Ovarian granular cell exosome preparation method and system based on directional cell differentiation

Abstract

The invention relates to the technical field of cell differentiation, in particular to an ovarian granular cell exosome preparation method and system based on cell directional differentiation, and the method comprises the following steps: reprogramming starting material cells to obtain initial induced pluripotent stem cells, carrying out cell identification to obtain induced pluripotent stem cells, and carrying out directional differentiation on the induced pluripotent stem cells to obtain the ovarian granular cell exosome. Obtaining an ovarian granular cell cluster, obtaining a culture supernatant, purifying the culture supernatant to obtain an initial exosome, carrying out quality detection on the initial exosome to obtain a detection result, if the detection result is that the detection does not reach the standard, obtaining an adjusted exosome, taking the adjusted exosome as the initial exosome, and taking the adjusted exosome as the initial exosome. And returning to the step of detecting the quality of the initial exosome until the detection result is that the detection reaches the standard, and confirming the initial exosome as the target exosome when the detection result is that the detection reaches the standard. The problem that the purity of the exosome is low in the preparation process of the ovarian granular cell exosome at present can be solved.

Description

Technical Field

[0001] This invention relates to the field of cell differentiation technology, and in particular to a method and system for preparing ovarian granulosa cell exosomes based on directed cell differentiation. Background Technology

[0002] With the continuous development of regenerative medicine and reproductive medicine, the role of ovarian granulosa cells in follicle development, hormone secretion and ovarian function maintenance is becoming increasingly prominent. As extracellular vesicles rich in bioactive molecules, ovarian granulosa cell exosomes are increasingly in demand for applications in reproductive medicine, ovarian function repair and targeted therapy of gynecological diseases. Therefore, the preparation of ovarian granulosa cell exosomes is of great significance.

[0003] Traditional methods for preparing ovarian granulosa cell exosomes typically rely on isolating ovarian granulosa cells from humans or animals, culturing them in vitro, and then extracting exosomes. While this method can obtain ovarian granulosa cell exosomes, it lacks precise quality control of the obtained exosomes, resulting in insufficient purity. Therefore, the current process for preparing ovarian granulosa cell exosomes suffers from the problem of low exosome purity. Summary of the Invention

[0004] This invention provides a method and system for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, the main purpose of which is to solve the problem of low exosome purity in the current process of preparing ovarian granulosa cell exosomes.

[0005] To achieve the above objectives, the present invention provides a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, comprising: Obtaining starting material cells; The starting material cells were reprogrammed to obtain initial induced pluripotent stem cells; Cell identification was performed based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; The induced pluripotent stem cells were directed to differentiate into ovarian granulosa cell clusters. Obtain the culture supernatant of the ovarian granulosa cell cluster; The culture supernatant was purified to obtain initial exosomes; The initial exosomes are subjected to quality testing to obtain test results, wherein the test results are either qualified or unqualified. If the test result is that the test does not meet the standard, then the adjusted exosomes are obtained, the adjusted exosomes are used as the initial exosomes, and the process of performing quality testing on the initial exosomes is returned until the test result meets the standard. When the test result is satisfactory, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

[0006] Optionally, the reprogramming of the starting material cells to obtain initial induced pluripotent stem cells includes: Obtain culture units; Based on the culture unit, the starting material cells are seeded to obtain a starting cell layer; The starting cell layer is activated to obtain an activated starting cell layer; The activated initiating cell layer is programmed using a preset reprogramming factor to obtain a mixed cell community; Initially induced pluripotent stem cells were identified from the mixed cell community.

[0007] Optionally, the activation treatment of the starting cell layer to obtain an activated starting cell layer includes: The activity of the initial cell layer is enhanced by using a preset cell activator to obtain an active cell layer; The metabolic state of the viable cell layer was detected to obtain a set of metabolic characteristic information. For each metabolic feature in the set of metabolic feature information, the following operation is performed: Based on the metabolic characteristic information, a metabolic determination result is obtained; Summarize the metabolic determination results to obtain the metabolic determination result set; Based on the metabolic determination result set, an active region set is selected from the active cell layer; Based on the set of active regions, the activation initiation cell layer is obtained.

[0008] Optionally, the metabolic state detection of the active cell layer to obtain a metabolic feature information set includes: The active cell layer was divided into regions to obtain multiple active cell regions; For each of the plurality of active cell regions, the following operation is performed: Based on the viable cell region, obtain viable cell samples; Obtain the first fluorescent probe and the second fluorescent probe; The viable cell sample was fluorescently labeled using the first fluorescent probe and the second fluorescent probe to obtain a fluorescently labeled sample. Fluorescence detection was performed on the fluorescently labeled sample to obtain a first fluorescence intensity sequence and a second fluorescence intensity sequence; Based on the first fluorescence intensity sequence and the second fluorescence intensity sequence, glucose uptake parameters and mitochondrial membrane potential parameters are obtained. The fluorescently labeled sample was lysed to obtain lysate; The lysed cell solution was incubated in the dark using a pre-set ATP fluorescent reagent to obtain the ATP concentration; Based on the glucose uptake parameters, mitochondrial membrane potential parameters, and ATP concentration, metabolic characteristic information is constructed. The metabolic feature information is summarized to obtain a metabolic feature information set.

[0009] Optionally, the step of cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells includes: Based on the initial induced pluripotent stem cells, obtain initial induced pluripotent stem cell samples; Identify pluripotency markers, wherein the pluripotency markers include: a first marker and a second marker; Based on the initial induced pluripotent stem cell samples, the positive expression of the first and second markers among the pluripotency markers was detected using a pre-set activity detection device to obtain the positive rate of the first marker and the positive rate of the second marker. Obtain alkaline phosphatase reagent and use alkaline phosphatase reagent to stain the initial induced pluripotent stem cell samples to obtain stained cell samples; The stained cell sample is scanned and imaged using a preset imaging unit to obtain a stained microscopic image; Based on the stained microscopic images, the phosphatase positivity rate was obtained; A set of identification indicators was constructed based on the positive rates of the first biomarker, the second biomarker, and the phosphatase. When the set of identification indicators does not meet the preset identification criteria, adjust induced pluripotent stem cells are obtained, and the adjusted induced pluripotent stem cells are used as initial induced pluripotent stem cells. Then, the above steps of obtaining initial induced pluripotent stem cell samples based on the initial induced pluripotent stem cells are returned until the set of identification indicators meets the identification criteria. When the set of identification indicators meets the identification criteria, the initial induced pluripotent stem cells are identified as induced pluripotent stem cells.

[0010] Optionally, the directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters includes: Obtain differentiation culture medium; Induced pluripotent stem cells were cultured using the aforementioned differentiation medium to obtain a pluripotent stem cell layer; Obtain the first-stage differentiation inducer and the second-stage differentiation inducer; The pluripotent stem cell layer was induced to differentiate using the first-stage differentiation inducer and the second-stage differentiation inducer to obtain a differentiated cell layer. The differentiated cell layer is divided into multiple differentiated cell regions; For each of the plurality of differentiated cell regions, the following operation is performed: Granulocyte markers were detected in the differentiated cell regions to obtain the marker expression levels; Based on the expression level of the marker, the regional detection result is determined, wherein the regional detection result includes: the region is qualified or the region is unqualified; By summarizing the detection results of the aforementioned regions, a set of region detection results is obtained; Based on the region detection result set, ovarian granulosa cell clusters were extracted from the multiple differentiated cell regions.

[0011] Optionally, obtaining the culture supernatant of the ovarian granulosa cell mass includes: Based on the ovarian granulosa cell clusters, the centrifugation medium was determined; The ovarian granulosa cell clusters were expanded and cultured using a pre-set exosome culture medium to obtain a high-density granulosa cell layer; A serum-free granulocyte culture medium was obtained, and the high-density granulocyte layer was statically cultured using the serum-free granulocyte culture medium to obtain a static cell system. Obtain a centrifuge unit, and based on the static cell system and centrifugation medium, use the centrifuge unit to perform centrifugation to obtain a preliminary clarified supernatant; The preliminarily clarified supernatant was subjected to vacuum filtration to obtain the culture supernatant.

