Method of isolating and culturing mesenchymal stem cell derived from umbilical cord blood
The present invention relates to a method of isolating and culturing mesenchymal stem cells using umbilical cord blood that is most ideal for cell therapy. The method comprises adding an anti-coagulant to umbilical cord blood having a volume of more than 45 ml per unit, which is obtained within 24 hours after parturition; diluting the resulting mixture of the anti-coagulant and umbilical cord blood with an αMEM (alpha-minimum essential medium), followed by centrifugation to harvest monocytes; and subjecting the obtained monocytes into suspension culture in the αMEM containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6).
1. A method for isolating and culturing mesenchymal stem cells from umbilical cord blood, comprising the steps of:
adding an anti-coagulant to pure umbilical cord blood obtained within 24 hours after parturition;
diluting the resulting mixture of the anti-coagulant and umbilical cord blood with an alpha-minimum essential medium (aMEM), followed by centrifugation to harvest monocytes; and
subjecting the obtained monocytes into suspension culture in the aMEM medium containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3), and IL-6 (interleukin-6).
2. The method as set forth in claim 1 , wherein the umbilical cord blood is diluted with 2-fold volume of the aMEM medium, overlapped on Ficoll-Hypaque and subjected to centrifugation so as to harvest monocytes.
3. The method as set forth in claim 1 , wherein aMEM medium for culturing monocytes further comprises at least one of an antibiotic, an anti-fungal, glutamine and fetal bovine serum.
4. The method as set forth in claim 3 , wherein the aMEM for culturing monocytes further comprises an antibiotic, an anti-fungal agent, glutamine and fetal bovine serum.
5. The method as set forth in claim 1 , wherein the umbilical cord blood has a volume of more than 45 ml per unit.
6. The method of claim 3 , wherein the antibiotic is selected from penicillin G, streptomycin sulfate, or a combination thereof.
7. The method of claim 3 , wherein the antifungal agent is amphotericin B.
8. The method of claim 1 , wherein the isolated mesenchymal stem cells are negative for CD14, CD34, CD45 indicators and are positive for SH2, SH3, CD29, CD44, CD90, and CD166 indicators.