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

Active Publication Date: 2015-08-19
浓孚雨医药河北有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to solve the practical problems of the currently commonly used peripheral blood DCs, such as the small number of DCs, poor antigen phagocytosis, low antigen extraction function, and large individual differences.

Method used

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  • Mass preparation method for cord blood CD34+ hematopoietic stem cell-derived dendritic cells
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  • Mass preparation method for cord blood CD34+ hematopoietic stem cell-derived dendritic cells

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention belongs to the field of cellular immunology, and discloses a methodological study for inducing mass generation of dendritic cells (DCs) from cord blood CD34+ hematopoietic stem cells. The method comprises the following main steps: separating mononuclear cells from cord blood, separating CD34+ hematopoietic stem cells through an immunomagnetic bead positive screening method, continuously amplifying the CD34+ cells for 30 days by using an amplification culture medium (IMDM, FBS, GM-CFS, SCF), periodically collecting mass CD34-DC precursor cells during the amplification process, and inducing the precursor cells to be differentiated into immaturate DCs through a GM-CFS/IL-4 culture medium. The method disclosed by the invention is capable of obtaining the DCs with 109 orders of magnitudes; compared with the traditional DCs induced by peripheral blood mononuclear cells, the CD34-DCs have high antigen phagocytic capacity and the capacity of inducing T lymphocyte proliferation.

Description

technical field [0001] The present invention relates to CD34 + A novel use of hematopoietic stem cells, specifically involving cord blood-derived CD34 + A method for preparing a large amount of dendritic cells induced by hematopoietic stem cells. Background technique [0002] Dendritic cells (DC) are professional antigen-presenting cells that exist in the human body and can induce immune responses. Currently, specific DC anti-tumor vaccines based on DC cells have been widely used in clinic. DC cells are mainly derived from bone marrow, umbilical cord blood, or peripheral blood mononuclear cells. By loading tumor antigens, DC can induce the body to produce a strong and specific anti-tumor immunity. More than 150 clinical trials have been launched around the world to verify the safety and effectiveness of DC vaccines against various tumors. [0003] At present, most DC biological preparation methods use autologous peripheral blood mononuclear cells of tumor patients, but t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784C12N5/0789
Inventor 蔡建辉刘刚崔红娟蔡颖
Owner 浓孚雨医药河北有限公司
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