Effect Of Different Cryopreservation Systems For The Olfactory Ensheathing Cells' Activity

ObjectiveSpinal cord injury (SCI) is a clinical common and frequently-occurring disease, not only leaving over high morbidity, but also the incidence of annual rise increased significantly. At present, in a variety treatment of SCI, efficacy of the olfactory ensheathing cells (OECs) transplantation is the best. In clinical transplantation, OECs derived from the olfactory bulb of aborted fetuses, not only its resource is few, and the specific time and location uncertain, but also there are uncertain factors of specific time and place on the clinical transplantation. Olfactory bulb of the aborted fetus need for timely cultivation, but OECs can not be unlimited passage and vulnerable to miscellaneous pollution in the training process, such as fibroblast cells, so cultivated for 10-14 days, OECs will be required to transplant. As differences of the specific time and place exist between cultivation and clinical transplantation, clinical transplantation of OECs has seriously affected. Because the source is fewer, in order to effectively use scarce resources and heal patients of SCI, The cryopreserve of OECs is very important. The purpose of this experiment explore proper cryopreservative systems for long-term preservation of OECs, as well as the time on cryopreserve, to solve these issues and provide a theoretical basis for the next clinical transplantation.Materials and methodsTaking 2-month-old male healthy Wistar rats'olfactory bulb, then undertake the culture of OECs, obtained OECs (logarithmic growth phase cells) were randomly divided into five groups. Group A and group D:put OECs into No.1 frozen liquid; group B and group E:No. use 2 frozen liquid suspension; group C:suspense OECs with No.3 frozen liquid (10% DMSO,5% DMSO-6% HES,5% DMSO respectively represent cryopreservative agent of No.1,2,3 frozen liquid). Group A, group B, group C were cooled with refrigerator and stored in liquid nitrogen; but group D and group E were cooled with autocontrolled programmed cryogenic system (PCS) and cryopreservatived in liquid nitrogen. Specimens were cryopreservation in one month, three months, and half-a-year, then the recovery of specimens, observed cell morphology under light microscope, undertake MTT assay and trypan blue staining method detecting activity of recovered cell, the index, contrast between the two groups or each group were performed.ResultsBy Microscopic observation, cell recovery of 5% DMSO-6% HES group is best, cells'adhesion and proliferation have short time, shrunken cells and cell debris or the bad cell of body's transmittance and three-dimensional has lest numbers in all groups; but in 5%DMSO group, you will be ease to see the shrink cells and apoptotic cells'fragments, by compared with the former, the OECs'number that the transmittance and three-dimensional sense is bad increases significantly, OECs spend a little longer time on adhesion and proliferation; while each index of 10%DMSO group is between the two formers.The specimens have been cryopreserved one month, three months and half-a-year, 1.Under the same cryopreservation system, has no significant difference in each group; 2.Under the same cryopreservative agent, Refrigerator cryopreservation with liquid nitrogen cryopreservation compared with PCS with liquid nitrogen cryopreservation, difference was not significant (P>0.05); 3. by the difference of cryopreservative agent, all indicators of 5% DMSO-6% HES group is better than 10% DMSO group and 5% DMSO group, the difference was statistically significant (P<0.05); all indicators of 10% DMSO group compared with 5% DMSO group is good, the difference was statistically significant (P<0.05).Samples of Refrigerator cryopreservation with liquid nitrogen cryopreservation preserved in three months, the recovery cells continue to foster five days before the cells will test, all indicators in B group is better than in A group and C group, significant difference (P<0.05); A group compared with C group, the cytoactive is good, difference is statistically significant (P<0.05).OECs randomly divide into three groups, separately foster two hours at room temperature with No.1 frozen liquid, No.2 frozen liquid and No.3 frozen liquid, then test cytoactive. Viability of each group decrease, of which 10%DMSO group of cell viability is significantly lower than the other two groups, significant difference (P<0.05), the latter two groups are not significantly different, no statistically significant (P<0.05).The specimens have been cryopreserved six months before being thawed, don't washing, and continue to exist on the original frozen liquid at the room temperature in different times, various indicators of the cytoactive decrease significantly, to 10% DMSO group most affect.Conclusion1. Selecting frozen liquid of OECs, we recommend to use 5% DMSO-6% HES as a cryopreservative agent;2. For small specimen of the OECs'preservation can use refrigerator cooling and liquid nitrogen storage methods;3. For large sample of the OECs'preservation may use autocontrolled programmed cryogenic system cooled or refrigerator cooled, then both preserved in liquid nitrogen4. Cryopreserved for half-a-year, OECs still has good cytoactive;5. Cryopreservative agent within the specimen should be diluted or removed immediately after Cells were thawed, then re-application of cell culture or transplantation.