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 clear within the same week in which we changed B27 batches. Furthermore, re-optimization of B27 leading to NS21 rectified the problem especially when holo-transferrin was used. These observations leave no doubt that it is the B27 supplement that is largely if not fully responsible for the low quality of our hippocampal cultures observed with recent B27.
Similarly, RGC and DRG culture quality was strongly impaired upon changing B27 batches from earlier to more recent products. Other components such as FBS, animals, and personnel were the same at the time of these changes. Furthermore, the different B27/NS21 batches were tested in parallel for RGC and DRG cultures as shown in and Figs. and , respectively. Because of the striking differences in the quality of cultures observed with earlier versus more recent B27 batches in the absence of any other obvious changes and because these dramatic results were obtained in three independent laboratories we conclude that it is the currently commercially available B27 that is problematic with respect to obtaining high quality hippocampal, RGC, and DRG cultures.
Several factors can contribute to this variability. First, the quality of the source and lot or batch of each of the 21 individual components can vary. Second, it might be important that the preparation of stocks of the individual components is performed with high care. This aspect might include the proper and careful storage of different components and stock solutions as detailed in . This factor is especially important for lipid stocks, some of which are sensitive to light or oxidation and need to be carefully prepared and stored at -80°C. Third, it might be important how the components are mixed together as detailed in with bovine serum albumin dissolved first before addition of other components. We avoid stirring and only gently but thoroughly swirl the solution after addition of all components. Temporal or local changes in pH or in the concentration of individual components could affect solubility or integrity of other components. It is obviously not possible for us to systematically test the importance of all these factors but given that the protocol outlined in yields NS21 of excellent quality it will be prudent to carefully follow it.
NS21 likely differs from B27 that for most if not all components different sources are used, which are clearly spelled out in for NS21. Furthermore, B27 contains apo-transferrin whereas NS21 contains holo-transferrin. The reason for using the latter is due to our observation that hippocampal cultures consistently looked healthier with more numerous dendritic branching, filopodia-like structure, and dendritic spines compared to those cultures that received NS21 in which holo-transferrin was replaced by apo-transferrin (). Axons and somata of DRG cells are less healthy if apo-transferrin is used for culturing instead of holo-transferrin (Figures and , respectively). We only used one source of apo-transferrin and one source of holo-transferrin and thus cannot be certain whether the superior effect with holo-transferrin is due to its overall rather low total content of iron or other factors such as a more intact batch of holo-transferrin. It is conceivable that transferrin is more stable or easier to purify in an intact form with its ligand iron present.
However, the largest difference with respect to neuronal survival and health is due to the use of a defined bovine serum albumin product. We do not know why bovine serum albumin is critical for the health of neuronal cultures but one possibility is its property to bind various lipids. It thus could serve as a critical carrier for lipids that are important for neuronal survival and development. As given in , we found that bovine serum albumin from Sigma with the ordering number A4919 is by far better than several other bovine serum albumin sources we tested including another one from Sigma. By now we have used three different charges of A4919 from Sigma over the last 3 years without noticing any obvious differences. These findings indicate that A4919 is a dependable component for NS21.
While it is certainly convenient to rely on commercially available B27, there are a number of disadvantages. Individual investigators have absolutely no information on or control over any of the above factors including origin and quality of the various components and their handling. The commercial supplier produces a number of different lots per year. It is difficult to carefully and thoroughly test a large number of lots to potentially identify one of suitable quality as would be necessary based on our own results without any certainty that a suitable lot can be identified. Even if this screen succeeds it then is not certain whether a sufficient amount of this same lot can be obtained after the time-consuming tests as the supplier changes lots frequently. Finally, as there is currently only one commercial supplier and the detailed nature of each of the components is confidential, individual investigators have no control over general availability of B27.
Hippocampal cultures are often used as a model to study the effect of oxygen and glucose deprivation. Our hippocampal cultures grown in NS21 are completely suitable for such studies. Cultures undergo massive degeneration within 24-48 hours following a six hour period of oxygen and glucose deprivation (Merrill, Strack, and Hell, data not shown) or a six hour period of glutamate treatment (Stein and Hell, data not shown).
We conclude that the growth of healthy neurons in several different culture systems including our hippocampal, RGC, and DRG cultures is not reliably supported by the currently commercially available B27. However a modified B27 formulation with explicitly defined components including their sources and holo-transferrin that we call NS21 allows culturing of high quality hippocampal, RGC, and DRG neurons.