CN113699103B - Preparation process and application of induced enhancement of exosomes of mesenchymal stem cells of birds - Google Patents
Preparation process and application of induced enhancement of exosomes of mesenchymal stem cells of birds Download PDFInfo
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Abstract
The invention discloses a preparation process for inducing and amplifying exosomes of mesenchymal stem cells of birds, which comprises the following steps: performing subculture on poultry mesenchymal stem cells after primary separation culture, taking the poultry mesenchymal stem cells, culturing the poultry mesenchymal stem cells by using a cell growth solution, discarding the culture solution, washing the cells for three times by using a phosphate buffer solution, then adding curcumin for stimulation and continuously culturing for 72 hours by using a serum-free culture solution, collecting the serum-free culture solution, and continuously performing the treatment on the cells which are not collected until a sufficient amount of supernatant is collected; centrifuging, extracting and concentrating the collected serum-free culture solution stimulated by curcumin, and sterilizing the obtained concentrated exosome filtrate. According to the invention, through improving a process formula, curcumin is utilized to stimulate and induce the poultry mesenchymal stem cells, so that exosomes which are amplified by the curcumin are generated, and the effect of the exosomes is better than that of exosomes which are not amplified by the curcumin.
Description
Technical Field
The invention relates to a preparation method of stem cell exosomes, in particular to an induced and amplified preparation process and application of an avian mesenchymal stem cell exosomes, and belongs to the technical field of stem cell biological medicines.
Background
The source of the poultry mesenchymal stem cells is wide, the materials are easy to obtain, the cell separation efficiency is high, the cell strain state is stable, and the problems of medical ethics are avoided, so that the poultry mesenchymal stem cells are paid attention to gradually. Exosomes are nanoscale vesicles that release outside the cell upon environmental stimulation or cell activation. The vesicle structures are classified into 3 classes according to their diameter sizes: exosomes (40-150 nm in diameter), microbubbles (100-1000 nm in diameter) and apoptotic bodies (1-4 μm in diameter). The exosome is formed by aggregating proteins, DNA fragments, miRNA and other substances in cytoplasm into multi-vesicle endosomes through endocytic pathway, and then releasing the substances out of the cells through fusion and cleavage of the multi-vesicle and plasma membrane. The internal structure of exosomes is mainly dependent on the cells from which they originate, but most exosomes are rich in integrins, heat shock proteins, the four transmembrane protein superfamily (CD 9, CD63, CD81 and CD 82), the cellular endogenous proteins alix and Rab protein family, which can serve as reliable markers for identifying exosomes.
Exosomes have the function of helping to remove old and useless cells, and are replaced with new cells, and are considered as important substances effective against aging and repair of skin. In addition to the development of cosmetic applications, exosomes have also been found to have an improved effect on allergic rhinitis caused by air pollution or seasonal changes. If the exosome is made into inhalation spray, the exosome can pass through nasal mucosa and relieve inflammatory reaction at the place by inhaling through nasal cavity and oral cavity, so that symptoms such as nasal obstruction, running nose and asthma are improved.
At present, although research has been conducted on the use of specific induction conditions to induce stem cells to secrete supernatant in vitro for medical treatment, cosmetics and the like, the use of specific cytokines to induce stem cells to secrete certain specific factors to achieve therapeutic effects has led to the fact that the cytokines themselves are expensive, which makes the use cost of stem cells abnormally high.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a preparation process and application of induced and amplified exosomes of mesenchymal stem cells of birds, which are stimulated by curcumin to induce the mesenchymal stem cells of birds, so that the exosomes amplified by the curcumin are generated, and the effect of the exosomes is more excellent than that of exosomes which are not amplified by the curcumin.
