Preparation method of purified and immortalized pilose antler stem cell line
By primary culture, purification and natural immortalization and domestication of deer antler stem cells, the problems of poor stability and low survival rate of deer antler stem cells in traditional methods were solved, and purified deer antler stem cell lines with high genetic stability and long lifespan were obtained, reducing costs , realizes large-scale commercial use.
Patent Information
- Application Number
- CN202411695957.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-02-18
AI Technical Summary
The antler stem cells cultured by traditional methods have a wide range of problems such as heterogeneity, poor stability, low survival rate, slow proliferation and short life span, which leads to high costs and limits their large-scale commercial use.
A single, naturally immortalized antler stem cell line with purified, infinite passages were obtained by primary culture, purification and natural immortalization of deer antler stem cells. The method includes collection of primary cells, screening of purified cell lines and domestication passages of natural immortalization until passages of more than 50 generations.
It has achieved the acquisition of purified antler stem cell lines with high genetic stability, long lifespan and strong proliferation ability, reducing costs and overcoming the problems of poor cell stability and low survival rates in traditional methods.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a purified and immortalized velvet antler stem cell system. Background Art
[0002] With the continuous development of life science research, stem cells have received widespread attention around the world due to their rapid proliferation, multidirectional differentiation, and regenerative repair potential. In recent years, stem cell therapy technology and industrialization have developed rapidly around the world, gradually becoming one of the important directions in the field of life sciences.
[0003] Deer antler is the only accessory organ in mammals that can be fully regenerated, and its growth rate can reach 2cm / day. Studies have found that deer antler regeneration is based on the process of stem cell division and differentiation. Deer antler stem cells support the complete regeneration of the entire deer antler through rapid division and differentiation. It has been identified that deer antler stem cells have strong self-renewal ability and can secrete a large number of cytokines and bioactive factors. Therefore, it can be used as an excellent cell source in the fields of tissue and organ damage, various forms of food and health products or anti-aging.
[0004] However, studies have found that antler stem cells cultured by traditional methods have extensive heterogeneity, with a mixture of multiple cell types and large differences in cell proliferation and differentiation capabilities, which leads to technical problems such as poor stability, low viability, slow proliferation, and short lifespan during cell expansion. At the same time, antler stem cells are collected from the tip of the antler and the periosteum tissue in the antler area, and the tissue source is expensive. Due to the disadvantages of short lifespan, the antler stem cells cultured by the above traditional methods need to continuously collect fresh antler and periosteum tissue during large-scale expansion and culture, which is costly and limits their large-scale commercial use. Although these cells can be artificially immortalized by introducing viral plasmids (such as large T antigen) or gene editing technology, the cell lines established by these methods change the cell gene expression pattern, have poor genetic stability, are prone to changes in cell characteristics, and have the risk of malignant transformation, posing a strong safety hazard, while immortalized cells in a natural state can completely avoid these problems.
[0005] Therefore, purifying antler stem cells and screening a single cell line that is immortalized in a natural state is an urgent problem to be solved. Summary of the invention
[0006] The purpose of the present invention is to provide a method for preparing a purified and immortalized antler stem cell line to obtain a purified antler stem cell line that can be infinitely propagated, thereby overcoming the technical problems of poor stability, low viability, slow proliferation, and short life span in the process of culturing antler stem cells using traditional methods.
[0007] The objective of the present invention is achieved through the following technical solutions: A method for preparing a purified and immortalized antler stem cell line comprises the following steps: S1. Primary cells are obtained by primary culturing antler stem cells; S2. Purify primary cells to obtain purified cell lines with strong proliferation ability; S3. A single naturally immortalized antler stem cell line is obtained by acclimating the purified cell line to natural immortality.
[0008] In the above scheme, by primary culture, purification and immortalization of antler stem cells, a pure antler stem cell line that can be passed on indefinitely under natural conditions can be obtained, which can be passed on continuously for more than 50 generations. At the same time, because the antler stem cell line is cloned from a single cell, it has strong genetic stability.
