferrin showed virtually no evidence of somal degeneration. Scale Bar is 50 μm.