CN117210397A - A method for preparing spontaneously immortalized velvet antler stem cells - Google Patents

Abstract

The invention discloses a method for preparing a spontaneously immortalized velvet antler stem cell line, which mainly includes the following steps: Obtain velvet antler stem cells: Separate and extract: select three-year-old sika deer two-bar antler, drain the blood and bring it to the laboratory in a refrigerator; wash with 75% alcohol Deer antler was washed with physiological saline and repeated twice; velvet antler was washed with PBS (2% double antibody). In order to solve the problems of slow value-added and low viability of velvet antler stem cells encountered in in vitro culture and amplification applications, the present invention uses a self-developed culture medium and adds a variety of plant-derived components to replace the components in the conventional culture medium. Enhance the viability of velvet antler stem cells, accelerate the value-added rate, and extend the number of cell passages, thereby achieving cell immortalization. At the same time, the biological characteristics of immortalized antler stem cells were identified through cell morphology identification, protein immunofluorescence, flow cytometry and other methods.

Description

A method for preparing spontaneously immortalized velvet antler stem cells

Technical field

The present invention relates to stem cell preparation, in particular to a method for preparing spontaneously immortalized velvet antler stem cells.

Background technique

As we all know, deer antler itself is a health food, and its efficacy has been studied and proven by many parties.

Deer antler is the only organ in mammals that can be completely regenerated periodically under natural conditions. It is rich in active peptides, proteins, cephalin and various trace elements and minerals necessary for the human body. It is also the most valuable Only a small amount of velvet antler stem cells exist in the tips of velvet antler. Traditional velvet antler is expensive, up to 70,000/kg. Using the method of the present invention, the stem cells that exert the greatest effect in velvet antler can be obtained, retaining the nutritional value of the original velvet antler without gene editing. It can be used in various forms of food and health products or anti-aging fields, and the cost can be reduced to one-tenth. The traditional preparation method of velvet antler stem cells has poor stability, and there are problems faced by velvet antler stem cells in large-scale in vitro culture and application. The viability rate is low, the value-added is slow, and the reserves need to be withdrawn repeatedly.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects of the existing technology and provide a method for preparing spontaneously immortalized velvet antler stem cells.

In order to solve the above technical problems, the present invention provides the following technical solutions:

A method for preparing spontaneously immortalized velvet antler stem cells of the present invention mainly includes the following steps:

1) Obtain deer antler stem cells:

Separation and extraction: Select the two-barred antler of three-year-old sika deer, drain the blood and bring it to the laboratory in a refrigerator; wash the antler with 75% alcohol, then wash with normal saline, repeat twice; wash the antler with PBS (2% double antibody), repeat Three times, transfer to the biosafety cabinet; wash again with PBS (2% double antibody) in the biosafety cabinet; slice the velvet antler tissue, remove the antler skin, and wash again at the same time, use a plastic tip dropper to absorb PBS, rinse the tissue block, collect and centrifuge at 400g ten minutes. Discard the supernatant and collect the pellet, resuspend in PBS and centrifuge again, repeat three times. Take the pellet, resuspend it in self-developed culture medium, plate it after counting, and place it in a 38.5c incubator for culture;

2) Self-developed medium suitable for the growth of velvet antler stem cells:

The self-developed medium is composed of DMEM basic medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, 3M 100-thiol Ethanol, in addition the culture medium contains salts, vitamins, energy sources, minerals, peptides and amino acids as well as factors suitable for the growth of velvet antler cells including GABA, pipecholic acid, lithium chloride and transforming growth factor beta and basic fibroblast growth factor, Platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor, neurotrophic factor, fibroblast growth factor;

3) Spontaneous immortalization process:

Leave one third of ADC and ASC for passage; use a 6cm culture dish to induce primary velvet antler cells, add 4ml PBS and 4ml methylene blue, avoid light for 30 minutes, illuminate for 30 minutes, then change to self-developed medium for culture; incubate in a 38.5c incubator. Leave for one day; repeatedly replace the self-developed culture medium, continue induction and passage, and continue to stand; select the cells and continue passage until the 50th generation.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1: This invention improves the stability of velvet antler stem cells. In order to solve the problems of slow value increase and low activity rate encountered by velvet antler stem cells in in vitro culture and amplification applications, it cooperates with self-developed culture media and adds a variety of plant-derived ingredients to replace conventional ones. The components in the culture medium are used to enhance the viability of velvet antler stem cells, accelerate the rate of proliferation, and extend the number of cell passages, thereby achieving cell immortalization. At the same time, the biological characteristics of immortalized antler stem cells were identified through cell morphology identification, protein immunofluorescence, flow cytometry and other methods.

