CN-121991882-A - Preparation optimization method and system of ovary granule cell exosome based on iPSC

CN121991882ACN 121991882 ACN121991882 ACN 121991882ACN-121991882-A

Abstract

The invention relates to the technical field of preparation of extracellular fluid, in particular to a preparation optimization method and system of ovarian granule extracellular fluid based on iPSC, comprising the following steps: obtaining human pluripotent stem cells, confirming optimal induction factors and optimal concentrations based on the human pluripotent stem cells, confirming basic culture parameters based on the human pluripotent stem cells, the optimal induction factors and the optimal concentrations, directionally culturing the human pluripotent stem cells by utilizing the basic culture parameters to obtain differentiated cell populations, detecting markers of the differentiated cell populations to obtain the anti-mullerian hormone positive rate and the second gene positive rate, performing amplification culture on target ovarian granule cells to obtain cell culture supernatant, separating the cell culture supernatant by utilizing the optimal separation parameters to obtain exosome extracts, and completing preparation optimization of exosome. The invention can improve the intelligent degree of the exosome preparation flow.

Inventors

CHEN LILING
ZHANG ZHAOYUAN
Chen Hengzi

Assignees

深圳市玖源细胞科技有限公司

Dates

Publication Date: 20260508
Application Date: 20260204

Claims (10)

