47 results about "Ovarian tissue" patented technology

The invention belongs to the technical field of biological medicine, and particularly relates to a reagent for improving ovarian development and a preparation method and application thereof. The reagent for improving ovarian development is prepared by mixing the following raw materials in parts by weight: 20-30 parts of N-acetylcysteine; 5 to 10 parts of tranexamic acid; 3 to 8 parts of creatine; and 3000 parts of water. According to the reagent, the mRNA expression of PKD1P6 and follicle stimulating hormone receptors (FSHR) in ovarian tissues is up-regulated, the activity and functions of cumulus granular cells are enhanced, and apoptosis of the cumulus granular cells is inhibited, so that the effects of effectively promoting follicle development and inhibiting the apoptosis of the cumulus granular cells are achieved.

The invention relates to an isolated culture method of functional Chinese softshell turtle ovarian granular cells. The isolated culture method comprises the following steps: S1, collecting ovarian tissues: collecting Chinese softshell turtle ovarian tissues; s2, separating and collecting a granular cell layer; s3, digesting and collecting granular cells; s4, primary culture of granulosa cells: transferring the collected cells into a cell culture bottle containing 4 ml of a complete culture medium for primary culture, wherein the complete culture medium comprises the following main components: a DMEM culture medium, 15% of fetal calf serum, 1% of penicillin streptomycin and 1% of an insulin-transferrin-selenium additive; and S5, subculturing the granular cells. The isolated culture method and the culture medium system provided by the invention are simpler and more time-saving and labor-saving; according to the method, the aromatase CYP19A1 can be expressed normally, the characteristics of the granular cells are kept, the aromatase CYP19A1 can be expressed normally, it is indicated that the in-vitro cultured cells can keep the functionality of the cells, and the in-vitro proliferation capacity of the granular cells is improved to a great extent.

The invention discloses a composition and a method for constructing ovarian organoid based on a clinical tissue sample. The composition comprises a sample preservation solution, a sample digestive juice and a sample culture medium, the method comprises the following operation steps: transporting a preservation solution A to a laboratory, and pretreating to obtain tissue fragment precipitates; carrying out resuspension incubation on the tissue fragment precipitate by using digestive juice B for digestion; collecting tissue blocks which are not completely digested and filtered, and performing secondary digestion to obtain single cells; combining the cells obtained in two times to obtain the required single cell; adding matrigel, then adding the mixture into a pore plate, and putting the pore plate into an incubator until the matrigel is solidified; and taking out the pore plate, adding a culture medium L2, and culturing to obtain the ovarian organ constructed based on the clinical sample. According to the method, the ovarian organoid is efficiently constructed from the ovarian tissue sample; the provided culture solution formula makes up the dilemma that normal ovarian tissue organoids are difficult to form and normal tissue-derived organoids are difficult to passage at present, and reduces the burden and experiment cost brought by sampling in the disease research process.

The application discloses a doxorubicin scavenger for protecting ovarian fertility and a preparation method thereof, comprising a DNA complex wrapped by a red blood cell membrane, wherein the red blood cell membrane is surface-modified by an anti-Mullerian duct antibody alphaAMH; the doxorubicin scavenger is specifically combined with AMH which is highly expressed in ovarian tissues through alphaAMH, is enriched in the ovarian part in a targeted manner, adsorbs doxorubicin through a DNA fragment in the DNA complex, reduces toxic damage of doxorubicin to ovarian cells, and further protects ovarian fertility. The doxorubicin scavenger can adsorb injected doxorubicin Dox, reduce side effects of doxorubicin Dox on ovarian granulosa cells, and further protect ovarian function and fertility.

