Preparation optimization method and system of ovarian granular cell exosome based on iPSC

Abstract

The invention relates to the technical field of cell exosome preparation, in particular to an optimization method and system for preparation of ovarian granular cell exosomes based on iPSC (induced pluripotent stem cells). Basic culture parameters are determined based on the human pluripotent stem cells, the optimal induction factors and the optimal concentration, directional culture is conducted on the human pluripotent stem cells through the basic culture parameters, a differentiated cell population is obtained, marker detection is conducted on the differentiated cell population, and the anti-mullerian hormone positive rate and the second gene positive rate are obtained. Performing multiplication culture on the target ovarian granular cells to obtain a cell culture supernatant, and separating the cell culture supernatant by utilizing the optimal separation parameters to obtain an exosome extract, thereby completing the optimization of exosome preparation. The intelligent degree of the exosome preparation process can be improved.

Description

Technical Field

[0001] This invention relates to the field of cell exosome preparation technology, and in particular to an optimized method and system for preparing ovarian granulosa cell exosomes based on iPSCs. Background Technology

[0002] Exosomes are small membrane vesicles secreted by cells that can be widely distributed in body fluids and play important biological functions.

[0003] Currently, there are various methods for preparing exosomes, including ultracentrifugation, immunoaffinity assays, and ultrafiltration, among which ultracentrifugation is the most commonly used method. Centrifugation allows for the efficient separation and purification of exosomes from culture media.

[0004] While the methods described above can achieve exosome preparation, traditional ultracentrifugation, although capable of yielding high-purity exosomes, is difficult to apply in large-scale production due to its complexity, time-consuming nature, and the need for expensive equipment. Furthermore, existing preparation methods often fail to effectively control exosome quality, leading to significant variations in exosome activity across different batches, and lack sufficient automation. Therefore, improving the automation level of exosome preparation processes has become an urgent problem to be solved. Summary of the Invention

[0005] This invention provides an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs and a computer-readable storage medium, the main purpose of which is to improve the intelligence level of the exosome preparation process.

[0006] To achieve the above objectives, this invention provides an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs, comprising: Obtain human pluripotent stem cells, and identify the optimal inducing factors and concentrations based on human pluripotent stem cells; The basic culture parameters were determined based on human pluripotent stem cells, the optimal inducing factor, and the optimal concentration. Human pluripotent stem cells were cultured in a targeted manner using basic culture parameters to obtain a differentiated cell population; Biomarker detection was performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone and second gene; Compare the positive rate of anti-Müllerian hormone with a preset hormone positive rate threshold, and compare the positive rate of the second gene with a preset second gene positive rate threshold; If the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold, and the positive rate of the second gene is greater than or equal to the gene positive rate threshold, then the differentiated cell population will be used as the target ovarian granulosa cells. Otherwise, the differentiated cell population is taken as human pluripotent stem cells, and the modified culture parameters are identified based on the basic culture parameters. The modified culture parameters are taken as the basic culture parameters, and the process is repeated until the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold and the positive rate of the second gene is greater than or equal to the gene positive rate threshold. The target ovarian granulosa cells were expanded and cultured to obtain cell culture supernatant. The optimal separation parameters were determined based on cell culture supernatant. The cell culture supernatant was separated using the optimal separation parameters to obtain exosome extracts, thus completing the optimization of exosome preparation.

[0007] Optionally, the identification of the optimal inducing factor and optimal concentration based on human pluripotent stem cells includes: Commonly used inducing factors were identified, including: factor one, factor two, and factor three. Based on human pluripotent stem cells, factor one, and a preset first sampling interval, the first optimal test score and the first optimal concentration were determined. Based on human pluripotent stem cells, factor 2, and a pre-defined second sampling range, a second optimal test score and a second optimal concentration were determined. Based on human pluripotent stem cells, the third factor, and the pre-defined third sampling range, the third optimal test score and the third optimal test concentration were determined. The optimal inducing factor and optimal concentration were determined based on the first optimal test score, the first optimal concentration, the second optimal test score, the second optimal concentration, the third optimal test score, and the third optimal test concentration.

[0008] Optionally, the step of determining the first optimal test score and the first optimal concentration based on human pluripotent stem cells, the first factor, and a preset first sampling interval includes: Once the concentration range of the first factor is identified, samples are taken uniformly within the first factor concentration range according to the first sampling interval to obtain multiple culture concentrations. Perform the following operation for each of the multiple culture concentrations: Human pluripotent stem cells were sampled to obtain test samples; Obtain standard culture medium, and identify the target culture medium based on the culture concentration and the standard culture medium; The test samples were cultured using the target culture medium to obtain cultured samples; Gene expression rates were detected in culture samples to obtain the first target expression rate, the second target expression rate, and the third target expression rate. The first test score is calculated based on the expression rates of the first, second, and third targets. The scores from the first test are combined to obtain multiple first test scores; The first best test score is determined based on multiple first test scores, where the first best test score is the largest first test score among multiple first test scores; The culture concentration corresponding to the first best test score is taken as the first best concentration.

[0009] Optionally, the determination of basic culture parameters based on human pluripotent stem cells, optimal inducing factors, and optimal concentrations includes: Obtain a second standard culture medium, and add the optimal inducing factor to the second standard culture medium at the optimal concentration to obtain the second target culture medium; Human pluripotent stem cells were briefly cultured using a second target culture medium to obtain cultured stem cells; Culture control parameters were identified based on human pluripotent stem cells, including: oxygen concentration range, seeding density range, and seeding time range. Multiple culture test groups were identified based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters. Perform the following operation on each of the multiple culture test groups: Samples were taken from the cultured stem cells to obtain stem cell samples; The test culture medium was identified based on the culture test group; Differentiated stem cells were obtained by culturing stem cell samples in a test culture medium. Basic tests were performed on differentiated stem cells to obtain test data; The cultivation effect score was determined based on the test data; The cultivation effect scores are summarized to obtain multiple cultivation effect scores; The optimal performance score was determined based on multiple performance scores, where the optimal performance score is the largest among the multiple performance scores. The basic culture parameters were determined based on the best performance score. These parameters included: basic oxygen concentration, basic inoculation density, and basic inoculation time.

[0010] Optionally, multiple culture test groups are identified based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters, including: The oxygen concentration range in the culture control parameters was divided into three levels to obtain low oxygen concentration, medium oxygen concentration and high oxygen concentration. The inoculation density range in the culture control parameters was divided into three levels to obtain low inoculation density, medium inoculation density and high inoculation density; Nine preliminary test combinations were obtained by pairing low oxygen concentration, medium oxygen concentration, and high oxygen concentration with low inoculation density, medium inoculation density, and high inoculation density. The vaccination time range is sampled evenly according to the preset second interval to obtain multiple vaccination times; The nine preliminary test combinations were combined with multiple inoculation times to obtain multiple culture test groups, each of which included: oxygen concentration, inoculation density, and inoculation time.

[0011] Optionally, the basic testing of differentiated stem cells to obtain test data includes: Differentiated stem cells were sampled to obtain stem cell samples to be tested; The stem cell samples to be tested are pretreated to obtain processed samples; The cell viability index was obtained by performing a viability test on the treated samples. RNA was extracted from the processed samples to obtain total RNA. Core gene detection was performed on the total RNA of the sample to obtain the first core expression level and the second core expression level. Gene expression indices were determined based on the first and second core expression levels, where the gene expression index is the geometric mean of the first and second core expression levels. Biochemical tests were performed on the samples to be treated to obtain the glucose consumption and lactic acid production. The cell metabolic activity index is calculated based on glucose consumption and lactate production. The cell viability index, gene expression index, and cell metabolic activity index were summarized to obtain the detection data.

