CN117844747A - Conditional medium for promoting secretion of mesenchymal stem cell exosome and application thereof - Google Patents
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Abstract
The invention discloses a conditional medium for promoting secretion of mesenchymal stem cell exosomes and application thereof, wherein the conditional medium comprises a DMEM/F12 medium, exosome-free serum, 5' -adenylate, silk cutting protein-1, annexin V, L-alpha-phosphatidylserine and N-acetylglucosamine. The conditioned medium can maintain the culture of the mesenchymal stem cells for 15-20 days and continuously stimulate the secretion of exosomes, and the secretion amount of the conditioned medium is 20-30 times that of the conditioned medium obtained by a conventional collection method. Purifying the exosome by combining a hollow fiber membrane column and an anion exchange chromatographic column, and the obtained exosome has the characteristics of high purity, uniform particle size and the like. The method has simple operation and low cost, is suitable for large-scale preparation of exosomes, and is beneficial to clinical application.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a conditional medium for promoting secretion of mesenchymal stem cell exosomes and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are taken as main members of stem cell families, have biological characteristics of multidirectional differentiation, immunoregulation, anti-fibrosis, anti-inflammatory, anti-apoptosis, pro-angiogenesis and the like, play an important role in tissue repair, cancer treatment and immunoregulation, and are widely used for research and treatment of various diseases of human beings. The function of MSCs is mainly achieved by the Paracrine (Paracrine) pathway, one of the key functional elements in Paracrine action being the exosomes (exosomes). Exosomes are Extracellular Vesicles (EVs) that are released outside the cell after fusion of the cell nucleus endosomes (also called multivesicular bodies) with the cell membrane. The exosomes are generally 30-150 nm in diameter and 1.10-1.18 g/mL in density. The cell membrane and cytoplasm components of the source cells contain various proteins, mRNA, miRNA, lipid and other components, and have important roles in maintaining the intracellular environment and mediating the intercellular communication.
The exosomes derived from MSCs not only have functions of MSCs, such as tissue injury repair and immune regulation, but also have a plurality of unique advantages and advantages, such as no immunogenicity, no risk of tumor formation, high stability, capability of penetrating the blood brain barrier, simple transportation and preservation, high administration safety, no fear of blocking micro blood vessels, lower manufacturing cost than stem cell preparations, and good clinical application prospect. Several preclinical studies currently demonstrate the potential of mesenchymal stem cell exosomes in the field of disease treatment, including immune diseases, neurological diseases, cardiovascular diseases, tumors, and the like.
At present, the restriction factor for preventing the clinical application of mesenchymal stem cell exosomes is low yield and cannot meet the clinical application. There are a number of techniques by which secretion of exosomes can be stimulated, for example: increasing the secretion of exosomes based on physical stimulation techniques, such as CN202011162504.5, rapidly increases the production of adipose mesenchymal stem cell exosomes by electromagnetic radiation or/and treatment with laser radiation, but these techniques typically require expensive and cumbersome equipment and are difficult to scale up; in addition, methods for increasing the secretion of exosomes based on biotechnology, such as CN202180092835.2, provide high concentration exosomes with enhanced anti-inflammatory function by pre-treating stem cells with Lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) while in culture to induce a mild inflammatory response, activating immune response of stem cells and then using stem cells induced with anti-inflammatory response. The biotechnology method is convenient for process amplification and is more beneficial to industrial production and clinical application.
Disclosure of Invention
The invention aims to solve the technical problem of improving a conditional medium for promoting secretion of mesenchymal stem cell exosomes and application thereof, and is used for improving the yield of the mesenchymal stem cell exosomes.
In order to solve the technical problems, the invention adopts the following technical scheme: a conditional medium for promoting secretion of mesenchymal stem cell exosome comprises DMEM/F12 medium, exosome-free serum, 5 '-adenylate, filaggrin-1, annexin V, L-alpha-phosphatidylserine and N-acetylglucosamine, wherein each 1L of DMEM/F12 medium contains 10-30 mL of exosome-free serum, 5-15 mg of 5' -adenylate, 0.5-2 mg of filaggrin-1, 5-20 mg of annexin V, 1-5 mg of L-alpha-phosphatidylserine and 100-500 mg of N-acetylglucosamine.
