Mass preparation method for cord blood CD34+ hematopoietic stem cell-derived dendritic cells
A technology of hematopoietic stem cells and dendritic cells, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of small number of DCs derived from peripheral blood, low antigen synthesis function, and large individual differences
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Embodiment 1
[0022] Example 1 Preparation of umbilical cord blood-derived DC
[0023] Isolation of cord blood mononuclear cells:
[0024] 1. Collect 50 ml of fresh umbilical cord blood from healthy full-term pregnant women after delivery with a sterile syringe, and anticoagulate with heparin;
[0025] 2. Divide 50ml of umbilical cord blood into two 50ml centrifuge tubes equally, and then centrifuge in a centrifuge for 10 minutes at a speed of 3000 r / min; after centrifugation, suck out the upper layer of plasma with a straw and discard it;
[0026] 3. According to the ratio of 1:1:1, add sterile saline and hydroxyethyl starch solution to the precipitated cord blood in the previous step to resuspend the cells; incubate in a 37°C incubator for 30 minutes to make the red blood cells precipitation;
[0027] 4. After the erythrocytes are precipitated, use a small pipette to suck out the upper layer liquid, and slowly transfer the cell suspension along the tube wall at a ratio of 2:1 (2 volumes...
Embodiment 2
[0043] Example 2 Preparation of peripheral blood-derived DC and preparation of lymphocytes
[0044] Isolation, induction and differentiation of peripheral blood DC (PBMC-DC)
[0045] 1. Peripheral blood was taken from the Hebei Provincial Blood Center. Obtain the discarded leukocyte filter after filtering 400ml of healthy human peripheral blood. After aspirating sterile saline with a sterile syringe, wash the leukocyte suspension in the opposite direction of the filter and collect it. To two 50ml centrifuge tubes, 30ml each;
[0046] 2. Slowly add the cell suspension to the surface of the human lymphocyte separation medium (Ficoll) along the tube wall according to the ratio of 2:1 (2 volumes of cell suspension: 1 volume of lymphatic separation medium) in the dark. Put it gently in the centrifuge, centrifuge at 900g / min without brake for 30min;
[0047] 3. After the centrifugation, the cells in the centrifuge tube are divided into 4 layers, suck out the buffy coat laye...
Embodiment 1、 Embodiment 2
[0063] DC mixed lymphocyte reaction in Example 1 and Example 2
[0064] 1. Cord blood CD34 + The ability of immature DC derived from CD34-imDC and immature DC derived from peripheral blood mononuclear cells (PBMC-imDC) to stimulate lymphocyte proliferation was detected by MTS method;
[0065] 2. Take the peripheral blood lymphocytes cultured on the 5th day and wash them twice with sterile saline, resuspend in 1640 medium containing 10% FBS, and mix according to the cell concentration (10 6 / per well) inoculated into 96-well plate;
[0066] 3. Take the CD34-imDC and PBMC-imDC cultured in GM-CSF / IL-4 medium until the fifth day, wash them twice with sterile saline, then add 1640 medium containing 10% FBS to resuspend the cells, ImDCs were inoculated into lymphocyte well plates according to the ratio of 1:10, 1:20, 1:40, 1:100 (imDC: lymphocytes). The lymphocytes not stimulated by imDC were set as the control group. Cultured in a 37°C, 5% CO2 stable humidity cell incubator.
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