Preparation method and application of iPS (induced pluripotent stem) cell exosome

Abstract

The invention discloses a preparation method and application of an iPS (induced pluripotent stem) exosome, and relates to the technical field of biologication.The preparation method comprises the steps of overexpression vector construction, nanoparticle preparation and transfection, iPS cell optimized culture, exosome separation, exosome purification, BMSC (bone mesenchymal stem cells) and exosome co-culture and bone defect repair experiment. The method has the advantages that the overexpression vector containing genes related to osteocyte differentiation, such as RUNX2 and ABC1, is constructed through gene editing, the vector is accurately introduced into the iPS cells by virtue of unique nano particle mediated transfection and magnetic field guidance, the transfection efficiency is greatly improved, the oxygen concentration is dynamically adjusted according to time in a culture link, and the osteocyte differentiation effect is improved. Meanwhile, substances such as a serum substitute and the like are added into the culture medium, a suitable growth environment is created for the iPS cells, the iPS cells are promoted to secrete high-quality exosomes, and the iPS cells are efficiently separated and purified by applying an advanced microfluidic technology and an affinity chromatography method, so that the yield and the quality of the exosomes are greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing iPS cell exosomes and an application thereof. Background Art

[0002] Chronic damage of locomotion system is a group of common clinical lesions. The tissues and structures involved in movement, whether bones, joints, muscles, tendons, ligaments, fascia, bursae and their adjacent blood vessels and nerves, can be damaged by repeated mechanical movements, etc., showing corresponding clinical symptoms and signs. At present, the treatment of sports injury repair mainly adopts non-steroidal anti-inflammatory drugs, physical therapy such as physical therapy and massage, and surgical treatment. Diseases of this system are prone to recurrence and are difficult to cure clinically. At present, new treatment options suitable for sports injury repair include: stem cell therapy, biomaterial therapy, gene therapy, new physical therapy technology therapy, nutritional support therapy, etc. Among them, stem cell therapy has made some important breakthroughs in clinical practice, bringing new hope for sports injury repair.

[0003] iPS cells, the full name of which is induced pluripotent stem cells (iPS cells), are a type of cell discovered by Japanese scientist Shinya Yamanaka in 2006, who won the Nobel Prize in Physiology or Medicine in 2012. This type of cell is similar to embryonic stem cells obtained by implanting specific transcription factors (Oct4, Sox2, Klf4 and c-Myc) into somatic cells to reprogram them. iPS cells have developmental pluripotency similar to embryonic stem cells and can differentiate into various cell types of the human body, such as blood cells, bone cells, nerve cells, etc., and can then culture various tissues and organs of the human body, which makes iPS cells PS cells have great potential in the medical field. They can be used for alternative treatments, research on pathogenesis, screening of new drugs, and treatment of clinical diseases such as neurological diseases and cardiovascular diseases. The morphological characteristics of IPS cells include: morphology similar to embryonic stem cells, round or oval cells, self-attaching and growing in culture dishes in the form of beads, containing a large amount of alkaline phosphatase in the cells, whose activity is significantly higher than that of adult cells, but lower than that of normal embryonic stem cells, and a relatively short survival time. The quality and differentiation ability of iPS cells are limited, and the cells contain chromosomal aberrations, because the generation of iPS cells is carried out by nuclear reprogramming, and the chromosomes The quality may change during the process. In the relevant technical fields of iPS cell exosome preparation and application in muscular system injury repair, the existing preparation methods and application strategies have defects such as low efficiency and insufficient precision. In the preparation process, the transfection technology is not effective, the optimization of culture conditions is not fine enough, and the separation and purification technology of exosomes is not advanced enough, resulting in the production and quality of exosomes difficult to meet clinical needs. When applied to muscular system injury repair, how to more effectively promote BMSC differentiation and improve the effect of bone defect repair still requires more innovative and efficient methods. To this end, we propose a method for preparing iPS cell exosomes and its application. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing iPS cell exosomes and applications thereof.

