Investagation On The Biological Character And Culture Of Human Umbilical Cord Bloodmesenchymal Stem Cells In Vitro And Transplantation Of MNC In HIBD Rats

Objective To investigate the isolation, purification and culture of humanumbilical cord blood (HUCB) mesenchymal stem cells (MSCs) in vitro, and built stable culturesystem to satisfy experimental and clinical needs, then the UCB-derived mononuclear cells(MNC) was transplanted into the brain of the neonate rats to observe its survival anddifferentiation. Methods The cord blood mononuclear cell was isolated by lymphocyteseparation medium using the Density-Gradient Technique.Each sample(n=16) was divided into 3groups,then the cells were set in six-well culture plates with 5 ml L-DMEM and the finalconcentration is 1×10~4/ml,1×10~6/mland 1×10~8/ml seperately. In remaining samples,eachsample(n=20) was divided into 3 groups with different fetal calf serum density(5%,10%,and20%),then the cells were set in six-well culture plates which was supplemented with 5% fetal calfserum (FBS) and the final concentration is 1×10~8/ml.Friedenstein method was used to cultureand amplify MSCs.Different factors was investigated: the final concentration, density of FBS andculture plates coated with FBS. Flow cytometry was used to test antigens associated with MSCs.30 rats were randomly divided into three groups: transplantation group, sham group and NSgroup, one group was subjected to hypoxic-ischemic brain damage (HIBD) and was transplantedwith UCB-derived mononuclear cells (MNC) labeled by bromodeoxyuridine (Brdu); in the othergroup, right arteria carotis communis was separated but not ligated, then they were nottransplanted with MNC; the third group was transplanted with NS. Neurological functionrecovery was observed by Y-maze test. Results MNC in uncoated group,when set in the densityof 1×10~4/ml1and 1×10~6/ml,gave rise to several adherent cells,which died before passage. Moreadherent cells could be seen when set in the density of 1×10~8/ml. More adherent cells could beseen when set in the density of 1×10~8/ml. MNC in coated group,when set in the density of 5%,10%,20%,gave rise to more adherent cells,which was mainly MSCs.In coated group, cellsadherented are less but the purity is higher than in uncoated group. Crucial points to cultureMSCs-like cells were a culture medium with 5% FBS, a culture flask coated with FBS and anaccount of more than 1×10~8MNC. Antigens associated with MSCs was tested in MSCs-likecells. The animals received MNC following the hypoxic-ischemic brain injury performedsignificantly better on measures of memory ability. The result of immunohistochemistry showedthat in the brain of HIE rats, some human MSCs could survive in the side of injury, cerebralcortex, seahorse et al. About 6% of them expressed glial fibrillary acidic protein (GFAP), and 8%of them expressed microtubule-associated protein. Conclusion In the condition of a culturemedium with 5% FBS, a culture flask coated with MSCs FBS and an account of more than1×10~8 MNC, MSCs in HUCB can be cultivated successfully in vitro with high achievement ratioand purity. The animals received MNC following the hypoxic-ischemic brain injury performedsignificantly better on measures of memory ability Mononuclearcell (MNC) from human cordblood could differentiate into neuron and neuroglial cells when it was transplanted into the brain of neonate rats.