Method for preparing spontaneously immortalized cervi cornu pantotrichum stem cells

Abstract

A method for preparing a spontaneously immortalized Cervi cornu pantotrichum stem cell line. The method mainly comprises the following steps: obtaining Cervi cornu pantotrichum stem cells by means of performing separation and extraction, which involves selecting two stalks of Cervi cornu pantotrichum from a three-year-old sika deer in its prime, draining out the blood and then refrigerating and taking same to a laboratory; washing the Cervi cornu pantotrichum with 75% alcohol, then washing same with physiological saline, and repeating twice; and washing the Cervi cornu pantotrichum with PBS (containing 2% of penicillin-streptomycin). In order to solve problems such as slow proliferation and low viability encountered in in-vitro culture and amplification applications of Cervi cornu pantotrichum stem cells, a self-developed culture medium is used and various plant-derived components are added to replace components in a conventional culture medium, so that the viability of the Cervi cornu pantotrichum stem cells is enhanced, the proliferation rate of the cells is increased, and the number of passages of the cells is increased, thereby achieving cell immortalization. Moreover, the biological characteristics of the immortalized Cervi cornu pantotrichum stem cell are identified by means of cell morphology identification, protein immunofluorescence, flow cytometry and other methods.

Description

A method for preparing spontaneously immortalized antler stem cells Technical Field

[0001] The present invention relates to the preparation of stem cells, and in particular to a method for preparing spontaneously immortalized antler stem cells. Background Art

[0002] As we all know, deer antler itself is a health food and its efficacy has been studied and confirmed by many parties.

[0003] Deer antler is the only organ in mammals that can fully regenerate periodically under natural conditions. It is rich in active peptides, proteins, cephalins, and a variety of nutrients such as trace elements and minerals necessary for the human body. The most valuable antler stem cells are only found in small quantities at the tip of the antler. Traditional antler is expensive, costing up to 70,000 yuan per kilogram. Using the method of the present invention, stem cells with the greatest efficacy in antler can be obtained, retaining the nutritional value of the original antler without gene editing, and can be applied to various forms of food and health products or in the anti-aging field, with the cost reduced to one-tenth. Traditional antler stem cell preparation methods have poor stability of antler stem cells, and there are problems with low activity rate, slow growth, and repeated extraction required for storage in large-scale in vitro culture and application.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a method for preparing spontaneously immortalized deer antler stem cells.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing spontaneously immortalized antler stem cells, which mainly comprises the following steps:

[0008] 1) Obtaining Deer Antler Stem Cells:

[0009] Separation and extraction: Select two-bar antlers from three-year-old sika deer, drain the blood, and refrigerate and bring to the laboratory; wash the antlers with 75% alcohol, then wash with physiological saline, repeat twice; wash the antlers with PBS (2% double antibody), repeat three times, and transfer to a biosafety cabinet; wash again with PBS (2% double antibody) in the biosafety cabinet; slice the antler tissue, remove the antler skin, and wash again. Use a rubber-tipped dropper to absorb PbS to rinse the tissue blocks, collect them, and centrifuge them at 400g for ten minutes. Discard the supernatant and take the precipitate, resuspend it with PBS, and centrifuge it again, repeat three times. Take the precipitate, resuspend it with self-developed culture medium, count it, and plate it, and culture it in a 38.5c incubator;

[0010] 2) Self-developed culture medium suitable for the growth of antler stem cells:

[0011] The self-developed culture medium is composed of DMEM basal medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, and 3M 100-mercaptoethanol. In addition, the culture medium contains salts, vitamins, energy sources, minerals, peptides and amino acids, as well as factors suitable for the growth of antler cells, including GABA, cholic acid, lithium chloride, transforming growth factor-β, basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor, neurotrophic factor, and fibroblast growth factor.

[0012] 3) Spontaneous immortalization process:

[0013] One-third of ADC and ASC were reserved for passage; primary antler cells were induced in 6 cm culture dishes by adding 4 ml PBS and 4 ml methylene blue, protecting from light for 30 minutes, exposing to light for 30 minutes, and then changing to self-developed culture medium for culture; incubating in a 38.5°C incubator for one day; repeatedly changing to self-developed culture medium, continuing induction and passage, and continuing to incubate; screening cells and continuing passage until the 50th passage.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention improves the stability of antler stem cells. To address the slow growth and low viability of antler stem cells during in vitro culture and expansion, a proprietary culture medium is developed that replaces conventional culture medium components with various plant-derived ingredients. This enhances antler stem cell viability, accelerates growth, and extends the number of cell passages, thereby achieving cell immortalization. The biological characteristics of immortalized antler stem cells are also identified through cell morphology, protein immunofluorescence, and flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] FIG1 is a comparison of the morphology of primary cells and immortalized cells of the present invention;

[0018] FIG2 is a cell identification diagram of the present invention; DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0020] Example 1

[0021] As shown in Figures 1-2, the present invention provides a method for preparing spontaneously immortalized antler stem cells, which mainly includes the following steps:

[0022] 1. Use 10mM methylene blue to induce ADC, ASC, and Myo differentiation;

[0023] 2. After differentiation, culture to obtain immortalized antler stem cell lines;

[0024] Specific implementation plan:

[0025] Methods for obtaining deer antler stem cells

[0026] Separation and extraction:

[0027] 1. Select two-bar antlers from three-year-old sika deer, drain the blood, refrigerate and bring to the laboratory;

[0028] 2. Clean the antlers with 75% alcohol, then wash with saline, and repeat twice.

