CN-121991899-A - Preparation method and system of ovarian granule extracellular fluid based on cell directional differentiation

CN121991899ACN 121991899 ACN121991899 ACN 121991899ACN-121991899-A

Abstract

The invention relates to the technical field of cell differentiation, in particular to a preparation method and a system of an ovarian granule cell exosome based on cell directional differentiation, which comprises the steps of reprogramming initial material cells to obtain initial induced pluripotent stem cells, carrying out cell identification to obtain induced pluripotent stem cells, carrying out directional differentiation on the induced pluripotent stem cells to obtain ovarian granule cell clusters, obtaining culture supernatant, purifying the culture supernatant to obtain an initial exosome, carrying out quality detection on the initial exosome to obtain a detection result, obtaining an adjusted exosome if the detection result is that the detection is not up to standard, taking the adjusted exosome as the initial exosome, and returning the step of carrying out quality detection on the initial exosome until the detection result is that the detection is up to standard, and confirming the initial exosome as a target exosome when the detection result is that the detection is up to standard. The invention can solve the problem of low purity of the exosome in the preparation process of the ovary granular cell exosome at present.

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: 20260211

Claims (10)

1. A method for preparing an ovarian granulosa extracellular fluid based on cell directed differentiation, the method comprising: obtaining a starting material cell; reprogramming the starting material cells to obtain initial induced pluripotent stem cells; performing cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; Performing directional differentiation on the induced pluripotent stem cells to obtain ovarian granulosa cell clusters; obtaining a culture supernatant of the ovarian granulosa cell mass; purifying the culture supernatant to obtain an initial exosome; Performing quality detection on the initial exosomes to obtain detection results, wherein the detection results are up to the standard or not up to the standard; if the detection result is that the detection does not reach the standard, acquiring an adjusted exosome, taking the adjusted exosome as the initial exosome, and returning to the step of detecting the quality of the initial exosome until the detection result is that the detection reaches the standard; And when the detection result is that the detection reaches the standard, confirming the initial exosome as a target exosome, and completing exosome preparation.
2. The method of preparing ovarian granulosa cell exosome based on cell-directed differentiation according to claim 1, wherein said reprogramming of the starting material cells results in an initially induced pluripotent stem cell comprising: Obtaining a culture unit; inoculating the initial material cells based on a culture unit to obtain an initial cell layer; activating the initial cell layer to obtain an activated initial cell layer; Programming the activated initial cell layer by using a preset reprogramming factor to obtain a mixed cell community; From the mixed cell population, initially induced pluripotent stem cells were confirmed.
3. The method for preparing an ovarian granulosa extracellular fluid based on cell-directed differentiation according to claim 2, wherein said activating said initial cell layer, obtaining an activated initial cell layer, comprises: the activity of the initial cell layer is improved by using a preset cell activator, so that an active cell layer is obtained; detecting the metabolic state of the active cell layer to obtain a metabolic characteristic information set; The following operations are performed on each of the metabolic feature information in the metabolic feature information set: based on the metabolism characteristic information, obtaining a metabolism judgment result; Summarizing the metabolism judgment results to obtain a metabolism judgment result set; selecting a set of active regions from the active cell layer based on the metabolic decision result set; based on the set of active regions, an activation initiating cell layer is obtained.
4. The method for preparing the ovarian granulosa extracellular fluid based on cell directional differentiation according to claim 3, wherein the metabolic state detection of the active cell layer to obtain a metabolic characteristic information set comprises: Dividing the active cell layer into areas to obtain a plurality of active cell areas; performing the following for each of the plurality of active cell regions: obtaining an active cell sample based on the active cell region; Acquiring a first fluorescent probe and a second fluorescent probe; performing fluorescent marking on the active cell sample by using the first fluorescent probe and the second fluorescent probe to obtain a fluorescent marked sample; Performing fluorescence detection on the fluorescence labeling sample to obtain a first fluorescence intensity sequence and a second fluorescence intensity sequence; acquiring a glucose uptake parameter and a mitochondrial membrane potential parameter based on the first fluorescence intensity sequence and the second fluorescence intensity sequence; lysing the fluorescent marked sample to obtain a lysed cell fluid; carrying out light-proof incubation reaction on the lysate by using a preset ATP fluorescent reagent to obtain ATP concentration; Constructing metabolism characteristic information according to the glucose uptake rate parameter, the mitochondrial membrane potential parameter and the ATP concentration; and summarizing the metabolism characteristic information to obtain a metabolism characteristic information set.
5. The method for preparing ovarian granulosa cell exosome based on cell directional differentiation according to claim 4, wherein said cell identification based on said initially induced pluripotent stem cells, resulting in induced pluripotent stem cells, comprises: Acquiring an initial induced pluripotent stem cell sample based on the initial induced pluripotent stem cell; Determining a pluripotency marker, wherein the pluripotency marker comprises a first marker and a second marker; Based on an initial induced pluripotent stem cell sample, carrying out positive expression detection on a first marker and a second marker in the pluripotent markers by using a preset activity detection device to obtain a first marker positive rate and a second marker positive rate; obtaining an alkaline phosphatase reagent, and performing dyeing treatment on the initially induced pluripotent stem cell sample by using the alkaline phosphatase reagent to obtain a dyed cell sample; scanning and imaging the stained cell sample by using a preset imaging unit to obtain a stained microscopic image; acquiring a phosphatase positive rate based on the staining microscopic image; Constructing an identification index set according to the first marker positive rate, the second marker positive rate and the phosphatase positive rate; When the identification index set does not meet the preset identification standard, acquiring the adjusted induced pluripotent stem cells, taking the adjusted induced pluripotent stem cells as initial induced pluripotent stem cells, and returning to the step of acquiring initial induced pluripotent stem cell samples based on the initial induced pluripotent stem cells until the identification index set meets the identification standard; when the identification index set meets the identification criteria, the initially induced pluripotent stem cells are identified as induced pluripotent stem cells.