The invention relates to an effect of an REV-ERBalpha agonist SR9009 in the aspect of delaying ovarian senescence, and aims at the problem of fertility decline caused by cliff-type decline of ovarian functions of women over 35 years old, the SR9009 is applied to activate an REV-ERBalpha circadian rhythm signal channel in ovarian tissues, inhibit excessive autophagy of granular cells and maintain a primordial follicle dormancy state, so that the agonist SR9009 can be used for delaying ovarian senescence. Therefore, the original follicle depletion speed is delayed, follicle storage is maintained, and the ovary reproductive function cycle is prolonged. The effect is verified by a mammal model: REV-ERBalpha protein expression in an ovary of an aging individual is reduced, rhythmicity expression is disordered, autophagy of granulosa cells can be effectively inhibited in the ovary of the aging individual intervened by the SR9009, and the number of residual primordial follicles and the number of total follicles are obviously greater than those of an untreated control group. The invention provides an innovative treatment strategy of targeted rhythm regulation and control for clinical intervention of ovarian aging.

This invention relates to the field of ovarian tissue in vitro culture technology, and discloses a formulation for activating and increasing follicles and its application. The formulation consists of a PI3K / Akt / mTOR pathway activator and / or a PTEN inhibitor SF1670. The PI3K / Akt / mTOR pathway activator is a 5-15 μM statin, and the PTEN inhibitor is a 20-50 μM SF1670. Using this formulation can achieve the follicle activation and increase effect of existing technologies, contributing to the development and selection of in vitro follicle activators in clinical practice. Furthermore, the use of this formulation opens up a new technological application area for statins.

Disclosed are a method and culture medium for performing an in vivo treatment on ovaries of mammals including humans or performing in vitro culturing on ex vivo ovaries or ovarian tissue by means of using 4-vinylcyclohexene diepoxide. The method or culture medium provided facilitates the retention and enhancement of the developmental and reproductive potential of mammalian ovaries or ovarian tissue, and is particularly capable of promoting follicle maturation and increasing the number of ovulations.

The invention discloses a stem cell exosome composition for improving premature ovarian failure as well as a preparation method and application of the stem cell exosome composition. The composition is composed of human umbilical cord mesenchymal stem cell exosomes obtained through hypoxia and TGF-beta1 combined pretreatment and resveratrol, and the human umbilical cord mesenchymal stem cell exosomes and the resveratrol are compounded according to a specific mass ratio. The pre-treatment condition is that 1% oxygen concentration and 10 ng / ml TGF-beta1 are co-cultured for 48 hours, and the condition can synergistically up-regulate the expression level of specific miRNA in the exosome. The composition prepared through physical incubation and an ultrafiltration method shows a clear synergistic treatment effect in vitro and in vivo, the combination index of the composition is 0.38-0.45, ovarian endocrine indexes can be effectively recovered, follicular reserve can be protected, and oxidative stress and inflammation states of ovarian tissues can be remarkably improved. The invention provides a novel treatment strategy with a novel mechanism and a remarkable curative effect for premature ovarian failure, especially chemotherapy-induced ovarian function impairment.

The invention belongs to the technical field of medicines, and particularly relates to application of a licorice extract in preparation of a medicine for treating premature ovarian failure. On the basis of network pharmacology prediction and animal experiment verification, through experimental exploration, it is clear that diammonium glycyrrhizinate can treat premature ovarian failure by improving ovarian tissue energy metabolism, reducing the oxidative stress level and inhibiting ovarian cell apoptosis, and also can treat premature ovarian failure by improving the ovarian growth and development microenvironment. According to the present invention, the growth and development rate of ovarian granular cells and membrane cells is improved, the sufficient quantity of granular cells and membrane cells are ensured to secrete female progestational hormone, the damaged ovarian tissue is further protected, the new strategy and hope are provided for the treatment of premature ovarian failure, and the pharmaceutical composition is expected to become the safe and effective treatment drug.

