Isolation, Culture And Identification Of The Mesenchymal Stem Cells Derived From Preterm And Full-term Infants Umbilical Cord Blood In Vitro

【Objective】To analyse the success rate of mesenchymal stem cells derived from preterm and full-term infants umbilical cord blood,and to study the related biological characteristics and differentiation ability,in order to explore the best way of obtaining umbilical cord blood mesenchymal stem cells(UCB-MSCs) and to provide theory basis for the further experimental study.【Methods】Umbilical cord bloods(n=54)were collected from full-term(n=32)and preterm infants (n=20) under aseptic condition.All specimens were anticoagulated with sodium citrate.Mononuclear cells(MNCs) were harvested from full-term infants UCB(n=12) using Ficoll lymphocytes separating solution and density gradient centrifugation,compared DMEM/F12 medium with Mesencult^TM medium to distinguish the influence of the success rate of UCB-MSCs under the same inoculation density; MNCs were got from full-term delivery infants UCB(n=20) using Ficoll lymphocytes separating solution and density gradient centrifugation,compared 1×10⁶/ml with 1×10⁷/ml inoculation density to distinguish the influence of the success rate of UCB-MSCs under the same medium; MNCs were harvested from preterm infants UCB(n=20),compared the success rate of mesenchymal stem cells that derived from preterm with full-term UCB-MSCs,and analysed the related biological characteristics and differentiation ability ,respectively. The morphology of UCB-MSCs and their growth characteristics were observed under an inverted phase contrast microscope;Growth curve was draw;The surface markers was analyzed by flow cytometry,including CD29,CD44,CD105,CD34,CD45;The osteogenic,adipogenic and chondrogenic medium were used to induce UCB-MSCs differentiate into osteoblasts,adipocytes and chondrocytes. Osteogenic potential was determined by alizarin red staining.Adipogenic potential was detected by oil-red O staining. Chondrogenic potential was detected by Alcian blue staining. The SPSS13.0 software package was used for all statistical analyses.【Results】1. The comparion of the success rate of preterm and full-term infants UCB-MSCs by using Mesencult^TM medium and DMEM/F12 medium,and by using 1×10⁷/ml and 1×10⁶/ml inoculation densityThere has significant differences between the success rate of UCB-MSCs which cultured with DMEM/F12 medium and Mesencult^TM medium, the success rate was 20% and 40%, respectively(P<0.05); There was statistically significant differences between the success rate of UCB-MSCs which inoculated in 1×10⁶/ml and 1×10⁷/ml,the success rate was 33.3% and 50% ,respectively(P<0.05).2. The comparion of the success rate derived from preterm and full-term infants UCB-MSCsThere had significant differences between the success rate of UCB-MSCs derived from preterm and full-term infants, the success rate was 85.00%(17/20)and 55.00%(11/20),respectively(P<0.05).3. The comparion of the biological characteristics derived from preterm and full-term infants UCB-MSCs3.1 There had statistically significant differences between the time of primary cells cultured for preterm and full-term infants, the time of primary cells cultured for preterm and full-term infants were 19.65±1.69d and 28.36±1.36d, respectively. (P<0.05); The time of subcultured from preterm and full-term infants was 14.94±1.75d and 19.27±2.10d respectively. There was statistically significant differences(P<0.05). 3.2 Flow cytometry showed that both of the UCB-MSCs expressed MSCs surface markers CD29,CD44 and CD105,but they failed to express hematopoictic stem cells surface markers,such as CD34,CD45.4. The comparion of the differentiation potential derived from preterm and full-term infants UCB-MSCsWhen the MSCs were induced to osteogenic differentiation for 2 weeks,the formation of mineralized matrix was detected by alizarin red staining;induced to osteogenic differentiation for 3 weeks,neutral lipid vacuoles was detected by oil-red O staining ; induced to chondrogenic differentiation for 3weeks ,proteoglycans was detected by Alcian blue staining.【Conclusions】1. UCB-MSCs can be isolated,purified and proliferated successfully in vitro.2. Choosing Mesencult^TM medium and 1×10⁷/ml inoculation density can improve the success rate of UCB-MSCs.3. The success rate of UCB-MSCs from preterm infants was higher than full-term deliveries.The time of cultured and subculture for primary cells from preterm infants were shorter.4. UCB-MSCs can be induced to differentiation to osteoblasts,lipoblasts and chondroblast in vitro.