[0012] Optionally, the step of determining the cultivation effect score based on the detection data includes: The culture effect score is calculated based on the cell viability index, gene expression index, and cell metabolic activity index from the test data. The calculation formula is shown below: in, Indicates the score for cultivation effectiveness. Indicates cell viability index, Indicates gene expression index, This represents the cell metabolic activity index.

[0013] Optionally, the step of determining the modified culture parameters based on the basic culture parameters includes: Culture defect types were identified based on the positive rates of anti-Müllerian hormone, second gene, hormone positivity threshold, and second gene positivity threshold. Among them, culture defect types include: insufficient anti-Müllerian hormone positivity rate, insufficient second gene positivity rate, or both. When the culture defect type is insufficient positive rate of anti-Müllerian hormone, the basic oxygen concentration in the basic culture parameters is increased by the preset first adjustment step to obtain the corrected oxygen concentration. The corrected culture parameters were obtained using the corrected oxygen concentration, the basic inoculation density in the basic culture parameters, and the basic inoculation time in the basic culture parameters. When the culture defect type is insufficient positive rate of the second gene, the basic inoculation density in the basic culture parameters is increased by a preset second adjustment step to obtain the corrected inoculation density; The corrected culture parameters were obtained using the corrected inoculation density, the basal oxygen concentration in the basal culture parameters, and the basal inoculation time in the basal culture parameters. When the culture defect type is that both are insufficient, the basic inoculation time in the basic culture parameters is increased by a preset third adjustment step to obtain the corrected inoculation time; The corrected culture parameters were obtained by using the corrected inoculation time, the basic inoculation density in the basic culture parameters, and the basic oxygen concentration in the basic culture parameters.

[0014] Optionally, the determination of optimal separation parameters based on cell culture supernatant includes: The cell culture supernatant was subjected to material analysis to obtain the total protein concentration and cell sap viscosity. Calculate the ultracentrifugal force based on the total protein concentration; The elution flow rate is calculated based on the cell fluid viscosity, using the following formula: in, Indicates the elution flow rate. The preset ideal maximum elution flow rate, Indicates cell sap viscosity, Indicates the preset reference cell sap viscosity; The optimal separation parameters were obtained by summing up the ultracentrifugal force and elution flow rate.

[0015] To achieve the above objectives, the present invention also provides an optimized system for the preparation of ovarian granulosa cell exosomes based on iPSCs, comprising: The basic parameter confirmation module is used to obtain human pluripotent stem cells, confirm the optimal inducing factor and optimal concentration based on human pluripotent stem cells, and confirm the basic culture parameters based on human pluripotent stem cells, optimal inducing factor and optimal concentration. The differentiation population acquisition module is used to perform targeted culture of human pluripotent stem cells using basic culture parameters to obtain a differentiated cell population. The differentiated cell population is then subjected to biomarker detection to obtain the positive rate of anti-Müllerian hormone and the positive rate of the second gene. The culture parameter correction module is used to compare the anti-Müllerian hormone positivity rate with a preset hormone positivity rate threshold and the second gene positivity rate with a preset second gene positivity rate threshold. If the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold, then the differentiated cell population is taken as the target ovarian granulosa cells; otherwise, the differentiated cell population is taken as human pluripotent stem cells. Based on the basic culture parameters, the corrected culture parameters are identified and used as the basic culture parameters. The process returns to the step of directional culture of human pluripotent stem cells using the basic culture parameters until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold. An optimization processing module is used to expand and culture target ovarian granulosa cells to obtain cell culture supernatant. Based on the cell culture supernatant, the optimal separation parameters are identified. The cell culture supernatant is then separated using the optimal separation parameters to obtain exosome extract, thus completing the optimization of exosome preparation.

[0016] To address the above problems, the present invention also provides an electronic device, the electronic device comprising: Memory, storing at least one instruction; and The processor executes the instructions stored in the memory to implement the above-described optimized method for preparing ovarian granulosa cell exosomes based on iPSCs.

[0017] 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 optimized method for preparing ovarian granulosa cell exosomes based on iPSCs.

[0018] To address the problems described in the background art, this invention obtains human pluripotent stem cells and identifies the optimal inducing factors and concentrations based on these cells. This invention intelligently matches the most efficient differentiation induction protocol to a specific stem cell line, laying the foundation for the subsequent targeted and efficient generation of target cell types. Furthermore, based on the human pluripotent stem cells, optimal inducing factors, and optimal concentrations, basic culture parameters are identified. This invention translates optimal induction conditions into precisely executable culture operation instructions, establishing a standardized initial differentiation process. Using these basic culture parameters, human pluripotent stem cells are directionally cultured to obtain a differentiated cell population. This invention, by initiating and executing the differentiation program under controlled conditions, enables stem cells to differentiate. Cells develop in a direction aligned with the fate of ovarian granulosa cells, yielding preliminary cell products. This improves the automation level of the exosome preparation process. Biomarker detection is performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone (AMH) and the second gene. This embodiment of the invention utilizes multiple molecular biomarkers for quantitative identification of differentiation products, objectively assessing differentiation efficiency and cell type accuracy. The positive rates of AMH and the second gene are compared with preset thresholds. This embodiment of the invention provides clear quantitative evidence for automated determination of differentiation success by setting strict quality standards. If the positive rate of AMH is greater than or equal to the threshold, and the second gene is positive... If the positive rate is greater than or equal to the gene positivity rate threshold, the differentiated cell population is used as the target ovarian granulosa cells. This embodiment of the invention automatically confirms the acquisition of high-quality, high-purity target cells for downstream preparation when the detection results simultaneously meet dual criteria, thus improving the intelligence level of the exosome preparation process. Otherwise, the differentiated cell population is used as human pluripotent stem cells, and corrected culture parameters are determined based on the basic culture parameters. These corrected culture parameters are then used as the basic culture parameters, and the process returns to the step of directional culture of human pluripotent stem cells using the basic culture parameters, until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold, and the second gene positivity rate is greater than or equal to the gene positivity rate threshold. This embodiment of the invention establishes a detection-judgment-optimization mechanism. The intelligent feedback loop automatically adjusts culture parameters and restarts the differentiation process when differentiation fails to meet the target until qualified cells are produced, ensuring the stability and reliability of the final cell product quality. This improves the intelligence level of the exosome preparation process. The target ovarian granulosa cells are expanded and cultured to obtain cell culture supernatant. This embodiment of the invention provides sufficient raw materials for exosome enrichment by scaling up the identified qualified cells and collecting the culture medium containing their secretions. Based on the cell culture supernatant, the optimal separation parameters are determined. This embodiment of the invention dynamically optimizes the core parameters of separation techniques such as ultracentrifugation and size exclusion chromatography according to the characteristics of the supernatant, such as volume and protein concentration, to achieve high yield and high purity recovery of exosomes.By using optimal separation parameters to separate the cell culture supernatant, exosome extracts were obtained, thus optimizing exosome preparation. It is evident that this invention, through the execution of an optimized separation and purification process, ultimately yields high-purity, highly active exosomes derived from ovarian granulosa cells. This achieves full-process optimization and quality control from intelligent cell differentiation to automated product preparation, improving the intelligence level of the exosome preparation process. Therefore, this invention can enhance the intelligence level of the exosome preparation process. Attached Figure Description

[0019] Figure 1 This is a schematic flowchart of an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs, according to an embodiment of the present invention. Figure 2 This is a functional block diagram of an optimized system for the preparation of ovarian granulosa cell exosomes based on iPSCs, provided in an embodiment of the present invention. Figure 3 This is a schematic diagram of an electronic device for implementing the optimized method for preparing ovarian granulosa cell exosomes based on iPSC, according to an embodiment of the present invention.

[0020] Explanation of reference numerals in the attached figures: 10. Electronic device; 11. Processor; 12. Memory; 13. Bus.

[0021] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] This application provides an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs. The execution entity of this optimized 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 optimized method for preparing ovarian granulosa cell exosomes based on iPSCs 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.