[0012] Optionally, the quality testing of the initial exosomes to obtain the test results includes: Multiple morphological images were obtained by using an imaging unit to perform multi-field observation of the initial exosomes; The morphological structure statistics of the multiple morphological images were performed to obtain the morphological qualification rate. The initial exosomes were subjected to particle detection using a preset particle analysis method to obtain particle size and particle concentration. By summarizing the morphological qualification rate, particle size, and particle concentration, a set of cell physical characteristics is obtained. If the set of cell physical characteristics meets the preset physical characteristic criteria, chemical characteristic detection is performed on the initial exosomes to obtain the detection results.

[0013] Optionally, the step of performing chemical characterization on the initial exosomes to obtain the detection results includes: Proteins were extracted from the initial exosomes to obtain exosome protein lysis buffer; Immunoblotting was performed on exosomal protein lysis buffer using a pre-defined antibody combination to obtain the expression levels of positive and negative markers. The exosome protein lysis buffer was subjected to protein detection to obtain the exosome protein concentration; Based on the exosomal protein concentration, positive marker expression level, and negative marker expression level, the chemical characteristic values ​​of the exosomes were calculated. If the chemical characteristic value of the exosome is less than the preset characteristic threshold, the detection failure will be confirmed as a detection result. If the chemical characteristic value of the exosome is greater than or equal to the characteristic threshold, the detection is considered to have met the standard and is thus confirmed as a detection result.

[0014] To achieve the above objectives, the present invention also provides a system for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, comprising: A cell reprogramming module is used to obtain starting material cells and reprogram the starting material cells to obtain initial induced pluripotent stem cells; The cell identification and differentiation module is used to identify cells based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells, and to perform directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters. The exosome purification module is used to obtain the culture supernatant of the ovarian granulosa cell cluster, and to purify the culture supernatant to obtain initial exosomes; The exosome quality detection module is used to perform quality detection on the initial exosomes and obtain detection results, wherein the detection results are either qualified or unqualified. If the detection result is unqualified, the adjusted exosomes are obtained and used as the initial exosomes. The process of performing quality detection on the initial exosomes is then repeated until the detection result is qualified. When the detection result is qualified, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

[0015] To address the above problems, the present invention also provides an electronic device, the electronic device comprising: A memory for storing at least one instruction; and a processor for executing the instruction stored in the memory to implement the above-described method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation.

[0016] To address the aforementioned problems, the present invention also provides a computer-readable storage medium storing at least one instruction, which is executed by a processor in an electronic device to implement the above-described method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation.

[0017] To solve the problems described in the background art, the present invention obtains starting material cells, which clarifies the basic raw materials for preparing exosomes from ovarian granulosa cells through this step. The starting material cells are reprogrammed to obtain initial induced pluripotent stem cells. Based on the initial induced pluripotent stem cells, cell identification is performed to obtain induced pluripotent stem cells. The induced pluripotent stem cells are directed to differentiate to obtain ovarian granulosa cell clusters. The present invention uses a first-stage differentiation inducer and a second-stage differentiation inducer to induce the differentiation of induced pluripotent stem cells, causing the induced pluripotent stem cells to differentiate into ovarian granulosa cells. By detecting granulosa cell markers in the differentiated cell regions, the purity of ovarian granulosa cells in each differentiated cell region is determined, and then a high-purity ovarian granulosa cell cluster is obtained. The culture supernatant of the ovarian granulosa cell cluster is purified to obtain initial exosomes, and the initial exosomes are quality-tested to obtain a test result. Among them, the test result is either qualified or unqualified. By quality-testing the initial exosomes, the present invention realizes the control of the quality of the initial exosomes and improves the purity of the finally obtained exosomes. If the test result is unqualified, adjusted exosomes are obtained, and the adjusted exosomes are used as the initial exosomes, and the above step of quality-testing the initial exosomes is repeated until the test result is qualified. When the test result is qualified, the initial exosomes are confirmed as target exosomes, and the exosome preparation is completed. Therefore, the present invention can solve the problem of low exosome purity in the current preparation process of ovarian granulosa cell exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 FIG. 6 is a flowchart of a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation provided by an embodiment of the present invention; Figure 2 FIG. 9 is a functional module diagram of a system for preparing ovarian granulosa cell exosomes based on cell-directed differentiation provided by an embodiment of the present invention; Figure 3 FIG. 12 is a schematic structural diagram of an electronic device for implementing the method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation provided by an embodiment of the present invention.

[0019] DESCRIPTION OF REFERENCE NUMERALS: 1. Electronic device; 10. Processor; 11. Memory; 12. Bus.

[0020] The realization, functional characteristics and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0021] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] This application provides a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation. The execution entity of this method includes, but is not limited to, at least one of the following electronic devices that can be configured to execute the method provided in this application: a server, a terminal, etc. In other words, the method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation can be executed by software or hardware installed on a terminal device or a server device, and the software can be a blockchain platform. The server includes, but is not limited to, a single server, a server cluster, a cloud server, or a cloud server cluster.

[0023] Reference Figure 1 The diagram shown is a schematic flowchart of a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation according to an embodiment of the present invention. In this embodiment, the method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation includes: S1. Obtain starting material cells, reprogram the starting material cells to obtain initial induced pluripotent stem cells.

[0024] Specifically, the reprogramming of the starting material cells to obtain initial induced pluripotent stem cells includes: Obtain culture units; Based on the culture unit, the starting material cells are seeded to obtain a starting cell layer; The starting cell layer is activated to obtain an activated starting cell layer; The activated initiating cell layer is programmed using a preset reprogramming factor to obtain a mixed cell community; Initially induced pluripotent stem cells were identified from the mixed cell community.

[0025] It should be noted that the starting material cells refer to human somatic cells isolated from the human body; optionally, human dermal fibroblasts are used as the starting material cells. The culture unit refers to equipment used for culturing the starting material cells, such as cell culture dishes, multi-well plates, culture flasks, etc. The inoculation treatment refers to the process of transferring the starting material cells into the culture unit, ensuring their uniform distribution on the surface of the culture unit. The starting cell layer refers to a monolayer of cells formed by the adherent growth of the starting material cells in the culture unit after inoculation treatment. The reprogramming factors refer to a set of transcription factors used to induce the reprogramming of the starting material cells into pluripotent stem cells; optionally, the set of transcription factors includes Oct4, Sox2, Klf4, and c-Myc. The programming treatment refers to the process of introducing the reprogramming factors into the activated starting cell layer through a preset introduction method (such as lentiviral transfection, electroporation transfection, liposome transfection, etc.), thereby reprogramming the starting material cells in the activated starting cell layer. The mixed cell community refers to a cell population with multiple cell types mixed together in the culture unit after reprogramming treatment, including: unreprogrammed starting material cells, reprogrammed starting material cells, starting material cells in the intermediate stage of reprogramming, and dead starting material cells. The initial induced pluripotent stem cells refer to the reprogrammed starting material cells extracted from the mixed cell community through morphological screening (e.g., screening out cell populations that meet the morphological characteristics of pluripotent stem cells from the mixed cell community based on the morphological characteristics of pluripotent stem cells, which can be obtained from existing literature). These reprogrammed cells possess the morphological characteristics of pluripotent stem cells, but their pluripotency has not been verified and needs to be confirmed through subsequent steps in this embodiment.

[0026] Specifically, the activation treatment of the starting cell layer to obtain an activated starting cell layer includes: The activity of the initial cell layer is enhanced by using a preset cell activator to obtain an active cell layer; The metabolic state of the viable cell layer was detected to obtain a set of metabolic characteristic information. For each metabolic feature in the set of metabolic feature information, the following operation is performed: Based on the metabolic characteristic information, a metabolic determination result is obtained; Summarize the metabolic determination results to obtain the metabolic determination result set; Based on the metabolic determination result set, an active region set is selected from the active cell layer; Based on the set of active regions, the activation initiation cell layer is obtained.