The application relates to a method for establishing an early ovarian failure rat model, which comprises the following steps: taking healthy rats, and performing gavage on the rats according to a dosage of 10-20 mg / Kg of a phytostilbene preparation, wherein the dosage is the ratio of phytostilbene to the weight of the rats, and obtaining the early ovarian failure rat model after four days of administration. The early ovarian failure rat model is obtained by performing gavage on the rats after the phytostilbene preparation, the model is simple to establish, and the model establishment time is short. Through ovarian pathological observation, it can be seen that the simulated animal model has obvious ovary tissue damage, follicular granulosa cell destruction and apoptosis, and follicle atresia and other symptoms. The obtained model has good stability and repeatability. The phytostilbene is used to successfully establish the early ovarian failure rat model, lays a good foundation for establishing a stable early ovarian failure model for basic research, and provides a reliable method for the construction of the early ovarian failure model.

The invention provides an ictalurus punctatus dnmt1 gene knockout RNAi target sequence, a sex regulation and control method and application, and belongs to the technical field of sex regulation and control. An RNAi sequence is designed based on the dnmt1 gene, and gonad differentiation intervention independent of exogenous hormones is realized in the species by inhibiting expression of the dnmt1 gene in the channel catfish. Experimental results show that dnmt1 expression down-regulation can effectively induce partial sex reversal of genetic female individuals, typical testis-like structures appear in ovarian tissues of the female individuals, it is proved that dnmt1 plays a key role in maintaining female gonad development stability, and it is hopeful that dnmt1 regulation is combined with a genetic screening or gene editing technology, so that the development stability of the female gonad is improved. A stable and efficient all-male channel catfish breeding system is established, and a new path is developed for all-male offspring seed production.

This invention belongs to the field of biomedical technology, specifically relating to the application of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome (PCOS). The composition comprises bovine spleen peptide and adenosine; the mass ratio of bovine spleen peptide to adenosine is 1:(1-2.5). Bovine spleen peptide, as a bioactive polypeptide, significantly improves insulin resistance associated with PCOS by targeting and activating the PI3K-Akt signaling pathway and promoting insulin receptor substrate phosphorylation. Adenosine, as a natural bioactive substance, significantly reduces serum luteinizing hormone and testosterone levels in rats and increases follicle-stimulating hormone levels, thereby effectively alleviating hyperandrogenism symptoms such as hirsutism and acne in PCOS. Furthermore, this composition can significantly improve polycystic pathological changes in the ovarian tissue of model rats, reduce the number of cystic dilated follicles, restore the granulosa cell layer structure, alleviate ovarian stromal hyperplasia, and promote normal follicular development.

The application relates to application of FOS / AP-1 inhibitors in preparation of drugs for improving vitrification freezing damage of ovary tissues. The application provides a new concept and intervention target for female fertility preservation based on ovary vitrification freezing, and has a good application prospect.

The invention relates to a traditional Chinese medicine effective component screening method based on a chemical fingerprint spectrum and multi-omics combined analysis. The method comprises the following steps: preparing a semen cuscutae-fructus lycii medicine pair extract and establishing a UPLC-Q-TOF-MS / MS chemical fingerprint spectrum; detecting the levels of estradiol, luteinizing hormone, anti-mullerian hormone and follicle-stimulating hormone in the premature ovarian insufficiency animal model; screening characteristic components related to the drug effect through multivariate statistical analysis; performing transcriptome sequencing on the ovarian tissue, and combining GO / KEGG analysis to determine a key pathway; integrating database prediction targets and differential genes, screening hub genes and constructing a protein interaction network; and finally verifying the binding activity of the candidate component and the core target protein through molecular docking and molecular dynamics experiments. According to the invention, systematic screening and action mechanism analysis of drug effect related components are realized, and a new technical approach is provided for mass marker determination and estrogen simulating action research of the dodder-wolfberry drug pair.

