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In vitro induction culture method for bone marrow-derived macrophages

A bone marrow-derived, macrophage technology, applied in the biological field, can solve the problems of short survival time, poor macrophage homogeneity, and low yield, and achieve the effect of improving purity, shortening the time required for differentiation and maturation, and improving purity.

Inactive Publication Date: 2012-10-17
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method of obtaining macrophages is simple and convenient, the obtained macrophages are often poor in uniformity, low in yield and short in survival time

Method used

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  • In vitro induction culture method for bone marrow-derived macrophages
  • In vitro induction culture method for bone marrow-derived macrophages
  • In vitro induction culture method for bone marrow-derived macrophages

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of bone marrow-derived cell mixture

[0029] Take 6-8 week old mice, let them die by taking off their necks, and immerse them in 70% ethanol by volume. Use tweezers to pick up the abdominal epithelium, cut a small opening horizontally under the mouse abdomen with scissors, clamp the two ends of the opening and separate them in different directions to expose the mouse legs, pull out the mouse femur and tibia by hand, and make the small The mouse femur was separated from the mouse body and tibia at the joint, leaving both ends intact. Use alcohol-soaked gauze to remove the remaining tissue and cartilage at the articular junction of the femur.

[0030] Rinse the removed femur in 70% alcohol for 2 to 3 times, and then rinse in PBS buffer; prepare 5 to 10ml of PBS in a centrifuge tube in advance, use a 1mL syringe to draw the PBS buffer in the centrifuge tube and insert it into the femur The cartilage at the two ends of the femur was quickly flushed o...

Embodiment 2

[0033] Example 2 Treatment of Cell Suspension

[0034] The bone marrow-derived cell mixture was gently pipetted several times to prepare a mononuclear cell suspension. Pipette the cell suspension through a 75 μm filter (multiple experiments have proved that a 40 μm filter can also be achieved), transfer it into a 10ml centrifuge tube, and centrifuge at 1000-1500rmp / min for no more than 10min. Remove the supernatant, and gently pipette the resuspended cell suspension with PRMI1640 complete medium containing M-CSF (20ng / ml) to prepare a mononuclear cell suspension. Bone marrow source used in "Ishii M, Wen HT, Corsa CAS, Liu TJ, Coelho AL, Allen RM, et al. Epigenetic regulation of the alternatively activated macrophage phenotype. Blood. 2009;114(15):3244-54" The in vitro induction culture method of macrophages does not have this step; adding this step will greatly improve the purity of bone marrow-derived progenitor cells, and more importantly, it will also reduce the chance of ...

Embodiment 3

[0035] Example 3 In vitro induction and culture of bone marrow-derived macrophages

[0036] Perform cell counting on the bone marrow-derived cells filtered in Example 2, determine the cell concentration, dilute the cell suspension and adjust the cell concentration to 1-2×10 6 pieces / ml. To plank, place 1 x 10 6 cells / ml of cell suspension was inoculated into 6 empty culture plates, 3ml / well. After incubating in a cell culture incubator at 37° C. and 5% for 30 hours, the supernatant was discarded, and new PRMI1640 complete medium containing M-CSF (20 ng / ml) was added. The step of aspirating and discarding the supernatant is very critical. The significance of this step is to purify macrophage progenitor cells and improve the purity of induced differentiation macrophages. The obtained single cell suspension is directly induced and cultured in vitro without the critical step of aspiration and discarding the supernatant. Some dead cells or non-macrophage progenitor cells are not...

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Abstract

The present invention belongs to the field of biotechnology, and relates to a cell culture method, and specifically to an in vitro induction culture method for bone marrow-derived macrophages. The method concretely comprises: culturing bone marrow-derived single cell suspension with an M-CSF-containing PRMI1640 medium in vitro, wherein pretreatment steps for the bone marrow-derived single cell suspension are performed before the medium is added, and the pretreatment steps comprise filtering the bone marrow-derived single cell suspension by a filter with a filter hole size of 40-75 mum and culturing the resulting filtering liquid. In embodiments of the present invention, the time required by macrophage differentiation mature is shortened by 1-2 days compared with the time required by the original method in the prior art; with treatment of the cell suspension, the bone marrow-derived progenitor cell purity in the filtered cell is improved compared with the bone marrow-derived progenitor cell purity in the unfiltered cells, wherein the bone marrow-derived progenitor cell purity in the unfiltered cells is only about 17-20%, and the bone marrow-derived progenitor cell purity is improved to 30-40% after filtering.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell culture method. Background technique [0002] Macrophages are widely distributed in various organs and tissues throughout the body, and play an important role in maintaining internal environment stability, body defense, inflammation regulation, and promoting tissue damage repair. Macrophages have different phenotypes in different microenvironments, which make macrophages in different functional states. These macrophages with different functional states exert different physiological functions under different physiological and pathological conditions; they are closely related to the occurrence and development of serious diseases such as tumors, metabolic syndrome, and diabetes. [0003] Resting macrophages can be activated and differentiated into two types with different phenotypes and functions under different stimulation conditions: M1 type and M2 type. M1-type macrophages m...

Claims

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Application Information

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IPC IPC(8): C12N5/0786
Inventor 张志仁杨琴杨晓风姜曼吴玉章
Owner ARMY MEDICAL UNIV
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