[0005] In order to solve the problems raised in the above background technology, the present invention provides the following technical solutions: a method for preparing exosomes from iPS cells, including constructing an overexpression vector, preparing and transfecting nanoparticles, optimizing iPS cell culture, isolating exosomes, purifying exosomes, co-culturing BMSCs and exosomes, and performing bone defect repair experiments. The specific steps of the method for preparing exosomes from iPS cells are as follows:

[0006] Step 1: Use gene editing technology to construct a specific overexpression vector, select genes related to osteoblast differentiation, use genomic DNA or cDNA containing the target gene as a template, use PCR technology to amplify RUNX2 gene and ABC1 gene fragments, and combine them with related genes that can enhance the differentiation-promoting ability of exosomes;

[0007] Step 2: Use transfection technology to introduce the overexpression vector into iPS cells. A nanoparticle-mediated transfection method is used to solidify the nanoparticles in the nanoemulsion by volatile organic solvents. The overexpression vector is combined with biocompatible nanoparticles, and the nanoparticles combined with the overexpression vector are added to the cultured iPS cells. At the same time, a magnetic field device is used to generate a magnetic field to guide the nanoparticles to gather around the cells and enter the cells.

[0008] Step 3: Optimize the culture of the transfected iPS cells, add appropriate amounts of serum replacement, small molecule compounds and growth factors to the basal culture medium to prepare an optimized culture medium, inoculate the transfected iPS cells into the optimized culture medium, and culture them in a culture container. Maintain the oxygen concentration at 21% within 24 hours of culture, and gradually reduce the oxygen concentration as the culture time goes by. Adjust the oxygen concentration to 10% within 2-4 days of culture, and reduce the oxygen concentration to 5% after 4 days of culture.

[0009] Step 4: Collect exosomes secreted by iPS cells, separate exosomes from cell culture supernatant using microfluidics technology, detect the particle size distribution and concentration of purified exosomes using nanoparticle tracking analyzer, detect the expression of exosome surface markers using Western blot to evaluate the purity and quality of exosomes, and collect and preserve the final exosomes.

[0010] As a further scheme of the present invention: In the nanoparticle-mediated transfection method, the preparation material of the nanoparticles includes a biodegradable high molecular polymer and magnetic nanoparticles, the surface of the nanoparticles is modified with a ligand that has specific binding ability to both the overexpression vector and the iPS cells, the overexpression vector and the nanoparticles are added to a buffer solution and mixed, the vector is bound to the nanoparticles by electrostatic adsorption, and the nanoparticles are guided to gather around the cells and enter the cells by an external magnetic field, the magnetic field strength is set to 0.01T-0.02T, and the action time is 15min-30min.

[0011] As a further solution of the present invention: in the optimized culture process, the culture system is 37°C and 5% CO2 concentration, the gene overexpression vector selects the DNA sequence of RUNX2 gene and other synergistic genes, and the synthesized DNA sequence is cloned into a suitable vector backbone to construct an overexpression vector.

[0012] As a further solution of the present invention: the microfluidic chip includes three groups of areas: a cell culture area, a secretion separation area, and a waste liquid collection area. The areas are connected by microchannels. The width of the microchannel is 50um-150um and the depth is 30um-150um.

[0013] As a further scheme of the present invention: After the exosomes are separated, they are purified by a purification method based on affinity chromatography, using an affinity medium with specific binding ability to specific markers on the surface of exosomes to purify them, designing and manufacturing a microfluidic chip, the chip uses polydimethylsiloxane, and the chip contains a cell culture area, an exosome separation area, and a waste liquid collection area. The cultured iPS cells and supernatant are introduced into the cell culture area of ​​the microfluidic chip, and the flow rate is controlled at 10uL / min-20uL / min to separate the exosomes.

[0014] As a further solution of the present invention: the prepared exosomes are co-cultured with BMSCs, an inducer is added and the culture conditions are adjusted to promote the differentiation of BMSCs into cell types related to the motor system.

[0015] An application of iPS cell exosomes, wherein the high-concentration exosomes carry biological information, and the exosome group can quickly promote the differentiation of BMSCs. The application steps of the iPS cell exosomes are as follows:

[0016] Step 1: construct an animal bone defect model, create a bone defect on its skull, expose the bone tissue using a surgical method, and use instruments to prepare a defect area with a diameter of 5 mm to 10 mm;

[0017] Step 2: pre-treat the bone defect area, inject a solution containing vascular endothelial growth factor and platelet-derived growth factor into the bone defect through a microneedle array, the microneedle length is 0.5mm-1mm, and the release time is 1h-2h;

[0018] Step 3: inject the prepared complex containing iPS cell exosomes into the defect site, and use an appropriate fixation method to fix the complex containing iPS cell exosomes on the bone tissue;

[0019] Step 4: Observe and monitor the animals after surgery, use X-rays and CT scans to observe the repair of bone defects, analyze the regeneration and structural changes of bone tissue through histological staining, and use biomechanical tests to evaluate the strength and functional recovery of the skull.