[0009] Preferably, the specific steps of S1 are as follows: S11. Antler stem cell collection: Collection of tissue containing antler stem cells; S12. Tissue separation and digestion: The tissues collected in S11 were washed with PBS solution until the blood stains were completely removed; the tissues were then poured into a disposable sterile plate, and the three tissues were cut into tissue fragments using a sterile scalpel; the tissues were then transferred into a centrifuge tube for digestion. When brush-like edges appeared on the edges of the tissue fragments, the digestion was terminated, and the supernatant was discarded after centrifugation. Complete cell culture medium was added for centrifugation, the supernatant was discarded, and the precipitate was collected; S13. Primary culture: Add the precipitate collected in S12 to complete culture medium and evenly spread it on the bottom of the cell culture plate. Then add DMEM complete culture medium along the edge of the culture dish and culture it in a cell culture incubator for 24 hours. Then slowly add fresh complete culture medium along the edge of the culture dish and continue to culture it statically for 3-7 days. S14. Primary cell collection: Observe the adhesion and cell divergence of the tissue blocks in S13 under a microscope. When the cell confluence reaches 70-80%, perform trypsin digestion and centrifugation to collect the primary cells.
[0010] Preferably, in S11, the tissue containing antler stem cells is the periosteum tissue of the antler-growing area of a healthy male deer, the periosteum tissue of the antler stalk, or the mesenchymal layer tissue at the top of the antler.
[0011] Preferably, the specific steps of S2 are as follows: S21. Cell inoculation: Count the primary cells collected in S14 with a cell counting plate, and inoculate the cells of each tissue source at a density of 1000 cells / dish into the bottom of 10 10 cm cell culture dishes. Mix the cells to ensure that the cells are fully separated, add 10 ml of complete cell culture medium, and culture them in a 37°C, 5% CO2 cell culture incubator for one week, during which the culture medium is changed once; S22. Monoclonal selection: Observe the growth of monoclonal cells cultured in S21 under a microscope. When a large monoclonal cell line appears, stop culturing and select monoclonal cells with good growth status and strong proliferation ability; S23. Monoclonal culture: The monoclonal cells selected in S22 were digested with trypsin, digestion was terminated with complete cell culture medium, and they were seeded into 48-well plates and cultured overnight. The complete culture medium was replaced and cultured for 3 days. Cells with confluence <80% were discarded, and cells with confluence >80% were passaged into 24-well plates. The above-mentioned passage steps were repeated, and the cells were gradiently amplified and screened through 24-well-12-well-6-well to obtain a purified cell line with strong proliferation ability.
[0012] Preferably, the diameter of the monoclonal cells with excellent growth status and strong proliferation ability selected in S22 is >1 mm.
[0013] Preferably, the specific steps of S3 are as follows: S31. Identification of stemness of purified cell lines: Using the stem cell tri-lineage differentiation system, the purified cell lines obtained in S23 were identified as stem cells, and purified cell lines with strong differentiation potential were screened; S32. Observe the tri-lineage differentiation ability of purified cell lines, screen purified cell lines that can successfully induce osteogenic, adipogenic and chondrogenic differentiation, and perform natural immortalization acclimatization culture as follows: 5x10 5 The cells were inoculated into T25 cell culture flasks at a density of 100 cells / flask, and complete cell culture medium was added. When the cell confluence was >90%, trypsin digestion was performed, and the digestion was terminated with DMEM complete culture medium. The cells were passaged into T25 flasks in a one-to-two manner, and the next passage was performed after 2 days of culture. The above-mentioned passaging process was repeated, and cell lines with cell confluence less than 90% were eliminated. The cells were screened and continued to be passaged until the 50th generation, thus obtaining a single naturally immortalized antler stem cell line.
[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The antler stem cells cultured by traditional methods have extensive heterogeneity and are mixed with various types of cells. The preparation method of the present invention can obtain a pure immortalized antler stem cell line, which is cloned from a single cell and has strong genetic stability.