In order to solve the technical problems, the invention provides a preparation process for inducing and amplifying exosomes of mesenchymal stem cells of birds, which comprises the following steps:
s1: performing primary isolated culture on poultry mesenchymal stem cells, and then performing subculture, and taking P3-P30 generation poultry mesenchymal stem cells subjected to subculture;
s2: culturing with cell growth liquid until 75-85% of cells are fused, discarding the culture liquid, washing the cells with phosphate buffer solution for three times, adding curcumin, culturing with serum-free culture liquid for 72 hours, and collecting serum-free culture liquid;
s3: the cells which are not collected in the step S2 are subjected to curcumin stimulation treatment in the step until serum-free culture solution is collected;
s4: collecting serum-free culture solution stimulated by curcumin in the steps S2 and S3, centrifuging, extracting and concentrating exosome filtrate, and finally preparing the needed curcumin-induced enhanced bird interstitial stem cell exosome after the obtained exosome concentrated filtrate is subjected to sterilization treatment;
s5: adding excipient and water into the exosome of the mesenchymal stem cells of the curcumin induced and amplified poultry, mixing, filtering, sterilizing, and freeze-drying to obtain the stem cell extract frozen crystal powder.
Although the curcumin-induced and amplified bird mesenchymal stem cell exosome has various biological activities, the application field is wide, but the biological activities are limited by various external conditions, and the preservation is inconvenient, so the invention also provides the curcumin-induced and amplified bird mesenchymal stem cell exosome frozen crystal powder, which is prepared by the curcumin-induced and amplified bird mesenchymal stem cell exosome prepared by a frozen crystal powder preparation method commonly used in the field; the frozen crystal powder is sterile powder prepared by freezing liquid medicine into solid state under aseptic environment, vacuumizing and sublimating and drying water, and the preparation method of the frozen crystal powder is a mature technology in the field.
According to a further defined technical scheme of the invention, the induction and amplification preparation process of the exosomes of the mesenchymal stem cells of the poultry is characterized in that the cell growth liquid is a DMEM culture liquid containing 5% fetal bovine serum, and the culture liquid also contains antibacterial antibiotics; the antibacterial antibiotics comprise penicillin and streptomycin, wherein the final concentration of the penicillin in the cell growth liquid is 200U/ml, the final concentration of the streptomycin in the cell growth liquid is 250U/ml, the DMEM culture solution containing 5% fetal bovine serum is obtained by adding the fetal bovine serum with the final concentration of 5% into a commercial basic DMEM culture solution, and the cell growth liquid is obtained by adding the penicillin with the final concentration of 200U/ml and the streptomycin with the final concentration of 250U/ml.
In the preparation process for inducing and amplifying the exosomes of the mesenchymal stem cells of the poultry, the phosphate buffer solution is a PBS solution with the pH value of 7.0-7.2; the excipient adopts any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced amplified bird mesenchymal stem cell exosome.
The above-mentioned preparation process of inducing and amplifying the exosome of the mesenchymal stem cells of birds, the water in said step S5 adopts the ultrapure water, the ultrapure water added is 100ml to the final volume; the freeze-drying treatment is to freeze-dry for 48 hours at the freeze-drying temperature of-35 to-20 ℃ and the vacuum degree of 50-200 Pa.
Further, in the aforementioned preparation process for induction and redundancy of the exosomes of the mesenchymal stem cells of the poultry, the centrifugation operation in the step S4 is a centrifugation process of 1,000xg for 10 minutes and 10,000xg for 10 minutes, collecting the supernatant after the completion, passing the collected supernatant through a hollow fiber membrane to perform tangential flow filtration, collecting the exosome solution concentrated by filtration, removing the exosome solution through an exosome extraction system, extracting the exosome solution with high purity, and the sterilization treatment is performed by filtration sterilization by using a sterile 0.22 μm filter membrane.
An application of exosomes of mesenchymal stem cells of poultry, which uses frozen crystal powder of stem cell extract to prepare wound healing medicine or cell repair medicine; preparing whitening products or fine chemical products for resisting skin wrinkle.
The beneficial effects of the invention are as follows: according to the invention, through improving a process formula, curcumin is utilized to stimulate and induce the poultry mesenchymal stem cells, so that exosomes which are amplified by the curcumin are generated, and the effect of the exosomes is more excellent than that of exosomes which are not amplified by the curcumin; meanwhile, exosomes secreted by the isolated cells are collected and extracted by a special technology, and are called curcumin-induced enhanced poultry mesenchymal stem cell exosomes; the curcumin induces the exosome of the stem cells of the bird to be freeze-dried to produce frozen crystal powder, thereby ensuring the biological activity and efficacy of the frozen crystal powder. The verification process of the invention mainly discusses the efficacy of curcumin to induce and strengthen the exosomes of the mesenchymal stem cells of the poultry on skin repair.