1. A method for optimizing preparation of an ovarian granulosa cell exosome based on iPSC, comprising: Obtaining human pluripotent stem cells, and confirming optimal induction factors and optimal concentrations based on the human pluripotent stem cells; Confirming basic culture parameters based on the human pluripotent stem cells, the optimal induction factors and the optimal concentration; Performing directional culture on the human pluripotent stem cells by using basic culture parameters to obtain differentiated cell populations; performing marker detection on the differentiated cell population to obtain the anti-mullerian hormone positive rate and the second gene positive rate; comparing the hormone positive rate of the anti-mullerian duct with a preset hormone positive rate threshold value, and comparing the second gene positive rate with a preset second gene positive rate threshold value; if the hormone positive rate of the anti-mullerian tube is greater than or equal to a hormone positive rate threshold value and the second gene positive rate is greater than or equal to a gene positive rate threshold value, taking the differentiated cell population as target ovarian granulosa cells; Otherwise, taking the differentiated cell population as the human pluripotent stem cells, confirming a corrected culture parameter based on the basic culture parameter, taking the corrected culture parameter as the basic culture parameter, and returning to the step of directionally culturing the human pluripotent stem cells by utilizing the basic culture parameter until the hormone positive rate of the anti-mullerian tube is greater than or equal to a hormone positive rate threshold value, and the second gene positive rate is greater than or equal to a gene positive rate threshold value; Amplifying and culturing target ovary granular cells to obtain cell culture supernatant; Confirming optimal separation parameters based on the cell culture supernatant; and separating the cell culture supernatant by utilizing the optimal separation parameters to obtain an exosome extract, and completing the preparation optimization of the exosome.
2. The method for optimizing preparation of iPSC-based ovarian granulosa extracellular fluid according to claim 1, wherein the identifying optimal induction factor and optimal concentration based on human pluripotent stem cells comprises: Identifying common induction factors, wherein the common induction factors comprise a first factor, a second factor and a third factor; Identifying a first optimal test score and a first optimal concentration based on the human pluripotent stem cells, the first factor, and a preset first sampling interval; identifying a second optimal test score and a second optimal concentration based on the human pluripotent stem cells, a second factor, and a preset second sampling range; Confirming a third optimal test score and a third optimal test concentration based on the human pluripotent stem cells, a third factor and a preset third sampling range; The optimal induction factor and optimal concentration are 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 method of optimizing preparation of iPSC-based ovarian granulosa extracellular fluid of claim 2, wherein the identifying the first optimal test score and the first optimal concentration based on the human pluripotent stem cells, the first factor, and the predetermined first sampling interval comprises: confirming a first factor concentration range, and uniformly sampling the first factor concentration range according to a first sampling interval to obtain a plurality of culture concentrations; the following is performed for each of the plurality of culture concentrations: Sampling the human pluripotent stem cells to obtain a test sample; Obtaining a standard culture medium, and confirming a target culture medium based on the culture concentration and the standard culture medium; culturing the test sample by using a target culture medium to obtain a culture sample; detecting the gene expression rate of the culture sample to obtain a first target expression rate, a second target expression rate and a third target expression rate; calculating a first test score according to the first target expression rate, the second target expression rate and the third target expression rate; Summarizing the first test scores to obtain a plurality of first test scores; identifying a first best test score based on the plurality of first test scores, wherein the first best test score is a largest first test score of the plurality of first test scores; the culture concentration corresponding to the first best test score was taken as the first best concentration.
4. The method for optimizing preparation of iPSC-based ovarian granulosa extracellular body according to claim 3, wherein the identifying basic culture parameters based on human pluripotent stem cells, optimal induction factors and optimal concentrations comprises: Obtaining a second standard culture medium, and adding an optimal induction factor into the second standard culture medium according to the optimal concentration to obtain a second target culture medium; Performing short-term culture on the human pluripotent stem cells by using a second target culture medium to obtain cultured stem cells; Identifying culture control parameters based on the human pluripotent stem cells, wherein the culture control parameters comprise an oxygen concentration range, an inoculation density range and an inoculation time range; Confirming a plurality of culture test groups based on the oxygen concentration range, the inoculation density range and the inoculation time range in the culture control parameters; the following is performed for each of the plurality of incubation test sections: sampling the cultured stem cells to obtain stem cell samples; confirming a test medium based on the culture test group; culturing the stem cell sample by using a test culture medium to obtain differentiated stem cells; Basic detection is carried out on the differentiated stem cells, and detection data are obtained; confirming a culture effect score based on the detection data; summarizing the culture effect scores to obtain a plurality of culture effect scores; Confirming the optimal effect score based on the plurality of culture effect scores, wherein the optimal effect score is the largest culture effect score in the plurality of culture effect scores; and confirming basic culture parameters based on the optimal effect score, wherein the basic culture parameters comprise basic oxygen concentration, basic inoculation density and basic inoculation time.
5. The method of optimizing preparation of iPSC-based ovarian granulosa extracellular fluid of claim 4, wherein identifying a plurality of culture test groups based on the oxygen concentration range, the seeding density range and the seeding time range in the culture control parameters comprises: Performing third-order division on the oxygen concentration range in the culture control parameter to obtain low oxygen concentration, medium oxygen concentration and high oxygen concentration; third-order division is carried out on the inoculation density range in the culture control parameters to obtain low inoculation density, medium inoculation density and high inoculation density; Combining the low oxygen concentration, the medium oxygen concentration and the high oxygen concentration with the low inoculation density, the medium inoculation density and the high inoculation density in pairs to obtain 9 preliminary test combinations; Uniformly sampling the inoculation time range according to a preset second interval to obtain a plurality of inoculation times; the 9 preliminary test combinations were combined with a plurality of inoculation times to obtain a plurality of culture test groups, wherein each culture test group included oxygen concentration, inoculation density and inoculation time.
