Cell culture medium and its application in preparing exosomes from antler stem cells

Abstract

The present invention relates to the field of cell culture technology, and in particular to a cell culture medium and its application in preparing antler stem cell exosomes. Compared with conventional culture medium, the use of the cell culture medium of the present invention can promote faster proliferation of antler stem cells, and the exosome production is increased by 2 to 3 times. Compared with other methods for promoting exosome production, the use of the cell culture medium of the present invention still maintains the original properties of AnSC-exos, and further promotes the ability of cell proliferation, inducing angiogenesis, and cell migration.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a cell culture medium and an application thereof in preparing antler stem cell exosomes. Background Art

[0002] Exosomes are nanoscale vesicles (30-150nm) present in cells or body fluids. They are produced by the inward budding of multivesicular bodies (MVBs) and secreted outside the cell after the fusion of multivesicular bodies with the plasma membrane. Since exosomes are important mediators of intercellular communication in many physiological and pathological processes, they are involved in a wide range of functional activities, such as protein clearance, immunity, inflammatory response and signal transduction.

[0003] Exosomes derived from stem cells have been shown to be useful for the treatment of a variety of diseases or tissue regeneration and repair, including osteoarthritis, skin injuries, burns, inhibition of tumor growth, and cognitive impairment, etc. This is mainly due to their inherent regenerative capacity, which can induce angiogenesis, promote proliferation, prevent cell apoptosis, and inhibit inflammatory responses.

[0004] However, the production of exosomes is one of the key reasons that restrict the large-scale application of exosomes. Although there are many methods that can increase the production of exosomes to a certain extent, such as changing gene expression, adjusting the pH of the culture medium, oxygen concentration, etc., the properties of exosomes have changed to a certain extent, deviating from the original application scenarios. Therefore, it is of great value and significance to allow limited cells to produce more exosomes without changing the original properties of exosomes. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a cell culture medium and its application in the preparation of antler stem cell exosomes. The cell culture medium provided by the present invention can effectively increase the production of antler stem cell exosomes, and the prepared antler stem cell exosomes can promote cell proliferation, cell migration and angiogenesis.

[0006] The invention provides a cell culture medium, which comprises norepinephrine bitartrate, penicillin, streptomycin, fetal bovine serum and a basic culture medium.

[0007] The cell culture medium provided by the present invention is suitable for the culture of antler stem cells and their exosomes. Compared with other culture media, the components in the cell culture medium provided by the present invention cooperate with each other, providing a more favorable nutritional system for the proliferation of antler stem cells, and can maximize the production efficiency of antler stem cell exosomes, thereby obtaining more accurate technical effects.

[0008] In some embodiments, a basal medium is included with the following components at the following concentrations:

[0009] Norepinephrine bitartrate 10~50μM, penicillin 50~150 U / mL, streptomycin 0.01~0.5 mg / mL, fetal bovine serum 5vol%~20vol%.

[0010] The cell culture medium with the above-mentioned components and concentration range was used to compare the exosome proliferation efficiency with the culture medium in the prior art. Within this concentration range, the cell culture medium provided by the present invention can efficiently amplify antler stem cells, further improve the production efficiency of antler stem cell exosomes, and thus obtain a more accurate technical effect.

[0011] In some embodiments, the basal culture medium is DMEM culture medium, and the fetal bovine serum is exosome-removed fetal bovine serum.

[0012] In some specific embodiments, the base medium includes the following components at the following concentrations:

[0013] Norepinephrine bitartrate 20 μM, penicillin 100 U / mL, streptomycin 0.1 mg / mL, fetal bovine serum 10 vol%.

[0014] Experiments show that the use of the above concentration of cell culture medium is most efficient in amplifying and preparing antler stem cell exosomes, and the prepared antler stem cell exosomes have the best ability to promote cell proliferation, cell migration and angiogenesis, thereby obtaining more accurate technical effects.

[0015] The present invention provides the use of the cell culture medium in preparing antler stem cell exosomes.

[0016] The present invention provides a method for preparing antler stem cell exosomes, wherein antler stem cells are cultured with the cell culture medium and extracted to obtain the antler stem cell exosomes.