[0015] 2. The tissues from which antler stem cells are derived are expensive. The present invention can obtain a pure, single, natural antler stem cell line that can be passed on indefinitely, and can be passed on continuously for more than 50 generations, effectively saving costs. The immortalization and domestication method is achieved through repeated passage, and the passage is a one-to-two method, and the repeated passage time is once every two days. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 :P50 cell morphology of three cell lines: AnSC-a, AnSC-p, and AnSC-r; Figure 2 :P50 karyotype analysis results of three cell lines AnSC-a, AnSC-p, and AnSC-r; Figure 3 :Immunofluorescence staining of stem cell markers in three cell lines: AnSC-a, AnSC-p, and AnSC-r; Figure 4 : Comparison of the proliferation rates of AnSC-a, AnSC-p, AnSC-r and deer antler stem cells cultured by traditional methods. DETAILED DESCRIPTION
[0017] Example 1: Primary culture of antler stem cells S11. Sample collection: Collect three tissues from healthy male deer: periosteal tissue of the antler area (AP; the tissue basis for the development of antler stalks and primary antlers), periosteal tissue of the antler stalk (PP; the tissue basis for antler regeneration), and antler apical mesenchymal layer tissue (RM; the tissue basis for the rapid growth of antlers). Place them in 8 times the volume of tissue preservation solution (DMEM culture medium + 5% double antibody) and transport them to the laboratory on ice within 1 hour for subsequent operations. Sampling method reference: Li and Suttie (2003) Tissue Collection Methods for Antler Research. European Journal of Morphology. 2003, Vol. 41, No. 1, pp. 23–30. S12. Tissue separation and digestion: Remove the freshly collected AP, PP or RM from the preservation solution and wash them repeatedly with PBS (containing 2% double antibody) solution until the blood stains are completely removed. Pour the tissue into a disposable sterile plate, cut the three tissues into 1-2mm tissue fragments with a sterile scalpel, transfer them into a 50mL centrifuge tube, add 5-10 times the volume of digestion solution (50-100U / mL type I collagenase solution) to the centrifuge tube, and slowly shake and digest for 30 minutes at 37℃. When the edges of the tissue fragments appear like brush edges, stop digestion, centrifuge at 1000rpm for 5min, discard the supernatant, add 10mL of complete cell culture medium (DMEM basal culture medium, containing 10 FBS, 1% double antibody), centrifuge at 1000rpm for 5min to wash away the digestion solution, discard the supernatant, and collect the precipitate.
[0018] S13. Primary culture: Take 3 g of the precipitate in S12, add 1 ml of complete culture medium, evenly spread it on the bottom of a 10 cm cell culture plate, add 2 mL of DMEM complete cell culture medium along the edge of the culture dish, place it in a 37°C, 5% CO2 cell culture incubator and culture it for 24 hours. Slowly add 3 ml of fresh complete cell culture medium along the edge of the culture dish and continue to culture it statically for 3-7 days.
[0019] S14. Cell collection: Observe the adhesion and cell proliferation of the above tissue blocks under a microscope. When the cell confluence reaches 70-80%, trypsinize and centrifuge at 1000 rpm for 5 minutes to collect primary cells.
[0020] Example 2: Purification of antler stem cells.
[0021] S21. Cell inoculation: Count the primary cells collected in S14 using a cell counting plate. Inoculate the cells of each tissue source at a density of 1000 cells / dish into the bottom of 10 10 cm cell culture dishes. Mix the cells to ensure that the cells are fully separated. Add 10 ml of complete cell culture medium and place at 37°C, 5% CO. 2 The cells were cultured in a cell culture incubator for one week, during which the culture medium was changed once.
[0022] S22. Monoclonal selection: Observe the growth of monoclonal cells cultured in S21 under a microscope, and stop culturing when a large monoclonal cell line has appeared. Due to the heterogeneity of antler stem cells, different types of cells have different morphologies after becoming monoclonal cells. Circle the cell monoclonals with the best cell growth and the largest monoclonal cell size (diameter>1mm) in each morphology under a microscope, select 5 monoclonal cells per dish, and select 50 clones from each tissue source.
