Human umbilical cord blood plasma can replace fetal bovine serum for primary culture, proliferation and cryopreservation of umbilical cord mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Fetal bovine serum based media used for expanding and cryopreserving human mesenchymal stem cells raise safety concerns in the clinical setting. OBJECTIVE:To investigate the feasibility of human umbilical cord blood plasma as a replacement for fetal bovine serum in culture and cryopreservation of human mesenchymal stem cells derived from umbilical cord. METHODS:Umbilical cord blood units were suitable for this research if they fulfil ed the donor selection criteria of the Guangzhou Cord Blood Bank strictly. Cord blood plasma was ready to use after col ected from the plasma reduction during the suitable cord blood units processing and pooling. Umbilical cord mesenchymal stem cells were harvested from the umbilical cord tissue of health ful-term newborns after delivery by enzyme digestion and were cultured in the presence of Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing either fetal bovine serum or pooled cord blood plasma. Morphology, proliferation, immunophenotype detected by flow cytometry and differentiation toward adipogenic and osteogenic lineages were utilized for investigating the effect of media on umbilical cord mesenchymal stem cells after 3-5 passages. Then cells were cryopreserved in media containing 10%dimethyl sulfoxide, 20%fetal bovine serum or 20%pooled cord blood plasma for at least 6 months. Viability, adhesion, proliferation, immunophenotype and osteogenic differentiation of the cells were assessed after thawing. RESULTS AND CONCLUSION:The morphology (spindle-shaped and plastic-adherent), phenotype and differentiation potential (osteogenic and adipogenic) were almost indistinguishable between cells cultured in fetal bovine serum or cord blood plasma medium, while cells grown in cord blood plasma medium demonstrated significantly higher proliferation rates than those in medium containing fetal bovine serum. After thawing, the cells maintained their adherence to the culture surface and differentiation potential to osteoblasts, but cells from cord blood plasma cryopreservation medium showed significantly better plastic attachment and produced greater cellnumbers than fetal bovine serum for the first three post-thaw passages. The results demonstrate that cord blood plasma can sever as an effective substitute to fetal bovine serum for growth, maintenance and differentiation of umbilical cord mesenchymal stem cells, and thus it wil be a safe choice for clinical-scale production of human mesenchymal stem cells.