[0024] Reference Figure 1 The diagram shown is a flowchart illustrating an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs, according to an embodiment of the present invention. In this embodiment, the optimized method for preparing ovarian granulosa cell exosomes based on iPSCs includes: S1. Obtain human pluripotent stem cells, and identify the optimal inducing factors and concentrations based on human pluripotent stem cells.

[0025] For example, Xiao Zhang is a staff member of an exosome preparation laboratory. He needs to use human pluripotent stem cells to prepare ovarian granulosa cell exosomes. Therefore, Xiao Zhang obtains human pluripotent stem cells to facilitate subsequent culture of human pluripotent stem cells and then prepare ovarian granulosa cell exosomes.

[0026] It should be explained that human pluripotent stem cells refer to human pluripotent cells.

[0027] Specifically, the identification of the optimal inducing factors and optimal concentrations based on human pluripotent stem cells includes: Commonly used inducing factors were identified, including: factor one, factor two, and factor three. Based on human pluripotent stem cells, factor one, and a preset first sampling interval, the first optimal test score and the first optimal concentration were determined. Based on human pluripotent stem cells, factor 2, and a pre-defined second sampling range, a second optimal test score and a second optimal concentration were determined. Based on human pluripotent stem cells, the third factor, and the pre-defined third sampling range, the third optimal test score and the third optimal test concentration were determined. The optimal inducing factor and optimal concentration were determined based on the first optimal test score, the first optimal concentration, the second optimal test score, the second optimal concentration, the third optimal test score, and the third optimal test concentration.

[0028] It should be explained that commonly used inducing factors are the most widely used substances in the culture of human pluripotent stem cells, and these commonly used inducing factors include: factor one, factor two, and factor three. Factor one is bone morphogenetic protein, factor two is follicle-stimulating hormone (FSH), and factor three is anti-Müllerian hormone (AMH). These commonly used inducing factors are common knowledge to those skilled in the art. The concentration range of factor one refers to the adjustable range of the amount of factor one added during the culture of human pluripotent stem cells.

[0029] It should be understood that the method for determining the second optimal test score and the second optimal concentration based on human pluripotent stem cells, the second factor, and the preset second sampling range, and the method for determining the third optimal test score and the third optimal test concentration based on human pluripotent stem cells, the third factor, and the preset third sampling range are all the same as the method for determining the first optimal test score and the first optimal concentration based on human pluripotent stem cells, the first factor, and the preset first sampling interval. These will not be repeated here. Furthermore, the specific method for determining the first optimal test score and the first optimal concentration based on human pluripotent stem cells, the first factor, and the preset first sampling interval can be found in subsequent embodiments.

[0030] It is understood that the determination of the optimal inducing factor and optimal concentration based on the first optimal test score, first optimal concentration, second optimal test score, second optimal concentration, third optimal test score, and third optimal test concentration means: firstly, the highest score is selected from the first optimal test score, second optimal test score, and third optimal test score, and the inducing factor corresponding to the highest score is taken as the optimal inducing factor; and the concentration corresponding to the highest test score among the first optimal concentration, second optimal concentration, and third optimal concentration is taken as the optimal concentration. If the first optimal test score is equal to the second optimal test score, then the concentration with the smaller optimal concentration is selected as the optimal concentration, and the inducing factor corresponding to the smaller optimal concentration is taken as the optimal inducing factor.

[0031] For example, if the first best test score is 80 and the first best concentration is 2%, the second best test score is 85 and the second best concentration is 3%, and the third best test score is 90 and the third best test concentration is 4%, then the best inducing factor determined based on the first best test score, first best concentration, second best test score, second best concentration, third best test score, and third best test concentration is the third factor, and the best concentration is 4%. If the first best test score is 80 and the first best concentration is 2%, the second best test score is 90 and the second best concentration is 3%, and the third best test score is 90 and the third best test concentration is 4%, then the best inducing factor determined based on the first best test score, first best concentration, second best test score, second best concentration, third best test score, and third best test concentration is the second factor, and the best concentration is 3%.

[0032] Specifically, the determination of the first optimal test score and the first optimal concentration based on human pluripotent stem cells, the first factor, and a preset first sampling interval includes: Once the concentration range of the first factor is identified, samples are taken uniformly within the first factor concentration range according to the first sampling interval to obtain multiple culture concentrations. Perform the following operation for each of the multiple culture concentrations: Human pluripotent stem cells were sampled to obtain test samples; Obtain standard culture medium, and identify the target culture medium based on the culture concentration and the standard culture medium; The test samples were cultured using the target culture medium to obtain cultured samples; Gene expression rates were detected in culture samples to obtain the first target expression rate, the second target expression rate, and the third target expression rate. The first test score is calculated based on the expression rates of the first, second, and third targets, using the following formula: in, This indicates the score of the first test. Indicates the expression rate of the first target. Indicates the expression rate of the second target. Indicates the expression rate of the third target; The scores from the first test are combined to obtain multiple first test scores; The first best test score is determined based on multiple first test scores, where the first best test score is the largest first test score among multiple first test scores; The culture concentration corresponding to the first best test score is taken as the first best concentration.

[0033] It should be explained that the concentration range of the first factor is an adjustable range of the concentration of the first factor added during exosome preparation. The concentration range of the first factor can be obtained from the product technical manual provided by the manufacturer of the first factor. For example, if the concentration range of the first factor is 1%–5% and the first sampling interval is 1%, then after uniformly sampling the concentration range of the first factor according to the first sampling interval, the multiple culture concentrations obtained are: 1%, 2%, 3%, 4%, 5%. Optionally, the first sampling interval is 1%.

[0034] Understandably, the sampling of human pluripotent stem cells refers to the process of extracting a certain mass (e.g., 50 mg) of human pluripotent stem cells from human pluripotent stem cells, and this certain mass (e.g., 50 mg) of human pluripotent stem cells is the test sample. The standard culture medium is a type of culture medium; optionally, HyCryo-STEM cryopreservation medium is used as the standard culture medium. The determination of the target culture medium based on the culture concentration and the standard culture medium involves: first, weighing the standard culture medium; then, calculating the weight of the first factor to be added based on the weight of the standard culture medium and the culture concentration, where the weight of the first factor to be added is the product of the weight of the standard culture medium and the culture concentration; finally, adding the first factor at the weight of the first factor to the standard culture medium to obtain the target culture medium.

[0035] It should be understood that culturing the test sample using the target culture medium refers to the process of inoculating the test sample onto the target culture medium to induce differentiation. The cultured sample refers to the test sample after culturing. Detecting the gene expression rate of the cultured sample refers to using gene detection technology (e.g., PCR detection) to detect the content of the FOXL2 gene, the content of anti-Müllerian hormone, and the content of aromatase in the test sample, respectively. The method for detecting these three contentes using gene detection technology (e.g., PCR detection) is existing technology and will not be elaborated here. The content of the FOXL2 gene is the first target expression rate, the content of anti-Müllerian hormone is the second target expression rate, and the content of aromatase is the third target expression rate. The first test score reflects the differentiation effect of the test sample; the higher the first test score, the better the differentiation effect. The first optimal concentration refers to the culture concentration corresponding to the first optimal test score.

[0036] S2. Based on human pluripotent stem cells, the optimal inducing factor, and the optimal concentration, the basic culture parameters were determined.