Drawings
FIG. 1 is a transmission electron microscope image of the invention before and after lyophilization.
FIG. 2 shows the particle size analysis and particle count of the present invention.
FIG. 3 is a purity test of the frozen powder of the present invention.
FIG. 4 shows the protein concentration contained in the frozen crystal powder of the present invention.
FIG. 5 shows the concentration of exosome RNA contained in the frozen powder of the present invention.
FIG. 6 shows the cell proliferation effect of the frozen crystal powder of the invention on human skin fibroblasts.
FIG. 7 shows the effect of the frozen powder of the invention on cell proliferation of human hair follicle cells.
FIG. 8 shows the wound healing effect of the frozen crystal powder of the invention on human dermal fibroblasts.
Description of the embodiments
The preparation process for inducing and amplifying the exosomes of the mesenchymal stem cells of the poultry provided by the embodiment comprises the following steps:
s1: performing primary isolated culture on poultry mesenchymal stem cells, and then performing subculture, and taking P3-P30 generation poultry mesenchymal stem cells subjected to subculture;
s2: culturing with cell growth liquid until 75-85% of cells are fused, discarding the culture liquid, washing the cells with phosphate buffer solution for three times, adding curcumin, culturing with serum-free culture liquid for 72 hours, and collecting serum-free culture liquid;
s3: the cells which are not collected in the step S2 are subjected to curcumin stimulation treatment in the step until serum-free culture solution is collected;
s4: collecting serum-free culture solution stimulated by curcumin in the steps S2 and S3, centrifuging, extracting and concentrating exosome filtrate, and finally preparing the needed curcumin-induced enhanced bird interstitial stem cell exosome after the obtained exosome concentrated filtrate is subjected to sterilization treatment; the centrifugation operation is to adopt a centrifugation process of 1,000Xg for 10 minutes and 10,000Xg for 10 minutes, collect supernatant after the end, flow the collected supernatant through a hollow fiber membrane for tangential flow filtration, collect, filter and concentrate exosome solution, then remove the exosome solution through an exosome extraction system, extract exosome solution with high purity, and carry out filtration sterilization by adopting a sterile 0.22 mu m filter membrane;
s5: adding excipient and ultrapure water into the exosome of the mesenchymal stem cells of the curcumin-induced amplified bird, mixing, filtering for sterilization, and then carrying out freeze-drying treatment to obtain the frozen crystal powder of the stem cell extract; adding ultrapure water to a final volume of 100ml; the freeze-drying treatment means that the freeze-drying temperature is minus 35 to minus 20 ℃, the vacuum degree is 50 to 200Pa, and the freeze-drying is carried out for 48 hours.
The cell growth liquid in this example is DMEM culture liquid containing 5% fetal bovine serum, and the culture liquid further contains antibacterial antibiotics; the antibacterial antibiotics comprise penicillin and streptomycin, wherein the final concentration of the penicillin in the cell growth liquid is 200U/ml, the final concentration of the streptomycin in the cell growth liquid is 250U/ml, the DMEM culture liquid containing 5% fetal calf serum is obtained by adding the fetal calf serum with the final concentration of 5% into the commercial basic DMEM culture liquid, and the penicillin with the final concentration of 200U/ml and the streptomycin with the final concentration of 250U/ml are added to obtain the cell growth liquid. The phosphate buffer solution is PBS solution with pH value of 7.0-7.2; the excipient adopts any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced amplified bird mesenchymal stem cell exosome.
