TW202140777A - Oviductal stem cell culture promotes oocyte maturation and embryo development in vitro, biopreparation comprining them the method of preparation and uses thereof. - Google Patents

Abstract

The oocytes in vitro maturation (IVM) rate and in vitro culture (IVC) development rate could be enhanced by the addition of serum, electron carrier, animo acids and anti-oxidants, but development rate still lower than in vivo. Also, the matured oocytes would suffer from polyspermy easily.

In IVM, porcine oocytes were matured under PFTSCs conditioned M199 and BFTSCs conditioned M199. In IVC, matured procine oocytes were cultured under PFTSCs conditioned PZM and BFTSCs conditioned PZM.

PFTSCs and BFTSCs promoted the porcine oocytes maturation rate, and had a trend to improve the blastocysts rate which had the potential to the clinic medical applications.

Description

Preparation, preparation method and application of oviduct stem cell for promoting oocyte maturation and embryo development in vitro

The invention relates to a preparation of oviduct stem cells to promote in vitro maturation and embryo development of oocytes, its use for preparing preparations for promoting in vitro maturation and embryo development of oocytes, and the use of establishing a therapeutic experimental platform system; in particular, it relates to a pig and cattle The use of oviduct stem cells for preparing preparations for promoting oocyte maturation and embryo development in vitro, and for preparing preparations for promoting oocyte maturation and embryo development in vitro, and for establishing a therapeutic experimental platform system.

Fallopian tube (Fallopian tube, FT) has secretory cells that can provide the functions required for egg maturation and development. Previous studies have confirmed that fallopian tube fluid can enhance egg maturity, and after co-cultivation with fallopian tube cells, in addition to enhancing egg maturity and development, It can also improve the hardening ability of the zona pellucida and reduce the problem of more sperm entering the egg.

In addition, previous studies have pointed out that stem cells have a paracrine effect, which can promote cell growth and repair. The fallopian tube is also a medical waste of the slaughterhouse. The source is sufficient and does not need to be obtained by invasive methods. Cells, FTSCs) have the potential for pluripotency and also have excellent proliferation and differentiation capabilities. The growth factors secreted by them can promote the repair of reproductive tract damage and egg development.

The US20160237402A1 patent mentions that this technology provides a method for directed differentiation of pluripotent cells, female germline stem cells or oocytes into oocytes, granule cells and/or granule precursor cells, that is, "synthetic granule cells". Synthetic granulosa cells can be used in methods for the growth and maturation of follicles or follicle-like structures and immature oocytes. In addition, synthetic granulosa cells can be used in methods to increase ovarian-derived hormones and growth factors in subjects in need. This technology provides a method for differentiating pluripotent cells into granule cells and/or granule precursor cells. The method includes culturing the pluripotent cells under culture conditions in which the pluripotent cells are differentiated into granules. Cells and/or granule precursor cells, in which the culture conditions are absent of mouse embryonic fibroblast (MEF) and leukemia inhibitory factor (LIF) and the presence of a GSK inhibitor.

The I322183 patent relates to a method for producing non-human mammal chimeric embryos (chimeric embryos), using non-human 1-cell stage embryos to morula from zona pellucida removed in a microcentrifuge tube ( Coculture with fresh or freshly thawed cells in Eppendorf vial to obtain non-human mammalian chimeric embryos; the aforementioned chimeric embryos are transferred to non-human embryos by embryo transfer, After the end of pregnancy, non-human chimeric animals with gonad heritability and animals formed from non-human embryonic stem cells (embryonic stem, ES, cell-derived animal) can be obtained. The patent is a method for producing chimeric embryos in mice. The steps include: obtaining mouse cells; obtaining mouse 1-cell stage embryos from zona pellucida to morula; The aforementioned mouse cells and the aforementioned zona pellucida-removed mouse 1-cell stage embryos to morula were mixed in a microcentrifuge tube (Eppendorf vial) to coculture (coculture) for a period of time to obtain small Mouse "embryo-cell aggregate"; and continue to cultivate the aforementioned mouse "embryo-cell" Polymerize to obtain mouse chimeric embryos.