Description of the drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. In the attached picture:

Figure 1 is a comparison of the morphology of primary cells and immortalized cells of the present invention;

Figure 2 is a cell identification diagram of the present invention;

Detailed ways

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

As shown in Figures 1-2, the present invention provides a method for preparing spontaneously immortalized velvet antler stem cells, which mainly includes the following steps:

1. Use 10 mm methylene blue to induce ADC, ASC, and Myo for differentiation;

2. After differentiation, culture and obtain immortalized velvet antler stem cell lines;

Specific implementation plan:

How to obtain deer antler stem cells

Separate extraction:

1. Select two-barred antler from a three-year-old sika deer, drain the blood and bring it to the laboratory in a refrigerator;

2. Wash the velvet antlers with 75% alcohol, then wash with saline, repeat twice.

3. Wash the velvet antler with PBS (2% double antibody), repeat three times, and transfer to the biological safety cabinet;

4. Clean the biological safety cabinet again with PBS (2% double antibody);

5. Slice the velvet antler tissue, remove the velvet antler skin, and wash it again. Use a plastic tip dropper to absorb PbS and rinse the tissue block, collect it, and centrifuge it at 400g for ten minutes. Discard the supernatant and collect the pellet, resuspend in PBS and centrifuge again, repeat three times. Take the precipitate, resuspend it in self-developed culture medium, plate it after counting, and place it in a 38.5c incubator for culture.

Self-developed medium suitable for the growth of velvet antler stem cells

The self-developed medium is composed of DMEM basic medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, 3M 100-thiol Ethanol, in addition the culture medium contains salts, vitamins, energy sources (such as glucose), minerals, peptides and amino acids as well as factors suitable for antler cell growth including GABA, pipecholic acid, lithium chloride and transforming growth factor beta (TGFR) and bFGF (basic fibroblast growth factor), PDGF (platelet derived growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), neurotrophic factor, fibroblast growth factor.

Yeast extract powder: promotes the growth of adherent cells and replaces part of serum, the concentration is 0.5g/L;

Wheat oligopeptide: promotes the proliferation of adherent cells and replaces part of serum, the concentration is 0.5g/L;

Whey protein peptide: Provides what is needed for normal cell growth, replacing part of serum, with a concentration of 2g/L;

PDGF (platelet-derived growth factor): Promotes cell proliferation and differentiation, the use concentration is 20ng/mL;

bFGF (basic fibroblast growth factor): Promotes the synthesis of beneficial biomolecules in velvet antler stem cells;

Minerals, peptides, amino acids: Promote telomerase and growth of velvet antler stem cells, maintain telomerase length and avoid aging.

spontaneous immortalization process

1. Leave one third of ADC and ASC for passage;

2. For induction of primary velvet antler cells in a 6cm culture dish, add 4ml PBS + 4ml methylene blue, avoid light for 30 minutes, illuminate for 30 minutes, and then change to self-developed medium for culture;

3. Let it stand for one day in a 38.5c incubator;

4. Repeatedly replace the self-developed culture medium, continue induction and passage, and continue to stand;

5. Screen the cells and continue passage until passage 50.

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or some of the technical features may be equivalently replaced. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (1)

1. A method for preparing spontaneously immortalized velvet antler stem cells, which mainly includes the following steps: 1) Obtain deer antler stem cells: Separation and extraction: Select the two-barred antler of three-year-old sika deer, drain the blood and bring it to the laboratory in a refrigerator; wash the antler with 75% alcohol, then wash with normal saline, repeat twice; wash the antler with PBS (2% double antibody), repeat Three times, transfer to the biosafety cabinet; wash again with PBS (2% double antibody) in the biosafety cabinet; slice the velvet antler tissue, remove the antler skin, and wash again at the same time, use a plastic tip dropper to absorb PBS, rinse the tissue block, collect and centrifuge at 400g ten minutes. Discard the supernatant and collect the pellet, resuspend in PBS and centrifuge again, repeat three times. Take the pellet, resuspend it in self-developed culture medium, plate it after counting, and place it in a 38.5c incubator for culture; 2) Self-developed medium suitable for the growth of velvet antler stem cells: The self-developed medium is composed of DMEM basic medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, 3M 100-thiol Ethanol, in addition the culture medium contains salts, vitamins, energy sources, minerals, peptides and amino acids as well as factors suitable for the growth of velvet antler cells including GABA, pipecholic acid, lithium chloride and transforming growth factor beta and basic fibroblast growth factor, Platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor, neurotrophic factor, fibroblast growth factor; 3) Spontaneous immortalization process: Leave one third of ADC and ASC for passage; use a 6cm culture dish to induce primary velvet antler cells, add 4ml PBS and 4ml methylene blue, avoid light for 30 minutes, illuminate for 30 minutes, then change to self-developed medium for culture; incubate in a 38.5c incubator. Leave for one day; repeatedly replace the self-developed culture medium, continue induction and passage, and continue to stand; select the cells and continue passage until the 50th generation.