The enrichment, separation and extraction method of the mesenchymal stem cell exosomes comprises the following steps:
(1) Inoculating mesenchymal stem cells into a culture bottle or a cell factory containing the conditional medium according to the inoculation density of 1X 105-2X 105/cm < 2 >, culturing in a cell incubator with 5% CO2 and 37 ℃ and saturated humidity of 95%, wherein the volume of the conditional medium is 0.1-0.6 mL/cm 2 ；
(2) After culturing for 15-20 days, collecting culture supernatant, and filtering by a 0.22 mu m filter membrane to remove aggregated protein particles and cell fragments, thereby obtaining mesenchymal stem cell exosome crude liquid;
(3) Concentrating the exosome crude liquid by using a 300-500 kD hollow fiber membrane column, and then performing 5-10 times of multiple ratio washing filtration by using DPBS;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, filtering and sterilizing by a membrane of 0.22 mu m to obtain mesenchymal stem cell exosome solution, and storing the solution in a refrigerator at the temperature of-80 ℃ for later use.
The mesenchymal stem cells are umbilical cord, placenta, fat, bone marrow and gingiva-derived mesenchymal stem cells, and the species source is human, canine, feline, equine, rat or mouse.
The beneficial effects of the invention are as follows: the conditioned medium of the invention can continuously maintain the activity of the cells after the cells are completely fused, and continuously stimulate the secretion of exosomes. The maintenance culture of the mesenchymal stem cells can reach 15-20 days, the stem cells are inhibited from proliferation in contact during the period, the cell activity rate is maintained to be above 80%, few cells fall off, and the capability of secreting exosomes is continuously activated. The stem cell exosome is purified by continuous secretion and enrichment, the secretion amount of the stem cell exosome is 20-30 times of that of a conventional collection method, and the exosome is purified by a method of combining a hollow fiber membrane column and an anion exchange chromatographic column. The method has simple operation and low cost, is suitable for large-scale preparation of exosomes, and is beneficial to clinical application.
Drawings
FIG. 1 shows the morphology of exosomes obtained by the method of the invention under electron microscopy.
FIG. 2 is a graph showing the particle size distribution of exosomes obtained by the method of the present invention.
FIG. 3 is a graph showing the expression of the exosome-specific marker CD63 obtained by the method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.
The conditioned medium for promoting the secretion of mesenchymal stem cell exosomes comprises a DMEM/F12 medium, exosome-free serum, 5 '-adenylate, filaggrin-1, annexin V, L-alpha-phosphatidylserine and N-acetylglucosamine, wherein each 1L of DMEM/F12 medium contains 10-30 mL of exosome-free serum, 5-15 mg of 5' -adenylate, 0.5-2 mg of filaggrin-1, 5-20 mg of annexin V, 1-5 mg of L-alpha-phosphatidylserine and 100-500 mg of N-acetylglucosamine.
The enrichment, separation and extraction method of the mesenchymal stem cell exosomes comprises the following steps:
(1) Inoculating mesenchymal stem cells into a culture bottle or a cell factory containing the conditional medium according to the inoculation density of 1X 105-2X 105/cm < 2 >, culturing in a cell incubator with 5% CO2 and 37 ℃ and saturated humidity of 95%, wherein the volume of the conditional medium is 0.1-0.6 mL/cm 2 ；
(2) After culturing for 15-20 days, collecting culture supernatant, and filtering by a 0.22 mu m filter membrane to remove aggregated protein particles and cell fragments, thereby obtaining mesenchymal stem cell exosome crude liquid;
(3) Concentrating the exosome crude liquid by using a 300-500 kD hollow fiber membrane column, and then performing 5-10 times of multiple ratio washing filtration by using DPBS;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, filtering and sterilizing by a membrane of 0.22 mu m to obtain mesenchymal stem cell exosome solution, and storing the solution in a refrigerator at the temperature of-80 ℃ for later use.
The mesenchymal stem cells are umbilical cord, placenta, fat, bone marrow and gingiva-derived mesenchymal stem cells, and the species source is human, canine, feline, equine, rat or mouse.
The present invention will be further described with reference to the accompanying drawings and examples, but the present invention is not limited to the following examples.
The material sources are as follows: the mesenchymal stem cells are derived from umbilical cord, placenta, fat, bone marrow and gingiva, and the species are derived from human, canine, feline, equine, rat and mouse. The following examples are given by way of example of canine placenta-derived mesenchymal stem cells.
DMEM/F12 medium is available from Sesameimer Feier technologies.
Exosome-free serum was purchased from sameidie science and technology.
5' -adenylate was purchased from sigma aldrich.
Silk fibroin-1 was purchased from sigma aldrich.
Annexin V was purchased from sigma aldrich.
L-alpha-phosphatidylserine is available from Sigma Aldrich.
N-acetylglucosamine was purchased from Sigma Aldrich.
0.22 μm filter membranes were purchased from Sidoris corporation.
Hollow fiber columns were purchased from Sidoris corporation.
Anion exchange chromatography columns were purchased from Sidoris corporation.