The US20190002919A1 patent and the EP3495470A1 patent mention a method for improving the fertilization ability of a female mammal. The method includes administering one or more bioenergetic agents to effectively enhance the oocyte or oocyte stem cell (OSC) of the female mammal. Female mammalian subjects, thereby improving the fertility of female mammalian subjects. The method of producing oocytes includes culturing stem cells in the presence of them, including but not limited to oocyte stem cells, embryonic stem cells, skin stem cells, pancreatic stem cells and induced pluripotent stem cells (iPS cells). The bioenergy reagent or its functional derivative is prepared under the conditions sufficient to differentiate the stem cells into the oocyte. The culture method is through the administration of one or more bioenergy reagents, so there are still technical differences. The main difference of the present invention is to verify differentiation through stem cells and gradually establish separation and culture methods, which have the effect of enhancing oocyte maturity and assisting egg development.

The problems to be solved by the present invention are as follows:

Although the previous technology can use serum, electron carriers, amino acids or antioxidants to improve the maturation rate of in vitro maturation (IVM) and the development rate of in vitro culture (IVC) in oocytes, due to The effect is not good, the growth rate of in vitro culture is 8.0%, and the rate of in vivo culture is 20.6%. The growth rate of in vitro culture is still not as good as the rate of in vivo culture (8.0% vs 20.6%), and in fact it is still prone to the shortcomings of getting too much sperm into the egg .

Follicular fluid (FF) is a fluid that fills the follicular cavity and surrounds the egg in the follicle of the ovary. It can be used for improved intracytoplasmic sperm injection.

Follicle-stimulating hormone (Follicle-stimulating hormone) is a hormone secreted and synthesized by the pituitary gland. It is a glycosylated protein hormone that regulates development, sexual maturity and production. Reproduction-related physiology.

Human chorionic gonadotropin (Human chorionic gonadotropin) is a glycoprotein hormone, which is secreted by the trophoblast cells of the placenta. Its main function is to stimulate the corpus luteum to promote the formation of the decidua of the uterus and make the placenta grow and mature.

M199 (Sigma, M4530), used in cell culture, was originally developed as a complete medium formulation. These media have a wide range of applicability, especially for untransformed cells.

Porcine oocytes are cultured with the following in vitro culture basic media, such as PZM series (PZM-3/PZM-4/PZM-5) medium and TCM-199. Used for in vitro development, metabolism and nutritional needs of pig oocytes.

M199 is used as oocyte maturation medium, and PZM-3 is used as porcine zygote medium-3 (PZM-3). PFTSCs are porcine fallopian tube stem cells, and BFTSCs are cattle fallopian tube stem cells.

The present invention discloses a preparation method of isolated porcine oviduct stem cells, which comprises the following steps: culturing porcine oocytes in IVM in M199 culture medium treated with porcine oviduct stem cells (PFTSCs), and culturing in IVC in PZM-treated PZM- treated with PFTSCs 3 Culture medium.

Sources of stem cells include cord blood stem cells, embryonic stem cells, skin cells, blood cells, and peripheral blood stem cells.

In 2006, the International Association for Cell Therapy (ISCT) standardized the definition of mesenchymal stem cells: adherent growth, expression of specific cell surface antigens (markers), such as CD44 and CD105, etc., in vitro (in vitro) , The ability of osteoblasts and chondrocytes to differentiate.

Among embryonic stem cells, OCT-4 is often used as a marker for stem cells, OCT-4 (Octamer-binding transcription factor 4): OCT-4 participates in the control of cell self-renewal and stem cell differentiation.

Cluster of differentiation (CD) antigens: CD antigens are cell surface proteins that can be divided into different types, such as receptors. Since different cells have different CD antigens, antibodies are used to identify the CD antigens on the cell surface for cell analysis.

The present invention discloses a preparation method of isolated porcine oviduct stem cells, which comprises the following steps: in vitro maturation and cultivation of porcine oocytes in M199 medium treated with porcine oviduct stem cells, and in vitro cultivation in PZM-treated porcine oviduct stem cells. 3 Culture medium.