In this example, a reagent instrument was used:
BCA protein assay kit: #23225; thermo Fisher Scientific, waltham, mass., USA;
RIPA buffer: 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 1% Na-deoxyplate, 0.1% SDS, 0.1 mM CaCl 2 , and 0.01 mM MgCl 2 ；
qEV RNA extraction kit: IZON Science LTD, christchurch, new Zealand;
protease inhibitor cocktail: thermo Fisher Scientific;
full wavelength microplate reader: molecular Devices San Jose, calif., USA;
hitachi HT7700 electron microscope: hitachi, tokyo, japan;
spectral luminance meter: nanoDrop ™ 2000c,Thermo Fisher Scientific;
silica gel insert: culture inserts, 2 well plates, ibiTreat; ibidi, martinsried, germany.
Curcumin-induced-enhanced bird mesenchymal stem cell exosome frozen crystal powder sample is prepared for transmission electron microscopy. Briefly, exosomes were immobilized on copper mesh, fixed with 1% glutaraldehyde in cold PBS for 5 min to stabilize the immune response, washed with sterile distilled water, aligned with uranium oxalate solution at pH7 for 5 min, and then embedded with methylcellulose on ice for 10 min. Excess cellulose was removed and the sample was dried for permanent storage. Exosome samples were imaged using a HitachiHT7700 electron microscope at 80kV voltage.
The pierceTMBCA protein assay kit was used for purity testing. A standard curve (range 0-2000. Mu.g/mL) was obtained from nine points of serial dilutions of Bovine Serum Albumin (BSA) and working reagents. All samples and standard points were repeated 3 times. Samples (10. Mu.l each) (100 mg curcumin induced enhanced frozen powder of the exosomes of the mesenchymal stem cells of the birds or of the control exosomes were dissolved in 500. Mu. lQH 2 O) was mixed with 200 μl of working reagent and incubated for 30 min at 65 ℃. After cooling to room temperature, each absorbance difference (minus the average absorbance of 562nm blank standard replicates) was measured by a full wavelength microplate reader and converted to μg/ml by a standard curve. If the protein concentration exceeds the upper limit of the standard curve by 2000. Mu.g/mL, the sample is diluted to an acceptable standardThe determination is made in the quasi-range and the final concentrate is calibrated taking into account the dilution factor.
Step of detecting the concentration of exosome protein curcumin induces the frozen crystal powder of the exosome of the enhanced bird interstitial stem cells 100mg and the frozen crystal powder of the control exosome 100mg to be resuspended in RIPA buffer solution, and protease inhibitor mixture is added. BCA protein assay kit was used for purity testing. Nine spots were serially diluted with Bovine Serum Albumin (BSA) and working reagent to give a standard curve (range 0-250. Mu.g/mL). All samples and standard points were repeated 3 times. Samples (10. Mu.l each) were mixed with 200. Mu.l of working reagent and incubated at 65℃for 30 minutes. After cooling to room temperature, each absorbance difference was measured by a full wavelength microplate reader, the average absorbance at 562nm of the blank standard repeat was subtracted, and the absorbance difference was converted to μg/ml by a standard curve.
Curcumin induced enhanced avian mesenchymal stem cell exosome frozen powder 100mg and control exosome frozen powder 100mg were resuspended in lysis buffer a of qEVRNA extraction kit. The detailed extraction method follows the user specification. The extracted exosome RNA concentrations of curcumin-induced-enhanced avian mesenchymal stem cell exosome frozen powder and control exosome were measured using a spectroluminometer.
To determine cell viability, human dermal fibroblasts and human hair follicle cells were cultured in their growth medium until they reached 80% dense. The cells were grown at 5X 10 3 A total volume of 100 μl of individual cells/wells was seeded on 96-well plates. After inoculation, the cells were incubated at 37℃and 5% CO 2 For 24 hours to allow cell attachment. Cells were then washed once with PBS and medium was replaced with medium containing 0.25% stripped FBS, starved for 24 hours. The birds were supplemented with different concentrations of curcumin-induced-amplified exosomatic frozen powder of mesenchymal stem cells and medium containing 0.25% exfoliated FBS and treated for 72 hours (human dermal fibroblasts) or 96 hours (human hair follicle cells). Curcumin induced-up poultry mesenchymal stem cell exosome frozen crystal powder culture medium mixture is updated every other day. After 72 hours or 96 hours of treatment, the medium containing 20% MTS solution was replaced with growth mediumAnd incubated for 2 hours. The absorbance at 490nm was measured for each well using a full wavelength microplate reader.
