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Culture medium and application thereof

A culture medium and basal medium technology, applied in the field of cells, can solve problems such as potential threats, unstable sources, and uncontrollable quality of conditioned medium, so as to achieve increased cell number and activity, better and richer nutrition, and improved protein acquisition volume effect

Inactive Publication Date: 2017-05-10
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of bovine serum is complex and the source is unstable, making the quality of conditioned medium uncontrollable and a potential threat

Method used

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  • Culture medium and application thereof
  • Culture medium and application thereof
  • Culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Take the P1-generation adipose-derived mesenchymal stem cells (use DMEMF12+10%FBS+NEAA to culture, when the cells are in the exponential growth phase and the confluence reaches 80%, remove the culture supernatant and wash twice with PBS;

[0030] Add 0.1ml / cm 2 After static culture of DMEMF12+0.1mM NEAA for 24 hours, the conditioned medium was collected; then 0.1ml / cm 2 DMEMF12+0.1mM NEAA was cultured for 24 hours, and the conditioned medium was collected; add 0.1ml / cm 2 DMEMF12+0.1mM NEAA was cultured for 24 hours, and the conditioned medium was collected for the last time. Conditional cultures collected over three days were pooled.

[0031] 2. The collected conditioned medium was centrifuged at 200 g for 5 min, the supernatant was taken, and passed through a 0.22 μm filter membrane.

[0032] 3. The 3KD membrane bag is concentrated by ultrafiltration to 1 / 5 of the original volume, and the obtained conditioned medium is the prepared conditioned medium.

Embodiment 3

[0039] Use the bradford method to detect the total protein amount obtained, and the results are shown in Table 1: (unit: μg / ml)

[0040] Table 1

[0041] Unit: μg / ml 1 2 3 Example 1 115 123 120 comparative example 76 69 81

[0042] The results showed that the amount of protein obtained by the invention group (the conditioned medium prepared in Example 1) was significantly higher than that of the contrast group (comparative example), P<0.01.

[0043] The adipose-derived mesenchymal stem cells of the P15 generation were cultured using conditioned medium, and their proliferation was detected:

[0044] Negative control: DMEMF12+10%FBS+0.1mM NEAA

[0045] Control group: DMEMF12+10%FBS+0.1mM NEAA+20% control group (comparative example) conditioned medium

[0046] Invention group: DMEMF12+10%FBS+0.1mM NEAA+20% condition medium of the invention group (Example 1),

[0047] Three repetitions were set up in each group, and the culture was continued for ...

Embodiment 4

[0052] 1. Take P3 generation umbilical cord mesenchymal stem cells and culture them with DMEMF12+10%FBS+0.1mMNEAA. When the cells are in the exponential growth phase and the confluence reaches 70%, remove the culture supernatant, wash twice with PBS, and add 0.2 ml / cm 2 DMEMF12+0.05mMNEAA After static culture for 24 hours, the conditioned medium was collected; add 0.2ml / cm 2 DMEMF12+0.05mM NEAA continued to culture for 24 hours, then collected the conditioned medium; then added 0.2ml / cm 2 DMEMF12+0.05mM NEAA was cultured for 24 hours, and the conditioned medium was collected for the last time. Conditional cultures collected over three days were pooled.

[0053] 2. The collected conditioned medium was centrifuged at 400 g for 10 min, the supernatant was taken, and passed through a 0.22 μm filter membrane.

[0054] 3. The 3KD membrane bag is concentrated by ultrafiltration to 1 / 20 of the original volume, and the obtained conditioned medium is the prepared conditioned medium. ...

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Abstract

The invention relates to the field of cells and particularly relates to a culture medium and application thereof. The culture medium comprises a basic culture medium and unnecessary amino acids. The culture medium utilizes DMEMF12+ and the unnecessary amino acids, so that the nutrients are more abundant, and more abundant and wide sources are provided for synthesizing various active proteins by cells, and furthermore, the yield of proteins is improved. A condition culture medium obtained by collecting mesenchymal stem cells cultured by the culture medium provided by the invention is used for culturing the mesenchymal stem cells, and the quantity and the activity of the cells are remarkably improved.

Description

technical field [0001] The invention relates to the field of cells, in particular to a culture medium and its application. Background technique [0002] Mesenchymal stem cells are a type of adult stem cells that exist in various tissues (such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc.), have multidirectional differentiation potential, and are non-hematopoietic stem cells. These stem cells have the potential to differentiate into a variety of mesenchymal series cells (such as osteoblast, chondrogenic and adipogenic cells, etc.) or non-mesenchymal series cells, and have unique cytokine secretion functions. Many clinical applications have confirmed that mesenchymal stem cells can solve a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, repair of partial meniscus resection of the knee joint, autoimmune diseases, etc. saved the lives of more patients. [0003] Many st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2500/32
Inventor 陈海佳王一飞葛啸虎梁美乐王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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