Cornu cervi pantotrichum stem cell exosome preparation and preparation method thereof

Abstract

The invention relates to the field of biology, and discloses a cornu cervi pantotrichum stem cell exosome preparation and a preparation method thereof.The preparation method of the cornu cervi pantotrichum stem cell exosome preparation comprises the following steps that after cornu cervi pantotrichum is disinfected, a semitransparent mesenchymal layer at the tip of the cornu cervi pantotrichum is separated out, the cornu cervi pantotrichum is cut into pieces and then flushed with PBS containing double antibodies, the cornu cervi pantotrichum is put into tissue digestion mixed enzyme, and the cornu cervi pantotrichum exosome preparation is obtained centrifuging and cleaning after digestion, and then spreading in a culture bottle for culture to obtain an isolate; when cells of the isolate grow to 70%-90% of a culture bottle, discarding an original culture solution, cleaning with PBS, adding pancreatin for digestion, terminating digestion with a complete medium, and performing cell passage to obtain a passage; and performing gene editing treatment on the cells of the passage. By obtaining the stem cells of the cornu cervi pantotrichum, damage to a donor when the stem cells are obtained by the donor is avoided, and due to the fact that the cornu cervi pantotrichum stem cells have stable biological characteristics, the difference between different sources is reduced, and gene editing is carried out on the subcultured cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a deer antler stem cell exosome preparation and a preparation method thereof. Background Art

[0002] The field of biotechnology has experienced rapid development in recent years, with emerging technologies such as cell therapy and gene therapy offering new hope for the treatment of a wide range of diseases. Exosomes, as crucial mediators of intercellular communication, have become a hot topic in biomedical research due to their unique biological properties and potential therapeutic applications. Exosomes are nanoscale vesicles secreted by cells, containing a variety of bioactive components, such as proteins, nucleic acids, and lipids. They can transmit information between cells and regulate their physiological functions. Stem cell-derived exosomes, due to their multipotential differentiation potential and immunomodulatory properties, exhibit even greater therapeutic potential, offering broad application prospects in areas such as tissue repair, immunomodulation, and tumor therapy.

[0003] In the field of cancer treatment, especially for gliomas, which are highly malignant and difficult to treat, traditional treatments such as surgical resection, radiotherapy, and chemotherapy have many limitations. Surgery is difficult to completely remove the tumor and can easily damage surrounding normal tissue. While radiotherapy and chemotherapy can inhibit tumor growth to a certain extent, their lack of targeting means that while they kill tumor cells, they can also cause severe damage to normal cells, triggering a series of adverse reactions.

[0004] Traditional stem cell exosome preparations, such as bone marrow mesenchymal stem cells, often cause certain damage to the donor during the acquisition process, and there are large differences between different donors, resulting in unstable exosome production and quality, making large-scale production of high-quality exosomes difficult and unable to meet the needs of clinical treatment. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a deer antler stem cell exosome preparation and a preparation method thereof, which solves the problem that the acquisition process of traditional stem cell exosome preparations often causes certain damage to the donor and there are large differences between different donors, resulting in unstable exosome yield and quality, making it difficult to produce high-quality exosomes on a large scale and unable to meet the needs of clinical treatment.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for preparing a deer antler stem cell exosome preparation, comprising the following steps:

[0007] After disinfecting the antler, the translucent mesenchymal layer at the tip was separated, minced, rinsed with PBS containing double antibodies, placed in a tissue digestion enzyme mixture, digested, centrifuged, washed, and then plated in a culture flask for culture to obtain the isolate;

[0008] When the cells of the isolate grow to 70%-90% of the culture flask, the original culture medium is discarded, the cells are washed with PBS, and then trypsin is added for digestion, and then the digestion is terminated with complete culture medium, and the cells are passaged to obtain the subculture;

[0009] The cells of the passaged material are subjected to gene editing treatment, digested and centrifuged, and then added into cell freezing solution for gradient freezing to obtain a stored material;

[0010] The stored material is revived and the three-dimensional culture system is pre-treated. The cells in the revived stored material are placed in the external circulation culture cylinder of the three-dimensional culture system, and the cells are repeatedly pumped to allow them to adhere evenly, and then cultured to obtain a culture;

[0011] The culture was subjected to ultraspeed gradient centrifugation, and then targeted functional modification was performed. The precipitate was resuspended with enzyme-free sterile PBS and stored at -80°C to complete the preparation.