[0023] S23. Monoclonal culture: Use a sterile separator (5 mm in diameter) to digest the monoclonal cells with good growth status and strong proliferation ability selected in S22 with trypsin, terminate the digestion with 200 ul of complete cell culture medium, inoculate into a 48-well plate and culture overnight, replace the complete culture medium, and continue to culture for 3 days. Discard cells with a confluence of <80%, and subculture cells with a confluence of >80% to a 24-well plate. Repeat the above subculture steps, and perform gradient amplification and screening of the cells through 24-well-12-well-6-well to obtain a purified cell line with strong proliferation ability.
[0024] Example 3: Domestication of naturally immortalized antler stem cells and identification of antler stem cells.
[0025] S31. Identification of stemness of purified cell lines: Using the stem cell tri-lineage differentiation system (osteogenic, adipogenic, and chondrogenic induction), the purified cell lines obtained in S23 were identified as stem cells and purified cell lines with strong differentiation potential were screened. Specific references for induction and differentiation operations: Datao Wang, Debbie Berg, Hengxing Ba, Hongmei Sun, Zhen Wang and Chunyi Li, Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ—deer antler. Cell Death and Disease, (2019) 10:443. S32. Observe the tri-lineage differentiation ability of purified cell lines, screen cell lines that can successfully induce osteogenic, adipogenic and chondrogenic differentiation, and carry out natural immortalization acclimatization culture. 5 The cells were inoculated into T25 cell culture flasks at a density of 100 cells / flask, and 5 ml of complete cell culture medium (DMEM basal medium, containing 10 FBS, 1% double antibody) was added. When the cell confluence was >90%, trypsin digestion was performed, and the digestion was terminated by DMEM complete medium. The cells were subcultured into T25 flasks in a one-to-two manner, and the next subculture was performed after 2 days of culture. The above subculture process was repeated, and cell lines with a cell confluence of less than 90% were eliminated. The cells were screened and continued to be subcultured until the 50th generation was reached, and a single naturally immortalized antler stem cell line was obtained, which was frozen for later use.
[0026] S33. Identification of antler stem cell line: Karyotype and stem cell characteristics of purified natural immortalized antler stem cell line passaged to 50 generations were identified. Cells were seeded into 6-well plates and grown to the logarithmic growth phase for 24 hours. After treatment with colchicine, hypotonic solution, fixation and staining, chromosomes were observed under a microscope to determine the chromosome stability of the immortalized cell line. Cell immunofluorescence staining was used to detect the expression of mesenchymal stem cell marker CD90 / CD73 / CD105 in the 50th generation immortalized antler stem cell line.
[0027] Experimental Results In the present application, three types of antler stem cells derived from AP, PP and RM were primary cultured, purified and immortalized in the manner of Examples 1-3, and ultimately one antler stem cell line AnSC-a / AnSC-p / AnSC-r that can be stably propagated was obtained from each source cell.
[0028] The three cell lines were subjected to P50 cell morphology analysis, P50 karyotype analysis, and stem cell marker immunofluorescence staining analysis. The analysis results are as follows: like Figure 1 As shown, the P50 cell morphology analysis confirmed that the three cell morphologies were normal; Figure 2 As shown in the figure, the karyotype analysis confirmed that the chromosome karyotype of the obtained antler stem cell line was normal, indicating that the immortalized cells obtained by this method can be stably inherited; Figure 3 As shown, through cell immunofluorescence staining analysis, the results confirmed that the 50-generation antler stem cell line can stably maintain stem cell characteristics, indicating that the immortalized cells obtained by the present invention can maintain normal cell characteristics; Figure 4 As shown, through the cell proliferation experiment, the results confirmed that the proliferation rate of the 50th generation antler stem cell line was similar to that of the primary cells, but significantly higher than the proliferation rate of the 10th generation cells cultured by the traditional method, indicating that the purified cell line obtained by the present invention has a strong proliferation ability.
Claims
1. A method for preparing a purified and immortalized velvet antler stem cell line, characterized in that: The steps include: S1. Primary cells are obtained by primary culturing antler stem cells; S2. Purify primary cells to obtain purified cell lines with strong proliferation ability; S3. A single naturally immortalized antler stem cell line is obtained by acclimating the purified cell line to natural immortality.