[0037] Specifically, the basic culture parameters determined based on human pluripotent stem cells, optimal inducing factors, and optimal concentrations include: Obtain a second standard culture medium, and add the optimal inducing factor to the second standard culture medium at the optimal concentration to obtain the second target culture medium; Human pluripotent stem cells were briefly cultured using a second target culture medium to obtain cultured stem cells; Culture control parameters were identified based on human pluripotent stem cells, including: oxygen concentration range, seeding density range, and seeding time range. Multiple culture test groups were identified based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters. Perform the following operation on each of the multiple culture test groups: Samples were taken from the cultured stem cells to obtain stem cell samples; The test culture medium was identified based on the culture test group; Differentiated stem cells were obtained by culturing stem cell samples in a test culture medium. Basic tests were performed on differentiated stem cells to obtain test data; The cultivation effect score was determined based on the test data; The cultivation effect scores are summarized to obtain multiple cultivation effect scores; The optimal performance score was determined based on multiple performance scores, where the optimal performance score is the largest among the multiple performance scores. The basic culture parameters were determined based on the best performance score. These parameters included: basic oxygen concentration, basic inoculation density, and basic inoculation time.

[0038] It should be explained that the second standard culture medium is a type of culture medium. Optionally, HyCryo-STEM cryopreserved culture medium can be used as the second standard culture medium. The addition of the optimal inducing factor to the standard culture medium at the optimal concentration means: first, weighing the standard culture medium; then, calculating the optimal inducing factor addition weight based on the weight of the standard culture medium and the optimal concentration, where the optimal inducing factor addition weight is the product of the weight of the standard culture medium and the optimal concentration; and finally, adding the optimal inducing factor to the standard culture medium at the optimal inducing factor addition weight. The second target culture medium refers to the target culture medium to which the optimal inducing factor has been added at the optimal inducing factor addition weight.

[0039] It should be understood that the method of briefly culturing human pluripotent stem cells using the second target culture medium is the same as the method of culturing test samples using the target culture medium, and will not be repeated here. The cultured stem cells refer to human pluripotent stem cells after brief culture.

[0040] Understandably, culture control parameters refer to the parameters that need to be regulated when culturing human pluripotent stem cells. These parameters include: oxygen concentration range, seeding density range, and seeding time range. The oxygen concentration range refers to the range of oxygen concentration in the culture medium during human pluripotent stem cell culture; optionally, the oxygen concentration range is 3%-21%. The seeding density range refers to the range of density at which human pluripotent stem cells are seeded onto the culture medium; optionally, the seeding density range is 0.3-1.2 × 10⁻⁶. 5 / mL, the inoculation time range refers to the range of time for culturing human pluripotent stem cells. Optionally, the inoculation time range is 2-6 days, and the oxygen concentration range, inoculation density range and inoculation time range are all common knowledge in the art.

[0041] It should be explained that the sampling of cultured stem cells refers to the process of selecting a certain mass (e.g., 50 mg) of cultured stem cells from them; this certain mass (e.g., 50 mg) of cultured stem cells is the stem cell sample. The test medium identified based on the culture test group refers to setting the oxygen concentration in the second basal medium to the oxygen concentration in the culture test group, setting the seeding density of human pluripotent stem cells in the second basal medium to the seeding density in the culture test group, and setting the culture time in the second basal medium to the seeding time in the culture test group.

[0042] It should be understood that the method for culturing stem cell samples using the test culture medium is the same as the method for short-term culturing of human pluripotent stem cells using standard culture medium, and will not be repeated here. Differentiated stem cells refer to stem cell samples after culture. The determination of basic culture parameters based on the optimal effect score means that the oxygen concentration, seeding density, and seeding time in the culture test group corresponding to the optimal effect score are used as basic culture parameters. The oxygen concentration in the culture test group corresponding to the optimal effect score is the basic oxygen concentration, the seeding density in the culture test group corresponding to the optimal effect score is the basic seeding density, and the seeding time in the culture test group corresponding to the optimal effect score is the basic seeding time.

[0043] Specifically, based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters, multiple culture test groups were identified, including: The oxygen concentration range in the culture control parameters was divided into three levels to obtain low oxygen concentration, medium oxygen concentration and high oxygen concentration. The inoculation density range in the culture control parameters was divided into three levels to obtain low inoculation density, medium inoculation density and high inoculation density; Nine preliminary test combinations were obtained by pairing low oxygen concentration, medium oxygen concentration, and high oxygen concentration with low inoculation density, medium inoculation density, and high inoculation density. The vaccination time range is sampled evenly according to the preset second interval to obtain multiple vaccination times; The nine preliminary test combinations were combined with multiple inoculation times to obtain multiple culture test groups, each of which included: oxygen concentration, inoculation density, and inoculation time.

[0044] For example, if the oxygen concentration range in the culture control parameters is 3%-21%, then dividing the oxygen concentration range into three levels results in a low oxygen concentration of 3%, a medium oxygen concentration of 12%, and a high oxygen concentration of 21%. If the inoculation density range in the culture control parameters is 0.3-1.2 × 10⁻⁶... 5 / mL, after dividing the inoculation density range in the culture control parameters into three levels, the low inoculation density obtained is 0.3×10 5 / mL, with an inoculation density of 0.75×10 5 / mL, with a high inoculation density of 1.2×10⁻⁶. 5 / mL. After pairing low, medium, and high oxygen concentrations with low, medium, and high inoculation densities, nine preliminary test combinations were obtained: (3%, 0.3×10⁻⁶). 5 ( / mL), (3%, 0.75×10 5 ( / mL), (3%, 1.2×105 ( / mL), (12%, 0.3×10 5 ( / mL), (12%, 0.75×10 5 ( / mL), (12%, 1.2×10 5 ( / mL), (21%, 0.3×10 5 ( / mL), (21%, 0.75×10 5 ( / mL), (21%, 1.2×10 5 / mL). If the inoculation time range is 2-6 days and the second interval is 1 day, then after uniformly sampling according to the second interval, multiple inoculation times are obtained: 2 days, 3 days, 4 days, 5 days, and 6 days. Then, by pairwise combining the 9 preliminary test combinations with the 6 inoculation times, 54 culture test groups are obtained: (3%, 0.3×10⁻⁶, 0.3×10⁻⁶). 5 / mL, 2 days), (3%, 0.75×10 5 / mL, 3 days), ..., (21%, 0.75×10 5 / mL, 6 days), (21%, 1.2×10 5 / mL, 6 days). Optional, the second interval is 1 day.

[0045] In detail, the basic testing of differentiated stem cells to obtain test data includes: Differentiated stem cells were sampled to obtain stem cell samples to be tested; The stem cell samples to be tested are pretreated to obtain processed samples; The cell viability index was obtained by performing a viability test on the treated samples. RNA was extracted from the processed samples to obtain total RNA. Core gene detection was performed on the total RNA of the sample to obtain the first core expression level and the second core expression level. Gene expression indices were determined based on the first and second core expression levels, where the gene expression index is the geometric mean of the first and second core expression levels. Biochemical tests were performed on the samples to be treated to obtain the glucose consumption and lactic acid production. The cell metabolic activity index is calculated based on glucose consumption and lactate production, using the following formula: in, Indicators of cellular metabolic activity This indicates the amount of glucose consumed. Indicates the amount of lactic acid produced; The cell viability index, gene expression index, and cell metabolic activity index were summarized to obtain the detection data.

[0046] It should be explained that the sampling of differentiated stem cells refers to extracting a certain mass (e.g., 50 mg) of differentiated stem cells from the differentiated stem cells. This certain mass (e.g., 50 mg) of differentiated stem cells is the stem cell sample to be tested. The pretreatment of the stem cell sample to be tested involves using a chemical detection reagent (e.g., CCK-8) to process the sample. The method of using this chemical detection reagent (e.g., CCK-8) to process the stem cell sample is existing technology and will not be elaborated here. The processed sample is the pretreated stem cell sample to be tested. The viability detection of the processed sample refers to testing the absorbance of the processed sample at a wavelength of 450 nm using a detection instrument (e.g., an ELISA reader). The method of using a detection instrument (e.g., an ELISA reader) to test the absorbance of the processed sample at a wavelength of 450 nm is existing technology and will not be elaborated here. The absorbance of the processed sample at a wavelength of 450 nm is the cell viability index.

[0047] It is understood that the RNA extraction from the treated sample involves using an RNA extraction reagent (e.g., TRIZOL) to transcribe the sample, thereby obtaining Total RNA. Total RNA is a mixture of RNA extracted from biological tissues or cells, containing all components such as mRNA, rRNA, and tRNA. The method of using an RNA extraction reagent (e.g., TRIZOL) to treat the sample to obtain Total RNA is existing technology and will not be elaborated here. TRIZOL is a novel total RNA extraction reagent that can directly extract total RNA from cells or tissues. The Total RNA is the sample's total RNA. The core gene detection of the sample's total RNA refers to using PCR technology (e.g., real-time quantitative PCR) to determine the content of Oct4 and NANOG in the sample's total RNA. The method of using PCR technology (e.g., real-time quantitative PCR) to determine the content of Oct4 and NANOG in the sample's total RNA is existing technology and will not be elaborated here. The Oct4 content in the total RNA of the sample is the first core expression level, and the NANOG content in the total RNA of the sample is the second core expression level.

[0048] It should be understood that the biochemical detection of the sample to be treated refers to the use of an analyzer (e.g., a biochemical analyzer) to detect the glucose and lactic acid content in the sample. The method for detecting these content using an analyzer is existing technology and will not be elaborated here. The glucose content in the sample represents glucose consumption, and the lactic acid content represents lactic acid production. The cell metabolic activity index reflects the intensity of cell metabolism; a higher index indicates greater metabolic intensity and better differentiation. The detection data refers to the data obtained by summing the cell viability index, gene expression index, and cell metabolic activity index.

[0049] Specifically, the process of determining the culture effect score based on the detection data includes: The culture effect score is calculated based on the cell viability index, gene expression index, and cell metabolic activity index from the test data. The calculation formula is shown below: in, Indicates the score for cultivation effectiveness. Indicates cell viability index, Indicates gene expression index, This represents the cell metabolic activity index.

[0050] It should be explained that the culture effect score reflects the culture effect of exosome culture; the higher the score, the better the culture effect. It is important to note that when incorporating cell viability index, gene expression index, and cell metabolic activity index into the calculation of the culture effect score, only their numerical values ​​are used, and their dimensions are not considered.

[0051] S3. Human pluripotent stem cells are cultured in a targeted manner using basic culture parameters to obtain a differentiated cell population.

[0052] It should be explained that the directed culture of human pluripotent stem cells using basic culture parameters refers to: preparing an inoculation medium according to the oxygen concentration specified in the basic culture parameters; then, inoculating the human pluripotent stem cells onto the inoculation medium at the inoculation density specified in the basic culture parameters; and culturing them according to the inoculation time specified in the basic culture parameters. The differentiated cell population refers to the human pluripotent stem cells after directed culture.

[0053] S4. Detect markers in the differentiated cell population to obtain the positive rates of anti-Müllerian hormone and second gene. Compare the positive rate of anti-Müllerian hormone with the preset hormone positive rate threshold, and compare the positive rate of second gene with the preset second gene positive rate threshold.

[0054] It should be explained that the marker detection of the differentiated cell population refers to the determination of the anti-Müllerian hormone (AMH) content and the FOXL2 gene content in the differentiated cell population using PCR technology (e.g., real-time quantitative PCR). The method for determining these two gene contents using PCR technology is existing technology and will not be elaborated here. The AMH content in the differentiated cell population is the AMH positivity rate, and the FOXL2 gene content is the FOXL2 gene positivity rate. The hormone positivity rate threshold is manually set by the staff of the exosome preparation laboratory based on the minimum AMH positivity rate of historically cultured, qualified differentiated cell populations. For example, if the minimum AMH positivity rate of historically cultured, qualified differentiated cell populations is 80%, then the hormone positivity rate threshold is 80%. The second gene positivity rate threshold is set manually by the staff of the exosome preparation laboratory based on the minimum second gene positivity rate of qualified differentiated cell populations in history. For example, if the minimum second gene positivity rate of qualified differentiated cell populations in history is 80%, then the hormone positivity rate threshold is 80%.

[0055] S5. If the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold, and the positive rate of the second gene is greater than or equal to the gene positive rate threshold, then the differentiated cell population will be used as the target ovarian granulosa cells.

[0056] It should be explained that target ovarian granulosa cells refer to a differentiated cell population with an anti-Müllerian hormone positivity rate greater than or equal to the hormone positivity rate threshold and a second gene positivity rate greater than or equal to the gene positivity rate threshold.

[0057] S6. Otherwise, treat the differentiated cell population as human pluripotent stem cells, and determine the modified culture parameters based on the basic culture parameters. Use the modified culture parameters as the basic culture parameters, and return to the step of using the basic culture parameters to perform targeted culture of human pluripotent stem cells until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold, and the second gene positivity rate is greater than or equal to the gene positivity rate threshold.

[0058] Specifically, the process of identifying modified culture parameters based on basic culture parameters includes: Culture defect types were identified based on the positive rates of anti-Müllerian hormone, second gene, hormone positivity threshold, and second gene positivity threshold. Among them, culture defect types include: insufficient anti-Müllerian hormone positivity rate, insufficient second gene positivity rate, or both. When the culture defect type is insufficient positive rate of anti-Müllerian hormone, the basic oxygen concentration in the basic culture parameters is increased by the preset first adjustment step to obtain the corrected oxygen concentration. The corrected culture parameters were obtained using the corrected oxygen concentration, the basic inoculation density in the basic culture parameters, and the basic inoculation time in the basic culture parameters. When the culture defect type is insufficient positive rate of the second gene, the basic inoculation density in the basic culture parameters is increased by a preset second adjustment step to obtain the corrected inoculation density; The corrected culture parameters were obtained using the corrected inoculation density, the basal oxygen concentration in the basal culture parameters, and the basal inoculation time in the basal culture parameters. When the culture defect type is that both are insufficient, the basic inoculation time in the basic culture parameters is increased by a preset third adjustment step to obtain the corrected inoculation time; The corrected culture parameters were obtained by using the corrected inoculation time, the basic inoculation density in the basic culture parameters, and the basic oxygen concentration in the basic culture parameters.

[0059] It should be explained that the determination of culture defect type based on the positive rate of anti-Müllerian hormone, the positive rate of the second gene, the hormone positive rate threshold, and the second gene positive rate threshold means: if the positive rate of anti-Müllerian hormone is less than the hormone positive rate threshold and the positive rate of the second gene is greater than or equal to the second gene positive rate threshold, then the culture defect type is determined to be insufficient anti-Müllerian hormone positive rate; if the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold and the positive rate of the second gene is less than the second gene positive rate threshold, then the culture defect type is determined to be insufficient second gene positive rate; if the positive rate of anti-Müllerian hormone is less than the hormone positive rate threshold and the positive rate of the second gene is less than the second gene positive rate threshold, then the culture defect type is determined to be insufficient in both.

[0060] It should be understood that increasing the basal oxygen concentration in the basal culture parameters by a preset first adjustment step size means adding the first adjustment step size to the basal oxygen concentration in the basal culture parameters. For example, if the basal oxygen concentration in the basal culture parameters is 3% and the first adjustment step size is 1%, then the corrected oxygen concentration is 4%. Optionally, the first adjustment step size is 1%, and the second adjustment step size is 0.1 × 10⁻⁶. 5 / mL, the third adjustment step is 1 day.

[0061] It should be understood that the method of increasing the basic inoculation density in the basic culture parameters by a preset second adjustment step and the method of increasing the basic inoculation time in the basic culture parameters by a preset third adjustment step are the same as the method of increasing the basic oxygen concentration in the basic culture parameters by a preset first adjustment step, and will not be described again here.

[0062] S7. Expand and culture the target ovarian granulosa cells to obtain cell culture supernatant, and determine the optimal separation parameters based on the cell culture supernatant.

[0063] It should be explained that the expansion culture of the target ovarian granulosa cells refers to the expansion of the target ovarian granulosa cells using expansion technology (e.g., gene amplification technology). The method for expanding the target ovarian granulosa cells using expansion technology (e.g., gene amplification technology) is existing technology and will not be elaborated here. The cell culture supernatant is the supernatant obtained after expanding the target ovarian granulosa cells.

[0064] Specifically, the determination of optimal separation parameters based on cell culture supernatant includes: The cell culture supernatant was subjected to material analysis to obtain the total protein concentration and cell sap viscosity. The ultracentrifugal force is calculated based on the total protein concentration, using the following formula: in, Indicates supercentrifugal force. This represents the preset basic centrifugal force. Indicates total protein concentration. This is the preset concentration influence coefficient. This indicates the preset reference protein concentration; The elution flow rate is calculated based on the cell fluid viscosity, using the following formula: in, Indicates the elution flow rate. The preset ideal maximum elution flow rate, Indicates cell sap viscosity, Indicates the preset reference cell sap viscosity; The optimal separation parameters were obtained by summing up the ultracentrifugal force and elution flow rate.

[0065] It should be explained that the material testing of cell culture supernatant to obtain total protein concentration and viscosity refers to: measuring the protein concentration in the cell culture supernatant using a protein concentration detection method (e.g., BCA protein concentration detection), and the method for measuring the protein concentration in the cell culture supernatant using this method is existing technology and will not be elaborated here. The protein content in the cell culture supernatant is the total protein concentration. The viscosity of the cell culture supernatant is measured using a viscosity testing device (e.g., a rotational viscometer), and the method for measuring the viscosity of the cell culture supernatant using this device is existing technology and will not be elaborated here. The viscosity of the cell culture supernatant is the cell fluid viscosity. Ultracentrifugation force refers to the centrifugal force required to separate the cell culture supernatant, and elution flow rate refers to the flow rate required to separate the cell culture supernatant. The reference protein concentration is manually set by the staff of the exosome preparation center based on the average total protein concentration in multiple historically processed cell culture supernatants. For example, if the average total protein concentration in multiple historically processed cell culture supernatants is 70%, then the reference protein concentration is 70%. The reference cell fluid viscosity is manually set by the staff of the exosome preparation center based on the average viscosity of multiple historically processed cell culture supernatants. For example, if the average viscosity of multiple historically processed cell culture supernatants is 80 Pascals per second, then the reference protein concentration is 80 Pascals per second.

[0066] Understandably, the baseline centrifugal force is manually set by the staff of the exosome preparation center based on the standard centrifugal force of the centrifugation equipment (e.g., centrifuge) during operation. For example, if the standard centrifugal force of the centrifugation equipment (e.g., centrifuge) during operation is 10N, then the baseline centrifugal force is 10N, and the standard centrifugal force can be obtained from the product technical manual provided by the centrifugation equipment (e.g., centrifuge) manufacturer. The ideal maximum elution flow rate is manually set by the staff of the exosome preparation center based on the maximum elution rate of the elution equipment (e.g., column pair) during operation. For example, if the maximum elution rate of the elution equipment (e.g., column pair) during operation is 10 cubic centimeters per second, then the ideal maximum elution flow rate is 10 cubic centimeters per second, and the maximum elution rate can be obtained from the product technical manual provided by the elution equipment (e.g., column pair) manufacturer. Optionally, the concentration influence coefficient is 1.1.

[0067] S8. Using the optimal separation parameters, the cell culture supernatant was separated to obtain exosome extract, thus completing the optimization of exosome preparation.

[0068] It should be explained that the separation of cell culture supernatant using optimal separation parameters refers to: using a centrifuge (e.g., a centrifuge) with the centrifugal force set to the ultracentrifugal force in the optimal separation parameters to centrifuge the cell culture supernatant to obtain preliminary centrifuged material; then, using an elution device (e.g., a chromatographic column pair) with the elution rate set to the elution flow rate in the optimal separation parameters to elute the material, and finally obtaining the eluted suspension, which is the exosome extract.

[0069] For example, once the exosome extract is obtained, Xiao Zhang can conduct subsequent research and development on the exosome extract, thereby completing the preparation and optimization of ovarian granulosa cell exosomes.

[0070] To address the problems described in the background art, this invention obtains human pluripotent stem cells and identifies the optimal inducing factors and concentrations based on these cells. This invention intelligently matches the most efficient differentiation induction protocol to a specific stem cell line, laying the foundation for the subsequent targeted and efficient generation of target cell types. Furthermore, based on the human pluripotent stem cells, optimal inducing factors, and optimal concentrations, basic culture parameters are identified. This invention translates optimal induction conditions into precisely executable culture operation instructions, establishing a standardized initial differentiation process. Using these basic culture parameters, human pluripotent stem cells are directionally cultured to obtain a differentiated cell population. This invention, by initiating and executing the differentiation program under controlled conditions, enables stem cells to differentiate. Cells develop in a direction aligned with the fate of ovarian granulosa cells, yielding preliminary cell products. This improves the automation level of the exosome preparation process. Biomarker detection is performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone (AMH) and the second gene. This embodiment of the invention utilizes multiple molecular biomarkers for quantitative identification of differentiation products, objectively assessing differentiation efficiency and cell type accuracy. The positive rates of AMH and the second gene are compared with preset thresholds. This embodiment of the invention provides clear quantitative evidence for automated determination of differentiation success by setting strict quality standards. If the positive rate of AMH is greater than or equal to the threshold, and the second gene is positive... If the positive rate is greater than or equal to the gene positivity rate threshold, the differentiated cell population is used as the target ovarian granulosa cells. This embodiment of the invention automatically confirms the acquisition of high-quality, high-purity target cells for downstream preparation when the detection results simultaneously meet dual criteria, thus improving the intelligence level of the exosome preparation process. Otherwise, the differentiated cell population is used as human pluripotent stem cells, and corrected culture parameters are determined based on the basic culture parameters. These corrected culture parameters are then used as the basic culture parameters, and the process returns to the step of directional culture of human pluripotent stem cells using the basic culture parameters, until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold, and the second gene positivity rate is greater than or equal to the gene positivity rate threshold. This embodiment of the invention establishes a detection-judgment-optimization mechanism. The intelligent feedback loop automatically adjusts culture parameters and restarts the differentiation process when differentiation fails to meet the target until qualified cells are produced, ensuring the stability and reliability of the final cell product quality. This improves the intelligence level of the exosome preparation process. The target ovarian granulosa cells are expanded and cultured to obtain cell culture supernatant. This embodiment of the invention provides sufficient raw materials for exosome enrichment by scaling up the identified qualified cells and collecting the culture medium containing their secretions. Based on the cell culture supernatant, the optimal separation parameters are determined. This embodiment of the invention dynamically optimizes the core parameters of separation techniques such as ultracentrifugation and size exclusion chromatography according to the characteristics of the supernatant, such as volume and protein concentration, to achieve high yield and high purity recovery of exosomes.By using optimal separation parameters to separate the cell culture supernatant, exosome extracts were obtained, thus optimizing exosome preparation. It is evident that this invention, through the execution of an optimized separation and purification process, ultimately yields high-purity, highly active exosomes derived from ovarian granulosa cells. This achieves full-process optimization and quality control from intelligent cell differentiation to automated product preparation, improving the intelligence level of the exosome preparation process. Therefore, this invention can enhance the intelligence level of the exosome preparation process.

[0071] like Figure 2 The diagram shown is a functional block diagram of an iPSC-based ovarian granulosa cell exosome preparation optimization system provided in an embodiment of the present invention.

[0072] The iPSC-based ovarian granulosa cell exosome preparation optimization system 100 described in this invention can be installed in an electronic device. Depending on the functions implemented, the iPSC-based ovarian granulosa cell exosome preparation optimization system 100 may include a basic parameter confirmation module 101, a differentiation population acquisition module 102, a culture parameter correction module 103, and a preparation optimization processing module 104. The module described in this invention can also be referred to as a unit, which refers to a series of computer program segments that can be executed by the processor of an electronic device and can perform a fixed function, and is stored in the memory of the electronic device.

[0073] The basic parameter confirmation module 101 is used to obtain human pluripotent stem cells, confirm the optimal inducing factor and optimal concentration based on human pluripotent stem cells, and confirm the basic culture parameters based on human pluripotent stem cells, optimal inducing factor and optimal concentration. The differentiation population acquisition module 102 is used to perform targeted culture of human pluripotent stem cells using basic culture parameters to obtain a differentiated cell population, and to perform biomarker detection on the differentiated cell population to obtain the positive rate of anti-Müllerian hormone and the positive rate of the second gene. The culture parameter correction module 103 is used to compare the anti-Müllerian hormone positivity rate with a preset hormone positivity rate threshold and compare the second gene positivity rate with a preset second gene positivity rate threshold. If the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold, then the differentiated cell population is taken as the target ovarian granulosa cells; otherwise, the differentiated cell population is taken as human pluripotent stem cells. Based on the basic culture parameters, the corrected culture parameters are identified and used as the basic culture parameters. The process returns to the step of using the basic culture parameters to perform targeted culture of human pluripotent stem cells until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold. The preparation optimization module 104 is used to expand and culture the target ovarian granulosa cells to obtain cell culture supernatant, identify the optimal separation parameters based on the cell culture supernatant, and use the optimal separation parameters to separate the cell culture supernatant to obtain exosome extract, thus completing the exosome preparation optimization.

[0074] In detail, the modules in the iPSC-based ovarian granulosa cell exosome preparation optimization system 100 described in this embodiment of the invention employ the same methods as described above. Figure 1 The preparation and optimization method of ovarian granulosa cell exosomes based on iPSC described herein uses the same technical means and can produce the same technical effect, so it will not be repeated here.

[0075] like Figure 3 The diagram shown is a schematic representation of an electronic device for implementing an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs, according to an embodiment of the present invention.

[0076] 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 an optimized method program for the preparation of ovarian granulosa cell exosomes based on iPSCs.

[0077] The memory 11 includes at least one type of readable storage medium, such as flash memory, portable hard drive, multimedia card, card-type memory (e.g., SD or DX memory), magnetic memory, magnetic disk, optical disk, etc. In some embodiments, the memory 11 can be an internal storage unit of the electronic device 1, such as a portable hard drive. 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 card (SD), 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 an optimized method for preparing ovarian granulosa cell exosomes based on iPSC, but also to temporarily store data that has been output or will be output.

[0078] 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., an optimized method for preparing ovarian granulosa cell exosomes based on iPSCs), and calls data stored in the memory 11 to perform various functions of the electronic device 1 and process data.

[0079] 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.

[0080] Figure 3 Only electronic devices with components are shown; it will be understood by those skilled in the art that... Figure 3 The 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] The optimized method program for the preparation of ovarian granulosa cell exosomes based on iPSCs, 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: Obtain human pluripotent stem cells, and identify the optimal inducing factors and concentrations based on human pluripotent stem cells; The basic culture parameters were determined based on human pluripotent stem cells, the optimal inducing factor, and the optimal concentration. Human pluripotent stem cells were cultured in a targeted manner using basic culture parameters to obtain a differentiated cell population; Biomarker detection was performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone and second gene; Compare the positive rate of anti-Müllerian hormone with a preset hormone positive rate threshold, and compare the positive rate of the second gene with a preset second gene positive rate threshold; If the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold, and the positive rate of the second gene is greater than or equal to the gene positive rate threshold, then the differentiated cell population will be used as the target ovarian granulosa cells. Otherwise, the differentiated cell population is taken as human pluripotent stem cells, and the modified culture parameters are identified based on the basic culture parameters. The modified culture parameters are taken as the basic culture parameters, and the process is repeated until the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold and the positive rate of the second gene is greater than or equal to the gene positive rate threshold. The target ovarian granulosa cells were expanded and cultured to obtain cell culture supernatant. The optimal separation parameters were determined based on cell culture supernatant. The cell culture supernatant was separated using the optimal separation parameters to obtain exosome extracts, thus completing the optimization of exosome preparation.

[0085] 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.

[0086] 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).

[0087] 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: Obtain human pluripotent stem cells, and identify the optimal inducing factors and concentrations based on human pluripotent stem cells; The basic culture parameters were determined based on human pluripotent stem cells, the optimal inducing factor, and the optimal concentration. Human pluripotent stem cells were cultured in a targeted manner using basic culture parameters to obtain a differentiated cell population; Biomarker detection was performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone and second gene; Compare the positive rate of anti-Müllerian hormone with a preset hormone positive rate threshold, and compare the positive rate of the second gene with a preset second gene positive rate threshold; If the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold, and the positive rate of the second gene is greater than or equal to the gene positive rate threshold, then the differentiated cell population will be used as the target ovarian granulosa cells. Otherwise, the differentiated cell population is taken as human pluripotent stem cells, and the modified culture parameters are identified based on the basic culture parameters. The modified culture parameters are taken as the basic culture parameters, and the process is repeated until the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold and the positive rate of the second gene is greater than or equal to the gene positive rate threshold. The target ovarian granulosa cells were expanded and cultured to obtain cell culture supernatant. The optimal separation parameters were determined based on cell culture supernatant. The cell culture supernatant was separated using the optimal separation parameters to obtain exosome extracts, thus completing the optimization of exosome preparation.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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. An optimized method for preparing ovarian granulosa cell exosomes based on iPSCs, characterized in that, The method includes: Obtain human pluripotent stem cells, and identify the optimal inducing factors and concentrations based on human pluripotent stem cells; The basic culture parameters were determined based on human pluripotent stem cells, the optimal inducing factor, and the optimal concentration. Human pluripotent stem cells were cultured in a targeted manner using basic culture parameters to obtain a differentiated cell population; Biomarker detection was performed on the differentiated cell population to obtain the positive rates of anti-Müllerian hormone and second gene; Compare the positive rate of anti-Müllerian hormone with a preset hormone positive rate threshold, and compare the positive rate of the second gene with a preset second gene positive rate threshold; If the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold, and the positive rate of the second gene is greater than or equal to the gene positive rate threshold, then the differentiated cell population will be used as the target ovarian granulosa cells. Otherwise, the differentiated cell population is taken as human pluripotent stem cells, and the modified culture parameters are identified based on the basic culture parameters. The modified culture parameters are taken as the basic culture parameters, and the process is repeated until the positive rate of anti-Müllerian hormone is greater than or equal to the hormone positive rate threshold and the positive rate of the second gene is greater than or equal to the gene positive rate threshold. The target ovarian granulosa cells were expanded and cultured to obtain cell culture supernatant. The optimal separation parameters were determined based on cell culture supernatant. The cell culture supernatant was separated using the optimal separation parameters to obtain exosome extracts, thus completing the optimization of exosome preparation.

2. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 1, characterized in that, The optimal inducing factors and concentrations identified based on human pluripotent stem cells include: Commonly used inducing factors were identified, including: factor one, factor two, and factor three. Based on human pluripotent stem cells, factor one, and a preset first sampling interval, the first optimal test score and the first optimal concentration were determined. Based on human pluripotent stem cells, factor 2, and a pre-defined second sampling range, a second optimal test score and a second optimal concentration were determined. Based on human pluripotent stem cells, the third factor, and the pre-defined third sampling range, the third optimal test score and the third optimal test concentration were determined. The optimal inducing factor and optimal concentration were determined based on the first optimal test score, the first optimal concentration, the second optimal test score, the second optimal concentration, the third optimal test score, and the third optimal test concentration.

3. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 2, characterized in that, The determination of the first optimal test score and the first optimal concentration based on human pluripotent stem cells, the first factor, and a preset first sampling interval includes: Once the concentration range of the first factor is identified, samples are taken uniformly within the first factor concentration range according to the first sampling interval to obtain multiple culture concentrations. Perform the following operation for each of the multiple culture concentrations: Human pluripotent stem cells were sampled to obtain test samples; Obtain standard culture medium, and identify the target culture medium based on the culture concentration and the standard culture medium; The test samples were cultured using the target culture medium to obtain cultured samples; Gene expression rates were detected in culture samples to obtain the first target expression rate, the second target expression rate, and the third target expression rate. The first test score is calculated based on the expression rates of the first, second, and third targets. The scores from the first test are combined to obtain multiple first test scores; The first best test score is determined based on multiple first test scores, where the first best test score is the largest first test score among multiple first test scores; The culture concentration corresponding to the first best test score is taken as the first best concentration.

4. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 3, characterized in that, The basic culture parameters, determined based on human pluripotent stem cells, optimal inducing factors, and optimal concentrations, include: Obtain a second standard culture medium, and add the optimal inducing factor to the second standard culture medium at the optimal concentration to obtain the second target culture medium; Human pluripotent stem cells were briefly cultured using a second target culture medium to obtain cultured stem cells; Culture control parameters were identified based on human pluripotent stem cells, including: oxygen concentration range, seeding density range, and seeding time range. Multiple culture test groups were identified based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters. Perform the following operation on each of the multiple culture test groups: Samples were taken from the cultured stem cells to obtain stem cell samples; The test culture medium was identified based on the culture test group; Differentiated stem cells were obtained by culturing stem cell samples using a test culture medium. Basic tests were performed on differentiated stem cells to obtain test data; The cultivation effect score was determined based on the test data; The cultivation effect scores are summarized to obtain multiple cultivation effect scores; The optimal performance score was determined based on multiple performance scores, where the optimal performance score is the largest performance score among the multiple performance scores. The basic culture parameters were determined based on the best performance score. These parameters included: basic oxygen concentration, basic inoculation density, and basic inoculation time.

5. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 4, characterized in that, Based on the oxygen concentration range, inoculation density range, and inoculation time range in the culture control parameters, multiple culture test groups were identified, including: The oxygen concentration range in the culture control parameters was divided into three levels to obtain low oxygen concentration, medium oxygen concentration and high oxygen concentration. The inoculation density range in the culture control parameters was divided into three levels to obtain low inoculation density, medium inoculation density and high inoculation density; Nine preliminary test combinations were obtained by pairing low oxygen concentration, medium oxygen concentration, and high oxygen concentration with low inoculation density, medium inoculation density, and high inoculation density. The vaccination time range is sampled evenly according to the preset second interval to obtain multiple vaccination times; The nine preliminary test combinations were combined with multiple inoculation times to obtain multiple culture test groups, each of which included: oxygen concentration, inoculation density, and inoculation time.

6. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 5, characterized in that, The basic testing of differentiated stem cells yields the following data: Differentiated stem cells were sampled to obtain stem cell samples to be tested; The stem cell samples to be tested are pretreated to obtain processed samples; The cell viability index was obtained by performing a viability test on the treated samples. RNA was extracted from the processed samples to obtain total RNA. Core gene detection was performed on the total RNA of the sample to obtain the first core expression level and the second core expression level. Gene expression indices were determined based on the first and second core expression levels, where the gene expression index is the geometric mean of the first and second core expression levels. Biochemical tests were performed on the samples to be treated to obtain the glucose consumption and lactic acid production. The cell metabolic activity index is calculated based on glucose consumption and lactate production. The cell viability index, gene expression index, and cell metabolic activity index were summarized to obtain the detection data.

7. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 6, characterized in that, The process of determining the cultivation effect score based on the detection data includes: The culture effect score is calculated based on the cell viability index, gene expression index, and cell metabolic activity index from the test data. The calculation formula is shown below: in, Indicates the score for cultivation effectiveness. Indicates cell viability index, Indicates gene expression index, This represents the cell metabolic activity index.

8. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 7, characterized in that, The process of determining the corrected culture parameters based on the basic culture parameters includes: Culture defect types were identified based on the positive rates of anti-Müllerian hormone, second gene, hormone positivity threshold, and second gene positivity threshold. Among them, culture defect types include: insufficient anti-Müllerian hormone positivity rate, insufficient second gene positivity rate, or both. When the culture defect type is insufficient positive rate of anti-Müllerian hormone, the basic oxygen concentration in the basic culture parameters is increased by the preset first adjustment step to obtain the corrected oxygen concentration. The corrected culture parameters were obtained using the corrected oxygen concentration, the basic inoculation density in the basic culture parameters, and the basic inoculation time in the basic culture parameters. When the culture defect type is insufficient positive rate of the second gene, the basic inoculation density in the basic culture parameters is increased by a preset second adjustment step to obtain the corrected inoculation density; The corrected culture parameters were obtained using the corrected inoculation density, the basal oxygen concentration in the basal culture parameters, and the basal inoculation time in the basal culture parameters. When the culture defect type is that both are insufficient, the basic inoculation time in the basic culture parameters is increased by a preset third adjustment step to obtain the corrected inoculation time; The corrected culture parameters were obtained by using the corrected inoculation time, the basic inoculation density in the basic culture parameters, and the basic oxygen concentration in the basic culture parameters.

9. The optimized method for preparing ovarian granulosa cell exosomes based on iPSCs as described in claim 8, characterized in that, The optimal separation parameters identified based on cell culture supernatant include: The cell culture supernatant was subjected to material analysis to obtain the total protein concentration and cell sap viscosity. Calculate the ultracentrifugal force based on the total protein concentration; The elution flow rate is calculated based on the cell fluid viscosity, using the following formula: in, Indicates the elution flow rate. The preset ideal maximum elution flow rate, Indicates cell sap viscosity, Indicates the preset reference cell sap viscosity; The optimal separation parameters were obtained by summing up the ultracentrifugal force and elution flow rate.

10. An optimized system for preparing ovarian granulosa cell exosomes based on iPSCs, characterized in that, The system includes: The basic parameter confirmation module is used to obtain human pluripotent stem cells, confirm the optimal inducing factor and optimal concentration based on human pluripotent stem cells, and confirm the basic culture parameters based on human pluripotent stem cells, optimal inducing factor and optimal concentration. The differentiation population acquisition module is used to perform targeted culture of human pluripotent stem cells using basic culture parameters to obtain a differentiated cell population. The differentiated cell population is then subjected to biomarker detection to obtain the positive rate of anti-Müllerian hormone and the positive rate of the second gene. The culture parameter correction module is used to compare the anti-Müllerian hormone positivity rate with a preset hormone positivity rate threshold and the second gene positivity rate with a preset second gene positivity rate threshold. If the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold, then the differentiated cell population is taken as the target ovarian granulosa cells; otherwise, the differentiated cell population is taken as human pluripotent stem cells. Based on the basic culture parameters, the corrected culture parameters are identified and used as the basic culture parameters. The process returns to the step of directional culture of human pluripotent stem cells using the basic culture parameters until the anti-Müllerian hormone positivity rate is greater than or equal to the hormone positivity rate threshold and the second gene positivity rate is greater than or equal to the gene positivity rate threshold. An optimization processing module is used to expand and culture target ovarian granulosa cells to obtain cell culture supernatant. Based on the cell culture supernatant, the optimal separation parameters are identified. The cell culture supernatant is then separated using the optimal separation parameters to obtain exosome extract, thus completing the optimization of exosome preparation.

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