[0020] As a further scheme of the present invention: exosomes are combined with a calcium carbonate scaffold to construct a complex, which is implanted into the bone defect site, and the biological activity of the exosomes promotes bone tissue regeneration and repair. The calcium carbonate scaffold needs to be surface treated before combining, and the monitoring time points are set at 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after surgery.

[0021] As a further scheme of the present invention: before the complex is implanted into the bone defect site, the bone defect area is pretreated, including removing necrotic tissue and using specific bioactive factors for local treatment to promote angiogenesis and cell migration. The pretreatment method uses a microneedle array-based drug delivery system to accurately deliver the bioactive factors to the bone defect site.

[0022] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. The present invention constructs an overexpression vector containing genes related to bone cell differentiation such as RUNX2 and ABC1 through gene editing, and accurately introduces the vector into iPS cells by means of unique nanoparticle-mediated transfection combined with magnetic field guidance, greatly improving the transfection efficiency. In the culture stage, the oxygen concentration is dynamically adjusted according to time, and serum substitutes and other substances are added to the culture medium to create a suitable growth environment for iPS cells and promote their secretion of high-quality exosomes. Advanced microfluidics technology and affinity chromatography methods are used to efficiently separate and purify them, which greatly improves the yield and quality of exosomes, providing a highly potential biologically active substance basis for the repair of motor system injuries;

[0024] 2. The present invention co-cultures the prepared exosomes with BMSCs, adds inducers and adjusts the culture conditions to promote the rapid differentiation of BMSCs into cell types related to the musculoskeletal system, thereby providing key cell resources for the injured site. After constructing an animal bone defect model, the bone defect area is pretreated with a microneedle array, and vascular endothelial growth factor and platelet-derived growth factor solutions are injected to promote angiogenesis and cell migration. Subsequently, a complex of exosomes combined with a surface-treated calcium carbonate scaffold is implanted into the defect site. The biological activity of the exosomes effectively promotes bone tissue regeneration and repair. After the operation, the repair effect is comprehensively monitored and evaluated by various means such as X-rays, CT scans, histological staining and biomechanical testing. This provides an innovative and efficient solution for the clinical treatment of chronic injuries of the musculoskeletal system, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the steps for preparing iPS cell exosomes in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The present invention provides a method for preparing exosomes from iPS cells, comprising constructing an overexpression vector, preparing and transfecting nanoparticles, optimizing iPS cell culture, isolating exosomes, purifying exosomes, co-culturing BMSCs and exosomes, and conducting a bone defect repair experiment. The specific steps of the method for preparing exosomes from iPS cells are as follows:

[0028] Step 1: Use gene editing technology to construct a specific overexpression vector, select genes related to osteoblast differentiation, use genomic DNA or cDNA containing the target gene as a template, use PCR technology to amplify RUNX2 gene and ABC1 gene fragments, and combine them with related genes that can enhance the differentiation-promoting ability of exosomes;

[0029] Step 2: Use transfection technology to introduce the overexpression vector into iPS cells. A nanoparticle-mediated transfection method is used to solidify the nanoparticles in the nanoemulsion by volatile organic solvents. The overexpression vector is combined with biocompatible nanoparticles, and the nanoparticles combined with the overexpression vector are added to the cultured iPS cells. At the same time, a magnetic field device is used to generate a magnetic field to guide the nanoparticles to gather around the cells and enter the cells.

[0030] Step 3: Optimize the culture of the transfected iPS cells, add appropriate amounts of serum replacement, small molecule compounds and growth factors to the basal culture medium to prepare an optimized culture medium, inoculate the transfected iPS cells into the optimized culture medium, and culture them in a culture container. Maintain the oxygen concentration at 21% within 24 hours of culture, and gradually reduce the oxygen concentration as the culture time goes by. Adjust the oxygen concentration to 10% within 2-4 days of culture, and reduce the oxygen concentration to 5% after 4 days of culture.