[0027] It should be explained that the cell activator refers to a compound or biological agent that can enhance the metabolic activity and proliferation capacity of the starting material cells, such as growth factors (e.g., basic fibroblast growth factor), small molecule activators (e.g., Wnt signaling pathway activators, AMPK activators), etc. The activity enhancement refers to the process of using a cell activator to increase the activity of the starting material cells in the starting cell layer, specifically manifested as increased intracellular ATP content, accelerated glucose uptake, and enhanced mitochondrial membrane potential parameters. Through activity enhancement, the risk of reprogramming failure due to insufficient activity in the starting material cells can be reduced. The method of using a cell activator to enhance the activity of the starting material cells in the starting cell layer is prior art and will not be described further here. The active cell layer refers to the starting cell layer after activity enhancement. The metabolic determination result set refers to the collection formed by summarizing all metabolic determination results. The metabolic determination results will be explained in the steps of subsequent embodiments. The active region set refers to the collection formed by summarizing active regions, and the active regions will be explained in the steps of subsequent embodiments. The process of obtaining the activated initiating cell layer based on the set of active regions refers to: collecting starting material cells from all active regions within the set of active regions, and transferring all collected starting material cells to a new culture unit for inoculation and cultivation. The resulting monolayer cell population is the activated initiating cell layer. The inoculation and cultivation method is the same as the inoculation treatment method and will not be described in detail here.

[0028] Furthermore, the metabolic state detection of the active cell layer to obtain a metabolic feature information set includes: The active cell layer was divided into regions to obtain multiple active cell regions; For each of the plurality of active cell regions, the following operation is performed: Based on the viable cell region, obtain viable cell samples; Obtain the first fluorescent probe and the second fluorescent probe; The viable cell sample was fluorescently labeled using the first fluorescent probe and the second fluorescent probe to obtain a fluorescently labeled sample. Fluorescence detection was performed on the fluorescently labeled sample to obtain a first fluorescence intensity sequence and a second fluorescence intensity sequence; Based on the first fluorescence intensity sequence and the second fluorescence intensity sequence, glucose uptake parameters and mitochondrial membrane potential parameters are obtained. The fluorescently labeled sample was lysed to obtain lysate; The lysed cell solution was incubated in the dark using a pre-set ATP fluorescent reagent to obtain the ATP concentration; Based on the glucose uptake parameters, mitochondrial membrane potential parameters, and ATP concentration, metabolic characteristic information is constructed. The metabolic feature information is summarized to obtain a metabolic feature information set.

[0029] It is understood that the region division refers to the process of dividing the viable cell layer into multiple independent regions of equal size using a grid division method. The viable cell region refers to an independent region obtained after dividing the viable cell layer into regions. The viable cell sample refers to a portion of the cell population composed of starting material cells extracted from the viable cell region. The first fluorescent probe refers to a fluorescent probe for detecting the glucose uptake capacity of the starting material cells. Optionally, 2-NBDG is used as the first fluorescent probe, which can be taken up by the starting material cells, and the glucose uptake rate parameter of the starting material cells is reflected by the change in the intensity of its fluorescence signal. The second fluorescent probe refers to a fluorescent probe for detecting the mitochondrial membrane potential parameter of cells. Optionally, JC-1 fluorescent probe is used as the second fluorescent probe. When the mitochondrial membrane potential parameter of the starting material cells changes, its fluorescence intensity changes accordingly. Therefore, the mitochondrial membrane potential parameter of the starting material cells can be reflected by the change in fluorescence intensity. The process of fluorescently labeling live cell samples using the first and second fluorescent probes to obtain fluorescently labeled samples involves adding the first and second fluorescent probes to the live cell samples, allowing the starting material cells to take up and bind to them. The resulting cell population composed of the starting material cells that have bound the first and second fluorescent probes is the fluorescently labeled sample. Fluorescence detection refers to the process of collecting the fluorescence signals emitted by the fluorescently labeled samples using fluorescence detection equipment (such as a fluorescence microscope or a fluorescence microplate reader). It is important to note that the fluorescence signals emitted by the first and second fluorescent probes must be collected separately to avoid interference between the two signals. The first fluorescence intensity sequence refers to the sequence formed by arranging the fluorescence intensity (i.e., the first fluorescence intensity) of the collected fluorescence signals emitted by the first fluorescent probe in chronological order from earliest to latest within a preset time period (e.g., 1 hour). The second fluorescence intensity sequence refers to the sequence formed by arranging the fluorescence intensity (i.e., the second fluorescence intensity) of the collected fluorescence signals emitted by the second fluorescent probe in chronological order from earliest to latest within the same time period.

[0030] It should be understood that obtaining the glucose uptake rate parameter and mitochondrial membrane potential parameter based on the first fluorescence intensity sequence and the second fluorescence intensity sequence means: subtracting all adjacent first fluorescence intensities in the first fluorescence intensity sequence, taking the absolute value, and then dividing by the acquisition time interval to obtain a sequence composed of the rate of change of the first fluorescence intensity (i.e., the first fluorescence intensity change rate sequence). The average first fluorescence intensity change rate is then obtained by averaging the first fluorescence intensity change rate sequence, and this average first fluorescence intensity change rate is used as the glucose uptake rate parameter. Similarly, the same operation is performed on the second fluorescence intensity sequence, and the average second fluorescence intensity change rate is used as the mitochondrial membrane potential parameter. The lysis refers to the process of using a lysis buffer (such as RIPA lysis buffer) to disrupt the membrane structure (including the plasma membrane and organelle membranes) of the starting material cells in the fluorescently labeled sample, causing the cell contents (such as proteins, nucleic acids, ATP, etc.) to flow out. The method of using a lysis buffer to disrupt the membrane structure of the starting material cells in the fluorescently labeled sample is existing technology and will not be described further here. The lysed cell fluid refers to the mixed liquid containing cell contents obtained after lysis. The ATP fluorescent reagent refers to a reagent that can specifically react with ATP and generate a fluorescent signal, such as an ATP detection kit. The light-protected incubation reaction refers to the process of mixing the ATP fluorescent reagent with cell lysis fluid in a dark environment, causing the ATP fluorescent reagent to react with the ATP in the cell lysis fluid and generate fluorescence. The ATP concentration refers to the concentration of ATP (in nmol / L) generated after measuring the fluorescence intensity produced during the light-protected incubation reaction and converting the fluorescence intensity into ATP concentration (in nmol / L) according to a pre-defined ATP concentration-fluorescence intensity comparison table. The ATP concentration-fluorescence intensity comparison table is a pre-defined two-dimensional table with fluorescence intensity as the horizontal axis and ATP concentration as the vertical axis. Each element in the table is represented as {fluorescence intensity, ATP concentration}. The ATP concentration-fluorescence intensity comparison table can be obtained by consulting existing literature. Constructing metabolic characteristic information based on the glucose uptake parameters, mitochondrial membrane potential parameters, and ATP concentration refers to integrating the glucose uptake parameters, mitochondrial membrane potential parameters, and ATP concentration into a set, which is the metabolic characteristic information. The metabolic characteristic information set refers to the set formed by summarizing the metabolic characteristic information. The metabolic determination result based on the metabolic feature information refers to comparing the glucose uptake rate parameter, mitochondrial membrane potential parameter, and ATP concentration in the metabolic feature information with the corresponding pre-set threshold values ​​for glucose uptake rate parameter, mitochondrial membrane potential parameter, and ATP concentration, respectively. If all three parameters are greater than or equal to the corresponding threshold values, the metabolic activity of the starting material cells in the active cell region is determined to meet the requirements and can be used as an active region.The active region refers to the active cell region within which the metabolic activity of the starting material cells meets the requirements (i.e., glucose uptake rate parameter greater than or equal to the glucose uptake rate parameter threshold, mitochondrial membrane potential parameter greater than or equal to the mitochondrial membrane potential parameter threshold, and ATP concentration greater than or equal to the ATP concentration threshold). Optionally, the requirements for metabolic activity (i.e., glucose uptake rate parameter threshold, mitochondrial membrane potential parameter threshold, and ATP concentration threshold) can be set according to industry standards in the field of cell reprogramming.