6. The method for preparing ovarian granulosa cell exosome based on cell directional differentiation according to claim 5, wherein said performing directional differentiation on said induced pluripotent stem cells to obtain an ovarian granulosa cell mass comprises: Obtaining a differentiation culture medium; culturing the induced pluripotent stem cells by using the differentiation medium to obtain a pluripotent stem cell layer; Obtaining a first-stage differentiation inducer and a second-stage differentiation inducer; Performing differentiation induction on the pluripotent stem cell layer by using the first-stage differentiation inducer and the second-stage differentiation inducer to obtain a differentiated cell layer; dividing the differentiated cell layer to obtain a plurality of differentiated cell areas; performing the following operation on each of the plurality of differentiated cell regions: performing granular cell marker detection on the differentiated cell region to obtain marker expression degree; Determining a region detection result based on the marker expression level, wherein the region detection result comprises whether a region is qualified or not; summarizing the region detection results to obtain a region detection result set; based on the region detection result set, an ovarian granulosa cell mass is extracted from the plurality of differentiated cell regions.
7. The method for preparing an ovarian granulosa cell exosome based on cell orientation differentiation according to claim 6, wherein said obtaining a culture supernatant of said ovarian granulosa cell mass comprises: Determining a centrifugation medium based on the ovarian granulosa cell mass; amplifying and culturing the ovary granular cell mass by using a preset exosome culture medium to obtain a high-density granular cell layer; obtaining a serum-free granulosa cell culture medium, and performing stationary culture on the high-density granulosa cell layer by using the serum-free granulosa cell culture medium to obtain a stationary cell system; Obtaining a centrifugal unit, and performing centrifugal treatment by using the centrifugal unit based on the static cell system and the centrifugal medium to obtain a primary clarified supernatant; and carrying out vacuum filtration treatment on the primary clear supernatant to obtain a culture supernatant.
8. The method for preparing ovarian granule extracellular fluid based on cell-directed differentiation as claimed in claim 7, wherein the performing the quality detection of the initial exosomes to obtain detection results comprises: performing multi-view observation on the initial exosome by using an imaging unit to obtain a plurality of morphological images; carrying out morphological structure statistics on the plurality of morphological images to obtain a morphological qualification rate; Carrying out particle detection on the initial exosomes by using a preset particle analysis method to obtain particle sizes and particle concentrations; Summarizing the morphological qualification rate, the particle size and the particle concentration to obtain a cell physical characteristic set; and if the cell physical characteristic set meets the preset physical characteristic standard, carrying out chemical characteristic detection on the initial exosomes to obtain a detection result.
9. The method for preparing ovarian granule extracellular fluid based on cell directional differentiation as claimed in claim 8, wherein the performing chemical characteristic detection on the initial exosomes to obtain detection results comprises: extracting protein from the initial exosome to obtain exosome protein lysate; Performing immunoblotting detection on the exosome protein lysate by using a preset antibody combination to obtain the positive marker expression level and the negative marker expression level; protein detection is carried out on the exosome protein lysate to obtain the exosome protein concentration; Calculating an exosome chemical characteristic value according to the exosome protein concentration, the positive marker expression level and the negative marker expression level; if the chemical characteristic value of the exosome is smaller than a preset characteristic threshold value, confirming that the detection does not reach the standard as a detection result; and if the exosome chemical characteristic value is greater than or equal to a characteristic threshold value, confirming that the detection reaches the standard as a detection result.
10. A system for preparing an ovarian granulosa extracellular fluid based on cell directed differentiation, said system comprising: the cell reprogramming module is used for obtaining initial material cells, reprogramming the initial material cells to obtain initial induced pluripotent stem cells; The cell identification differentiation module is used for carrying out cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells, and carrying out directional differentiation on the induced pluripotent stem cells to obtain ovarian granulosa cell clusters; the exosome purification module is used for obtaining culture supernatant of the ovary granular cell mass, and purifying the culture supernatant to obtain an initial exosome; The exosome quality detection module is used for carrying out quality detection on the initial exosome to obtain a detection result, wherein the detection result is that the detection is up to standard or the detection is not up to standard, if the detection result is that the detection is not up to standard, the adjustment exosome is obtained, the adjustment exosome is used as the initial exosome, the above steps for carrying out quality detection on the initial exosome are returned until the detection result is that the detection is up to standard, and when the detection result is that the detection is up to standard, the initial exosome is confirmed to be a target exosome, and the exosome preparation is completed.

