Culture of Neural Stem Cells in Alginate Microbeads

Neural Stem Cells(NSCs) with the capacity of self-renewal and differentiation into neurons and glial cells have obtained 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 repair.The shortage of the source and the amount of NSCs,however,is the main obstacle 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 would 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 of 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 diameter,reactant concentration,and gelation time were determined firstly to form the proper bead structure for cell growth.The diffusion experiment was performed to get the diffusion data for different Ca-Alg MBs and a diffusive mathematical model was set up to find the diffusion coefficient(D) 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 were 2mm in bead diameter,(1.5%) sodium alginate,(3.5%)CaCl-2,and the gelation time of 10 min.The culture of encapsulated NSCs with different cell density was then conducted. The results showed that the culture effect with the encapsulation density of(0.08)×10~6cells·mL~(-1) was the best among the testing samples.The recovered NSCs were stained by immunofluorescence and the majority of the expanded NSCs in Ca-Alg MBs were nestin-positive.The experimental results demonstrated that mouse hippocampus-derived NSCs could survive and be expanded in Calcium-alginate Microbeads.