[0031] S2. Based on the initial induced pluripotent stem cells, cell identification is performed to obtain induced pluripotent stem cells.

[0032] In detail, the process of cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells includes: Based on the initial induced pluripotent stem cells, obtain initial induced pluripotent stem cell samples; Identify pluripotency markers, wherein the pluripotency markers include: a first marker and a second marker; Based on the initial induced pluripotent stem cell samples, the positive expression of the first and second markers among the pluripotency markers was detected using a pre-set activity detection device to obtain the positive rate of the first marker and the positive rate of the second marker. Obtain alkaline phosphatase reagent and use alkaline phosphatase reagent to stain the initial induced pluripotent stem cell samples to obtain stained cell samples; The stained cell sample is scanned and imaged using a preset imaging unit to obtain a stained microscopic image; Based on the stained microscopic images, the phosphatase positivity rate was obtained; A set of identification indicators was constructed based on the positive rates of the first biomarker, the second biomarker, and the phosphatase. When the set of identification indicators does not meet the preset identification criteria, adjust induced pluripotent stem cells are obtained, and the adjusted induced pluripotent stem cells are used as initial induced pluripotent stem cells. Then, the above steps of obtaining initial induced pluripotent stem cell samples based on the initial induced pluripotent stem cells are returned until the set of identification indicators meets the identification criteria. When the set of identification indicators meets the identification criteria, the initial induced pluripotent stem cells are identified as induced pluripotent stem cells.

[0033] It should be noted that the initial induced pluripotent stem cell sample refers to a portion of the cell population extracted from the initial induced pluripotent stem cells. The pluripotency marker refers to a biomolecule characterizing the pluripotency of induced pluripotent stem cells, including a first marker and a second marker. The first marker refers to a surface antigen of a pluripotent cell. The second marker refers to a surface antigen of a pluripotent cell other than the first marker. Optionally, the first marker may be SSEA-4, and the second marker may be TRA-1-60. The process of detecting positive expression of the first and second pluripotency markers using a pre-set activity detection device involves adding fluorescently labeled antibodies against the first and second markers (i.e., the first marker antibody) and the second marker antibody (i.e., the second marker antibody) to the initial induced pluripotent stem cell sample. The antibodies against the first and second markers bind to the first and second markers respectively, resulting in fluorescent labeling of both markers. Flow cytometry is then used to analyze the initial induced pluripotent stem cell sample containing the first and second marker antibodies. The total number of cells in the initial induced pluripotent stem cell sample is counted, and the number of cells fluorescently labeled with each marker is counted separately. The positive rates of the first and second markers are then calculated. The positive rate of the first marker refers to the proportion of cells fluorescently labeled with the first marker to the total number of cells in the initial induced pluripotent stem cell sample. The positive rate of the second marker refers to the proportion of cells fluorescently labeled with the second marker to the total number of cells in the initial induced pluripotent stem cell sample.

[0034] It should be explained that the alkaline phosphatase reagent refers to the reagent used to induce a colorimetric reaction of alkaline phosphatase. Optionally, the BCIP / NBT alkaline phosphatase colorimetric kit can be used as the alkaline phosphatase reagent. The staining treatment refers to the process of mixing the alkaline phosphatase reagent with the initial induced pluripotent stem cell sample, causing a colorimetric reaction between the alkaline phosphatase and the reagent. The stained cell sample refers to the cell population formed after the initial induced pluripotent stem cell sample has undergone alkaline phosphatase staining treatment, containing alkaline phosphatase-positive cells (i.e., cells that appear blue-purple) and alkaline phosphatase-negative cells (i.e., cells that do not appear blue-purple). The imaging unit refers to the instrument used to acquire images of the stained cell sample. Optionally, an optical microscope can be used as the imaging unit. The scanning imaging refers to the process of using the imaging unit to acquire images of the entire area containing the stained cell sample. The stained microscopic image refers to a microscopic image of the stained cell sample obtained through scanning imaging, which can show the color of all cells in the stained cell sample. The phosphatase positivity rate refers to the proportion of alkaline phosphatase-positive cells to the total number of cells in the stained cell sample. The construction of the identification index set based on the positive rates of the first biomarker, the second biomarker, and the phosphatase positive rate refers to integrating these three positive rates into a single set, which constitutes the identification index set. The identification criteria refer to a series of pre-defined standards used to determine whether the pluripotency of initially induced pluripotent stem cells meets user requirements. Specifically, these include: a threshold for the positive rate of the first biomarker, a threshold for the positive rate of the second biomarker, and a phosphatase positive rate. Optionally, the methods for setting the thresholds for the positive rates of the first biomarker, the second biomarker, and the phosphatase positive rate are consistent with the methods for setting the thresholds for glucose uptake rate, mitochondrial membrane potential, and ATP concentration, and will not be elaborated further here. The adjustment of induced pluripotent stem cells refers to the process of re-acquiring initial induced pluripotent stem cells by adjusting the acquisition method to make the identification index set meet the identification criteria when the identification index set is determined to be unsatisfactory (satisfactory identification criteria mean that the positive rates of the first marker, the second marker, and the phosphatase are all greater than or equal to the corresponding thresholds for the first marker, the second marker, and the phosphatase; otherwise, it is considered unsatisfactory). The adjustments include, but are not limited to, changing the type of reprogramming factor and adjusting culture conditions (such as changing the cell activator, changing the culture unit, and adjusting the duration of the programming treatment). It should be noted that the first and second markers are both cell surface antigens located outside the cell membrane, and therefore can directly bind to fluorescently labeled antibodies through antigen-antibody reactions, generating fluorescent signals that can be directly detected by flow cytometry. Alkaline phosphatase, however, is an intracellular enzyme that does not produce fluorescent signals; it requires an enzymatic reaction to generate a blue-purple insoluble precipitate, from which the phosphatase positivity rate can be statistically calculated.

[0035] S3. The induced pluripotent stem cells are directed to differentiate to obtain ovarian granulosa cell clusters.

[0036] Specifically, the directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters includes: Obtain differentiation culture medium; Induced pluripotent stem cells were cultured using the aforementioned differentiation medium to obtain a pluripotent stem cell layer; Obtain the first-stage differentiation inducer and the second-stage differentiation inducer; The pluripotent stem cell layer was induced to differentiate using the first-stage differentiation inducer and the second-stage differentiation inducer to obtain a differentiated cell layer. The differentiated cell layer is divided into multiple differentiated cell regions; For each of the plurality of differentiated cell regions, the following operation is performed: Granulocyte markers were detected in the differentiated cell regions to obtain the marker expression levels; Based on the expression level of the marker, the regional detection result is determined, wherein the regional detection result includes: the region is qualified or the region is unqualified; By summarizing the detection results of the aforementioned regions, a set of region detection results is obtained; Based on the region detection result set, ovarian granulosa cell clusters were extracted from the multiple differentiated cell regions.

[0037] It should be explained that the differentiation medium refers to a cell culture medium used to cultivate induced pluripotent stem cells (i.e., DMEM medium). The cultivation of i.e., using the differentiation medium, means that i.e., seeding i.e., culturing ... The differentiated cell layer refers to the pluripotent stem cell layer after differentiation induction, which includes ovarian granulosa cells, ovarian granulosa cell precursor cells, and undifferentiated induced pluripotent stem cells. Subsequent granulosa cell marker detection is required to ensure the extraction of pure ovarian granulosa cells.

[0038] It is understood that dividing the differentiated cell layer into multiple differentiated cell regions means dividing the differentiated cell layer into multiple independent regions of equal size. These independent regions are the differentiated cell regions. The method for dividing the differentiated cell layer is the same as the method for dividing the active cell layer, and will not be elaborated further here. Granulosa cell marker detection refers to the process of using fluorescent staining to fluorescently label antibodies (i.e., granulosa cell marker antibodies) corresponding to ovarian granulosa cell markers (such as FSHR, AMH, etc.), and then detecting the expression level of granulosa cell markers within the differentiated cell regions using the fluorescently labeled granulosa cell marker antibodies. The expression level is defined as the ratio of fluorescently labeled cells in the differentiated cell region to the total number of cells in the differentiated cell region. The method for obtaining the number of fluorescently labeled cells and the total number of cells in the differentiated cell region is the same as the method for obtaining the total number of cells in the initial induced pluripotent stem cell sample, and will not be elaborated further here. The marker expression level refers to the expression level of the granulosa cell markers obtained after granulosa cell marker detection. The region detection result refers to the conclusion drawn after performing region detection and comparing the expression level of markers in differentiated cell regions with a preset expression level threshold (the expression level threshold can be determined according to the user's accuracy requirements for the region detection step), including regions that are qualified and regions that are unqualified. A qualified region is defined as a conclusion where the marker expression level is greater than or equal to the expression level threshold, indicating that the purity of ovarian granulosa cells in the differentiated cell region is high and meets the user's needs. An unqualified region is defined as a conclusion where the marker expression level is less than the expression level threshold, indicating that the purity of ovarian granulosa cells in the differentiated cell region is insufficient. The region detection result set refers to the collection formed by summarizing all region detection results. Extracting ovarian granulosa cell clusters from the multiple differentiated cell regions based on the region detection result set means: based on the region detection results, identifying all qualified analysis regions from multiple analysis regions, collecting ovarian granulosa cells from all qualified analysis regions, and the cell population composed of ovarian granulosa cells from all collected qualified analysis regions is the ovarian granulosa cell cluster.

[0039] S4. Obtain the culture supernatant of the ovarian granulosa cell cluster, and purify the culture supernatant to obtain the initial exosomes.

[0040] Furthermore, obtaining the culture supernatant of the ovarian granulosa cell cluster includes: Based on the ovarian granulosa cell clusters, the centrifugation medium was determined; The ovarian granulosa cell clusters were expanded and cultured using a pre-set exosome culture medium to obtain a high-density granulosa cell layer; A serum-free granulocyte culture medium was obtained, and the high-density granulocyte layer was statically cultured using the serum-free granulocyte culture medium to obtain a static cell system. Obtain a centrifuge unit, and based on the static cell system and centrifugation medium, use the centrifuge unit to perform centrifugation to obtain a preliminary clarified supernatant; The preliminarily clarified supernatant was subjected to vacuum filtration to obtain the culture supernatant.

[0041] It should be understood that determining the centrifugation medium based on the ovarian granulosa cell cluster means selecting a suitable liquid medium for subsequent centrifugation steps according to the biological characteristics of ovarian granulosa cells (such as cell size and density). Optionally, PBS buffer is used as the liquid medium. Expanding the ovarian granulosa cell cluster using a pre-set exosome culture medium to obtain a high-density granulosa cell layer means inoculating the ovarian granulosa cell cluster into an exosome culture medium for proliferation culture, increasing the number of ovarian granulosa cells contained in the exosome culture medium. The resulting monolayer or three-dimensional cell population is the high-density granulosa cell layer. The exosome culture medium refers to a culture medium used to promote the proliferation of ovarian granulosa cells. Optionally, DMEM / F12 medium supplemented with growth factors (such as insulin-like growth factor, transferrin, etc.) is used as the exosome culture medium. The serum-free granulosa cell culture medium refers to a culture medium that does not contain animal serum or serum derivatives, but only contains the basic nutrients (such as amino acids, glucose, vitamins, and inorganic salts) and cell growth factors (such as bFGF) required for the growth and survival of ovarian granulosa cells. This avoids contamination of the subsequent culture supernatant by impurities and proteins in serum, ensuring the purity of exosomes in the culture supernatant. The static cell system obtained by statically culturing the high-density granulosa cell layer using the serum-free granulosa cell culture medium refers to: placing the high-density granulosa cell layer in the serum-free granulosa cell culture medium and culturing it under static conditions (such as without shaking or agitation), allowing the exosomes and cytokines secreted by the ovarian granulosa cells to enter the culture medium. The resulting liquid system containing the secreted products (exosomes, cytokines, etc.) is the static cell system.

[0042] It should be noted that the centrifugation unit refers to the equipment used for centrifuging the static cell system. Optionally, a benchtop high-speed centrifuge can be used as the centrifugation unit. The centrifugation process refers to the process of centrifuging the static cell system using the centrifugation unit. By centrifuging the static cell system, the centrifugal force causes the solid components such as intact cells and cell debris in the static cell system to settle to the bottom of the centrifugation unit, thereby separating the solid components from the suspension containing exosomes. The method of centrifuging the static cell system using the centrifugation unit is existing technology and will not be described in detail here. The preliminary clarified supernatant refers to the suspension containing exosomes obtained after centrifugation. This suspension may contain incompletely settled cell debris and particulate impurities, which require further filtration. The vacuum filtration process refers to filtering the preliminary clarified supernatant using a vacuum filtration device equipped with a filter membrane (such as a vacuum filter equipped with a polyethersulfone microporous filter membrane). Specifically, the preliminary clarified supernatant is added to the vacuum filtration device, and the supernatant is forced through a sterile filter membrane using the vacuum negative pressure to remove residual cell debris and particulate impurities from the culture supernatant. The culture supernatant refers to the preliminary clarified supernatant after vacuum filtration.

[0043] It should be explained that the purification of the culture supernatant to obtain initial exosomes refers to the following: using ultracentrifugation, the centrifugal force generated during high-speed centrifugation is used to separate the exosomes from the remaining components by utilizing the density and size differences between the exosomes and the small molecule impurities and soluble proteins remaining in the culture supernatant. The separated exosomes are the initial exosomes. The remaining components refer to the small molecule impurities and soluble proteins remaining in the culture supernatant. The method of separating exosomes from the remaining components by ultracentrifugation is existing technology and will not be described in detail here.

[0044] S5. Perform quality testing on the initial exosomes to obtain test results, wherein the test results are either qualified or unqualified.

[0045] In detail, the quality testing of the initial exosomes and the resulting test results include: Multiple morphological images were obtained by using an imaging unit to perform multi-field observation of the initial exosomes; The morphological structure statistics of the multiple morphological images were performed to obtain the morphological qualification rate. The initial exosomes were subjected to particle detection using a preset particle analysis method to obtain particle size and particle concentration. By summarizing the morphological qualification rate, particle size, and particle concentration, a set of cell physical characteristics is obtained. If the set of cell physical characteristics meets the preset physical characteristic criteria, chemical characteristic detection is performed on the initial exosomes to obtain the detection results.

[0046] It is understood that the multi-field observation of the initial exosomes using the imaging unit to obtain multiple morphological images refers to: dividing the initial exosomes into multiple equal parts (by dividing the initial exosomes into multiple equal parts, the number of particles in each morphological image can be reduced, simplifying the subsequent morphological structure statistics), obtaining multiple equally divided exosomes, and scanning and imaging each equally divided exosome using the imaging unit; the resulting images of the multiple equally divided exosomes are the multiple morphological images. The morphological structure statistics refer to the process of analyzing the multiple morphological images and calculating the proportion of particles in each morphological image that conform to the morphological characteristics of exosomes (such as circular or cup-shaped vesicles with complete membrane structures) to the total number of particles in the morphological image. The morphological qualification rate refers to the proportion of particles in the obtained morphological images that conform to the morphological characteristics of exosomes to the total number of particles in the morphological images after morphological structure statistics. The process of using a preset particle analysis method to detect particles in the initial exosomes and obtain particle size and concentration involves: adding the initial exosomes to PBS buffer to form a suspension containing the initial exosomes (i.e., exosome suspension); employing nanoparticle tracking analysis to analyze the Brownian motion of particles in the exosome suspension to calculate the particle size distribution and particle concentration (i.e., the total number of exosome particles per unit volume (in mL)); obtaining the number of particles corresponding to each particle size based on the particle size distribution; multiplying the particle size by the corresponding number of particles; and then dividing by the total number of particles to obtain the average particle size. The cell physical characteristic set refers to the set formed by summing the morphological conformity rate, particle size, and particle concentration. The physical characteristic standards refer to pre-set standards used to determine whether the initial exosome physical characteristics are qualified. These standards include: a morphological qualification rate threshold, a particle size range, and a particle concentration threshold. Only when the morphological qualification rate is greater than or equal to the morphological qualification rate threshold, the particle size is within the particle size range, and the particle concentration is greater than or equal to the particle concentration threshold, can the cell physical characteristic set be considered to meet the physical characteristic standards. Otherwise, it is considered not to meet the physical characteristic standards, and the process of obtaining the culture supernatant of the ovarian granulosa cell cluster needs to be repeated. By adjusting the parameters of the centrifugation unit and the pore size of the vacuum filtration device, the static cell system is centrifuged and vacuum filtered again to obtain a new culture supernatant until the cell physical characteristic set of the new culture supernatant meets the physical characteristic standards. Optionally, the morphological qualification rate threshold and particle concentration threshold can be determined according to the user's accuracy requirements for the quality detection steps, and the particle size range can be obtained by consulting existing literature.

[0047] Specifically, the chemical characterization of the initial exosomes to obtain the detection results includes: Proteins were extracted from the initial exosomes to obtain exosome protein lysis buffer; Immunoblotting was performed on exosomal protein lysis buffer using a pre-defined antibody combination to obtain the expression levels of positive and negative markers. The exosome protein lysis buffer was subjected to protein detection to obtain the exosome protein concentration; Based on the exosomal protein concentration, positive marker expression level, and negative marker expression level, the chemical characteristic values ​​of the exosomes were calculated. If the chemical characteristic value of the exosome is less than the preset characteristic threshold, the detection failure will be confirmed as a detection result. If the chemical characteristic value of the exosome is greater than or equal to the characteristic threshold, the detection is considered to have met the standard and is thus confirmed as a detection result.

[0048] It should be understood that protein extraction refers to the process of disrupting the membrane structure of exosomes using a lysis buffer (such as RIPA lysis buffer), allowing proteins in the exosomes to flow out. The method of protein extraction is consistent with the method used for lysing fluorescently labeled samples and will not be elaborated further here. The exosome protein lysis buffer refers to the mixed liquid containing proteins from exosomes obtained after protein extraction. The antibody combination refers to a set of antibodies consisting of antibodies against positive markers (i.e., proteins enriched only in exosomes, such as Alix, CD81, etc.) and antibodies against negative markers (i.e., proteins that should not be present in high-purity exosomes, such as Calnexin, reflecting the purity of exosomes in the exosome protein lysis buffer). Immunoblot detection refers to a method for measuring the protein expression levels of positive and negative markers (represented by the detected signal intensity), which is existing technology and will not be elaborated further here. The positive marker expression level refers to the signal intensity of the positive marker obtained by immunoblotting detection, used to characterize the expression level of characteristic proteins in exosomes. The expression level of the negative biomarker refers to the signal intensity of the negative biomarker obtained by immunoblotting, used to characterize the expression level of proteins that should not be present in exosomes. By obtaining the expression levels of both positive and negative biomarkers, the purity of the exosomes contained in the initial exosomes can be determined. The protein detection refers to the process of determining the total protein content in the exosome protein lysate using a protein quantification method (such as the Coomassie Brilliant Blue method). The method for determining the total protein content in the exosome protein lysate using a protein quantification method (such as the Coomassie Brilliant Blue method) is existing technology and will not be described further here. The exosome protein concentration refers to the concentration of the total protein in the exosome protein lysate, expressed in micrograms per milliliter (μg / mL).

[0049] It should be noted that the exosome chemical characteristic value refers to a quantitative indicator used to represent the overall quality of the initial exosomes. A higher value indicates better quality and higher purity of the initial exosomes. The exosome chemical characteristic value is calculated as follows: The exosome protein concentration is normalized using the Z-score normalization method to obtain the normalized exosome protein concentration, which is then expressed by the formula: Calculate the chemical characteristic values ​​of exosomes, where, Indicates the expression level of positive biomarkers. Indicates the expression level of negative biomarkers. This represents the zero-prevention constant (an extremely small constant, such as 0.001, pre-set to prevent the denominator from being zero). This represents the normalized exosome protein concentration. The characteristic threshold refers to a pre-set critical value for exosome characteristic values. This can be achieved by acquiring multiple exosomes that meet user requirements, performing chemical characteristic detection on each exosome, obtaining multiple exosome chemical characteristic values, and setting the minimum value among these values ​​as the characteristic threshold. The detection result refers to the conclusion drawn after comparing the exosome chemical characteristic value with the characteristic threshold, including whether the detection meets the standard or not. A failure to meet the standard refers to a conclusion obtained when the exosome chemical characteristic value is less than the characteristic threshold, indicating that the initial exosome quality does not meet the user's requirements. A success to meet the standard refers to a conclusion obtained when the exosome chemical characteristic value is greater than or equal to the characteristic threshold, indicating that the initial exosome quality meets the user's requirements.

[0050] S6. If the test result is that the test does not meet the standard, then obtain the adjusted exosomes, use the adjusted exosomes as the initial exosomes, and return to the above steps of performing quality testing on the initial exosomes until the test result meets the standard.

[0051] It should be noted that the adjusted exosomes refer to the step of purifying the culture supernatant when the test result is substandard. By adjusting the relevant process parameters (such as adjusting the speed of the centrifuge unit during ultracentrifugation, changing the lysis buffer during protein extraction, etc.), new initial exosomes are obtained again. The new initial exosomes are the adjusted exosomes.

[0052] S7. When the test result is satisfactory, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

[0053] It should be explained that the target exosomes refer to the initial exosomes whose chemical characteristic values ​​are greater than or equal to the characteristic threshold. When the detection result is that the detection meets the standard, it means that the overall quality of the initial exosomes meets the user's requirements and can be used as the target exosomes.

[0054] To solve the problems described in the background art, the present invention obtains starting material cells, thereby identifying the basic raw materials for preparing exosomes from ovarian granulosa cells through this step. The starting material cells are reprogrammed to obtain initial induced pluripotent stem cells. Based on the initial induced pluripotent stem cells, cell identification is performed to obtain induced pluripotent stem cells. The induced pluripotent stem cells are induced to differentiate directionally to obtain ovarian granulosa cell clusters. The present invention uses a first-stage differentiation inducer and a second-stage differentiation inducer to induce the differentiation of induced pluripotent stem cells, causing the induced pluripotent stem cells to differentiate into ovarian granulosa cells. By detecting granulosa cell markers in the differentiated cell regions, the purity of ovarian granulosa cells in each differentiated cell region is determined, and then a high-purity ovarian granulosa cell cluster is obtained. The culture supernatant of the ovarian granulosa cell cluster is purified to obtain initial exosomes, and quality detection is performed on the initial exosomes to obtain a detection result. Among them, the detection result is either qualified or unqualified. By performing quality detection on the initial exosomes, the present invention controls the quality of the initial exosomes and improves the purity of the finally obtained exosomes. If the detection result is unqualified, adjusted exosomes are obtained, the adjusted exosomes are used as the initial exosomes, and the above step of performing quality detection on the initial exosomes is repeated until the detection result is qualified. When the detection result is qualified, the initial exosomes are confirmed as target exosomes, and the exosome preparation is completed. Therefore, the present invention can solve the problem of low exosome purity in the current preparation process of ovarian granulosa cell exosomes.

[0055] As Figure 2 shown, it is a functional module diagram of an ovarian granulosa cell exosome preparation system based on cell-directed differentiation provided by an embodiment of the present invention.

[0056] The ovarian granulosa cell exosome preparation system 100 based on cell-directed differentiation described in the present invention can be installed in an electronic device. According to the functions achieved, the ovarian granulosa cell exosome preparation system 100 based on cell-directed differentiation can include a cell reprogramming module 101, a cell identification and differentiation module 102, an exosome purification module 103, and an exosome quality detection module 104. The modules described in the present invention can also be referred to as units, which refer to a series of computer program segments that can be executed by a processor of an electronic device and can complete fixed functions, and are stored in the memory of the electronic device.

[0057] The cell reprogramming module 101 is used to obtain starting material cells and reprogram the starting material cells to obtain initial induced pluripotent stem cells; The cell identification and differentiation module 102 is used to identify cells based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells, and to perform directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters. The exosome purification module 103 is used to obtain the culture supernatant of the ovarian granulosa cell cluster, and to purify the culture supernatant to obtain initial exosomes; The exosome quality detection module 104 is used to perform quality detection on the initial exosomes and obtain a detection result, wherein the detection result is either qualified or unqualified. If the detection result is unqualified, an adjusted exosome is obtained and used as the initial exosome. The process of performing quality detection on the initial exosome is then repeated until the detection result is qualified. When the detection result is qualified, the initial exosome is confirmed as the target exosome, and the exosome preparation is completed.

[0058] In detail, the modules in the ovarian granulosa cell exosome preparation system 100 based on cell-directed differentiation described in this embodiment of the invention employ the same methods as described above. Figure 1 The method used is the same as the method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation described in the article, and can produce the same technical effect, so it will not be repeated here.

[0059] like Figure 3 The diagram shown is a schematic representation of an electronic device for implementing a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, according to an embodiment of the present invention.

[0060] The electronic device 1 may include a processor 10, a memory 11 and a bus 12, and may also include a computer program stored in the memory 11 and executable on the processor 10, such as a method program for preparing ovarian granulosa cell exosomes based on cell-directed differentiation.

[0061] The memory 11 includes at least one type of readable storage medium, including flash memory, portable hard drive, multimedia card, card-type memory (e.g., SD or DX memory), magnetic memory, disk, optical disk, etc. In some embodiments, the memory 11 can be an internal storage unit of the electronic device 1, such as the portable hard drive of the electronic device 1. In other embodiments, the memory 11 can be an external storage device of the electronic device 1, such as a plug-in portable hard drive, smart media card (SMC), secure digital (SD) card, flash card, etc., equipped on the electronic device 1. Furthermore, the memory 11 includes both internal storage units and external storage devices of the electronic device 1. The memory 11 can be used not only to store application software and various types of data installed on the electronic device 1, such as the code of a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, but also to temporarily store data that has been output or will be output.

[0062] In some embodiments, the processor 10 may be composed of integrated circuits, such as a single packaged integrated circuit or multiple integrated circuits with the same or different functions, including combinations of one or more central processing units (CPUs), microprocessors, digital processing chips, graphics processors, and various control chips. The processor 10 is the control unit of the electronic device, connecting various components of the entire electronic device through various interfaces and lines. It executes programs or modules stored in the memory 11 (e.g., a method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation) and calls data stored in the memory 11 to perform various functions of the electronic device 1 and process data.

[0063] The bus 12 can be a peripheral component interconnect (PCI) bus or an extended industry standard architecture (EISA) bus, etc. The bus 12 can be divided into an address bus, a data bus, a control bus, etc. The bus 12 is configured to realize the connection and communication between the memory 11 and at least one processor 10, etc.

[0064] Figure 3 Only electronic devices with components are shown; those skilled in the art will understand that... Figure 3The structure shown does not constitute a limitation on the electronic device 1, and may include fewer or more components than shown, or combine certain components, or have different component arrangements.

[0065] For example, although not shown, the electronic device 1 may also include a power supply (such as a battery) to power the various components. Preferably, the power supply can be logically connected to the at least one processor 10 through a power management device, thereby enabling functions such as charging management, discharging management, and power consumption management. The power supply may also include one or more DC or AC power supplies, recharging devices, power fault detection circuits, power converters or inverters, power status indicators, and other arbitrary components. The electronic device 1 may also include various sensors, Bluetooth modules, Wi-Fi modules, etc., which will not be described in detail here.

[0066] Furthermore, the electronic device 1 may also include a network interface. Optionally, the network interface may include a wired interface and / or a wireless interface (such as a Wi-Fi interface, a Bluetooth interface, etc.), which is typically used to establish communication connections between the electronic device 1 and other electronic devices.

[0067] Optionally, the electronic device 1 may further include a user interface, which may be a display, an input unit (such as a keyboard), and optionally, a standard wired interface or a wireless interface. Optionally, in some embodiments, the display may be an LED display, a liquid crystal display, a touch-sensitive liquid crystal display, or an OLED (Organic Light-Emitting Diode) touchscreen, etc. The display may also be appropriately referred to as a screen or display unit, used to display information processed in the electronic device 1 and to display a visual user interface.

[0068] The program for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, stored in the memory 11 of the electronic device 1, is a combination of multiple instructions. When run in the processor 10, it can achieve the following: Obtaining starting material cells; The starting material cells were reprogrammed to obtain initial induced pluripotent stem cells; Cell identification was performed based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; The induced pluripotent stem cells were directed to differentiate into ovarian granulosa cell clusters. Obtain the culture supernatant of the ovarian granulosa cell cluster; The culture supernatant was purified to obtain initial exosomes; The initial exosomes are subjected to quality testing to obtain test results, wherein the test results are either qualified or unqualified. If the test result is that the test does not meet the standard, then the adjusted exosomes are obtained, the adjusted exosomes are used as the initial exosomes, and the process of performing quality testing on the initial exosomes is returned until the test result meets the standard. When the test result is satisfactory, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

[0069] Specifically, the processor 10's implementation method for the above instructions can be found in [reference needed]. Figures 1 to 3 The descriptions of the relevant steps in the corresponding embodiments are not repeated here.

[0070] Furthermore, if the modules / units integrated in the electronic device 1 are implemented as software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. The computer-readable storage medium can be volatile or non-volatile. For example, the computer-readable medium may include: any entity or device capable of carrying the computer program code, a recording medium, a USB flash drive, a portable hard drive, a magnetic disk, an optical disk, a computer memory, or a read-only memory (ROM).

[0071] The present invention also provides a computer-readable storage medium storing a computer program, which, when executed by a processor of an electronic device, can perform the following: Obtaining starting material cells; The starting material cells were reprogrammed to obtain initial induced pluripotent stem cells; Cell identification was performed based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; The induced pluripotent stem cells were directed to differentiate into ovarian granulosa cell clusters. Obtain the culture supernatant of the ovarian granulosa cell cluster; The culture supernatant was purified to obtain initial exosomes; The initial exosomes are subjected to quality testing to obtain test results, wherein the test results are either qualified or unqualified. If the test result is that the test does not meet the standard, then the adjusted exosomes are obtained, the adjusted exosomes are used as the initial exosomes, and the process of performing quality testing on the initial exosomes is returned until the test result meets the standard. When the test result is satisfactory, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

[0072] In the embodiments provided by this invention, it should be understood that the disclosed devices, systems, and methods can be implemented in other ways. For example, the system embodiments described above are merely illustrative, and actual implementations may have other classification methods.

[0073] The modules described as separate components may or may not be physically separate. The components shown as modules may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs.

[0074] Furthermore, the functional modules in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or in the form of hardware plus software functional modules.

[0075] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, characterized in that, The method includes: Obtaining starting material cells; The starting material cells were reprogrammed to obtain initial induced pluripotent stem cells; Cell identification was performed based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; The induced pluripotent stem cells were directed to differentiate into ovarian granulosa cell clusters. Obtain the culture supernatant of the ovarian granulosa cell cluster; The culture supernatant was purified to obtain initial exosomes; The initial exosomes are subjected to quality testing to obtain test results, wherein the test results are either qualified or unqualified. If the test result is that the test does not meet the standard, then the adjusted exosomes are obtained, the adjusted exosomes are used as the initial exosomes, and the process of performing quality testing on the initial exosomes is returned until the test result meets the standard. When the test result is satisfactory, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

2. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 1, characterized in that, The reprogramming of the starting material cells to obtain initial induced pluripotent stem cells includes: Obtain culture units; Based on the culture unit, the starting material cells are seeded to obtain a starting cell layer; The starting cell layer is activated to obtain an activated starting cell layer; The activated initiating cell layer is programmed using a preset reprogramming factor to obtain a mixed cell community; Initially induced pluripotent stem cells were identified from the mixed cell community.

3. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 2, characterized in that, The activation treatment of the initiating cell layer to obtain an activated initiating cell layer includes: The activity of the initial cell layer is enhanced by using a preset cell activator to obtain an active cell layer; The metabolic state of the viable cell layer was detected to obtain a set of metabolic characteristic information. For each metabolic feature in the set of metabolic feature information, the following operation is performed: Based on the metabolic characteristic information, a metabolic determination result is obtained; Summarize the metabolic determination results to obtain the metabolic determination result set; Based on the metabolic determination result set, an active region set is selected from the active cell layer; Based on the set of active regions, the activation initiation cell layer is obtained.

4. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 3, characterized in that, The metabolic state detection of the active cell layer yields a set of metabolic feature information, including: The active cell layer was divided into regions to obtain multiple active cell regions; For each of the plurality of active cell regions, the following operation is performed: Based on the viable cell region, obtain viable cell samples; Obtain the first fluorescent probe and the second fluorescent probe; The viable cell sample was fluorescently labeled using the first fluorescent probe and the second fluorescent probe to obtain a fluorescently labeled sample. Fluorescence detection was performed on the fluorescently labeled sample to obtain a first fluorescence intensity sequence and a second fluorescence intensity sequence; Based on the first fluorescence intensity sequence and the second fluorescence intensity sequence, glucose uptake parameters and mitochondrial membrane potential parameters are obtained. The fluorescently labeled sample was lysed to obtain lysate; The lysed cell solution was incubated in the dark using a pre-set ATP fluorescent reagent to obtain the ATP concentration; Based on the glucose uptake parameters, mitochondrial membrane potential parameters, and ATP concentration, metabolic characteristic information is constructed. The metabolic feature information is summarized to obtain a metabolic feature information set.

5. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 4, characterized in that, The process of cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells includes: Based on the initial induced pluripotent stem cells, obtain initial induced pluripotent stem cell samples; Identify pluripotency markers, wherein the pluripotency markers include: a first marker and a second marker; Based on the initial induced pluripotent stem cell samples, the positive expression of the first and second markers among the pluripotency markers was detected using a pre-set activity detection device to obtain the positive rate of the first marker and the positive rate of the second marker. Obtain alkaline phosphatase reagent and use alkaline phosphatase reagent to stain the initial induced pluripotent stem cell samples to obtain stained cell samples; The stained cell sample is scanned and imaged using a preset imaging unit to obtain a stained microscopic image; Based on the stained microscopic images, the phosphatase positivity rate was obtained; A set of identification indicators was constructed based on the positive rates of the first biomarker, the second biomarker, and the phosphatase. When the set of identification indicators does not meet the preset identification criteria, adjust induced pluripotent stem cells are obtained, and the adjusted induced pluripotent stem cells are used as initial induced pluripotent stem cells. Then, the above steps of obtaining initial induced pluripotent stem cell samples based on the initial induced pluripotent stem cells are returned until the set of identification indicators meets the identification criteria. When the set of identification indicators meets the identification criteria, the initial induced pluripotent stem cells are identified as induced pluripotent stem cells.

6. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 5, characterized in that, The directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters includes: Obtain differentiation culture medium; Induced pluripotent stem cells were cultured using the aforementioned differentiation medium to obtain a pluripotent stem cell layer; Obtain the first-stage differentiation inducer and the second-stage differentiation inducer; The pluripotent stem cell layer was induced to differentiate using the first-stage differentiation inducer and the second-stage differentiation inducer to obtain a differentiated cell layer. The differentiated cell layer is divided into multiple differentiated cell regions; For each of the plurality of differentiated cell regions, the following operation is performed: Granulocyte markers were detected in the differentiated cell regions to obtain the marker expression levels; Based on the expression level of the marker, the regional detection result is determined, wherein the regional detection result includes: the region is qualified or the region is unqualified; By summarizing the detection results of the aforementioned regions, a set of region detection results is obtained; Based on the region detection result set, ovarian granulosa cell clusters were extracted from the multiple differentiated cell regions.

7. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 6, characterized in that, The process of obtaining the culture supernatant of the ovarian granulosa cell mass includes: Based on the ovarian granulosa cell clusters, the centrifugation medium was determined; The ovarian granulosa cell clusters were expanded and cultured using a pre-set exosome culture medium to obtain a high-density granulosa cell layer; A serum-free granulocyte culture medium was obtained, and the high-density granulocyte layer was statically cultured using the serum-free granulocyte culture medium to obtain a static cell system. Obtain a centrifuge unit, and based on the static cell system and centrifugation medium, use the centrifuge unit to perform centrifugation to obtain a preliminary clarified supernatant; The preliminarily clarified supernatant was subjected to vacuum filtration to obtain the culture supernatant.

8. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 7, characterized in that, The quality testing of the initial exosomes, and the resulting test results, include: Multiple morphological images were obtained by using an imaging unit to perform multi-field observation of the initial exosomes; The morphological structure statistics of the multiple morphological images were performed to obtain the morphological qualification rate. The initial exosomes were subjected to particle detection using a preset particle analysis method to obtain particle size and particle concentration. By summarizing the morphological qualification rate, particle size, and particle concentration, a set of cell physical characteristics is obtained. If the set of cell physical characteristics meets the preset physical characteristic criteria, chemical characteristic detection is performed on the initial exosomes to obtain the detection results.

9. The method for preparing ovarian granulosa cell exosomes based on cell-directed differentiation as described in claim 8, characterized in that, The chemical characterization of the initial exosomes, and the resulting detection results, include: Proteins were extracted from the initial exosomes to obtain exosome protein lysis buffer; Immunoblotting was performed on exosomal protein lysis buffer using a pre-defined antibody combination to obtain the expression levels of positive and negative markers. The exosome protein lysis buffer was subjected to protein detection to obtain the exosome protein concentration; Based on the exosomal protein concentration, positive marker expression level, and negative marker expression level, the chemical characteristic values ​​of the exosomes were calculated. If the chemical characteristic value of the exosome is less than the preset characteristic threshold, the detection failure will be confirmed as a detection result. If the chemical characteristic value of the exosome is greater than or equal to the characteristic threshold, the detection is considered to have met the standard and is thus confirmed as a detection result.

10. A system for preparing ovarian granulosa cell exosomes based on cell-directed differentiation, characterized in that, The system includes: A cell reprogramming module is used to obtain starting material cells and reprogram the starting material cells to obtain initial induced pluripotent stem cells; The cell identification and differentiation module is used to identify cells based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells, and to perform directed differentiation of the induced pluripotent stem cells to obtain ovarian granulosa cell clusters. The exosome purification module is used to obtain the culture supernatant of the ovarian granulosa cell cluster, and to purify the culture supernatant to obtain initial exosomes; The exosome quality detection module is used to perform quality detection on the initial exosomes and obtain detection results, wherein the detection results are either qualified or unqualified. If the detection result is unqualified, the adjusted exosomes are obtained and used as the initial exosomes. The process of performing quality detection on the initial exosomes is then repeated until the detection result is qualified. When the detection result is qualified, the initial exosomes are identified as the target exosomes, and the exosome preparation is completed.

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