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Serum-free bone marrow culture medium as well as preparation method and application thereof

A culture medium and serum-free technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve problems such as difficulty in obtaining human lymphoma cell culture, unstable medium quality, and different serum quality standards , to achieve the effects of easy promotion and use, convenient quality control, and reduced production costs

Active Publication Date: 2020-12-11
上海培晖生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But above-mentioned bone marrow culture medium all contains fetal calf serum, and composition is complex, easily occurs the pollution such as fungus, mycoplasma; In addition, the serum quality standard is different, and this is easy to cause medium quality unstable for mass production, and The price is also higher
In addition, the above two bone marrow culture media contain human lymphoma cell culture at a concentration of 60-140ul / ml, which is relatively high and used for mass production. It is difficult to obtain human lymphoma cell culture that meets the needs of production, and the cost is also high. high

Method used

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  • Serum-free bone marrow culture medium as well as preparation method and application thereof
  • Serum-free bone marrow culture medium as well as preparation method and application thereof
  • Serum-free bone marrow culture medium as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The serum-free bone marrow culture medium of this embodiment includes: basal medium RPMI 1640, and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL- 2. CpG oligonucleotides, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, NaH 2 PO 4 、Na 2 HPO 4 , human lymphocyte culture supernatant cultured by rhu-EPO; wherein:

[0026] The concentration of L-ascorbic acid in the basal medium is 5mg / L; the concentration of vitamin E in the basal medium is 15mg / L; the concentration of penicillin in the basal medium is 1×10 5U / L; the concentration of streptomycin in the basal medium is 50mg / L; the concentration of sodium pyruvate in the basal medium is 80mg / L; the concentration of sodium selenite in the basal medium is 20nM; The concentration of amides in the basal medium is 30mM; the concentration of rhIL-2 in the basal medium is 0.01mg / L; the concentration of CpG oligonucleotides in the basal m...

Embodiment 2

[0035] Serum-free bone marrow cell culture medium without human lymphocyte culture supernatant

[0036] The serum-free bone marrow cell culture medium without human lymphocyte culture supernatant was prepared according to the method of Example 1. The difference between the serum-free bone marrow cell culture medium of this example and Example 1 was that it did not contain human lymphocyte culture supernatant.

[0037] Specifically, the serum-free bone marrow cell medium that does not contain human lymphocyte culture supernatant includes basal medium RPMI 1640, and L-ascorbic acid, vitamin E, penicillin, streptomycin, pyruvate added to the basal medium Sodium, Sodium Selenite, Glutamine, rhIL-2, CpG Oligonucleotides, Linoleic Acid, Linolenic Acid, Stearic Acid, BSA, Sodium Glycerophosphate, HEPES, NaH 2 PO 4 、Na 2 HPO 4 ;in:

[0038] The concentration of L-ascorbic acid in the basal medium is 5mg / L; the concentration of vitamin E in the basal medium is 15mg / L; the concentra...

Embodiment 3

[0041] Serum-free bone marrow medium without rhu-EPO-cultured human lymphocyte culture supernatant

[0042] According to the method of Example 1, the serum-free bone marrow culture medium without the culture supernatant of human lymphocytes cultured with rhu-EPO was prepared, but not without the culture supernatant of human lymphocytes.

[0043] Specifically, the serum-free bone marrow culture medium without rhu-EPO cultured human lymphocyte culture supernatant includes basal medium RPMI 1640, and L-ascorbic acid, vitamin E, penicillin, streptavidin added to the basal medium Sodium pyruvate, sodium selenite, glutamine, rhIL-2, CpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, NaH 2 PO 4 、Na 2 HPO 4 , human lymphocyte culture supernatant; wherein:

[0044] The concentration of L-ascorbic acid in the basal medium is 5mg / L; the concentration of vitamin E in the basal medium is 15mg / L; the concentration of penicillin in the ...

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Abstract

The invention provides a serum-free bone marrow culture medium as well as a preparation method and application thereof. The culture medium comprises a basal culture medium, and human lymphocyte culture supernatant which is cultured by adding L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, CpG oligonucleotide, linoleic acid, linolenic acid,stearic acid, BSA, sodium glycerophosphate, HEPES, NaH2PO4, Na2HPO4 and rhu-EPO into the basal culture medium. Quality control between batches is facilitated. The human lymphocyte culture supernatantcultured by the rhu-EPO is used for providing growth factors required by bone marrow growth, so that the production cost is reduced. The sodium glycerophosphate and the like are used as a pH buffer system, the pH value is more stable, and the pH value is stable in the storage process. rhIL-2 and CpG oligonucleotides are adopted to stimulate the activity of B cells, and more karyotypes can be obtained when the oligonucleotides are used for karyotype analysis.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a serum-free bone marrow culture medium and its preparation method and application. Background technique [0002] Chromosomal aberrations are closely related to blood system diseases. In recent years, with the development of technologies such as cell culture and chromosome banding, cytogenetics has become an indispensable and important content in the analysis and research of blood system diseases. Bone marrow karyotype analysis plays a very important role in the diagnosis, treatment, prognosis and pathogenesis research of blood system diseases. However, the preparation process of bone marrow chromosome karyotype is complex, with many influencing factors, low success rate and high cost, few cleavage phases obtained, thick and short chromosomes, and poor dispersion. [0003] The traditional bone marrow culture medium is composed of RPMI1640 basal medium, fetal bovine serum, antibiotic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077G01N1/28G01N1/30
CPCC12N5/0669G01N1/2813G01N1/30C12N2500/38C12N2500/30C12N2500/32C12N2501/2302C12N2500/40C12N2500/36C12N2500/42C12N2500/12C12N2500/84C12N2501/998C12N2500/90Y02A50/30
Inventor 李晓辉王亦平
Owner 上海培晖生物科技发展有限公司
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