Efficient multiplication culture system of neutrophil and application of efficient multiplication culture system

A technology of neutrophils and neutrophils, applied in the biological field, can solve the problems of neutropenia, high risk of infection in patients, and inability to take immediate effect, achieving remarkable effects and no risk of tumorigenesis Effect

Pending Publication Date: 2022-07-01
BIOPHARMAGEN CORP FANGZHOU SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some treatment options for fungal or bacterial infections unresponsive to antimicrobial therapy in the presence of diffuse lesions of the skin, mucous membranes, or radiological examination can only rely on clinical infusion of neutrophils.
[0003] High-dose chemoradiotherapy can cause severe neutropenia during which patients are at high risk of infection
Although the injection of granulocyte colony-stimulating factor can alleviate and treat neutropenia to a certain extent, the use of granulocyte colony-stimulating factor cannot immediately treat critically ill patients, especially those who have not recovered from bone marrow hematopoietic stem cell transplantation. To play a role, neutrophil infusion must be used to rescue the patient
At present, cells in neutrophil infusion therapy are mainly isolated from human peripheral blood. Donors who donate neutrophils need to use G-CSF to stimulate the number of neutrophils in peripheral blood. The complicated donation process is serious. Limits the method availability and source of neutrophils

Method used

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  • Efficient multiplication culture system of neutrophil and application of efficient multiplication culture system
  • Efficient multiplication culture system of neutrophil and application of efficient multiplication culture system
  • Efficient multiplication culture system of neutrophil and application of efficient multiplication culture system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Efficient expansion and culture of human umbilical cord blood-derived neutrophils.

[0040] (1) Extraction of CD34 from umbilical cord blood + cell

[0041] 1) Mix equal volume of fresh umbilical cord blood with sterile PBS (pH 7.2), and calculate the volume ratio of the total volume of umbilical cord blood after mixing to the lymphocyte separation liquid (Ficoll) of 4:3, and put it in a sterile centrifuge tube. First add Ficoll, then slowly add the cord blood to the Ficoll to make it float on the top of the Ficoll;

[0042] 2) After all the addition is complete, carefully take out the centrifuge tube and centrifuge at 2000rpm for 30 minutes. After the centrifugation is completed, take out the mononuclear cell layer and use a 1000μl micropipette to aspirate the white mononuclear cell layer into an empty 50ml centrifuge. spare in the tube;

[0043] 3) Add sterile PBS to make up the volume to 40ml and mix well, take a small amount of solution and count the c...

Embodiment 2

[0063] Example 2 In vitro expansion and differentiation of neutrophils using peripheral blood hematopoietic stem cells.

[0064] The process of mobilizing peripheral blood to isolate hematopoietic stem cells and the above-mentioned use of magnetic bead sorting to sort umbilical cord blood CD34 + The process of hematopoietic stem cells is the same, and the culture method is basically the same. It should be noted that CD34 in peripheral blood during the culture process + The expansion and differentiation speed of hematopoietic stem cells is slightly slower than that of umbilical cord blood hematopoietic stem cells. Counting and changing the medium at each stage should be carried out according to the cell density (the first stage maintains the cell density at 2 × 10). 5 to 1×10 6 cells / mL, the cell seeding density in the second stage was 1.5×10 5 / mL~3×10 5 / mL, then keep no more than 2 × 10 7 / mL). The proportion of neutrophils in the total cells obtained will be much highe...

Embodiment 3

[0066] Example 3: Mobilization of peripheral blood CD34 in non-human primate cynomolgus monkeys + Isolation, in vitro expansion and differentiation of hematopoietic stem cells.

[0067] 1) Cynomolgus monkey peripheral blood hematopoietic stem cell mobilization and culture process: Cynomolgus monkeys were injected subcutaneously with G-CSF (200 μg / kg) and SCF (200 μg / kg) every day for five consecutive days to mobilize bone marrow stem cells in the humerus and femur. into peripheral blood. 15ml of peripheral blood was collected on the 6th and 7th day of injection respectively;

[0068] 2) Use Miltenyi's MACS magnetic bead sorting system to separate CD34 + cells, isolated hematopoietic stem cells (about 2-3 × 10 6 a) The sorting process is the same as the method for human umbilical cord blood hematopoietic stem cells, and the magnetic beads are replaced with non-human primate anti-CD34 magnetic beads;

[0069] 3) Use the culture formula for efficient expansion of hematopoieti...

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Abstract

The invention discloses an efficient multiplication culture system for neutrophil. The preparation method comprises the following steps: adding one or a combination of more of a cell growth factor SCF, a human FMS-like tyrosine kinase 3 ligand Flt-3L, a granulocyte colony stimulating factor G-CSF, a granulocyte-macrophage colony stimulating factor GM-CSF, an interleukin-3 IL-3, a thrombopoietin TPO, fetal bovine serum and human serum albumin HSA into a basic culture medium according to different culture stages, the basic culture medium is obtained by adding nutritional ingredients into an IMDM culture medium, and the nutritional ingredients comprise putrescine, selenium, insulin, transferrin and a B-27 supplement. The method realizes large-scale in-vitro efficient amplification and differentiation of hematopoietic stem cells (HSC) into neutrophils, and further confirms that the obtained neutrophils have the same efficacy as the neutrophils separated from human peripheral blood.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-efficiency expansion and culture system and application of neutrophils. Background technique [0002] Neutrophils are specialized phagocytic cells that grow, store, and release into the bloodstream in the bone marrow. During the initial (acute) phase of inflammation, especially as bacterial infection, environmental exposure, and the development of some cancers, neutrophils are the first responders of inflammatory cells that migrate to the site of injury. Patients receiving intensive chemotherapy in the clinic often experience frequent and prolonged neutropenia, a major risk factor for serious bacterial and fungal infections. Although the use of modern antibiotics and / or blood cell stimulating growth factors has shortened the period of treatment for neutropenia, infection remains a major cause of morbidity and mortality in these patients. Normal neutrophil counts ...

Claims

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Application Information

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IPC IPC(8): C12N5/0787
CPCC12N5/0642C12N2501/125C12N2501/26C12N2501/22C12N2501/2303C12N2501/145C12N2500/46C12N2500/25Y02A50/30
Inventor 蒋永平王含璐接振旺
Owner BIOPHARMAGEN CORP FANGZHOU SUZHOU
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