6. The method for optimizing preparation of an ovarian granulosa cell exosome based on iPSC according to claim 5, wherein the performing basic detection on the differentiated stem cells to obtain detection data comprises: Sampling the differentiated stem cells to obtain stem cell samples to be detected; pretreating a stem cell sample to be detected to obtain a treated sample; performing activity detection on the treated sample to obtain a cell activity index; extracting RNA from the processed sample to obtain total RNA of the sample; core gene detection is carried out on the total RNA of the sample to obtain a first core expression quantity and a second core expression quantity; Confirming a gene expression index based on the first core expression quantity and the second core expression quantity, wherein the gene expression index is a geometric mean of the first core expression quantity and the second core expression quantity; Carrying out biochemical detection on a sample to be processed to obtain glucose consumption and lactic acid generation; calculating a cell metabolic activity index according to the glucose consumption and the lactic acid production; And summarizing the cell vitality index, the gene expression index and the cell metabolic activity index to obtain detection data.
7. The method for optimizing preparation of iPSC-based ovarian granulosa extracellular fluid of claim 6, wherein the identifying a culture effect score based on the test data comprises: calculating a culture effect score according to the cell viability index, the gene expression index and the cell metabolic activity index in the detection data, wherein the calculation formula is as follows: Wherein, the The score of the culture effect is indicated, Represents the cell viability index of the cells, Represents the expression index of the gene, Indicating the index of the metabolic activity of the cells.
8. The method for optimizing preparation of iPSC-based ovarian granulosa extracellular body according to claim 7, wherein the identifying the corrected culture parameters based on the basic culture parameters comprises: confirming a culture defect type based on the anti-mullerian hormone positive rate, the second gene positive rate, the hormone positive rate threshold and the second gene positive rate threshold, wherein the culture defect type comprises insufficient anti-mullerian hormone positive rate, insufficient second gene positive rate or both; When the culture defect type is insufficient in the anti-mullerian hormone positive rate, increasing the basic oxygen concentration in the basic culture parameters by a preset first adjustment step length to obtain the corrected oxygen concentration; obtaining corrected culture parameters by utilizing 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 that the second gene positive rate is insufficient, increasing the basic inoculation density in the basic culture parameters by a preset second adjustment step length to obtain the corrected inoculation density; Obtaining corrected culture parameters by utilizing corrected inoculation density, basic oxygen concentration in the basic culture parameters and basic inoculation time in the basic culture parameters; When the culture defect types are insufficient, increasing the basic inoculation time in the basic culture parameters by a preset third adjustment step length to obtain corrected inoculation time; And obtaining the corrected culture parameters 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 method for optimizing preparation of iPSC-based ovarian granulosa extracellular fluid according to claim 8, wherein said identifying optimal separation parameters based on cell culture supernatant comprises: performing material testing on the cell culture supernatant to obtain total protein concentration and cell fluid viscosity; calculating an overspeed centrifugal force according to the total protein concentration; The elution flow rate is calculated according to the cell sap viscosity, and the calculation formula is as follows: Wherein, the Represents the elution flow rate, For a preset desired maximum elution flow rate, The viscosity of the cell fluid is indicated, Representing a preset reference cell fluid viscosity; and summarizing the overspeed centrifugal force and the elution flow rate to obtain the optimal separation parameters.
10. An iPSC-based preparation optimization system of ovarian granulosa cell exosomes, the system comprising: The basic parameter confirmation module is used for acquiring the human pluripotent stem cells, confirming the optimal induction factors and the optimal concentrations based on the human pluripotent stem cells, and confirming basic culture parameters based on the human pluripotent stem cells, the optimal induction factors and the optimal concentrations; The differentiated population acquisition module is used for directionally culturing the human pluripotent stem cells by utilizing basic culture parameters to obtain differentiated cell populations, and carrying out marker detection on the differentiated cell populations to obtain the anti-mullerian hormone positive rate and the second gene positive rate; The culture parameter correction module is used for comparing the anti-mullerian hormone positive rate with a preset hormone positive rate threshold value, comparing the second gene positive rate with a preset second gene positive rate threshold value, if the anti-mullerian hormone positive rate is greater than or equal to the hormone positive rate threshold value and the second gene positive rate is greater than or equal to the gene positive rate threshold value, taking the differentiated cell population as target ovarian granulosa cells, otherwise taking the differentiated cell population as human pluripotent stem cells, confirming a correction culture parameter based on a basic culture parameter, taking the correction culture parameter as the basic culture parameter, and returning to the step of directionally culturing the human pluripotent stem cells by using the basic culture parameter until the anti-mullerian hormone positive rate is greater than or equal to the hormone positive rate threshold value and the second gene positive rate is greater than or equal to the gene positive rate threshold value; The preparation optimization processing module is used for carrying out amplification culture on target ovary granular cells to obtain cell culture supernatant, confirming optimal separation parameters based on the cell culture supernatant, separating the cell culture supernatant by utilizing the optimal separation parameters to obtain an exosome extract, and completing preparation optimization of the exosome.