Example 1
Preparation of conditioned Medium: adding 30mL of exosome-free serum, 5mg of 5' -adenylate, 0.5mg of filaggrin-1, 5mg of annexin V, 1mg of L-alpha-phosphatidylserine and 100mg of N-acetylglucosamine into 1L of DMEM/F12 medium, and blowing and uniformly mixing;
example 2
Preparation of conditioned Medium: 1L of DMEM/F12 medium is added with 10mL of serum without exosomes, 15mg of 5' -adenylate, 2mg of silk cutting protein-1, 20mg of annexin V, 5mg of L-alpha-phosphatidylserine and 500mg of N-acetylglucosamine, and the mixture is blown and mixed uniformly.
Example 3
Preparation of conditioned Medium: 1L of DMEM/F12 medium is added with 20mL of serum without exosomes, 10mg of 5' -adenylate, 1mg of silk cutting protein-1, 10mg of annexin V, 2mg of L-alpha-phosphatidylserine and 200mg of N-acetylglucosamine, and the mixture is blown and evenly mixed.
Example 4
The enrichment, separation and extraction method of the mesenchymal stem cell exosomes comprises the following steps:
(1) Mesenchymal stem cells were prepared according to 2X 10 5 /cm 2 Is inoculated into a culture flask containing the conditioned medium obtained in example 1, the conditioned medium volume being 0.1mL/cm 2 Placed in 5% CO 2 Culturing in a cell incubator at 37 ℃ with a saturation humidity of 95%;
(2) After 20 days of culture, 500mL of culture supernatant is collected, and aggregated protein particles and cell fragments are removed by filtration through a 0.22 mu m filter membrane, so that coarse mesenchymal stem cell exosome liquid is obtained;
(3) Concentrating the filtrate to 100mL by using a 500 kD hollow fiber membrane column, and then performing 5-fold-ratio washing filtration by using DPBS to obtain a 10mL washed-filtered sample;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, filtering by a 0.22 mu m membrane to obtain 15mL of sterile mesenchymal stem cell exosomes, and storing in a refrigerator at-80 ℃ for later use.
Example 5
The enrichment, separation and extraction method of the mesenchymal stem cell exosomes comprises the following steps:
(1) Mesenchymal stem cells were prepared according to 1X 10 5 /cm 2 Is inoculated into a culture flask containing the conditioned medium obtained in example 3, the conditioned medium volume being 0.3mL/cm 2 Placed in 5% CO 2 Culturing in a cell incubator at 37 ℃ with a saturation humidity of 95%;
(2) After 20 days of culture, 1000mL of culture supernatant is collected, and aggregated protein particles and cell fragments are removed by filtration through a 0.22 mu m filter membrane, so that a crude mesenchymal stem cell exosome solution is obtained;
(3) Concentrating the filtrate to 100mL by using a 300 kD hollow fiber membrane column, and then performing 10-time multiple ratio washing filtration by using DPBS to obtain a 15mL washed-filtered sample;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, filtering by a 0.22 mu m membrane to obtain 25mL of sterile mesenchymal stem cell exosome, and storing in a refrigerator at-80 ℃ for later use.
Example 6
The enrichment, separation and extraction method of the mesenchymal stem cell exosomes comprises the following steps:
(1) Mesenchymal stem cells were prepared according to 1X 10 5 /cm 2 Is inoculated in a cell factory containing the conditioned medium obtained in example 2, the conditioned medium volume being 0.6mL/cm 2 Placed in 5% CO 2 Culturing in a cell incubator at 37 ℃ with a saturation humidity of 95%;
(2) After culturing for 20 days, collecting 5L of culture supernatant, and filtering by a 0.22 mu m filter membrane to remove aggregated protein particles and cell fragments, thereby obtaining mesenchymal stem cell exosome crude liquid;
(3) Concentrating the filtrate to 200mL by using a 500 kD hollow fiber membrane column, then performing 6 times of multiple ratio washing filtration by using DPBS, and concentrating the sample volume to 40mL after washing filtration;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, and filtering and sterilizing the sample by a 0.22 mu m membrane to obtain 60mL of sterile mesenchymal stem cell exosome solution;
(5) The exosomes are observed by electron microscopy to show typical tea tray-shaped structure, as shown in figure 1, the particle size distribution of mesenchymal stem cell exosomes is detected by using nanosight_ns300 (Malvern) as shown in figure 2, the average particle size is 104.8nm plus or minus 0.7nm, and the exosomes concentration is 4.8x10 13 Particles/mL, exosome purity 8.63X10 8 particles/mug. Experimental results show that the mesenchymal stem cell exosomes prepared in large scale have moderate particle size, high particle size uniformity and high concentration content.