[0012] By adopting the above technical solution, stem cells can be obtained from the unique source of deer antler, which reduces damage to the donor. In addition, since the biological characteristics of deer antler stem cells themselves are relatively stable, the differences between different sources are reduced. Gene editing can regulate the function and composition of exosomes secreted by stem cells. The three-dimensional culture system simulates the in vivo environment, improves the yield and quality of exosomes, and targeted functional modification enhances the specificity of exosomes, thereby achieving the preparation of stable and high-quality deer antler stem cell exosome preparations. This solves the problem that traditional stem cell exosome preparations often cause certain damage to the donor during the acquisition process and there are large differences between different donors, resulting in unstable exosome yield and quality, making large-scale production of high-quality exosomes difficult and difficult to meet the needs of clinical treatment.

[0013] Preferably, the antler is the antler of a three-year-old healthy male sika deer, the separation environment is a sterile environment, the rinsing with PBS containing double antibody is rinsing with PBS containing 2% double antibody until the blood is washed away, and the tissue digestion mixed enzyme is prepared by adding 64 mg of type I collagenase, 52 mg of type II collagenase and 44 mg of type IV collagenase to every 80 mL of DMEM, and filtering with a 0.22 μm filter membrane after fully dissolving.

[0014] Preferably, the digestion is performed by heating in a 37° C. water bath for 30-40 minutes, shaking every 5 minutes, and terminating the digestion when a layer of gelatinous mass appears on the surface of the shredded translucent mesenchymal layer.

[0015] Preferably, the addition of trypsin digestion is to add 0.25% trypsin for digestion, and the trypsin is discarded when the cells shrink and become round. The complete culture medium is prepared by adding 10% FBS and 1% penicillin-streptomycin to DMEM. The gene editing treatment includes knocking in exosome secretion-promoting genes or anti-tumor functional genes, or knocking out cancer-promoting genes through CRISPR / Cas9 technology. The exosome secretion-promoting genes include Rab27a and Rab27b, the anti-tumor functional genes include PTEN and P53, and the cancer-promoting genes include EGFR and K-Ras.

[0016] Preferably, the gradient freezing conditions are 4°C for 5 min, -20°C for 20 min, and -80°C overnight before long-term storage in liquid nitrogen. The thawing is performed by placing the tube in a water bath at 37°C and starting to melt. When the cryopreserved liquid in the tube gradually melts to a state of ice-water mixture, complete culture medium is added to the tube in a sterile clean bench, and after centrifugation at 1000 rpm for 5 min, the original supernatant is discarded and complete culture medium is added. After pipetting evenly, the tube is transferred to a culture flask and placed in a cell culture incubator.

[0017] Preferably, the three-dimensional culture system comprises a hollow fiber bioreactor, and the pretreatment is to sequentially subject the three-dimensional culture system to internal and external circulation using enzyme-free and sterile PBS, DMEM, and complete culture medium containing 10% DMEM, with a circulation time of 24-48 hours.

[0018] Preferably, during the culture, the external circulation culture tube is turned upside down every 30 minutes, and the power is turned on after 2 hours and 30 minutes to connect the external circulation complete culture medium. The sugar consumption in the complete culture medium is detected every other day, and new complete culture medium is replaced in time. The targeted functional modification includes connecting tumor cell-specific antibody fragments, aptamers or targeting peptides to the surface of the culture after ultraspeed gradient centrifugation through a chemical coupling method. The tumor cell-specific antibody fragments include anti-EGFR single-chain antibodies and anti-HER2 single-chain antibodies. The aptamers include nucleic acid aptamers and polypeptide aptamers. The targeting peptides include iRGD peptides and TAT peptides.

