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A proliferation medium and proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow mesenchymal stem cells

A technology of proliferation medium and mammals, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of limited clinical application, toxic side effects, etc., to improve clinical efficacy, maintain growth, and promote rapid growth. The effect of massive amplification

Active Publication Date: 2022-07-01
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The medium is an important factor in the in vitro culture environment of cells. Although the complete medium prepared by adding an appropriate amount of serum to the basal medium can meet the requirements of most cells including BM-MSCs, the clinical application is limited due to the above-mentioned toxic and side effects.

Method used

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  • A proliferation medium and proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow mesenchymal stem cells
  • A proliferation medium and proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow mesenchymal stem cells
  • A proliferation medium and proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation of BGJb medium:

[0026] According to the following ingredients: glycine (800mg / L), L-alanine (250mg / L), L-arginine (175mg / L), L-aspartic acid (150mg / L), L-cysteine Amino acid (101mg / L), L-glutamine (200mg / L), L-histidine (150mg / L), L-isoleucine (30mg / L), L-leucine (50mg / L) L), L-Lysine (240mg / L), L-Methionine (50mg / L), L-Phenylalanine (50mg / L), L-Proline (400mg / L), L-Serine ( 200mg / L), L-threonine (75mg / L), L-tryptophan (40mg / L), L-tyrosine disodium salt (58mg / L) and L-valine (65mg / L) ); vitamin C (50mg / L), biotin (0.2mg / L) choline chloride (50mg / L), calcium pantothenate (0.2mg / L), DL-α-tocopherol (1.0mg / L) folic acid (0.2mg / L), nicotinamide (20mg / L), p-aminobenzoic acid (2.0mg / L), pyridoxal hydrochloride (0.2mg / L), riboflavin (0.2mg / L), thiamine hydrochloride (4mg / L), vitamin B12 (0.04mg / L) and inositol (0.2mg / L); magnesium sulfate (98mg / L) potassium chloride (400mg / L), potassium dihydrogen phosphate (160mg / L), Sodium bicarbonate (3500mg / L),...

Embodiment 2

[0028] Example 2: Preparation of proliferation medium (ZEzh medium)

[0029] Medium composition: glycine (30mg / L), alanine (2mg / L), L-arginine hydrochloride (84mg / L), L-asparagine-H 2 O (0.83mg / L), cysteine ​​(31.5mg / L), L-histidine hydrochloride-H 2 O (42.0mg / L), L-isoleucine (105mg / L), L-leucine (105mg / L), L-lysine hydrochloride (146mg / L), L-methionine (30mg / L), L-phenylalanine (66mg / L), proline (7.76 / L), L-serine (42mg / L), threonine (95mg / L), L-tryptophan (16mg / L), tyrosine (72mg / L) and valine (94mg / L); choline chloride (4mg / L), calcium D-pantothenate (4mg / L), folic acid (4mg / L), tobacco Amide (4mg / L), Pyridoxal Hydrochloride (4mg / L), Riboflavin (0.4mg / L), Thiamine Hydrochloride (4mg / L), Vitamin B12 (0.0068) and Inositol (7.2); Chlorine Calcium chloride (anhydrous) (200mg / L), iron nitrate (0.1mg / L), magnesium chloride (anhydrous) (77.3mg / L), potassium chloride (400mg / L), sodium bicarbonate (2200mg / L) , sodium chloride (4000mg / L), sodium dihydrogen phosphate (125mg / L) ...

Embodiment 3

[0031] Example 3: Acquisition of mammalian BM-MSCs:

[0032] Acquisition of bone marrow BM-MSCs from large (pig, etc.) and small (large, mouse) animals:

[0033] After the C57BL / 6 pure line mice were killed by dislocation, they were soaked in 75% ethanol for 1-3 minutes, and the bilateral femur and tibia were separated under sterile conditions. 2 Remove the musculature around the femur in a glass dish, cut off the epiphysis on both sides including the epiphyseal plate, use a 10ml syringe (with a 4.5 gauge needle) to extract phosphate buffered saline (PBS) to flush the bone marrow cavity, and flush the bone marrow into the culture flask. The bone marrow cell suspension was repeatedly pipetted with a 7-gauge needle and a 4.5-gauge needle (or 21, 23, and 25 in turn) to prepare a single-cell suspension. The cell suspension was centrifuged at 1000 rpm for 5 min and the cells were collected or subjected to density gradient centrifugation. Add the single cell suspension at a ratio ...

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Abstract

The invention provides a proliferation medium and a proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow-derived meschymal stem cells (BM-MSCs). The medium comprises amino acids, vitamins, inorganic Salts, carbon sources, buffers and indicators. The proliferation medium of the present invention can be used for the massive expansion of mammalian bone marrow mesenchymal stem cells BM-MSCs before clinical application. The mammalian BM-MSCs are cultured by using the proliferation medium without allogeneic serum of the present invention, and 10 BM-MSCs can be obtained within 24 hours. 10 about 10 cells, more than the dose usually required for clinical cell therapy by 10 9 The proliferation rate is about 70 times higher than that of the conventional BGjB medium; the mammalian bone marrow mesenchymal stem cells cultured by the proliferation culture method of the present invention maintains a complete stem cell property and better proliferation activity, which can satisfy both the quantity and quality. clinical requirements.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a proliferation medium and a proliferation culture method for rapidly improving the proliferation efficiency of mammalian bone marrow mesenchymal stem cells. Background technique [0002] Domestic and foreign studies have proved that mammalian BM-MSCs cultured in vitro have the potential of multi-directional or trans-blast differentiation, and can be differentiated into a variety of functional cells for repair, such as liver, gallbladder, pancreas, lung, kidney, cartilage , brain, retina, intestine, spleen, blood, skin and other functional cells. This indicates that BM-MSCs can be induced to differentiate into multi-directional functions in an appropriate culture environment, thereby promoting repair and regeneration. At present, clinical cases have demonstrated that BM-MSCs can be transplanted into patients and successfully repair damaged diseases, such as inducing bone ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2500/32C12N2500/38C12N2500/35C12N2500/14C12N2500/24C12N2500/16C12N2500/12C12N2500/22C12N2500/34C12N2500/30C12N2500/60
Inventor 白莲花将师放林恒赖洁娟张玉君陈泉余张宏宇张雷达
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV