A culture medium and method for in vitro stem expansion of umbilical cord blood hematopoietic stem cells
A technology of hematopoietic stem cells and umbilical cord blood, applied in the direction of cell culture active agents, blood/immune system cells, animal cells, etc., can solve problems such as shortage, and achieve the effect of promoting expansion, avoiding excessive differentiation and high expansion multiple.
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Embodiment 1
[0045] Inject 100ml of fresh healthy neonatal umbilical cord blood sample used to provide hematopoietic stem cells to be expanded into a 250ml centrifuge tube under aseptic conditions, centrifuge at 1200rpm for 10 minutes at room temperature, discard the upper plasma, and add normal saline to dilute the blood cell pellet to the original volume. 2 times, mix with a pipette.
[0046] Slowly load the blood cell suspension onto the pre-prepared 0.4 volume of lymphocyte separation medium, taking care to keep the layers clear. Centrifuge at 2000 rpm for 20 minutes at room temperature. Due to different densities, the centrifuge tube is divided into four layers. The upper layer is saline and plasma; the lower layer is junctional layer cells, mainly composed of lymphocytes and monocytes, and the third layer is lymphocytes. Cell separation medium, the fourth layer is the sedimented erythrocytes and granulocytes.
[0047] Aspirate most of the uppermost liquid with a pipette and discard,...
Embodiment 2
[0052] The fresh healthy neonatal umbilical cord blood sample used to provide hematopoietic stem cells to be expanded uses the same CD34 as in Example 1 + Cell isolation methods are processed to obtain CD34 + Hematopoietic stem cells, counted.
[0053] The fresh healthy neonatal umbilical cord blood sample was subjected to the above steps to obtain CD34 + Cells, inoculated in cell culture medium for expansion culture, cell culture medium using serum-free medium F12 medium, adding thrombopoietin to a concentration of 109ng / ml, adding stem cell growth factor to a concentration of 8.2ng / ml, adding FMS sample Tyrosine kinase 3 ligand to a concentration of 110ng / ml, polyvinyl alcohol to 5mg / ml, insulin, transferrin, selenium and ethanolamine to final concentrations of 11.8mg / L, 4.9mg / L, 0.0062mg / ml, respectively L and 2.1 mg / L, add glutamine to a final concentration of 289 mg / L.
[0054] The amount of cells inoculated in a 24-well plate is 5 × 10 4 / ml, placed at 37°C, 5% CO 2...
Embodiment 3
[0056] The fresh healthy neonatal umbilical cord blood sample used to provide hematopoietic stem cells to be expanded uses the same CD34 as in Example 1 + Cell isolation methods are processed to obtain CD34 + Hematopoietic stem cells, counted.
[0057] The fresh healthy neonatal umbilical cord blood sample was subjected to the above steps to obtain CD34 + Cells, inoculated in cell culture medium for expansion culture, cell culture medium using serum-free medium HIPP-T009 medium, adding thrombopoietin to a concentration of 101ng / ml, adding stem cell growth factor to a concentration of 10.3ng / ml, adding FMS-like tyrosine kinase 3 ligand was added to a concentration of 102.1 ng / ml, polyvinyl alcohol was added to 1 mg / ml, and insulin, transferrin, selenium, and ethanolamine were added to final concentrations of 10 mg / L, 5.5 mg / L, and 0.0067, respectively mg / L and 2.0 mg / L, add glutamine to a final concentration of 292 mg / L.
[0058] The amount of cells inoculated in a 24-well p...
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