[0031] Step 4: Collect exosomes secreted by iPS cells, separate exosomes from cell culture supernatant using microfluidics technology, detect the particle size distribution and concentration of purified exosomes using nanoparticle tracking analyzer, detect the expression of exosome surface markers using Western blot to evaluate the purity and quality of exosomes, and collect and preserve the final exosomes.

[0032] In one embodiment of the present invention: In the nanoparticle-mediated transfection method, the preparation materials of the nanoparticles include biodegradable high molecular polymers and magnetic nanoparticles, the surface of the nanoparticles is modified with a ligand that has specific binding ability to both the overexpression vector and the iPS cells, the overexpression vector and the nanoparticles are added to a buffer solution and mixed, the vector is bound to the nanoparticles by electrostatic adsorption, and the external magnetic field is used to guide the nanoparticles to gather around the cells and enter the cells, the magnetic field strength is set to 0.01T-0.02T, and the action time is 15min-30min.

[0033] In one embodiment of the present invention: during the optimized culture process, the culture system is 37°C and 5% CO2 concentration, the gene overexpression vector selects the DNA sequence of RUNX2 gene and other synergistic genes, and the synthesized DNA sequence is cloned into a suitable vector backbone to construct an overexpression vector.

[0034] In one embodiment of the present invention: the microfluidic chip includes three groups of areas, namely, a cell culture area, a secretion separation area, and a waste liquid collection area. The areas are connected by microchannels. The width of the microchannel is 50um-150um and the depth is 30um-150um.

[0035] In one embodiment of the present invention: after the exosomes are separated, they are purified by a purification method based on affinity chromatography, using an affinity medium with specific binding ability to specific markers on the surface of exosomes to purify them, designing and manufacturing a microfluidic chip, the chip uses polydimethylsiloxane, and the chip contains a cell culture area, an exosome separation area, and a waste liquid collection area. The cultured iPS cells and supernatant are introduced into the cell culture area of ​​the microfluidic chip, and the flow rate is controlled at 10uL / min-20uL / min to separate the exosomes.

[0036] In one embodiment of the present invention: the prepared exosomes are co-cultured with BMSCs, an inducer is added and the culture conditions are adjusted to promote the differentiation of BMSCs into cell types related to the motor system.

[0037] An application of iPS cell exosomes. High-concentration exosomes carry biological information. The exosome group can quickly promote the differentiation of BMSCs. The application steps of iPS cell exosomes are as follows:

[0038] Step 1: construct an animal bone defect model, create a bone defect on its skull, expose the bone tissue using a surgical method, and use instruments to prepare a defect area with a diameter of 5 mm to 10 mm;

[0039] Step 2: pre-treat the bone defect area, inject a solution containing vascular endothelial growth factor and platelet-derived growth factor into the bone defect through a microneedle array, the microneedle length is 0.5mm-1mm, and the release time is 1h-2h;

[0040] Step 3: inject the prepared complex containing iPS cell exosomes into the defect site, and use an appropriate fixation method to fix the complex containing iPS cell exosomes on the bone tissue;

[0041] Step 4: Observe and monitor the animals after surgery, use X-rays and CT scans to observe the repair of bone defects, analyze the regeneration and structural changes of bone tissue through histological staining, and use biomechanical tests to evaluate the strength and functional recovery of the skull.

[0042] In one embodiment of the present invention: exosomes are combined with a calcium carbonate scaffold to construct a complex, which is implanted into a bone defect site, and the biological activity of the exosomes promotes bone tissue regeneration and repair. The calcium carbonate scaffold needs to be surface treated before combining, and the monitoring time points are set at 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after surgery.

[0043] In one embodiment of the present invention: before the composite is implanted into the bone defect, the bone defect area is pretreated, including removing necrotic tissue and using specific bioactive factors for local treatment to promote angiogenesis and cell migration. The pretreatment method uses a microneedle array-based drug delivery system to accurately deliver the bioactive factors to the bone defect site.