Description

Preparation method and system of ovarian granule extracellular fluid based on cell directional differentiation Technical Field The invention relates to the technical field of cell differentiation, in particular to a preparation method and a system of ovarian granule extracellular fluid based on cell directional differentiation. Background Along with the continuous development of regenerative medicine and reproductive medicine, the role of the ovarian granulosa cells in follicular development, hormone secretion and ovarian function maintenance is increasingly highlighted, and the exosomes of the ovarian granulosa cells are used as extracellular vesicles rich in bioactive molecules, so that the application demands of the ovarian granulosa cells in the fields of reproductive medicine, ovarian function repair, gynecological disease targeted therapy and the like are increasing, and the ovarian granulosa cells have great significance in preparation of the exosomes of the ovarian granulosa cells. The conventional preparation method of the oophoral granulosa cell exosomes generally relies on separating oophoral granulosa cells of human or animals, extracting exosomes after in vitro culture, and although the oophoral granulosa cell exosomes can be obtained, the quality of the obtained exosomes is not accurately detected, so that the purity of the obtained exosomes is insufficient, and therefore, the problem of low purity of the exosomes in the preparation process of the oophoral granulosa cell exosomes exists currently. Disclosure of Invention The invention provides a preparation method and a system of an ovarian granule extracellular body based on cell directional differentiation, and mainly aims to solve the problem of low purity of the ovarian granule extracellular body in the preparation process of the ovarian granule extracellular body at present. In order to achieve the above object, the present invention provides a method for preparing ovarian granule extracellular fluid based on cell directional differentiation, comprising: obtaining a starting material cell; reprogramming the starting material cells to obtain initial induced pluripotent stem cells; performing cell identification based on the initial induced pluripotent stem cells to obtain induced pluripotent stem cells; Performing directional differentiation on the induced pluripotent stem cells to obtain ovarian granulosa cell clusters; obtaining a culture supernatant of the ovarian granulosa cell mass; purifying the culture supernatant to obtain an initial exosome; Performing quality detection on the initial exosomes to obtain detection results, wherein the detection results are up to the standard or not up to the standard; if the detection result is that the detection does not reach the standard, acquiring an adjusted exosome, taking the adjusted exosome as the initial exosome, and returning to the step of detecting the quality of the initial exosome until the detection result is that the detection reaches the standard; And when the detection result is that the detection reaches the standard, confirming the initial exosome as a target exosome, and completing exosome preparation. Optionally, the reprogramming of the starting material cells to obtain initially induced pluripotent stem cells comprises: Obtaining a culture unit; inoculating the initial material cells based on a culture unit to obtain an initial cell layer; activating the initial cell layer to obtain an activated initial cell layer; Programming the activated initial cell layer by using a preset reprogramming factor to obtain a mixed cell community; From the mixed cell population, initially induced pluripotent stem cells were confirmed. Optionally, the activating treatment is performed on the initial cell layer to obtain an activated initial cell layer, which includes: the activity of the initial cell layer is improved by using a preset cell activator, so that an active cell layer is obtained; detecting the metabolic state of the active cell layer to obtain a metabolic characteristic information set; The following operations are performed on each of the metabolic feature information in the metabolic feature information set: based on the metabolism characteristic information, obtaining a metabolism judgment result; Summarizing the metabolism judgment results to obtain a metabolism judgment result set; selecting a set of active regions from the active cell layer based on the metabolic decision result set; based on the set of active regions, an activation initiating cell layer is obtained. Optionally, the detecting the metabolic state of the active cell layer to obtain a metabolic characteristic information set includes: Dividing the active cell layer into areas to obtain a plurality of active cell areas; performing the following for each of the plurality of active cell regions: obtaining an active cell sample based on the active cell region; Acquiring a first fluorescent probe and a second fluo