2. The method for preparing a purified and immortalized velvet antler stem cell line according to claim 1, characterized in that: The specific steps of S1 are as follows: S11. Antler stem cell collection: Collection of tissue containing antler stem cells; S12. Tissue separation and digestion: The tissues collected in S11 were washed with PBS solution until the blood stains were completely removed; the tissues were then poured into a disposable sterile plate, and the three tissues were cut into tissue fragments using a sterile scalpel; the tissues were then transferred into a centrifuge tube for digestion. When brush-like edges appeared on the edges of the tissue fragments, the digestion was terminated, and the supernatant was discarded after centrifugation. Complete cell culture medium was added for centrifugation, the supernatant was discarded, and the precipitate was collected; S13. Primary culture: Add the precipitate collected in S12 to complete culture medium and evenly spread it on the bottom of the cell culture plate. Then add DMEM complete culture medium along the edge of the culture dish and culture it in a cell culture incubator for 24 hours. Then slowly add fresh complete culture medium along the edge of the culture dish and continue to culture it statically for 3-7 days. S14. Primary cell collection: Observe the adhesion and cell divergence of the tissue blocks in S13 under a microscope. When the cell confluence reaches 70-80%, perform trypsin digestion and centrifugation to collect the primary cells.
3. The method for preparing a purified and immortalized velvet antler stem cell line according to claim 2, characterized in that: In S11, the tissue containing antler stem cells is the periosteum tissue of the antler-growing area of a healthy male deer, the periosteum tissue of the antler stalk, or the mesenchymal layer tissue at the top of the antler.
4. The method for preparing a purified and immortalized velvet antler stem cell line according to claim 2, characterized in that: The specific steps of S2 are as follows: S21. Cell inoculation: Count the primary cells collected in S14 with a cell counting plate, and inoculate the cells of each tissue source at a density of 1000 cells / dish into the bottom of 10 10 cm cell culture dishes. Mix the cells to ensure that the cells are fully separated, add 10 ml of complete cell culture medium, and culture them in a 37°C, 5% CO2 cell culture incubator for one week, during which the culture medium is changed once; S22. Monoclonal selection: Observe the growth of monoclonal cells cultured in S21 under a microscope. When a large monoclonal cell line appears, stop culturing and select monoclonal cells with good growth status and strong proliferation ability; S23. Monoclonal culture: The monoclonal cells selected in S22 were digested with trypsin, digestion was terminated with complete cell culture medium, and they were seeded into 48-well plates and cultured overnight. The complete culture medium was replaced and cultured for 3 days. Cells with confluence <80% were discarded, and cells with confluence >80% were passaged into 24-well plates. The above-mentioned passage steps were repeated, and the cells were gradiently amplified and screened through 24-well-12-well-6-well to obtain a purified cell line with strong proliferation ability.
5. The method for preparing a purified and immortalized velvet antler stem cell line according to claim 4, characterized in that: The diameter of the monoclone with excellent growth status and strong proliferation ability selected in S22 is >1 mm.
6. The method for preparing a purified and immortalized velvet antler stem cell line according to claim 4, characterized in that: The specific steps of S3 are as follows: S31. Identification of stemness of purified cell lines: Using the stem cell tri-lineage differentiation system, the purified cell lines obtained in S23 were identified as stem cells, and purified cell lines with strong differentiation potential were screened; S32. Observe the tri-lineage differentiation ability of purified cell lines, screen purified cell lines that can successfully induce osteogenic, adipogenic and chondrogenic differentiation, and perform natural immortalization acclimatization culture as follows: 5x10 5 The cells were inoculated into T25 cell culture flasks at a density of 100 cells / flask, and complete cell culture medium was added. When the cell confluence was >90%, trypsin digestion was performed, and the digestion was terminated with DMEM complete culture medium. The cells were passaged into T25 flasks in a one-to-two manner, and the next passage was performed after 2 days of culture. The above-mentioned passaging process was repeated, and cell lines with cell confluence less than 90% were eliminated. The cells were screened and continued to be passaged until the 50th generation, thus obtaining a single naturally immortalized antler stem cell line.