The invention discloses an ovarian tissue low-temperature cryopreservation solution as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The invention relates to an ovarian tissue low-temperature cryopreservation solution as well as a preparation method and application thereof. Every 100 ml of the ovarian tissue low-temperature cryopreservation solution comprises 15-25 ml of dimethyl sulfoxide, 15-25 ml of ethylene glycol, 15-20 g of polysucrose, 38-46 g of sucrose, 40-45 ml of a phosphate buffer solution and 0.12-0.13 g of human serum albumin. According to the low-temperature cryopreservation solution for the ovarian tissues, the dimethyl sulfoxide and the ethylene glycol are used as permeable low-temperature protective agents to prevent the ovarian tissues from being damaged at low temperature, and the polysucrose and the sucrose are used for regulating the cell osmotic pressure to improve the cell dehydration effect, reduce the formation of ice crystals in cells and increase the viscosity of the solution; according to the low-temperature cryopreservation solution for the ovarian tissue, the solution forms an amorphous glassy state instead of ice crystals in the freezing process, the human serum albumin maintains osmotic pressure and nutrition, the tissue survival rate and the functional integrity are guaranteed, the ovarian tissue can be efficiently cryopreserved through the low-temperature cryopreservation solution for the ovarian tissue, and the follicle survival rate and the functional integrity after ovarian transplantation are effectively improved.

The application provides application of a hydrogen sulfide donor SPRC in improving early-onset ovarian dysfunction caused by a chemotherapy drug, and relates to the technical field of biomedicine, and the technical key points are as follows: the application provides application of the hydrogen sulfide donor SPRC in preparing a drug for improving early-onset ovarian dysfunction. The present application aims at the technical problems of early-onset ovarian dysfunction causing the decline of female ovarian function and reproductive capacity, including the decline of ovarian index, the reduction of the number of follicles at all levels, and the significant increase of the number of apoptotic cells in ovarian tissue. The specific verification experiments prove that supplementing the hydrogen sulfide donor SPRC in a mouse model of early-onset ovarian dysfunction can improve the body weight and ovarian index of the mouse, and maintain the normal morphology of the ovary. Specifically, the SPRC can improve the number and quality of follicles in the ovary by reducing cell apoptosis, and ultimately rescue the ovarian damage caused by POI.

The invention provides an application of a hydrogen sulfide donor SPRC in improving early-onset ovarian insufficiency caused by chemotherapeutic drugs, relates to the technical field of biomedicine, and has the technical key point that the application of the hydrogen sulfide donor SPRC in preparing drugs for improving the early-onset ovarian insufficiency is provided. The application aims to solve the technical problems that the ovarian function and reproductive capacity of women are reduced, including the ovarian index is reduced, the number of follicles at all levels is reduced and the number of apoptotic cells in ovarian tissues is remarkably increased, caused by premature ovarian insufficiency at present. Specific verification experiments prove that by supplementing a hydrogen sulfide donor SPRC in a premature ovarian insufficiency mouse model, the weight and the ovarian index of the mouse can be increased, the normal form of the ovarian can be maintained, specifically, the SPRC can improve the number and the quality of follicles in the ovarian by reducing cell apoptosis, and finally ovarian injury caused by POI is rescued.

The invention belongs to the field of bioelectromagnetism, and particularly discloses a method for improving functions of frozen and thawed ovarian tissues by adopting an mT-level pulsed magnetic field. According to the method, ovarian tissue which is subjected to vitrification freezing and resuscitation is exposed in a pulsed magnetic field with specific parameters for intervention treatment; wherein the intensity of the pulsed magnetic field is 5 mT, the current period is 1000-6000 [mu] s, the duty ratio is 50%, the pulse repetition number is 30, and the pulse interval is 60 ms. According to the application, a non-intrusive and drug-residue-free physical intervention means is adopted, inflammatory response can be effectively inhibited, oxidative stress injury can be relieved, the number of functional mitochondria is increased by up-regulating mitochondrial fusion genes (such as Mfn1 and Mfn2), the energy metabolism state of cells is fundamentally improved, the survival rate of transplanted ovarian tissue is remarkably increased, the functional life of the ovarian tissue is prolonged, and the survival rate of the ovarian tissue is increased. The method is simple to operate, low in cost and good in clinical application prospect.