[0019] Preferably, the ultraspeed gradient centrifugation treatment is to centrifuge at 4°C and 300×g for 15 minutes, take the supernatant and transfer it to a new centrifuge tube, and then centrifuge at 4°C and 2000×g for 30 minutes, collect the supernatant again, and then centrifuge at 4°C and 10000×g for 30 minutes, take the supernatant and filter it through a 0.22μm filter membrane, centrifuge the filtered supernatant at 4°C and 120000×g for 2 hours, discard the supernatant, and collect the bottom precipitate.

[0020] A deer antler stem cell exosome preparation is prepared by the above-mentioned method for preparing a deer antler stem cell exosome preparation.

[0021] Preferably, a deer antler stem cell exosome preparation is used as a drug carrier for treating glioma.

[0022] The present invention provides a deer antler stem cell exosome preparation and a preparation method thereof. It has the following beneficial effects:

[0023] 1. The present invention obtains stem cells from velvet antlers to avoid damage to the donor when obtaining stem cells. Since velvet antler stem cells themselves have stable biological characteristics, the differences between different sources are reduced. The cells after passage are gene-edited to knock in genes that promote exosome secretion and anti-tumor functional genes, and knock out cancer-promoting genes. A three-dimensional culture system is used to simulate the in vivo environment to culture stem cells, thereby improving the yield and quality of exosomes. The obtained exosomes are subjected to targeted functional modification to enhance their specificity, so that the stem cell exosome preparation can meet the needs of clinical treatment.

[0024] 2. The present invention performs gene editing on the cells of the passage, knocks in genes that promote exosome secretion to increase exosome production, knocks in anti-tumor functional genes to give exosomes stronger tumor-suppressing ability, knocks out cancer-promoting genes to reduce tumor-related adverse factors, optimizes the functional characteristics of exosomes, and makes the final exosome preparation more targeted and effective in treating diseases such as glioma, thereby enhancing the therapeutic value of the preparation.

[0025] 3. The present invention creates a microenvironment for antler stem cells that is closer to the body by adopting a three-dimensional culture system. The cells grow better in such an environment, the secretion of exosomes is greatly increased, and the secreted exosomes are of better quality and contain richer bioactive ingredients. This not only meets the clinical demand for large quantities of high-quality exosome preparations, but also provides a strong guarantee for the preparations to exert better therapeutic effects.

[0026] 4. The present invention performs targeted functional modification and connects tumor cell-specific antibody fragments, aptamers or targeting peptides, so that the exosome preparation can accurately identify and enrich around glioma cells, and can more efficiently deliver therapeutic ingredients to the target site, thereby improving the treatment effect while reducing damage to normal cells, greatly improving the accuracy and safety of the preparation in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of a method for preparing a deer antler stem cell exosome preparation proposed in the present invention;

[0028] Figure 2This is a diagram of ASCs migrating from tissues according to the present invention;

[0029] Figure 3 This is a diagram of the third generation of ASCs of the present invention;

[0030] Figure 4 The IFA analysis of ASCs surface marker factor map of the present invention;

[0031] Figure 5 This is an Oil Red O staining diagram of ASCs according to the present invention;

[0032] Figure 6 This is an Alizarin Red staining diagram of ASCs according to the present invention;

[0033] Figure 7 This is a diagram of ASCs stained with Alcian blue according to the present invention. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] Please see the attached Figure 1 The present invention provides a method for preparing a deer antler stem cell exosome preparation, comprising the following steps:

[0036] After disinfecting the antler, the translucent mesenchymal layer at the tip was separated, chopped, rinsed with PBS containing double-antibody antibodies, and placed in a tissue digestion enzyme mix. After digestion, the antler was centrifuged, washed, and then plated in a culture flask for culture to obtain the isolate. The antler was from a healthy three-year-old male sika deer. The isolation environment was sterile. The rinsing with PBS containing 2% double-antibody antibodies was performed until all blood was washed away. The tissue digestion enzyme mix was prepared by adding 64 mg of type I collagenase, 52 mg of type II collagenase, and 44 mg of type IV collagenase to 80 mL of DMEM, fully dissolving the mixture, and filtering it through a 0.22 μm filter. Digestion was performed in a 37°C water bath for 30-40 minutes, shaking it every 5 minutes, until a layer of gelatinous mass appeared on the surface of the chopped translucent mesenchymal layer.

[0037] Specifically, healthy three-year-old male sika deer are selected and anesthetized. Under a strict sterile environment, antlers are quickly harvested using sterilized instruments. After harvesting, the antlers are disinfected with iodine and alcohol cotton to ensure that there is no microbial contamination on the surface.

[0038] On a sterile operating table, the translucent mesenchymal layer at the tip of the antler, rich in stem cells, is isolated. The isolated translucent mesenchymal layer is minced into small pieces and repeatedly rinsed in PBS buffer containing 2% double-streptomycin. During the rinse, the pieces are carefully observed until no blood is present in the rinse solution to ensure cleanliness.

[0039] Transfer the cleaned tissue blocks to a container containing a tissue digestion enzyme mix. This enzyme mix is prepared by adding 64 mg of type I collagenase, 52 mg of type II collagenase, and 44 mg of type IV collagenase to every 80 mL of DMEM medium. After thorough stirring to dissolve, the mixture is filtered through a 0.22 μm filter. Place the container in a 37°C water bath for digestion. Gently shake the container every 5 minutes during digestion to ensure full contact between the tissue blocks and the digestive enzymes. The digestion time is controlled within 30-40 minutes. When a layer of gelatinous clumps is observed on the surface of the shredded translucent mesenchymal layer, the digestion has reached the desired level and digestion is terminated.

[0040] After digestion, the digestion solution was transferred to a centrifuge tube and centrifuged at room temperature for 15 min at a centrifugal force of 300 × g. After centrifugation, the supernatant was carefully discarded and the tissue sediment at the bottom was collected. An appropriate amount of culture fluid containing 10% FBS (fetal bovine serum) was added to the sediment, and the culture fluid was gently pipetted evenly. The cells were centrifuged at a centrifugal force of 300 × g for 5 min, and this cleaning step was repeated 2-3 times to thoroughly remove residual digestive enzymes and impurities. The cleaned tissue fragments were evenly spread in a culture bottle, and each T75 culture bottle added 2 mL of culture fluid. The culture bottle was placed in a cell culture incubator and cultivated under a 37°C, 5% CO2 environment. During the incubation process, the growth of the cells was observed under a microscope every day. When the cells migrated out of the tissue block and gradually covered 70%-90% of the culture bottle bottom, the cell growth state was good and the next step was performed. This resulted in the separation of the cells.

[0041] When the cells of the isolate grow to 70%-90% of the culture flask, the original culture medium is discarded, and the cells are washed with PBS and then digested with trypsin. The digestion is then terminated with complete culture medium, and the cells are passaged to obtain passages; the trypsin digestion is performed by adding 0.25% trypsin for digestion, and the trypsin is discarded when the cells shrink and become round. The complete culture medium is prepared by adding 10% FBS and 1% penicillin-streptomycin to DMEM.

[0042] Specifically, when the cells of the isolate have grown to cover 70%-90% of the bottom of the culture flask, remove the culture flask from the cell culture incubator and place it on a sterile workbench. Carefully discard the original culture medium and slowly rinse the cells 2-3 times with PBS buffer to remove residual culture medium and cellular metabolites.