(6) Western blot is used for detecting the expression condition of the mesenchymal stem cell exosome specific protein CD 63. The experimental results show that mesenchymal stem cell exosomes specifically express exosome marker protein CD63 (see fig. 3).
Example 7
Comparison with the prior art
(1) Mesenchymal stem cells were prepared according to 2X 10 5 /cm 2 Is inoculated into a culture flask containing the conditioned medium obtained in example 3, the conditioned medium volume being 0.4mL/cm 2 Placed in 5% CO 2 Culturing in a cell incubator at 37 ℃ with a saturation humidity of 95%;
(2) After culturing for 20 days, collecting culture supernatant, and filtering by a 0.22 mu m filter membrane to remove aggregated protein particles and cell fragments, thereby obtaining 100mL (group (1)) of mesenchymal stem cell exosome crude liquid;
(3) Mesenchymal stem cells were cultured using conventional medium at an seeding density of 2×10 5 /cm 2 The conventional culture medium is a DMEM/F12 culture medium containing 10% of exosome-free serum, cells start to fall off continuously after the culture is completely fused, 80% of cells fall off after the complete fusion is carried out for 3 days, then culture supernatant is collected, and aggregated protein particles and cell fragments are removed by filtration through a 0.22 mu m filter membrane to obtain 100mL (group (2)) of mesenchymal stem cell exosome crude liquid;
(4) Culturing mesenchymal stem cells according to the technology disclosed in chinese patent CN201910381006.0 and obtaining 100mL of mesenchymal stem cell exosome crude liquid (group (3));
(5) Concentrating the three groups of filtrate with equal volume through a 500 kD hollow fiber membrane column, performing 6 times of multiple ratio washing filtration by using DPBS, purifying by using an anion exchange chromatographic column, and performing membrane filtration sterilization by using a 0.22 mu m membrane to obtain three groups of mesenchymal stem cell exosome solutions with the volume of 5mL respectively;
(6) The particle size distribution and concentration of exosomes obtained by different methods were determined using nanosight_ns300. The specific data are shown in Table 1, and the results show that the particle size distribution obtained by the three methods is not greatly different, wherein the exosome concentration of the group (1) is the highest. The highest concentration of exosomes obtained by the method disclosed in the present invention was demonstrated.
TABLE 1 particle size and concentration of exosomes obtained by different methods
In view of the foregoing, the present invention is not limited to the above-described embodiments, and other embodiments can be easily proposed by those skilled in the art within the scope of the technical teaching of the present invention, but such embodiments are included in the scope of the present invention.
Claims (3)
1. A conditional medium for promoting secretion of mesenchymal stem cell exosomes is characterized by comprising a DMEM/F12 medium, exosome-free serum, 5 '-adenylate, filaggrin-1, annexin V, L-alpha-phosphatidylserine and N-acetylglucosamine, wherein each 1L of DMEM/F12 medium contains 10-30 mL of exosome-free serum, 5-15 mg of 5' -adenylate, 0.5-2 mg of filaggrin-1, 5-20 mg of annexin V, 1-5 mg of L-alpha-phosphatidylserine and 100-500 mg of N-acetylglucosamine.
2. The enrichment, separation and extraction method of the mesenchymal stem cell exosomes is characterized by comprising the following steps:
(1) Mesenchymal stem cells were prepared according to 1X 10 5 ～2×10 5 /cm 2 Is inoculated in a flask or cell factory containing the conditioned medium of claim 1, placed in 5% CO 2 Culturing in incubator at 37 deg.c and saturation humidity of 95% under the condition of medium volume of 0.1-0.6 mL/cm 2 ；
(2) After culturing for 15-20 days, collecting culture supernatant, and filtering by a 0.22 mu m filter membrane to remove aggregated protein particles and cell fragments, thereby obtaining mesenchymal stem cell exosome crude liquid;
(3) Concentrating the exosome crude liquid by using a 300-500 kD hollow fiber membrane column, and then performing 5-10 times of multiple ratio washing filtration by using DPBS;
(4) Purifying the washed and filtered sample by an anion exchange chromatographic column, filtering and sterilizing by a membrane of 0.22 mu m to obtain mesenchymal stem cell exosome solution, and storing the solution in a refrigerator at the temperature of-80 ℃ for later use.
3. The method for enriching and isolating mesenchymal stem cell exosomes according to claim 2, wherein the mesenchymal stem cells are umbilical cord, placenta, fat, bone marrow, gingival derived mesenchymal stem cells, and the species source is human, canine, feline, equine, rat or mouse.
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