Description

Preparation optimization method and system of ovary granule cell exosome based on iPSC Technical Field The invention relates to the technical field of preparation of extracellular fluid, in particular to a preparation optimization method and system of ovarian granule extracellular fluid based on iPSC. Background Exosomes are a class of small membrane vesicles secreted by cells, which can be widely present in body fluids and perform important biological functions. Currently, the preparation methods of exosomes are various, including ultracentrifugation, immunoaffinity, ultrafiltration, etc., with ultracentrifugation being one of the most commonly used methods. By centrifugation, the exosomes can be efficiently isolated and purified from the culture medium. Although the above method can realize the preparation of exosomes, the conventional ultracentrifugation method can obtain exosomes of higher purity, but is difficult to apply in mass production due to its complicated operation, long time and high cost of equipment. In addition, the quality of the exosomes cannot be controlled well in the existing preparation method, so that great difference may exist in functional activities of the exosomes in different preparation batches, and the degree of intellectualization is insufficient, so that how to improve the degree of intellectualization of the exosome preparation process becomes a problem to be solved urgently. Disclosure of Invention The invention provides an iPSC-based preparation optimization method of an ovarian granule cell exosome and a computer-readable storage medium, and mainly aims to improve the intelligentization degree of an exosome preparation process. In order to achieve the above purpose, the preparation optimization method of the ovarian granule cell exosome based on iPSC provided by the invention comprises the following steps: Obtaining human pluripotent stem cells, and confirming optimal induction factors and optimal concentrations based on the human pluripotent stem cells; Confirming basic culture parameters based on the human pluripotent stem cells, the optimal induction factors and the optimal concentration; Performing directional culture on the human pluripotent stem cells by using basic culture parameters to obtain differentiated cell populations; performing marker detection on the differentiated cell population to obtain the anti-mullerian hormone positive rate and the second gene positive rate; comparing the hormone positive rate of the anti-mullerian duct with a preset hormone positive rate threshold value, and comparing the second gene positive rate with a preset second gene positive rate threshold value; if the hormone positive rate of the anti-mullerian tube is greater than or equal to a hormone positive rate threshold value and the second gene positive rate is greater than or equal to a gene positive rate threshold value, taking the differentiated cell population as target ovarian granulosa cells; Otherwise, taking the differentiated cell population as the human pluripotent stem cells, confirming a corrected culture parameter based on the basic culture parameter, taking the corrected culture parameter as the basic culture parameter, and returning to the step of directionally culturing the human pluripotent stem cells by utilizing the basic culture parameter until the hormone positive rate of the anti-mullerian tube is greater than or equal to a hormone positive rate threshold value, and the second gene positive rate is greater than or equal to a gene positive rate threshold value; Amplifying and culturing target ovary granular cells to obtain cell culture supernatant; Confirming optimal separation parameters based on the cell culture supernatant; and separating the cell culture supernatant by utilizing the optimal separation parameters to obtain an exosome extract, and completing the preparation optimization of the exosome. Optionally, the identifying the optimal induction factor and optimal concentration based on the human pluripotent stem cells comprises: Identifying common induction factors, wherein the common induction factors comprise a first factor, a second factor and a third factor; Identifying a first optimal test score and a first optimal concentration based on the human pluripotent stem cells, the first factor, and a preset first sampling interval; identifying a second optimal test score and a second optimal concentration based on the human pluripotent stem cells, a second factor, and a preset second sampling range; Confirming a third optimal test score and a third optimal test concentration based on the human pluripotent stem cells, a third factor and a preset third sampling range; The optimal induction factor and optimal concentration are 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. Optionally,