[0044] In one embodiment of the present invention: the cell culture area is designed as a microchannel network structure so that the cells are evenly distributed and grown, the exosome separation area adopts a special filtering structure, which can separate the exosomes from the culture supernatant according to their size and physical properties, and the waste liquid collection area is used to collect the separated waste liquid. The cultured iPS cells and the supernatant are introduced into the cell culture area of ​​the microfluidic chip, and the cells and the supernatant are made to flow in the chip through the fluid dynamics in the microchannel to ensure that the exosomes can be effectively separated.

[0045] In one embodiment of the present invention: BMSC culture and co-culture with exosomes, BMSC are extracted from animals, separated and purified by density gradient centrifugation or adherent culture, BMSC are inoculated in conventional culture medium for culture, the culture medium can be DMEM culture medium containing 10% fetal bovine serum, the culture conditions are 37°C, 5% CO2, when BMSC grow to a certain density, they are inoculated on a three-dimensional scaffold material, the scaffold material is a composite material of polylactic acid-glycolic acid copolymer and hydroxyapatite, the shape of the scaffold is cylindrical, the prepared iPS cell exosomes are added to the three-dimensional scaffold material inoculated with BMSC, the exosomes are fully in contact with the BMSC, some inducers that promote BMSC differentiation are added to the exosome solution, the inducer is a mixture of dexamethasone, sodium β-glycerophosphate and vitamin C, and the concentrations are 0.1umo l, 10mmo l and 50ug per milliliter of culture medium, respectively, and then the scaffold material is placed in an incubator for co-culture, the culture conditions are 37°C, 5% CO2, and the culture time is 7d-14d.

[0046] In one embodiment of the present invention: an optimized culture medium containing specific small molecule compounds and growth factors is prepared, the concentration of the cell autophagy inducer is 0.05umo l-0.1umo l per milliliter of culture medium as the culture medium, the concentration of the bone morphogenetic protein signaling pathway activator is 0.1umo l-0.2umo l per milliliter of culture medium, the transfected iPS cells are inoculated into the optimized culture medium, and cultured under specific culture conditions, the culture conditions are 37°C, 5% CO2, and the culture time is 3-5 days. During the culture process, the culture conditions are dynamically adjusted according to the metabolic state of the cells and the amount of exosome secretion, and the oxygen concentration is appropriately reduced in the later stage of culture to simulate the physiological environment in vivo to promote the maturation and secretion of exosomes.

[0047] Embodiment 1

[0048] During the gene overexpression vector construction phase, the sequence information of RUNX2 gene and candidate gene ABC1 was obtained by consulting the gene database. Based on this information, specific primers were designed. The genomic DNA or cDNA containing the target gene was used as a template to amplify the RUNX2 gene and ABC1 gene fragments using PCR technology. During the PCR reaction, the pre-denaturation temperature was set at 95°C for 5 minutes, the denaturation temperature was set at 95°C for 30 seconds, the annealing temperature was set at 58°C for 30 seconds, and the extension temperature was set at 72°C for 1 minute. A total of 30 cycles were performed, and the final extension temperature was set at 72°C for 10 minutes. The amplified gene fragments were quenched with restriction endonucleases. The gene fragment and the gene cloning vector are double-digested with enzymes. The enzyme digestion reaction system is prepared according to the enzyme instructions, and the reaction is carried out at 37°C for 1h-2h. The gene fragment and the vector after enzyme digestion are connected by DNA ligase. The connection reaction system is reacted at 16°C overnight. The connection product is transformed into DH5α competent cells, and the cells are spread on LB solid culture medium containing ampicillin, and inverted in a constant temperature incubator at 37°C overnight. Positive clones are selected from the cultured colonies, and colony PCR identification and sequencing verification are performed to ensure that the gene is correctly inserted into the vector. The correct recombinant plasmid is extracted and purified, which is the constructed overexpression vector;