[0043] Add an appropriate amount of 0.25% trypsin solution to the culture flask, ensuring that the trypsin solution can completely cover the cell surface. Place the culture flask under a microscope and observe. When the cells begin to shrink and round, the cell digestion process is appropriate. At this point, quickly discard the trypsin solution and add an appropriate amount of complete culture medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) to terminate the digestion. Gently pipette the cells to detach them from the bottom of the culture flask and form a uniform cell suspension.

[0044] Transfer the cell suspension to a new culture flask and add an appropriate amount of complete culture medium at a ratio of 1:2 or 1:3. Gently shake the suspension and return it to the cell culture incubator for continued culture. During the culture process after subculturing, regularly observe the growth status of the cells and replace the culture medium every 2-3 days to ensure that the cells have adequate nutrient supply and obtain subcultured products.

[0045] The cells in the passages were gene-edited, digested, centrifuged, and then cryopreserved in a gradient freezing solution to obtain the stock. Gene editing involved knocking in exosome-promoting genes or anti-tumor genes, or knocking out oncogenes, using CRISPR / Cas9 technology. Exosome-promoting genes included Rab27a and Rab27b, anti-tumor genes included PTEN and P53, and oncogenes included EGFR and K-Ras. The gradient freezing conditions were 4°C for 5 minutes, -20°C for 20 minutes, and -80°C overnight before being placed in liquid nitrogen for long-term storage.

[0046] Specifically, when the passaged cells grow to a confluence of more than 80% in the culture flask, gene editing is performed. Under sterile operating conditions, the cells in the culture flask are trypsinized to a single-cell suspension, which is then transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant is discarded and the cell pellet is collected.

[0047] CRISPR / Cas9 technology is used to gene-edit cells. To knock in a gene that promotes exosome secretion, such as Rab27a or Rab27b, an expression vector containing the target gene is introduced into the cells along with the CRISPR / Cas9 system, where it is integrated into the cell genome through homologous recombination. To knock in an anti-tumor gene, such as PTEN or P53, the corresponding expression vector and CRISPR / Cas9 system are also used. To knock out a cancer-promoting gene, such as EGFR or K-Ras, sgRNAs targeting these genes are designed to guide the Cas9 protein to cleave the target gene, achieving gene knockout. After gene editing, the cells are placed in a culture flask containing complete medium and cultured in a cell culture incubator for 24-48 hours to allow the cells to recover.

[0048] After the cells have stabilized, trypsinize them again, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and collect the cell pellet. Add an appropriate amount of cell freezing solution to the cell pellet and gently pipette to evenly suspend the cells in the freezing solution. Cryopreserve according to the gradient freezing conditions: first place the cell suspension at 4°C for 5 minutes to acclimate the cells to the low temperature; then transfer to -20°C for 20 minutes; then place in a -80°C freezer overnight; and finally transfer to liquid nitrogen for long-term storage.

[0049] The stored material was resuscitated and the 3D culture system was preconditioned. Cells from the resuscitated material were inoculated into the external circulation culture cylinder of the 3D culture system and repeatedly pumped to ensure uniform cell attachment. Cultures were then cultured. Resuscitation was initiated by placing the cryopreserved material in a 37°C water bath. Once the cryopreserved material in the tube had gradually thawed to a mixture of ice and water, complete culture medium was added to the tube in a sterile laminar flow hood. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and complete culture medium was added. After pipetting to evenly distribute the culture medium, the tube was transferred to a culture flask and placed in a cell culture incubator. The 3D culture system consisted of a hollow fiber bioreactor. Preconditioning involved sequentially circulating the 3D culture system with enzyme-free sterile PBS, DMEM, and complete culture medium containing 10% DMEM for 24-48 hours. During culture, the external circulation culture cylinder was inverted every 30 minutes. After 2 hours and 30 minutes, the power supply was connected to the external circulation complete culture medium. Sugar consumption in the complete culture medium was monitored every other day and replaced with new complete culture medium.