This invention discloses the application of a DMSO-free cryopreservation solution in the cryopreservation of organs and / or tissues. The cryopreservation solution, per 100 mL volume, contains 0.01-50.0 g of a biomimetic ice-controlling material, 5.0-30 mL of polyol, 1-30 g of water-soluble sugar, 0-30 mL of serum, with the remainder being buffer solution. The biomimetic ice-controlling material is selected from PVA and / or amino acid-based biomimetic ice-controlling materials. The cryopreservation solution and cryoequilibration solution of this invention do not contain DMSO, exhibiting high preservation efficiency when used for cryopreservation of ovarian tissue or organs. Furthermore, the DMSO- and serum-free cryopreservation solution further solves the problems of poor stability and the potential introduction of parasitic biological contaminants caused by the presence of serum in currently widely used commercial cryopreservation solutions. The cryopreservation solution of this invention has a simple composition, readily available raw materials, and low cost, and can be widely used for the cryopreservation of various organs or tissues.

The invention relates to a sea cucumber ovum oligopeptide composition for increasing female AMH value and improving ovarian function and application of the sea cucumber ovum oligopeptide composition, and belongs to the field of medicines, and the sea cucumber ovum oligopeptide composition comprises, by weight, 15-25 parts of sea cucumber ovum oligopeptide; 10 to 15 parts of mussel oligopeptide; and 10-20 parts of a polyphenol extract with homology of medicine and food. The sea cucumber ovum oligopeptide has the effects of improving the levels of hormones such as AMH, improving the energy metabolism and oxidative stress levels of oocytes and ovarian granular cells and promoting the proliferation of ovarian stem cells. The medicinal and edible polyphenol extract has the effects of reducing chronic ovarian cell inflammation, improving ovarian tissue blood supply, increasing uterus visceral organ coefficient and relieving ovarian immune injury, and compared with direct intake of an existing traditional Chinese medicine formula, the medicinal and edible polyphenol extract has the advantage that the absorption rate is remarkably improved. All the components improve the ovarian function from different aspects, the ovarian reserve improving effect better than that of single or few components is achieved under the synergistic effect of all the components, and the composition is safe and free of toxic and side effects.

The invention discloses a mare follicle development noninvasive real-time monitoring method and system based on multi-parameter fusion, and relates to the crossing field of animal breeding technology and medical imaging technology. The method comprises the following steps: transmitting ultrasonic waves to mare ovarian tissues through a monitoring assembly, receiving reflection echoes, generating two-dimensional / three-dimensional ultrasonic image data of ovarian follicles and endometrium, and collecting temperature data and light intensity time sequence data; the received ultrasonic image data, temperature data and light intensity time sequence data are preprocessed respectively, and follicle parameters, endometrial parameters, pelvic cavity temperature parameters and ovarian peripheral tissue blood flow related parameters are obtained; and constructing a multi-parameter fusion model, inputting the obtained follicle parameters, endometrial parameters, pelvic cavity temperature parameters and blood flow related parameters into the model, carrying out feature fusion by adopting a deep learning architecture, and outputting a mare follicle development stage evaluation result and an ovulation time prediction value. According to the invention, injury, infection and stress risks caused by invasive operation can be avoided, and long-term multi-time monitoring can be realized.

A grading and counting method for follicles and corpus luteum growing in ovaries of rats relates to the technical field of image analysis, and comprises the following steps: scanning ovary staining sections; identifying needed ovarian tissues and converting the needed ovarian tissues into a first region of interest ROI1; screening out growing follicles and corpus luteum meeting requirements on ROI1; and identifying and marking the screened growing follicles meeting the requirements as primary follicles, secondary follicles, tertiary follicles and follicles which cannot be graded, identifying and marking the screened corpus luteum meeting the requirements as newborn corpus luteum, recent corpus luteum, old corpus luteum and corpus luteum which cannot be graded, and finally counting to obtain the number of the follicles and corpus luteum of each level. According to the invention, artificial intelligence of classification and counting of ovarian growth follicles and corpus luteum of rats can be realized, and rapid and accurate batch operation is realized.