[0017] In some embodiments, the method for preparing antler stem cells comprises the following steps:

[0018] Step 1, taking periosteum tissue from the antler area of ​​deer, washing and mincing it, adding basal culture medium, centrifuging, and discarding the supernatant to obtain a first precipitate;

[0019] Step 2, taking the first precipitate in step 1, digesting it with a digestion solution, centrifuging it, and discarding the supernatant to obtain a second precipitate;

[0020] Step 3, taking the second precipitate in step 2, terminating the digestion with FBS, centrifuging, and discarding the supernatant to obtain a third precipitate;

[0021] Step 4: Take the third precipitate described in step 3 and inoculate it into a culture dish, invert it and place it in a saturated humidity incubator for incubation, add complete culture medium and culture it upright until the cell growth confluence reaches 70% to 80%, so as to obtain the antler stem cells.

[0022] In some specific embodiments,

[0023] In the step 1, the deer antler area periosteal tissue is taken from a healthy 8-month-old male sika deer, and the antler tissue that begins to develop and thicken is collected above the frontal bone and stored in DMEM containing 2-5 times the volume of double antibody. The mincing includes using a sterile small cutter to evenly mince the deer antler area periosteal tissue into 1mm 2 Left and right tissue blocks, the basal culture medium is DMEM basal culture medium;

[0024] In the step 2, the digestion solution is 10-100 U / mL collagenase I, the added volume of the digestion solution is 10-25 mL, the shaking digestion is performed in a 37°C water bath until burrs appear around most tissue blocks, and the centrifugation is performed at 1000 rpm for 5 min;

[0025] In the step 3, the digestion termination solution includes 10-25 mL of a complete medium containing 10 vol% FBS, and the centrifugation is performed at 1000 rpm for 5 min;

[0026] In step 4, the inoculation includes taking the third precipitate and evenly coating it on a 10 cm 2 Add about 1-3 mL of complete culture medium to the culture dish to keep the cells moist, then invert the culture dish and incubate in an incubator at 37°C, 5% CO2, and saturated humidity for 2-4 h, add 5-10 mL of complete culture medium, and continue culturing for 5-7 days after placing it upright.

[0027] In some embodiments, the culturing step includes inoculating the antler stem cells into a culture bottle, adding culture medium and culturing for 2 to 5 days, and collecting the culture supernatant.

[0028] In some specific embodiments, the seeding density of the antler stem cells is 1.0-1.5×10 6 / 75 cm 2 The volume of the complete culture medium is 15 mL, and the culture conditions include culturing in a cell culture incubator at 37° C., 5% CO 2 and saturated humidity for 72 h.

[0029] In some embodiments, the extraction includes taking the culture supernatant and centrifuging it multiple times to sequentially remove dead cells, cell debris, and free organelles, and then obtaining the antler stem cell exosomes by ultra-high-speed centrifugation.

[0030] In some specific embodiments, the multiple centrifugations sequentially include 300 g centrifugation for 10 min to remove dead cells, 2000 g centrifugation for 15 min to remove cell debris, and 10000 g centrifugation for 30 min to remove free organelles, and the ultrahigh-speed centrifugation includes 100000 g centrifugation for 90 min to precipitate and obtain the antler stem cell exosomes.

[0031] In some specific embodiments, the number of ultra-high speed centrifugation is 1 to 3 times.

[0032] The present invention provides antler stem cell exosomes prepared by the preparation method.

[0033] The present invention provides the use of the velvet antler stem cell exosomes in the preparation of a drug for promoting cell proliferation, cell migration and / or angiogenesis.

[0034] The present invention provides a drug for promoting cell proliferation, cell migration and / or angiogenesis, comprising the antler stem cell exosomes.

[0035] The present invention provides a cell culture medium and its application in preparing antler stem cell exosomes. Compared with the prior art, the present invention has the following effects:

[0036] 1. Compared with conventional culture medium, the cell culture medium of the present invention can promote faster proliferation of antler stem cells and increase the exosome production by 2 to 3 times;

[0037] 2. Compared with other methods to promote exosome production, such as changing gene expression and changing the pH of the culture medium, the cell culture medium developed in this study still maintains the original properties of AnSC-exos, such as morphology, diameter, distribution range, ability to promote cell proliferation, induce angiogenesis, and cell migration. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of stem cell marker detection of antler stem cells (AnSCs) using flow cytometry are shown;

[0039] Figure 2 Figure 2 shows the flow chart of exosome extraction;

[0040] Figure 3 Figure 2 shows the structure of AnSC-exos collected by electron microscopy using conventional culture medium and antler stem cell culture medium used in the present invention, wherein Figure A shows the structure of AnSC-exos collected by electron microscopy using conventional culture medium, and Figure B shows the structure of AnSC-exos collected by electron microscopy using antler stem cell culture medium of the present invention;