[0049] In the nanoparticle preparation and transfection stage, polylactic acid-glycolic acid copolymer and magnetic nanoparticles are dissolved in dichloromethane to form a uniform mixed solution, the concentration of polylactic acid-glycolic acid copolymer is controlled to contain 10ug-20ug per milliliter of solvent, and the concentration of magnetic nanoparticles is controlled to contain 5ug-10ug per milliliter of solvent. The mixed solution is added dropwise into an aqueous solution containing a surfactant to form a nanoemulsion by ultrasonic emulsification. The ultrasonic power is set at 200w-300w and the ultrasonic time is 5min-10min. The nanoemulsion is made of nanoparticles by evaporating the organic solvent. The rice particles are solidified to form polylactic acid-glycolic acid copolymer nanoparticles containing magnetic nanoparticles, and the oligonucleotide chains and RGD polypeptides with specific chemical groups are connected to the surface of the nanoparticles through chemical reactions. The reaction conditions are 25°C and the reaction is carried out in a reaction system containing an appropriate phosphate buffer for 2h-3h. The prepared overexpression vector and the nanoparticles are mixed in a buffer containing an appropriate amount of sodium chloride, so that the overexpression vector is combined with the nanoparticles through the chemical groups on the oligonucleotide chain. The reaction is carried out at room temperature for 30min-60min. The nanoparticles combined with the overexpression vector are added to the cultured iPS cells, and a magnetic field device is used to generate a magnetic field to guide the nanoparticles to gather around the cells and enter the cells. The magnetic field strength is set at 0.01T-0.015T, and the action time is 20min-30min.

[0050] During the iPS cell optimization culture stage, an appropriate amount of serum replacement, small molecule compounds and growth factors were added to the basal culture medium to prepare an optimized culture medium, in which the concentration of the novel cell autophagy inducer XYZ1 was controlled to contain 0.05umo l-0.1umo l per milliliter of culture medium, and the concentration of the bone morphogenetic protein signaling pathway activator BMP analog was controlled to contain 0.1umo l-0.2umo l per milliliter of culture medium. l. Inoculate the transfected iPS cells into the optimized culture medium and culture them in the culture container. The culture conditions are set at 37°C and 5% CO2. The oxygen concentration is adjusted according to the culture stage. In the first 24 hours of culture, the oxygen concentration is maintained at 21% to promote cell metabolism and adapt to the new culture environment. As the culture time goes by, the oxygen concentration is gradually reduced. From the 2nd to the 4th day of culture, the oxygen concentration is adjusted to 10% to simulate the microenvironment of certain tissues in the body and promote the secretion of exosomes by cells. In the late stage of culture, after the 4th day, the oxygen concentration is reduced to 5% to promote the maturation and secretion of exosomes. At the same time, according to the growth status of the cells and the secretion of exosomes, the culture medium is replaced every 2-3 days;

[0051] In the exosome separation stage, the cultured mixture containing iPS cells and cell culture supernatant is introduced into the cell culture area of ​​the microfluidic chip through a microchannel. A microinjection pump is used to control the flow rate at 10uL-20uL per minute. The cells and supernatant flow in the microchannel of the cell culture area. Since the microchannel is like a curved serpentine structure, the cells can be evenly distributed in it and continue to grow and secrete exosomes. As the fluid flows, the supernatant containing exosomes enters the exosome separation area. The microchannel in the exosome separation area is designed with a special filtering structure, such as a nanofiltration membrane with a pore size of 40nm-100nm or a structure with a filter column. When the fluid containing exosomes passes through the filtering structure, the exosomes are retained and gradually enriched according to the size and physical properties of the exosomes, while other larger cell components and impurities are filtered out and enter the waste liquid collection area. The enriched exosomes are collected from the exosome separation area and transferred to a collection container.

[0052] During the exosome purification stage, the affinity chromatography column was balanced with PBS buffer to keep the affinity medium in a suitable working state. The equilibrium flow rate was controlled at 1 mL / min-5 mL / min. The separated exosome solution was slowly added to the affinity chromatography column to allow the exosomes to fully contact the affinity medium. The flow rate was controlled at 0.5 mL / min-2 mL / min. The exosomes were allowed to stay in the affinity chromatography column for 15 min-30 min to allow the specific markers on their surface to fully bind to the antibodies on the affinity medium. The affinity chromatography column was rinsed with PBS buffer to remove unbound impurities and other contaminants. The rinse flow rate was controlled at 2 mL / min-5 mL / min per minute. The exosomes bound to the affinity medium were eluted with an eluent containing 0.5 mol / L-1 mol l / L sodium chloride buffer or low pH buffer was used for elution, and the elution flow rate was controlled at 0.5mL / min-1mL / min. The eluted exosomes were collected in a collection container, and the particle size distribution and concentration of the purified exosomes were detected by nanoparticle tracking analyzer. The expression of exosome surface markers was detected by Western blot to evaluate the purity and quality of the exosomes.