A novel medical use of ingenol HEP14, a dosage form of an HEP14-based drug, and a preparation method for the dosage form. The dosage form of the HEP14-based drug is an HEP14 / PLGA microsphere sustained-release dosage form formed by encapsulating the HEP14-based drug with a polylactic acid-glycolic acid copolymer, and has the functions of high HEP14 drug loading rate, high encapsulation rate, and long-acting HEP14 sustained-release. It has been found for the first time that HEP14 can promote production and autophagy of mitochondria in cells and enhance the function of mitochondria in cells, promote the secretion of antioxidant enzymes and remove reactive oxygen species, promote the growth and viability of ovarian follicle granulosa cells and restore the growth and viability of aging granules, promote the secretion of angiogenic factors, reduce the fibrosis of ovarian tissues, promote the angiogenesis of ovarian tissues, and promote the regeneration of aged ovarian follicles and ovarian endocrine functions, and thus has a good effect of treating and / or preventing related diseases caused by mitochondrial dysfunction, especially the effect of treating age-related ovarian decline.

The invention discloses an exosome preparation for treating premature ovarian failure, and belongs to the technical field of biology. And adding leonurine and estradiol into the umbilical cord mesenchymal stem cell culture solution, and co-culturing to prepare the exosome preparation for treating premature ovarian failure. The conventional exosome and the leonurine and estradiol co-cultured exosome with the same dosage can weaken the inhibition effect of the 4-OHCP on the KGN cell activity, and the leonurine and estradiol co-cultured exosome has a better human ovarian granular cell damage repair effect than the conventional exosome. When the leonurine and estradiol co-cultured exosome is used for treating a rat with premature ovarian failure, inflammatory cell infiltration in ovarian tissue interstitial substances is improved, follicle granular cell layers are increased, follicles in the growing period are increased, and mature follicles develop. Compared with a premature ovarian failure model group, the leonurine and estradiol co-cultured exosome group has the advantages that the serum FSH and LH levels of rats are reduced, the AMH level is increased, and the treatment effect on the premature ovarian failure rats is superior to that of a conventional exosome group.

The application provides a method for separating and culturing porcine preantral follicles, and belongs to the technical field of preantral follicle separation and culture. The preantral follicles are separated by a mechanical-enzyme compound method, and the preantral follicles are cultured in vitro by a three-dimensional culture method coated by a sodium alginate gel system. The separation method of the porcine preantral follicles comprises the following steps: mixing porcine ovarian tissue fragments with follicle separation solution and digesting for 0.5-2 hours, discarding the supernatant, stopping the digestion, and passing the digested tissue through a 400-micron mesh screen to obtain the preantral follicles. The preantral follicle separation and in-vitro culture system are optimized, the in-vitro separation efficiency of the preantral follicles is improved, and the in-vitro antrum formation culture of the preantral follicles is achieved. Not only can the method provide sufficient egg sources for porcine embryo engineering research and application, but also can provide a reference for further tapping the genetic potential of pigs.

The present application belongs to the technical field of pharmaceutical preparations, and specifically relates to the new use of Tongren Wuji Baifeng Pills and a pharmaceutical composition in the preparation of a drug for treating premature ovarian failure, a pharmaceutical preparation comprising Tongren Wuji Baifeng Pills, a method for preparing same, and a method for preparing a drug for treating premature ovarian failure. In the present application, a rat model of chemotherapy-induced premature ovarian failure via intraperitoneal injection of cisplatin and a mouse model of immune-mediated premature ovarian failure via multi-point subcutaneous injection of mouse zona pellucida peptide 3 (pZP3) are established. By means of observing the effects of Tongren Wuji Baifeng Pills on the estrous cycles, organ coefficients, serum sex hormone levels, and ovarian histopathological changes in animals in both models, the therapeutic effect of Tongren Wuji Baifeng Pills on premature ovarian failure is verified and clarified. This expands the new clinical use of the drug and provides a new therapeutic drug and means for treating premature ovarian failure, thereby offering technical support for clinical precision medication.