[0050] Specifically, when the stored cells need to be used, remove the cryovial from the liquid nitrogen and quickly place it in a 37°C water bath. Gently shake the cryovial to quickly thaw the cryopreservation solution until it reaches a mixture of ice and water. In a sterile laminar flow hood, transfer the cell suspension in the cryovial to a centrifuge tube, add an appropriate amount of complete culture medium, centrifuge at 1000 rpm for 5 minutes, discard the original supernatant, and collect the cell pellet. Add an appropriate amount of complete culture medium to the cell pellet, gently pipette to evenly distribute the cell suspension, transfer it to a culture flask, and place it in a cell incubator to allow the cells to resume growth.

[0051] While the cells are recovering, the three-dimensional culture system (hollow fiber bioreactor) is pretreated by sequentially circulating enzyme-free, sterile PBS, DMEM, and complete culture medium containing 10% DMEM, with a cycle time of 24-48 hours to clean and balance the culture system and provide a suitable growth environment for the cells.

[0052] After the cells have recovered, they are connected to the outer circulation culture tube of the three-dimensional culture system. Use a pipette to repeatedly pump the cell suspension so that the cells are evenly attached to the inner wall of the culture tube. During the culture process, turn the outer circulation culture tube upside down every 30 minutes so that the cells can be evenly distributed and fully contact the culture medium. After 2h30min, turn on the power to connect the outer circulation complete culture medium, and check the sugar consumption in the complete culture medium every other day. According to the test results, replace the new complete culture medium in time to ensure an adequate supply of nutrients required for cell growth, maintain the cells in a good growth state, and obtain a culture. During the culture process, the growth morphology and distribution of cells in the three-dimensional culture system can be observed under a microscope to evaluate the culture effect.

[0053] The culture is subjected to ultraspeed gradient centrifugation, followed by targeted functionalization, and the pellet is resuspended in enzyme-free sterile PBS and stored at -80°C to complete the preparation. Targeted functionalization involves chemically coupling tumor cell-specific antibody fragments, aptamers, or targeting peptides to the surface of the culture after ultraspeed gradient centrifugation. Tumor cell-specific antibody fragments include anti-EGFR single-chain antibodies and anti-HER2 single-chain antibodies. Aptamers include nucleic acid aptamers and polypeptide aptamers. Targeting peptides include iRGD peptides and TAT peptides. Ultraspeed gradient centrifugation treatment is as follows: centrifuge at 4°C and 300×g for 15 minutes, transfer the supernatant to a new centrifuge tube, and then centrifuge at 4°C and 2000×g for 30 minutes, collect the supernatant again, and then centrifuge at 4°C and 10000×g for 30 minutes, take the supernatant and filter it through a 0.22μm filter membrane, centrifuge the filtered supernatant at 4°C and 120000×g for 2 hours, discard the supernatant, and collect the bottom precipitate.

[0054] Specifically, the culture medium in the three-dimensional culture system was collected and subjected to ultraspeed gradient centrifugation to extract exosomes. First, the culture medium was centrifuged at 4°C and 300×g for 15 minutes to pellet larger cells and cell debris, and the supernatant was transferred to a new centrifuge tube. Next, the supernatant was centrifuged at 4°C and 2000×g for 30 minutes to further remove smaller impurities, and the supernatant was collected again. The supernatant was then centrifuged at 4°C and 10,000×g for 30 minutes to further concentrate the exosomes. The supernatant was collected and filtered through a 0.22μm filter to remove residual cell debris and impurities. Finally, the filtered supernatant was centrifuged at 4°C and 120,000×g for 2 hours. At this time, the exosomes precipitated at the bottom of the centrifuge tube. The supernatant was discarded and the bottom precipitate was collected.

