CD133/prominin-positive neural stem cells derived from mouse or human brain can be maintained in a proliferative stage when grown in the presence of growth factors and serum-free medium. These cells grow into floating neurospheres due to clonal expansion as well as some agglutination. It is essential to dissociate these neurospheres at each passage for expansion. If not dissociated, the neurospheres grow to be excessively large; when the cells in middle of the neurospheres do not get enough oxygen, this leads to necrosis and cell death. The cells in the neurospheres are generally very tightly packed and dissociation of these cells into a single cell suspension is not an easy task.
Until recently, the most commonly used (or perhaps the only) method used to dissociate neurospheres was by mechanical means. In this, the scientists follow a procedure called “trituration” where the neurospheres are manually disrupted by repeated pipeting using a fire polished glass Pasteur pipet or a P200 micropipettor. This harsh treatment of the neurospheres results in a huge cell death and damage. StemCell Technologies have come up with a kit which involves chemical, rather than mechanical or enzymatic, dissociation of the neurospheres. The kit is primarily made to dissociate mouse neurospheres. However, we have been using the kit to dissociate fetal human brain derived neurospheres for the past 3 years and have been very successful. Although there is chemical treatment involved, it appears that the cells are not damaged and the cell viability is not compromised. We have been able to continually propagate neural stem cells after using this kit for at least 4 months.
The procedure for this kit is very simple and easy to follow. The kit comes with 3 solutions, A, B and C, which can be stored at room temperature. After washing the neurospheres and pelleting by centrifugation, solutions A and B are added to the cell pellet in the specified amount depending on the tissue type. This is followed by pipeting (not trituration) of the neurospheres for 8 to 10 times. The pipeting is repeated every few minutes for 10 minutes. Finally, solution C is added and the resultant cell suspension is washed and ready for propagation or future experiments. The fact that this procedure is completed in less than half an hour is very attractive. Further, the same kit can be used for both human and mouse neurospheres and this makes it very convenient for laboratories working with both types of tissues.
In summary, NeuroCult® Chemical Dissociation Kit is a fast and convenient method to disrupt both human and mouse neurospheres. The procedure is simple and can be completed in less than half an hour. The treatment of the cells is gentle therefore, cell viability is good. However, as with most proprietary products, the composition of the solutions in the kit is not revealed.
Scientist
Children’s Research Center of Michigan
Children’s Hospital of Michigan