[0029] 3. Wash the antlers with PBS (2% double antibody) three times and transfer to a biosafety cabinet;

[0030] 4. Wash again with PBS (2% double antibody) in the biosafety cabinet;

[0031] 5. Section the antler tissue, remove the antler skin, and wash again. Use a rubber-tipped pipette to aspirate PBS to rinse the tissue blocks, collect them, and centrifuge them at 400g for 10 minutes. Discard the supernatant, collect the pellet, resuspend it in PBS, and centrifuge it again three times. Collect the pellet, resuspend it in a custom-made culture medium, count the cells, plate them, and incubate them in a 38.5°C incubator.

[0032] Self-developed culture medium suitable for the growth of antler stem cells

[0033] The self-developed culture medium is composed of DMEM basal culture medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, 3M 100-mercaptoethanol. In addition, the culture medium contains salts, vitamins, energy sources (such as glucose), minerals, peptides and amino acids, as well as factors suitable for the growth of antler cells including GABA, cholic acid, lithium chloride and transforming growth factor β (TGFR) and bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), neurotrophic factors, and fibroblast growth factor.

[0034] Yeast extract powder: promotes the growth of adherent cells and replaces part of the serum at a concentration of 0.5g / L;

[0035] Wheat oligopeptide: Promotes the proliferation of adherent cells and replaces part of the serum at a concentration of 0.5g / L;

[0036] Whey protein peptide: provides the requirements for normal cell growth and replaces part of the serum at a concentration of 2g / L;

[0037] PDGF (platelet-derived growth factor): promotes cell proliferation and differentiation, used at a concentration of 20 ng / mL;

[0038] bFGF (basic fibroblast growth factor): promotes the synthesis of beneficial biological molecules in antler stem cells;

[0039] Minerals, peptides, amino acids: promote telomerase and growth of antler stem cells, maintain telomerase length and avoid aging.

[0040] Spontaneous immortalization process

[0041] 1. ADC, ASC keep one-third for passage;

[0042] 2. For induction of primary antler cells, add 4 ml PBS + 4 ml methylene blue to a 6 cm culture dish, protect from light for 30 minutes, expose to light for 30 minutes, and then switch to self-developed culture medium for culture;

[0043] 3. Place in a 38.5°C incubator for one day;

[0044] 4. Repeatedly replace the self-developed culture medium, continue induction and passage, and continue to stand;

[0045] 5. Select the cells and continue to subculture until the 50th generation.

[0046] Finally, it should be noted that the above descriptions are merely 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 aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing spontaneously immortalized antler stem cells, mainly comprising the following steps: 1) Obtaining antler stem cells: Separation and extraction: Select two-bar antlers of three-year-old sika deer, drain the blood and bring to the laboratory in cold storage; wash the antlers with 75% alcohol, then wash with saline, repeat twice; wash the antlers with PBS (2% double antibody), repeat three times, and transfer to the biosafety cabinet; Wash again with PBS (2% double antibody) in the biosafety cabinet; slice the antler tissue, remove the antler skin, and wash again. Use a rubber-tipped dropper to absorb PbS to rinse the tissue block, collect it, and centrifuge it at 400g for ten minutes. Discard the supernatant and take the precipitate, resuspend it with PBS, and centrifuge it again. Repeat three times. Take the precipitate, resuspend it with self-developed culture medium, count it, and plate it, and place it in a 38.5c incubator for culture; 2) Self-developed culture medium suitable for the growth of antler stem cells: The self-developed culture medium is composed of DMEM basal culture medium and other components, supplemented with yeast extract powder, wheat oligopeptides, whey protein peptides, 5mM non-essential amino acids, 5mM L-glutamine, 3M 100-mercaptoethanol, and the culture medium contains salts, vitamins, energy sources, minerals, peptides and amino acids, and factors suitable for the growth of antler cells including GABA, piperacholic acid, lithium chloride and transforming growth factor β and basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor, neurotrophic factor, fibroblast growth factor; 3) Spontaneous immortalization process: One-third of ADC and ASC were reserved for passage; primary antler cells were induced in 6 cm culture dishes, 4 ml PBS and 4 ml methylene blue were added, protected from light for 30 minutes, illuminated for 30 minutes, and then cultured with self-developed culture medium; incubated in a 38.5°C incubator for one day; self-developed culture medium was repeatedly replaced, induction and passage were continued, and the cells were continued to be allowed to stand; cells were screened and passaged until the 50th passage was reached.

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