The invention discloses an application of a sheep placenta extract in preparation of a medicine for improving polycystic ovarian syndrome, and relates to the technical field of biological medicine, the medicine is used for improving endocrine disorder and abnormal inflammation caused by the polycystic ovarian syndrome, and particularly for reducing the plasma androgen level and increasing the plasma estrogen level and the plasma progestational hormone level; the level of an inflammatory factor TNF-alpha is reduced, and the level of an inflammatory factor IL-10 is increased. The sheep placenta extract can improve ovarian polycystic lesions caused by polycystic ovarian syndrome (PCOS) in a targeted manner, and experiments show that after intervention of the sheep placenta extract, the number of macroscopic cystic follicles in rat ovary is reduced, the weight of the ovary with abnormal weight gain is remarkably reduced, the cystic follicle ratio in ovarian histopathology detection is greatly reduced, and the sheep placenta extract has a good application prospect. The ratio of the number of the corpus luteum is closer to normal, and the pathological injury of the polycystic ovarian syndrome is repaired from the organ level.

This application relates to an ovarian tissue cutting table and cutting assembly. The ovarian tissue cutting table includes a base plate and a laminated plate stacked on top of the base plate. The laminated plate comprises two layers, each layer having a cutting window and graduations. Ovarian tissue is pressed onto the laminated plate, allowing the cut ovarian tissue to protrude beyond the cutting window. Gaps are provided between the laminated plates and between the laminated plate and the base plate to allow a thin-blade cutter to laterally cut the ovarian tissue protruding beyond the cutting window. The ovarian tissue cutting table and cutting assembly of this invention facilitate cutting operations and allow for intuitive measurement of the cut dimensions of the ovarian tissue, providing necessary data for the cryopreservation of ovarian tissue.

The application discloses a method for separating and culturing ovary granulosa cells of Chinese soft-shelled turtles, which comprises the steps of selecting female Chinese soft-shelled turtles in the positive oviposition period, executing cervical dislocation after anesthesia, killing the turtles, separating ovary tissues, removing redundant parts after washing, separating II-III stage follicles in a basic culture medium, keeping follicle integrity, performing enzyme digestion treatment, removing supernatant through centrifugation, obtaining a precipitate containing ovules with a diameter of 0.1-0.5 cm, resuspending the precipitate by using a mixed culture medium, and inoculating and culturing. The ovary granulosa cells obtained by the separation method have a high yield, the ovary granulosa cells are cultured in the specific mixed culture medium, the method can replace many factors, the amount of added factors is small, the NaHCO3-free culture medium is used, the use of CO2 is reduced, and the ovary granulosa cells obtained are consistent with the in-vivo environment, so that the cost is reduced and the in-vitro survival period of the ovary granulosa cells is prolonged.

The invention discloses a graphene-heated ovarian tissue cryopreservation tube and a use method thereof, and relates to the technical field of medical instruments. The ovarian tissue cryopreservation tube heated through graphene comprises a tube body, a tube cap and a fixing piece arranged on the tube cap, the fixing piece comprises a containing part, a first fixing part arranged on the containing part and used for fixing ovarian tissue and a connecting part connected with the first fixing part, and the connecting part is fixedly connected with the tube cap; a temperature sensor close to one side of the first fixing part is arranged in the placing part, the temperature sensor is adjacent to a heating assembly, the pipe cap is provided with an electric connection assembly used for supplying power to the heating assembly, the electric connection assembly is electrically connected with an external power supply end, and the heating assembly is electrically connected with the electric connection assembly through a connecting part.