As mentioned above, the porcine oviduct stem cells express CD44 highly and CD105 moderately.

As mentioned above, the porcine oviduct stem cells have the potential to induce differentiation into sclerocytes, adipocytes and chondrocytes.

The present invention discloses a porcine fallopian tube stem cell preparation, which is prepared by a preparation method of porcine fallopian tube stem cells as described above, and the preparation comprises a composition prepared by a preparation method of porcine fallopian tube stem cells.

The invention discloses a preparation method of isolated porcine oviduct stem cells. The porcine oviduct stem cells are used to prepare preparations for promoting oocyte maturation and embryo development in vitro and to establish a therapeutic experimental platform system.

The present invention discloses a method for preparing oviduct stem cells isolated from yellow cattle, which comprises the following steps: maturation of porcine oocytes in vitro and processing of oviduct stem cells from cattle In the M199 culture medium, the PZM-3 culture medium matured in vitro and processed by cattle fallopian tube stem cells.

As mentioned above, the cattle fallopian tube stem cells are highly expressing CD44.

As mentioned above, the cattle fallopian tube stem cells have the potential to induce differentiation into sclerocytes, adipocytes and chondrocytes.

The present invention discloses a porcine fallopian tube stem cell preparation, which is prepared by a preparation method of bovine fallopian tube stem cells as described above, and the preparation comprises a composition prepared by a preparation method of bovine fallopian tube stem cells.

The present invention discloses a preparation method of oviduct stem cells isolated from cattle. The use of the cattle oviduct stem cells for preparing preparations for promoting oocyte maturation and embryo development in vitro and establishing a therapeutic experimental platform system.

The main effect of the present invention lies in:

1. After adding the conditioned medium of porcine oviduct stem cells and cattle oviduct stem cells, the maturation rate of oocytes was significantly increased (P<0.05). When added to IVC, the conditioned medium of the two cells significantly reduced the development rate of blastocysts (P <0.05).

2. Experiments prove that cattle fallopian tube stem cells have the potential of repotting stem cells and the ability of mesoderm-related cells to differentiate.

3. Experiments show that porcine oviduct stem cells and cattle oviduct stem cells can enhance oocyte maturation.

4. Porcine fallopian tube stem cells and cattle fallopian tube stem cells, which can be used to prepare preparations that promote oocyte maturation and embryo development in vitro and establish a therapeutic experimental platform system.

S101~S102: Porcine fallopian tube stem cells promote oocyte maturation and embryo development in vitro

S201~S202: Yellow cattle fallopian tube stem cells promote oocyte maturation and embryo development in vitro

Figure 1-1 shows the steps of porcine fallopian tube stem cells promoting oocyte maturation and embryo development in vitro;

Figure 1-2 shows the steps of the yellow cattle fallopian tube stem cells promoting oocyte maturation and embryo development in vitro;

Figure 2 is a cell culture state diagram of porcine oviduct stem cells and cattle oviduct stem cells of the present invention;

Figure 3 and Figure 4 are the RT-PCR analysis diagrams of the present invention;

Figures 5 and 6 are analysis diagrams of the flow cytometer of the present invention;

Figures 7 and 8 are diagrams of cells of the present invention in osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation.

Experimental example:

Material method

Animal source:

Collect Taiwanese cattle (Bos indicus) and pig fallopian tubes, and immediately ^{wash them with saline containing 1% penicillin/streptomycin (P/S, Gibco TM} , 15140122, euApproved, south American) from the slaughterhouse, and within 2 hours These tissues are brought to the cell laboratory.

Isolation of Fallopian Tube Stem Cells Experiment:

The fallopian tubes were washed twice with saline containing 1% penicillin/streptomycin, finely cut into ^{a size of 0.1~0.5mm 2} and placed in the tubes supplemented with 10% fetal bovine serum (FBS, Gibco ^TM 10270106 and 1% penicillin/ The streptomycin α-MEM cell culture medium (Sigma M0894) is placed in a 1.5ml microcentrifuge tube (Eppendorf, or microcentrifuge tube) tube at 37°C. Next, the small tissues are placed in a 10cm culture plate (TPP 93100), and ^{Cultivation (Thermo Scientific TM} ) under cell culture conditions of 38.5°C and 5% CO _2.

