Comparison of different methods for separation and ex vivo expansion of cord blood progenitor cells

• Published in: Annals of hematology 1999-08, Vol.78 (8), p.364-370
• Main Authors: SCHWINGER, W, BENESCH, M, LACKNER, H, KERBL, R, WALCHER, M, URBAN, C
• Format: Article
• Language: English
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Antigens, CD34 - blood; Biological and medical sciences; Blood Component Removal - methods; Bone marrow, stem cells transplantation. Graft versus host reaction; Cell Separation - methods; Colony-Forming Units Assay; Cytapheresis; Erythrocyte Transfusion - methods; Erythroid Precursor Cells - cytology; Female; Fetal Blood - cytology; Granulocytes - cytology; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - immunology; Humans; Macrophages - cytology; Medical sciences; Megakaryocytes - cytology; Monocytes - cytology; Pregnancy; Transfusions. Complications. Transfusion reactions. Cell and gene therapy
• ISSN: 0939-5555; 1432-0584
• DOI: 10.1007/s002770050530

Umbilical cord blood is capable of hematopoietic stem cell reconstitution in children. However, the major limitation of cord blood is a relatively low content of pluripotent progenitor cells. Thus, safe engraftment for adolescents and for adults is still not predictable and a technology for ex vivo expansion of umbilical cord blood cells is desirable. In a first step, four different methods of red cell depletion followed by magnetic cell sorting of CD34+ cells were evaluated in this study in order to assess the efficacy and safety of optimal stem cell recovery. A modified two-step Ficoll gradient separation and a hydroxyethyl starch separation tended to produce a better WBC/MNC recovery (median 94.2+/-2.44% vs. 90.2+/-5. 8%) as compared with standard Ficoll gradient separation and a gelatin-based procedure (median 78.35+/-7.1% vs. 67.2+/-5.5%). However, the recovery of CD34+ cells after magnetic cell sorting did not reach a statistically significant difference after the four different methods of red cell depletion, indicating that the recovery of WBC/MNC is not predictably correlated with the recovery of stem cells within these fractions. In a second step, we established three different cytokine combinations by adding the megakaryocyte growth and development factor +/- erythropoietin and granulocyte colony-stimulating factor to a fetal calf serum containing medium with Flt 3, stem cell factor, and interleukin-3. Net expansion of total colony-forming cells 20- to 50-fold and expansion of colony-forming cells after 5 weeks of culture 1.5- to 3-fold were obtained over a period of 7-14 days. These results demonstrate that cord blood stem cells can be expanded substantially in this short-term culture system.