[0053] Embodiment 2

[0054] In the stage of promoting differentiation by co-culturing BMSC and exosomes, bone marrow samples of animals are obtained by bone marrow puncture. After mixing the bone marrow samples with an appropriate amount of PBS buffer, they are slowly added to the pre-prepared density gradient centrifuge for density gradient centrifugation. The centrifugation conditions are set to 1500r / min-2000r / min, and the centrifugation time is 20min-30min. BMSCs located in the middle layer of the density gradient centrifuge are collected and inoculated into a culture container containing BMSC culture medium. They are cultured in a cell culture incubator under the conditions of 37°C and 5% CO2. When BMSCs grow to a cell fusion degree of 70%-80%, they are inoculated onto a three-dimensional scaffold material. A micropipette can be used to evenly drop the cell suspension onto the scaffold material, or the scaffold material can be immersed in the cell suspension for a period of time so that the BMSCs can fully attach to the scaffold. The prepared iPS cell exosomes are added to the culture system containing BMSC and scaffold material so that the final concentration of the exosomes reaches 10 per milliliter of culture medium. 8 -10 9The induction agent that promotes BMSC differentiation is added, and the culture container is placed in a cell culture incubator for co-culture. The culture conditions are maintained at a temperature of 37°C and 5% CO2 for 7-14 days. During the culture process, the morphological changes and differentiation of BMSCs are regularly observed. The changes in cell morphology can be observed under a microscope, and the degree of differentiation of BMSCs into osteoblasts or chondrocytes can be evaluated by using alkaline phosphatase activity detection and calcium nodule staining.

[0055] During the bone defect repair experiment, the animals were anesthetized and fixed on the operating table. The bone tissue of the femur or skull of the animals was exposed using instruments such as scalpels and tweezers. A dental drill was used to carefully create a bone defect of a predetermined size and shape on the bone tissue. The size of the defect was accurately measured using a vernier caliper to ensure that it met the experimental requirements. A solution containing VEGF and PDGF was accurately delivered to the bone defect site using a microneedle array-based drug delivery system. The microneedle array was inserted into the tissue around the bone defect, and the bioactive factor solution was slowly injected through the tiny channels of the microneedles. The injection time was controlled at 10-20 minutes. The injection volume was adjusted according to the size of the defect and the weight of the animal. 0.1uL-0.2uL was injected per gram of body weight. The exosome complex was implanted into the bone defect site so that the complex was in close contact with the edge of the bone defect. If necessary, biodegradable sutures or screws could be used to fix the complex to the bone tissue to ensure its stability. The animals were observed and monitored regularly after surgery. At different time points after surgery (1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after surgery), X-ray imaging equipment was used to observe the repair of the bone defect. The regeneration and structural changes of the bone tissue were evaluated by histological analysis, including HE staining and Masson staining, observing the morphology of the bone tissue and the distribution of collagen fibers. Biomechanical testing equipment was used to evaluate the strength and functional recovery of the bone and measure the compressive strength of the bone. Based on the results of monitoring and evaluation, the role and effect of exosomes in bone defect repair were analyzed.

[0056] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

[0057] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0058] The above contents are merely examples and explanations of the present invention. Various modifications or additions to the specific embodiments described or replacements in similar ways by technicians in the technical field shall fall within the protection scope of the present invention as long as they do not deviate from the invention or exceed the scope defined by the claims.

[0059] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing exosomes from iPS cells, comprising constructing an overexpression vector, preparing and transfecting nanoparticles, optimizing iPS cell culture, isolating exosomes, purifying exosomes, co-culturing BMSCs and exosomes, and conducting a bone defect repair experiment, characterized in that: The specific operation steps of the iPS cell exosome preparation method are as follows: Step 1: Use gene editing technology to construct a specific overexpression vector, select genes related to osteoblast differentiation, and combine them with related genes that can enhance the differentiation-promoting ability of exosomes; Step 2: Use transfection technology to introduce the overexpression vector into iPS cells, using a nanoparticle-mediated transfection method that uses biocompatible nanoparticles combined with the overexpression vector; Step 3: Optimize the culture of the transfected iPS cells, add small molecule compounds and growth factors to the culture system, and define the reaction conditions; Step 4: Collect exosomes secreted by iPS cells, separate exosomes from cell culture supernatant using microfluidics technology, and collect and preserve them.