To examine the wound healing effect of curcumin-induced-enhanced bird mesenchymal stem cell exosome jelly powder for treating human dermal fibroblasts, wound healing experiments were performed using a silica gel insert, and cells were placed in 24-well plates. Specifically, cells were grown at 2.5X105 cells/cm 2 Is inoculated in two compartments of the silica gel insert. Cells were grown for 24 hours, then medium was changed to starvation medium (serum free) and cultured for an additional 24 hours. The culture insert was removed using sterile forceps, creating a 500 μm wide gap. Curcumin-induced-enhanced bird interstitial stem cell exosome frozen crystal powder and 0.25% FBS-stripped medium supplemented with different concentrations were added and incubated for 24 hours. In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.
Claims (7)
1. The preparation process of induced enhancement of the exosome of the mesenchymal stem cells of the poultry is characterized by comprising the following steps:
s1: performing primary isolated culture on poultry mesenchymal stem cells, and then performing subculture, and taking P3-P30 generation poultry mesenchymal stem cells subjected to subculture;
s2: culturing with cell growth liquid until 75-85% of cells are fused, discarding the culture liquid, washing the cells with phosphate buffer solution for three times, adding curcumin, culturing with serum-free culture liquid for 72 hours, and collecting serum-free culture liquid;
s3: the cells which are not collected in the step S2 are subjected to curcumin stimulation treatment in the step until serum-free culture solution is collected;
s4: collecting serum-free culture solution stimulated by curcumin in the steps S2 and S3, centrifuging, extracting and concentrating exosome filtrate, and finally preparing the needed curcumin-induced enhanced bird interstitial stem cell exosome after the obtained exosome concentrated filtrate is subjected to sterilization treatment;
s5: adding excipient and water into the exosome of the mesenchymal stem cells of the curcumin induced and amplified poultry, mixing, filtering, sterilizing, and freeze-drying to obtain the stem cell extract frozen crystal powder.
2. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps: the cell growth liquid is DMEM culture liquid containing 5% of fetal calf serum, and the culture liquid also contains antibacterial antibiotics; the antibacterial antibiotics comprise penicillin and streptomycin, wherein the final concentration of the penicillin in the cell growth liquid is 200U/ml, the final concentration of the streptomycin in the cell growth liquid is 250U/ml, the DMEM culture solution containing 5% fetal bovine serum is obtained by adding the fetal bovine serum with the final concentration of 5% into a commercial basic DMEM culture solution, and the cell growth liquid is obtained by adding the penicillin with the final concentration of 200U/ml and the streptomycin with the final concentration of 250U/ml.
3. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps: the phosphate buffer solution is PBS solution with pH value of 7.0-7.2.
4. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps: the excipient adopts any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced amplified bird mesenchymal stem cell exosome.
5. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps: the water in the step S5 is ultrapure water, and the ultrapure water is added to a final volume of 100ml.
6. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps: the freeze-drying treatment is to freeze-dry for 48 hours at the freeze-drying temperature of-35 to-20 ℃ and the vacuum degree of 50-200 Pa.
7. The process for preparing induced rescue of the exosomes of the mesenchymal stem cells of the poultry according to claim 1, wherein the process comprises the following steps:
the centrifugation operation in the step S4 is to adopt a centrifugation process of 1,000Xg for 10 minutes and 10,000Xg for 10 minutes, collect supernatant after the completion, flow the collected supernatant through a hollow fiber membrane for tangential flow filtration, collect, filter and concentrate exosome solution, then remove the exosome solution through an exosome extraction system, extract high-purity exosome solution, and the sterilization treatment adopts a sterile 0.22 mu m filter membrane for filtration sterilization.
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Effective date of registration: 20231222 Address after: Taipei City, Taiwan, China Patentee after: Shengyi Biotechnology Co.,Ltd. Address before: Unit G1-1101-023, Artificial Intelligence Industrial Park, No. 88 Jinjihu Avenue, Suzhou Industrial Park, Suzhou City, Jiangsu Province, 215000 Patentee before: Xie Rusheng |