Embryonic Rat Spinal Cord-derived Neural Stem Cell Transplantation For Treatment Of Spinal Cord Nearly Half Of The Cut Injury In Rats

Objectives: We take the advantage of floating culture to isolate neural stem cells from spinal cord of fetal rats. Study their selfrenew and multipotential capacity by passaging their progenies.Methods:1 Get materials We isolated the tissue mass of spinal cord from embryonic day 13 (E13) under the anatomical microscope.2 Digest The tissue mass was digested with digest solution(1.34mg/ml hyalidase / 0.25%trypsin 1:1+ 0.2mg/ml kynurenic acid).3 Disperse and culture The tissue mass was dispersed with pipettes, cells were inoculated with 3 different densities(4× 10⁴/ml,4× 10⁵/ml and 4×10⁶/ml) to 24-well culture plate after the number of vital cells had been determined with trypan blue. The culture condition is 37℃, saturated humidity, 5%CO₂ open floating culture without substrate. The formula of culture solution is DMEM( high glucose) /F12 1:1 , EGF 10ng/ul, bFGF 10ng/ul, B27 2%(V/V), gentamycin 100u/ml.4 Conventional mechanical passaging The 6-7 days old primary neurospheres were triturated with fire polished glass pipette, in this way the neurospheres could be dissolved into single cells or quarters of smaller cell masses. The progeny neurospheres were also dissolved in this way.5 Differentiation5.1 Differentiation in the very solution of neural stem cells Cover slips pasted with polylysine were casted to culture wells, only the slips with single neurophere wereselected to new wells and covered with the very solution of neural stem cells. Its migration, proliferation and differentiation will be recorded.5.2 Effects of serum on differentiation EGF and bFGF were removed from the very solution of neural stem cells, additional 10% calf serum was contained in this solution..6 Identification of neural stem cells and their differentiated progenies with immunocytochemistry The specific antibodies matching neural stem cell cell skeleton (Nestin), neuron(MAP2), astrocyte(GFAP) and oligodendrocyte(GC) were applied to identify the primary and passaged neural stem cells and the three major cell types of central nervous system.Results and Conclusion1. The vast majority of the cell clones originate from single cell proliferation .2 Neural stem cells' proliferating abilities vary according to different cell densityUnder the density of 4 X 10 /ml, cell reunion wouldn't affect the proliferation of neural stem cells and recording of experiment data. It's a ideal cell density.3 Effects of conventional mechanical passaging on the neural stem cell proliferative ability.The mechanical trituration produced secondary neurospheres were active and tolerate passging. It should be suggested as a routine passaging method.4 Identification of neural stem cells and their multipotentalty with immunocytochemistry methods4.1 Identification of neural stem cells A lot of cells within the primary and passaged neurospheres could be stained with specific antibody against cell skeleton protein(Nestin) of mammalian neural stem cells. This result verified that we have not only got the neural stem cells from fetal spinal cord, these neural stem cells also possess the ability of proliferation and selfreproduction in vitro.4.2 Multipotentialty of primary and lower neurospheres 4.2.1 Differentiation of neural stem cells in the very solution...