[0041] Figure 4The effect of AnSC-exos collected using antler stem cell culture medium of the present invention on the proliferation of HUVECs was detected by CCK8;

[0042] Figure 5 The scratch test was used to detect the effect of AnSC-exos collected using antler stem cell culture medium on HUVECs migration;

[0043] Figure 6 The HUVECs tube formation experiment is shown to detect the angiogenesis ability of AnSC-exos collected using antler stem cell culture medium of the present invention. DETAILED DESCRIPTION

[0044] The present invention provides a cell culture medium and its application in the preparation of antler stem cell exosomes. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0046] Example

[0047] 1. Preparation of Deer Antler Stem Cells

[0048] Select healthy male sika deer (about 8 months old) and collect the antler tissue that begins to develop and thicken above the frontal bone. Put it in DMEM containing 2-5 times double antibody and bring it to the laboratory for primary culture. The specific method is: take out the collected tissue, wash away the remaining blood, and use a sterile small knife to evenly chop it into 1mm 2 Add DMEM basal medium to the left and right tissue blocks, centrifuge and collect the precipitate; discard the supernatant, add 10-25mL (10-100 U / mL) collagenase I, and digest in a 37℃ water bath with shaking. Observe every 10 minutes. When burrs appear around most tissue blocks, centrifuge at 1000 rpm for 5 minutes and discard the supernatant; add 10-25mL complete medium (containing 10% FBS) to stop digestion, centrifuge at 1000 rpm for 5 minutes; discard the supernatant, aspirate the bottom precipitate, and evenly spread it on a 10 cm 2Add about 1-3 mL of complete culture medium to the culture dish to keep the cells moist, then invert the culture dish and incubate it in a 37°C, 5% CO2, saturated humidity incubator for 2-4 h, add 5-10 mL of complete culture medium, and continue to culture after turning it upright. After 5-7 days, a large number of cells will grow around the tissue, and when the confluence reaches 70%-80%, the cells will be collected for subsequent research.

[0049] The expression of mesenchymal stem cell markers in the above cells was detected by flow cytometry. The results showed that the prepared antler stem cells highly expressed mesenchymal stem cell positive markers CD44 (96%) and CD105 (99.7%), and did not express mesenchymal stem cell negative markers CD34 and CD45 ( Figure 1 ), the test results meet the definition of antler stem cells.

[0050] 2. Preparation of complete culture medium for antler stem cell exosomes

[0051] Preparation of exosome-free fetal bovine serum: Use ultracentrifugation to remove the exosome components in the culture fetal bovine serum, that is, 120,000 g, 90 min, and collect the supernatant for later use.

[0052] Complete medium preparation:

[0053] Control group: 90% DMEM basal medium, 10% exosome-free fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin.

[0054] Experimental Example 1: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 10-100 μM norepinephrine bitartrate (NBM for short).

[0055] Experimental Example 2: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 10-100 μM forskolin (FK).

[0056] Experimental Example 3: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 10-100 μM N-methyldopamine hydrochloride (NMH).

[0057] Experimental Example 4: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 10-100 μM fenoterol hydrobromide (FH).

[0058] Experimental Example 5: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 20 μM NBM, 10 μM FH.

[0059] Experimental Example 6: 90% DMEM basal medium, 10% of the above exosome-free fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 20 μM NBM, 10 μM NMH.

[0060] 3. Cultivation of antler stem cells and preparation of exosomes

[0061] Deer antler stem cells are 1.0-1.5×10 6 / 75 cm 2 The cells were inoculated at a density of 1.50 μg / mL in a culture flask, incubated with 15 mL of complete culture medium in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 72 h, and the culture supernatant was collected.

[0062] 4. Extraction of exosomes from the supernatant

[0063] The ultra-high-speed centrifugation method was used to extract exosomes. The specific steps were as follows: centrifuge at 300 g for 10 min to remove dead cells; centrifuge at 2000 g for 15 min to remove cell debris; centrifuge at 10000 g for 30 min to remove free organelles; centrifuge at 100000 g for 90 min to precipitate exosomes; remove the supernatant and resuspend the exosomes in PBS for washing, and centrifuge at 100000 g for 90 min to precipitate the exosomes again; all centrifugation processes were performed at 4°C. Pour off the supernatant, add an appropriate amount of PBS to resuspend the precipitate, and a high-purity exosome solution is obtained (the specific process is shown in Figure 2 as shown).