2. The method for preparing iPS cell exosomes according to claim 1, characterized in that: In the nanoparticle-mediated transfection method, the preparation materials of the nanoparticles include biodegradable high molecular polymers and magnetic nanoparticles, the surface of the nanoparticles is modified with a ligand that has specific binding ability with both the overexpression vector and the iPS cells, the overexpression vector and the nanoparticles are added to a buffer solution and mixed, the vector is bound to the nanoparticles by electrostatic adsorption, and the nanoparticles are guided to gather around the cells and enter the cells by an external magnetic field, the magnetic field strength is set to 0.01T-0.02T, and the action time is 15min-30min.

3. The method for preparing iPS cell exosomes according to claim 1, characterized in that: In the optimized culture process, the culture system is 37° C. and 5% CO 2 concentration, the gene overexpression vector uses the DNA sequence of RUNX2 gene and other synergistic genes, and the synthesized DNA sequence is cloned into a suitable vector backbone to construct an overexpression vector.

4. The method for preparing iPS cell exosomes according to claim 1, characterized in that: The microfluidic chip includes three groups of areas, namely, a cell culture area, a secretion separation area and a waste liquid collection area. The areas are connected by microchannels. The width of the microchannel is 50um-150um and the depth is 30um-150um.

5. The method for preparing iPS cell exosomes according to claim 1, characterized in that: After the exosomes are separated, they are purified by a purification method based on affinity chromatography, using an affinity medium with specific binding ability to specific markers on the surface of exosomes to purify them, designing and manufacturing a microfluidic chip, the chip uses polydimethylsiloxane, and the chip contains a cell culture area, an exosome separation area, and a waste liquid collection area. The cultured iPS cells and supernatant are introduced into the cell culture area of ​​the microfluidic chip, and the flow rate is controlled at 10uL / min-20uL / min to separate the exosomes.

6. The method for preparing iPS cell exosomes according to claim 1, characterized in that: The prepared exosomes are co-cultured with BMSCs, inducers are added and culture conditions are adjusted to promote the differentiation of BMSCs into cell types related to the motor system.

7. An application of iPS cell exosomes applicable to the method for preparing iPS cell exosomes according to any one of claims 1 to 6, characterized in that: The high-concentration exosomes carry biological information, and the exosome group can quickly promote the differentiation of BMSCs. The application steps of the iPS cell exosomes are as follows: Step 1: construct an animal bone defect model, create a bone defect on its skull, expose the bone tissue using a surgical method, and use instruments to prepare a defect area with a diameter of 5 mm to 10 mm; Step 2: pre-treat the bone defect area, inject a solution containing vascular endothelial growth factor and platelet-derived growth factor into the bone defect through a microneedle array, the microneedle length is 0.5mm-1mm, and the release time is 1h-2h; Step 3: inject the prepared complex containing iPS cell exosomes into the defect site, and use an appropriate fixation method to fix the complex containing iPS cell exosomes on the bone tissue; Step 4: Observe and monitor the animals after surgery, use X-rays and CT scans to observe the repair of bone defects, analyze the regeneration and structural changes of bone tissue through histological staining, and use biomechanical tests to evaluate the strength and functional recovery of the skull.

8. The use of iPS cell exosomes according to claim 7, characterized in that: The exosomes are combined with the calcium carbonate scaffold to construct a complex, which is implanted into the bone defect. The biological activity of the exosomes promotes bone tissue regeneration and repair. The calcium carbonate scaffold needs to be surface treated before combining. The monitoring time points are set at 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after surgery.

9. The use of iPS cell exosomes according to claim 7, characterized in that: Before the composite is implanted into the bone defect, the bone defect area is pretreated, including removing necrotic tissue and using specific bioactive factors for local treatment to promote angiogenesis and cell migration. The pretreatment method uses a microneedle array-based drug delivery system to accurately deliver the bioactive factors to the bone defect site.

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