[0055] Chemical conjugation is used to modify exosomes for targeted functionalization. If tumor cell-specific antibody fragments, such as anti-EGFR single-chain antibodies or anti-HER2 single-chain antibodies, are chosen for attachment, the exosomes are first mixed with a buffer containing an activating group (e.g., carbodiimide, EDC) to activate the carboxyl groups on the exosome surface. The anti-EGFR single-chain antibody or anti-HER2 single-chain antibody is then added and reacted to covalently bind the antibody fragments to the activated carboxyl groups on the exosome surface. If aptamers, such as nucleic acid aptamers or peptide aptamers, are chosen for attachment, conjugation can be performed using a biotin-avidin system. The aptamer is first labeled with biotin and then mixed with exosomes modified with avidin. The specific binding of biotin and avidin allows the aptamer to attach to the exosome surface. If targeting peptides, such as iRGD peptide or TAT peptide, are chosen, chemical conjugation agents can also be used to attach the targeting peptide to the exosome surface.

[0056] Resuspend the targeted exosome pellet in enzyme-free, sterile PBS and store the resuspended exosome preparation at -80°C to complete the preparation of the deer antler stem cell exosome preparation. During the extraction and modification process, strict aseptic techniques must be adhered to to prevent contamination of the exosomes. Furthermore, regular quality testing of the exosome preparation can be performed, such as observing the exosome morphology using a transmission electron microscope and measuring the particle size distribution using a nanoparticle size analyzer.

[0057] A deer antler stem cell exosome preparation is prepared by the above-mentioned method for preparing a deer antler stem cell exosome preparation.

[0058] Application of a deer antler stem cell exosome preparation as a drug carrier for treating glioma.

[0059] Specifically, this deer antler stem cell exosome preparation contains a variety of bioactive components, such as proteins, mRNA, and miRNA, and possesses a unique vesicle structure. These bioactive components include a variety of factors with potential anti-tumor effects, such as signaling molecules that regulate cell growth, differentiation, and apoptosis. Furthermore, the vesicle structure protects the bioactive components within from degradation by enzymes within the body, ensuring their stable presence and effectiveness. The exosome preparation has specific targeting molecules on its surface, such as tumor cell-specific antibody fragments, aptamers, or targeting peptides. These targeting molecules can specifically recognize antigens or receptors on the surface of glioma cells, enabling the exosome preparation to be precisely enriched around glioma cells. Furthermore, it exhibits excellent biocompatibility and low immunogenicity. Once in the body, it is less likely to induce immune rejection, allowing it to circulate smoothly and reach the tumor site. In this way, the biologically active ingredients carried can be accurately delivered to glioma cells, and by regulating the physiological processes of tumor cells, inhibiting tumor cell proliferation, inducing tumor cell apoptosis, inhibiting tumor angiogenesis, etc., the purpose of treating glioma is achieved, and it can be used as a drug carrier for the treatment of glioma.

[0060] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a deer antler stem cell exosome preparation, characterized in that: The following steps are involved: After disinfecting the antler, the translucent mesenchymal layer at the tip was separated, minced, rinsed with PBS containing double antibodies, placed in a tissue digestion enzyme mixture, digested, centrifuged, washed, and then plated in a culture flask for culture to obtain the isolate; When the cells of the isolate grow to 70%-90% of the culture flask, the original culture medium is discarded, the cells are washed with PBS, and then trypsin is added for digestion, and then the digestion is terminated with complete culture medium, and the cells are passaged to obtain the subculture; The cells of the passaged material are subjected to gene editing treatment, digested and centrifuged, and then added into cell freezing solution for gradient freezing to obtain a stored material; The stored material is revived and the three-dimensional culture system is pre-treated. The cells in the revived stored material are placed in the external circulation culture cylinder of the three-dimensional culture system, and the cells are repeatedly pumped to allow them to adhere evenly, and then cultured to obtain a culture; The culture was subjected to ultraspeed gradient centrifugation, and then targeted functional modification was performed. The precipitate was resuspended with enzyme-free sterile PBS and stored at -80°C to complete the preparation.

2. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The antler is from a healthy three-year-old male sika deer. The separation environment is a sterile environment. The rinsing with PBS containing double antibody is rinsing with PBS containing 2% double antibody until the blood is rinsed away. The tissue digestion mixed enzyme is prepared by adding 64 mg of type I collagenase, 52 mg of type II collagenase, and 44 mg of type IV collagenase to 80 mL of DMEM, fully dissolving the mixture, and filtering it with a 0.22 μm filter membrane.

3. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The digestion was performed by heating in a 37° C. water bath for 30-40 minutes, shaking every 5 minutes, and stopping the digestion when a layer of gelatinous mass appeared on the surface of the shredded translucent mesenchymal layer.

4. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The trypsin digestion is performed by adding 0.25% trypsin for digestion, and the trypsin is discarded when the cells shrink and become round. The complete culture medium is prepared by adding 10% FBS and 1% penicillin-streptomycin to DMEM. The gene editing treatment includes knocking in exosome secretion-promoting genes or anti-tumor functional genes, or knocking out cancer-promoting genes through CRISPR / Cas9 technology. The exosome secretion-promoting genes include Rab27a and Rab27b, the anti-tumor functional genes include PTEN and P53, and the cancer-promoting genes include EGFR and K-Ras.

5. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The gradient freezing conditions are as follows: placing at 4°C for 5 minutes, placing at -20°C for 20 minutes, and placing at -80°C overnight before placing in liquid nitrogen for long-term storage. The resuscitation is to place the tube in a water bath at 37°C and begin to thaw. When the cryopreserved liquid in the tube gradually melts to a state of ice-water mixture, complete culture medium is added to the tube in a sterile clean bench, and after centrifugation at 1000 rpm for 5 minutes, the original supernatant is discarded and complete culture medium is added. After pipetting evenly, the tube is transferred to a culture flask and placed in a cell culture incubator.

6. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The three-dimensional culture system includes a hollow fiber bioreactor. The pretreatment is to sequentially use enzyme-free and sterile PBS, DMEM, and a complete culture medium containing 10% DMEM for internal and external circulation of the three-dimensional culture system, with a circulation time of 24-48 hours.

7. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: During the culture, the external circulation culture tube is turned upside down every 30 minutes, and the power is turned on after 2 hours and 30 minutes to connect the external circulation complete culture medium. The sugar consumption in the complete culture medium is detected every other day, and new complete culture medium is replaced in time. The targeted functional modification includes connecting tumor cell-specific antibody fragments, aptamers or targeting peptides to the surface of the culture after ultraspeed gradient centrifugation through a chemical coupling method. The tumor cell-specific antibody fragments include anti-EGFR single-chain antibodies and anti-HER2 single-chain antibodies. The aptamers include nucleic acid aptamers and polypeptide aptamers. The targeting peptides include iRGD peptides and TAT peptides.

8. The method for preparing a deer antler stem cell exosome preparation according to claim 1, characterized in that: The ultraspeed gradient centrifugation treatment is as follows: centrifugation at 4°C and 300×g for 15 minutes, transferring the supernatant to a new centrifuge tube, and then centrifuging at 4°C and 2000×g for 30 minutes, collecting the supernatant again, and then centrifuging at 4°C and 10,000×g for 30 minutes, taking the supernatant and filtering it through a 0.22 μm filter membrane, and centrifuging the filtered supernatant at 4°C and 120,000×g for 2 hours, discarding the supernatant, and collecting the bottom precipitate.

9. A deer antler stem cell exosome preparation, characterized by: The antler antler stem cell exosome preparation is prepared by the preparation method of the antler antler stem cell exosome preparation according to any one of claims 1 to 8.

10. Use of the deer antler stem cell exosome preparation according to claim 9 as a drug carrier for treating glioma.

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