Observation Of Neurons Differentiated In Vitro Cultured In Thermosensitive Chitosan-based Gelatum

Objective: Irreversible neuronal network breakdown and glial cell loss after brain injury block the rebuild of the organization structure of the brain, made the relevant function of the brain lose. With the development of neural tissue engineering has shown attractive prospects in the rebuild of central nervous system .It is important that set up three-dimensional tissues with the seed cells and biomaterials. A thermogelling chitosan solution which has better biocompatibility , degradation ,avirulence was prepared by mixing solutions ofβ-glycerophosphate (GP)and chitosan. When it injected in vivo the liquid formulations turn into gel implants in situ in response to temperature and pH changes. It offers specific advantages such as: possibility of a minimally invasive implantation, an ability to fill a desired shape, and easy incorporation of various growth factors. We put the neuron-like cells induced by MSCs combined with C/GP in vitro .The proliferation and the differentiation of neuron-like cells in C/GP were observed by inverted microscope. The objective to explore the biocompatibility of biodegradable material of C/GP and neuron-like cells. Offer experimental evidence to look for injectable scaffold for transplanation of neuron-like cells induced by MSCs into brain .Method: 1 MSCs can be obtained from femurs and shin-bone of the Sprague-Dawley rat about 50-60g under anesthesia. MSCs were purified from the bone marrow by density gradient centrifugation and adhering to the culture flasks. Growth property and cell morphologic change were obtained continuously.2 CTP were droped into the passage 3 culture with the concentration about 0.2mg/ml. Observe the changes of the cells everyday. The cells were identify by examined though NSE,GFAP by immunocytochemistry after 10 days .3 Typical C/GP solution was obtained by dissolving 200mg of chitosan (with medium viscosity and a degree of the deacetylation of 91%)in 9ml of HCl solution (0.1M). To the resulting solution, 560mg of glycerophosphate disodium salt dissolved in 1ml of distilled water according to different ratio, was carefully added drop by drop to obtain a clear and homogeneous liquid solution. Measure the pH value and solidify time of the mixture at 37℃in order to choose the best ratio.4 Gently mixing C/GP solution at room temperature with cell suspension(cell density, 2×105 cells/ml) until homogeneous. The thermogelling cell suspension which divided into 3 groups was then poured into 96-well culture plates(30μl/well). The experimental group was composed of 20μl cells suspension with 10μl C/GP ,The cell comparison group was composed of 20μl cells suspension with 10μl complete medium, The C/GP comparison group was composed of 20μl complete medium with 10μl C/GP. Put the 96-well culture plates in to incubator. After gelation, each well was rinsed twice with fresh complete medium. Exchange complete medium and observe the growth of the neuron-like cells in C/GP every day. 5 pores of the experimental groups and cell comparison groups were taken out and counted by MTT , 7 days later, the growth curve was drawn. T-test was used in comparison of the OD of the two groups, with 0.05 set as having a significant difference .Result: 1 The isolated MSCs were circular and some of them began to adherent to the culture flask after 24h. After 72h most of isolated cells were adherent to the bottom of the culture flask. Primary culture reached 90% of confluence in 8-10 days and the cells were like shuttle and ranked as barrier shape The passage cells grew faster than the primary cells and re- ached 90% of confluence in 3-7 days.2 The deformation of MSCs were began at 48h later of the revulsant was added. The neurites of the cells elongated gradually and connected with each others as time goes by After 10 days Most MSCs were differentiated into neuronlike cells and the immunocytochemistry showed positive expression of NSE and GFAP .3 The physical characters of injectable hydrogel :The chitosan-Hcl solution (pH value:5.65 )was strow yellow clearing liquid .56% GP solution was achromatic color clearing liquid (pH value:8.91) .Different vol ratio of chitosan and GP made different pH value and at 37℃(Table 1). We find the best vol ratio of the chitosan and GP is 3:2 (pH value:7.2, solidify time:10min).4 Neuron-like cells cultured in C/GP : We found that most of the cells holding roundness in the C/GP gel at fist 24 hours . cell morphology and neurite outgrowth were more distinct in 3D culture conditions. More and more amoebocyte had been found especially close to the underside of the 96-well culture plates from then on. Neuron-like cells exhibited larger cell bodies and sent out neurites within the C/GP gel and cell morphology and neurite outgrowth were more distinct in 3D culture conditions .5 Cells viability was assessed by the MTT assay. It was obvious that a great deal of brown crystal granules in C/GP gel .It was also shown indirectly the numbers , shape and distribution of the cells . Growth curve of neuron-like cells was measured using MTT method. A classical sigmoidal growth curves of the two groups were quite similar:the first two days was latent period ,3-5 days was logarithm trophophase,then the numbers of the cells decrease.6 Statistics analyse is that the OD of the experimental groups is similar to the cell control groups. There was no significant difference in statistics(P>0.05). Conclusion: 1MSCs has a powerful capable of multiplication. MSCs can differentiate into neuron like cells that has steady characters.2 CTP can induce MSCs into neuron-like cells.3 The best vol ratio of C and GP is 3:2. It needs about 10min that C/GP sol turn into gel at 37℃which propitious to the inject operation and the convergence of the cells .4 Neuron-like cells were able to grow very well in the C/GP . Biocompatibility between C/GP and neuron-like cells is very good. C/GP is a kind of perfect frame.