[0064] 5. Exosome identification

[0065] Detection of exosome particle number using a particle size analyzer

[0066] The instrument connecting tube was rinsed with PBS, and then diluted samples of different concentrations were extracted and slowly injected into the connecting tube. The single particle nanobiological detector detected the number of particles and exported the data.

[0067] The results of particle size analysis showed that the addition of 20 μM NBM to the antler stem cell culture medium had the greatest increase in exosome production, which was about 2.5 times that of the conventional exosome culture medium (Control) (Table 1). The diameters of the exosomes collected from the two culture media were distributed in the range of 50–150 nm, which was consistent with the average diameter size of the exosomes (Table 2). Exosomes from the antler stem cell culture medium (added with 20 μM NBM) will be used for subsequent property and function analysis.

[0068] Table 1

[0069]

[0070] Table 2

[0071]

[0072] (3) Observation of the morphology of exosomes by transmission electron microscopy: 20 μl of high-purity exosome solution was added to the carbon-coated copper mesh. The copper mesh was inverted on the uranyl acetate staining solution and negatively stained for 30 s. After drying, it was placed under white light and observed and photographed under a -80 KV transmission electron microscope to analyze the exosome structure.

[0073] Transmission electron microscopy showed that the morphology of AnSC-exos collected from the two culture media was round or oval double-layer vesicle structure, and no abnormal changes occurred ( Figure 3 Figures A and B show the structures of AnSC-exos collected by electron microscopy using conventional culture medium and antler stem cell culture medium used in the present invention, respectively.

[0074] 6. Detection of the properties of AnSC-exos in promoting cell proliferation, migration and angiogenesis

[0075] The present invention detected the effects on proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) by adding AnSC-exos (50 μg / mL) to the cell culture medium. The CCK8 results showed that compared with Blank, the exosomes derived from conventional culture medium (AnSC-exos Control) and the exosomes derived from the antler stem cell culture medium of the present invention (AnSC-exos 20 μM) could promote the proliferation of HUVECs, and there was no significant difference ( Figure 4 ). The scratch test results showed that both the AnSC-exos Control group and the AnSC-exos 20 μM group could promote the migration of HUVECs, and there was no significant difference between the two groups ( Figure 5 In addition, the results of the angiogenesis experiment showed that both the AnSC-exos Control group and the AnSC-exos 20 μM group had strong angiogenesis ability, and there was no significant difference between the two in the three main indicators of trunk length, tube formation area and branch length ( Figure 6 ).

[0076] In summary, AnSC-exos have typical exosome characteristics, and there is no significant difference between AnSC-exos from the two culture media. However, the use of the antler stem cell exosome culture medium of the present invention can increase the exosome yield by about 2.5 times compared with the conventional exosome culture medium, greatly saving cell culture costs, and is conducive to the large-scale application of antler stem cell exosomes.

[0077] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing exosomes from velvet antler stem cells, characterized in that: The antler stem cells are cultured and extracted with a cell culture medium to obtain the antler stem cell exosomes, wherein the cell culture medium is composed of a DMEM culture medium and the following components at the following concentrations: Norepinephrine bitartrate 20 μM, penicillin 100 U / mL, streptomycin 0.1 mg / mL and fetal bovine serum 10 vol%, wherein the fetal bovine serum is exosome-removed fetal bovine serum.

2. The preparation method according to claim 1, characterized in that: The preparation method of antler stem cells comprises the following steps: Step 1, taking periosteum tissue from the antler area of ​​deer, washing and mincing it, adding basal culture medium, centrifuging, and discarding the supernatant to obtain a first precipitate; Step 2, taking the first precipitate in step 1, digesting it with a digestion solution, centrifuging it, and discarding the supernatant to obtain a second precipitate; Step 3, taking the second precipitate in step 2, terminating the digestion with FBS, centrifuging, and discarding the supernatant to obtain a third precipitate; Step 4: Take the third precipitate described in step 3 and inoculate it into a culture dish, invert it and place it in a saturated humidity incubator for incubation, add complete culture medium and culture it upright until the cell growth confluence reaches 70% to 80%, so as to obtain the antler stem cells.

3. The preparation method according to claim 1, characterized in that: The culturing step includes inoculating the antler stem cells into a culture bottle, adding the cell culture medium and culturing for 2 to 5 days, and collecting the culture supernatant.

4. The preparation method according to claim 3, characterized in that: The extraction comprises taking the culture supernatant and subjecting it to multiple centrifugations to sequentially remove dead cells, cell debris, and free organelles, and then subjecting it to ultra-high-speed centrifugation to obtain the antler stem cell exosomes.

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