| Purpose: Since the great potentiality and foreseeableinfluence of Mesenchymal stem cells(MSCs) on the treatment ofhuman disease, in resent years, many researchers have been devotingefforts to the basic and clinic study of Mesenchymal stem cells(MSCs)on disease treatments, among which the research of Umbilical cordblood mesenchymal stem cells（UCB-MSCs）becomes one of theprevailing field of studies. UCB-MSCs have been successfullyinduced and differentiated into a variety of cells. Researchers haveexplored many methods for it’s separation and purification and thetechniques and conditions for culturing have been improved. There is,however, merely academic literature on the influence of differentwashing centrifugal forces on the purification of UCB-MSCs. Theauthor did not acquire satisfactory results of adherence and growth ofUCB-MSCs while with the traditional washing techniques suggestedin many literatures[1](Washing stem cells by1500rpm*10min, r=15cm,relative centrifugal field=378g). Analysis suggests that it was due tothe low cell purity result from the traditional washing technique.Further experiments and exploration is essential since the dramaticinfluences of different centrifugal forces on the purification of cells.The experiment was conducted under the same testing conditions, the process can be illustrated as follows: effectively layering umbilicalcord blood; washing UCB-MSCs with different centrifugal forces;documenting and comparing the influence of different centrifugalforces on the purification of UCB-MSCs; choose the centrifugal forceswhich is more conducive to the purification of UCB-MSCs and providbetter culturing environment for the growth and differentiation ofUCB-MSCs. Methods: Select eight pregnant women who meet theconditions of UCB-MSCs collection; then collect about40mlumbilical cord blood under sterile conditions; Diluting the cord bloodin1:1by0.9%physiological saline；slowly adding the blood to thelymphocyte isolation liquid（1.077g/ml）；Layering the cord bloodeffectively by the centrifugal rotational speed of2000rpm*20min（671g）；Collecting30ml sample of the White Coat, which is then mixedwith0.9%physiological saline to50ml；Blend the mixture and divideit into five lots；Centrifugalize and wash the mixture by the centrifugalrotational speed of1500r/min*10min（378g）、1300r/min*10min（284g）、1200r/min*10min（242g）、1000r/min*10min（168g）、800r/min*10min（107g） respectively；Discard the supernatant andobtain2ml sediments；Mix the medium with L-DMEM-10%fetalbovine serum-gentamicin to10ml; then blend the mixture andinoculate into the medium of T25; Observe the cells with invertedphase contrast microscope（the amplification is10×20）; Choose the field whose number of the impurities is intercalated and obtain imagesand contrast the differences in each group；Analyze the cell suspensionby sequential multiple autocytometer(XT-1800i)；and record thedensity of monocytes(MNCs)（10~9/L）、red blood cells(RBCs)（10~9/L）and blood platelet （10~9/L）and the rate of monocytes(MNCs)（%）.Analyze the data with SPSS15.0statistic software and draw curvecharts；Obtain one drop from each cell suspension using a pipette gun；Observe the impurities by Wright’s staining under oil immersion lens（the amplification is10×1000） and sample images. Discard all theculture medium at7thday after cell seeding；Choose five fields whichhave moderate number of adherent cells from every culture bottle andcount the number of adherent cells and spindle cells each,then analyzethe data using SPSS15.0statistic software；Observe growth conditionsof cells in every culture bottle at14thday after cell seeding and sampleimages which have moderate number of adherent cells.Result:1、There was massive impurities besides cells in the bottles in which thecells were washed by the centrifugal rotational speed of1500r/min*10min and1300r/min*10min when we observed andcompared the situation using inverted phase contrast microscope whenUCB-MSCs were just seeded. However there was significantly lessimpurities besides cells in the bottles labeling1200r/min*10min、1000r/min*10min and800r/min*10min, among which the phenomenon was most significant in bottles labeling800r/min*10mins; The result of cells and impurities under oil immersionlens by Wright’s staining was the same as the result under invertedphase contrast microscope.2、The curve charts of the density ofmonocytes(MNCs（)10~9/L）、red blood cells(RBCs（)10~9/L）and bloodplatelet（PLT）（10~9/L）and the rate of monocytes(MNCs)（%） underdifferent centrifugal forces showed that: the density of MNCs（10~9/L）increased and the rate of MNCs（%） decreased with the decreasingof the centrifugal forces, which has little statistical significance（P>0.05）. However the results demonstrated reduction in the densityof RBC（s10~9/L）and PLT（10~9/L）with the decreasing of the centrifugalforces，which has statistical significance（P<0.05）.3、Seven days aftercell seeding, chose five fields which had moderate number of adherentcells in each culture bottle；the number of adherent cells were countedunder inverted phase contrast microscope and the data wereanalyzed.The differences had statistical significance（P<0.05）.Observed the spindle cells in the bottle which was marked800r/min*10min,there were5-8fields which had above5spindlecells.In the bottles which were marked1200r/min*10min、1000r/min*10min, there were about1-3fields which meet the demand,andthere was no fields met the demand in the bottle marked1500r/min*10min and1300r/min*10min4.Fourteen days after cell seeding, the cells in the bottle marked800r/min*10min grew best,and we could find2-5fields in which the spindle cells fused andembraced (the number of the cells aboves7in each field ofmicroscope). However the cells in other bottles did not fuse andembrace and were detached.Conclusions：1、When purifying theUCB-MSCs with the lymphocyte isolation liquid（1.077g/ml） bydensity gradient centrifugation, the cell purity we acquired by thetradition washing method that1500r/min*10min（378g）is low,and it isdisadvantageous to the adherence、growth and differentiation of theUCB-MSCs.The situation will be improved by decreasing theCentrifugal Force for washing，and the situation will be best by800r/min*10min（107g）;2、The influence of the density of the residualRBCs on the adherent of UCB-MSCs is much less than that of PLTwhen purifying the UCB-MSCs with the lymphocyte isolation liquidby density gradient centrifugation.May be the the density of theresidual PLT is the most important factor which influence the adherentof UCB-MSCs,and we must focus on removing PLT when purifyingthe cells. |
PDF Full Text Request