Culture Of Neural Stem Cells In Calcium Alginate Microbeads

Neural stem cells (NSCs) with the capacity of self-renewal and differentiation into neurons and glial cells, have gotten more and more worldwide attention. The finding, research and application of NSCs will play an important role in the nerve disease treatment and nerve injury reparation. The shortage of the source and the number of NSCs, however, is the main challenge for its clinic application.Large-scale three-dimensional culture of NSCs in vitro provides a feasible answer for this problem. But the shear stress in bioreactors can cause serious cell damage especially for the shear sensitive cells like NSCs. To avoid the shear stress, encapsulation of NSCs and then cultivation in bioreactors is thus worth investigating. Therefore, we explored the method of culturing NSCs in calcium alginate microbeads (Ca-Alg MBs).To provide the optimum growth conditions for NSCs in Ca-Alg MBs, The gelation parameters, such as the diameter, reactant concentration, and gelation time should be determined firstly to form the proper beads structure for cell growth. In the experiment different Ca-Alg MBs were formed in different gelation conditions. Then, the diffusion experiment was performed to get the diffusion data for different Ca-Alg MBs. After that, a diffusive mathematical model was set up to find the diffusion coefficient (D_AB) of glucose in Ca-Alg MBs by fitting the experimental data. Base on the orthogonal test, the optimum gelation conditions for preparing microbeads with a proper diffusivity were determined. And the gelation parameters are 2 mm in beads diameter, 1.5% (wt%) sodium alginate, 3.5% (wt%) CaCl₂, and the gelation time of 10 min.The biocompatibility of Ca-Alg MBs for NSCs was tested by coculturing NSCs with Ca-Alg MBs up to 21 days. The cells were collected and assayed with the immunocytochemical methods. The results indicate that Ca-Alg MBs have a good biocompatibility for NSCs. After that the culture of the encapsulated NSCs with different cell density was then experimented. The results show that the culture effect with the encapsulation density of 8×10⁴ cells-mL-1 is the best. The recovered NSCs were stained with immunofluorescence and the majority of the expanded NSCs in Ca-Alg MBs were nestin-positive. Finally cell recovery method was got by dissociating Ca-Alg MBs with 55 mmol.L-1 sodium citrate for 10 min. The mean cell recovery rate was about 90%.The above explorations demonstrate that mouse hippocampus-derived NSCs cansurvive and be expanded in calcium alginate microbeads.