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Stem cell culture medium and stem cell separating method

A separation method and stem cell technology, which is applied in the field of stem cell culture medium and stem cell culture, can solve the problems of low cell yield, affecting cell activity, difficult to grasp the digestion time, etc.

Active Publication Date: 2017-08-22
沃昕生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the isolation methods of adipose stem cells are collagenase digestion method, which leads to low cell yield, difficult to grasp the digestion time, and easy to affect the activity of cells
Moreover, the culture system mostly adopts the serum-containing culture method. Since the serum contains other animal-derived components, the culture system has the risk of introducing exogenous viruses, so that there is a big bottleneck in the clinical application of adipose-derived stem cells.

Method used

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  • Stem cell culture medium and stem cell separating method
  • Stem cell culture medium and stem cell separating method
  • Stem cell culture medium and stem cell separating method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Stem Cell Medium Configuration

[0036] Use DMEM / F12 as the base medium, add platelet lysate, L-glutamine, mitomycin c, so that the volume percentage of platelet lysate in the stem cell medium is 5%; L-glutamine and mitomycin c The final concentrations of prime c in the stem cell culture medium were 50ng / ml, 4mmol / L and 50ng / ml respectively, and they were stored at 4°C in the dark for future use.

Embodiment 2

[0038] Adipose stem cell isolation, the steps are as follows:

[0039] Step 1. Adipose tissue is obtained through liposuction by a professional beauty institution or professional hospital. Transfer 50ml of the separated adipose tissue to a sterile collection bottle, store at 2-8°C, and separate the adipose stem cells within 12 hours.

[0040] Step 2, the adipose tissue was washed 3 times with normal saline containing double antibodies (1% penicillin and 1% streptomycin by mass percentage), and blood cells were washed away until the adipose tissue suspension was clear and bloodless after adding normal saline.

[0041] Step 3: centrifuge at 800r / min for 5min, and remove the lower liquid. Aspirate the upper layer of tissue pieces into a T75 culture bottle, tilt the culture bottle slightly to remove the physiological saline brought in during the washing step 2, spread the tissue pieces evenly in the culture bottle, and store at 37°C, 5% CO 2 cultured in an incubator. After 30 m...

Embodiment 3

[0043] Adipose stem cell culture, the steps are as follows, the medium used in this example is the stem cell medium configured in Example 1:

[0044] The adipose stem cells obtained in Step 3 of Example 2 were fully replaced on the second day, and then half of the liquid was replaced every 2-3 days; when the adipose stem cells were cultured in the stem cell medium until the ninth day, the adipose stem cells crawled out of the tissue After scraping off the tissue block, let the adipose stem cells continue to culture until the cell confluence reaches 80%-90%, remove the medium in the culture flask, wash the cells twice with normal saline, add 0.25% EDTA-trypsin for digestion, and wait for the cells to Add an equal volume of the stem cell culture medium prepared in Example 1 to stop digestion when it becomes round; after centrifugal collection, resuspend and count with the stem cell culture medium of the present invention, and calculate the viability. As shown in Table 1, 6.5×10 ...

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Abstract

The invention belongs to the field of stem cell culture, and particularly relates to a cell culture medium and a stem cell culture method. The invention provides a stem cell culture medium. The stem cell culture medium comprises a basic culture medium, a platelet lysate, an L-Glutamine and a mitomycin c. The invention also provides a stem cell separating method. The stem cell separating method comprises the following steps that the in-vitro tissue and the stem cell culture medium are mixed to be placed in a culture flask for culturing until cells creep out from the in-vitro tissue, the in-vitro tissue is scraped; the culture of the cells is continued, when the fusion degree of stem cells in the culture flask reaches 80% to 90%, the cells are passage-cultured after digestion. The stem cell culture medium can avoid the risk that the existing stem cell culture medium contains mostly animal serum so as to introduce exogenous virus; the stem cell separating method is simple to operate.

Description

technical field [0001] The invention belongs to the field of stem cell culture, and in particular relates to a stem cell culture medium and a stem cell culture method. Background technique [0002] Stem cells (StemCell) are cells with self-renewal ability and multi-directional differentiation potential. According to the differentiation potential, stem cells can be divided into three categories: totipotent stem cells, pluripotent stem cells, and unipotent stem cells. According to the stage of development, stem cells can be divided into: embryonic stem cells and adult stem cells. Due to the limitation of ethics, most of the stem cells used in clinical research and application are adult stem cells. Adult stem cells are derived from many tissues and organs of adult animals, including hematopoietic stem cells, neural stem cells, adipose stem cells, bone marrow mesenchymal stem cells, umbilical cord stem cells, cord blood stem cells, etc. [0003] Adipose stem cells are adipose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/32C12N2500/84C12N2501/603C12N2509/00
Inventor 林词雄王旭林洁璇李陶朱刚
Owner 沃昕生物科技(深圳)有限公司
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