Flow cytometry analysis:

PFTSCs and BFTSCs of stem cells cultured to the fifth generation (THE PASSAGE 5, P5) were added to 0.25% trypsin-EDTA (Gibco ^TM , 25200072) for 3 minutes, followed by centrifugation at 270G for 5 minutes. Subsequent addition of 100μl Dulbecco's phosphate buffered saline (DPBS) (Gibco ^TM , 21600010) suspension cells, anti-porcine CD4 (Bio-rad, MCA1749GA), anti-porcine CD44 (anti-bovine) CD44) (Thermo Fisher Scientific, MA1-19277), anti-bovine CD44 (Bio-rad, MCA1653F), anti-bovine CD44 (Bio-rad, MCA2433F), anti-porcine CD105 (anti-porcine CD105) ( Thermo Fisher Scientific, MA5-11854) and anti-bovine CD105 (Thermo Fisher Scientific, MA5-11854), anti-mouse IgG1-PE (PE anti-mouse IgG1 Antibody) (eBioscience ^TM , 12-4015- 82) Add appropriate concentration and place on ice for 30 minutes. Then, 800 μl DPBS was added and the antibody was removed by centrifugation. Finally, the cells were suspended in 500 μl DPBS, and the background value of the surface antigen and the addition of the second antibody were analyzed by flow cytometry.

RT-PCR analysis:

PFTSCs and BFTSCs cultured to the fifth passage (THE PASSAGE 5, P5) were suspended in 0.25% trypsin cell digestion solution and washed twice with DPBS. Follow the instructions of Qiagen RNeasy Micro Kit (74004) for RNA extraction and cDNA production. In the PCR experiment, referring to Mierni et al., Hu et al., and Gao et al. literature, respectively, using pig and cattle primers (primers) to analyze GAPDH, Oct 4, CD29 and CD34, and at 95℃-45 seconds, 55℃-30 seconds and 30 proliferation cycles (PCR cycles) were carried out at 72°C-50 seconds. Please refer to the following table 1 for the porcine primer sequence and the cattle primer sequence.

Table 1 The primer sequence of pig and the primer sequence of cattle

Tri-lineage differentiation:

Osteogenic differentiation: For osteogenic differentiation, 1x10 ⁴ P5 PFTSCs and BFTSCs are cultured in a differentiation medium in 6 well dishes. The differentiation medium is composed of 10% FBS α-MEM, 10mM glycerol phosphate (Sigma 17766), 50μM ascorbic acid (Sigma, 33034) and 0.1μM dexamethasone (Sigma, 31381), maintained culture for 21 days.

Adipogenic differentiation: For adipogenic differentiation, 1 ^{×10 5} P5 PFTSCs and BFTSCs are cultured in a differentiation medium in 6 well dishes (TPP 92006). The differentiation medium consists of α-containing 10% FBS. MEM, 0.5mM isobutyl-methylxanthine (Sigma 15879), 100μM indomethacin (USpharmacopeia,1341001), 10μg/mL beef insulin (USpharmacopeia,1342208) and 1μM dexamethasone were maintained in culture for 21 days.

Chondrogenic differentiation: For chondrogenic differentiation, 5x10 ⁵ P5 PFTSCs and BFTSCs are cultured in a differentiation medium in a 15ml centrifuge tube. The components of the differentiation medium are α-MEM containing only 1% FBS, 50μM ascorbic acid, 10ng/mL Transforming growth factor-β2 (Sigma, T5300) and 6.25μg/mL beef insulin, maintained culture for 21 days.

Differentiation verification: In 21 days, the differentiation medium was changed every 3 days by using Oil red O (Sigma, O0625), Alizarin red S (Sigma, A5533) and toluidine blue O (Toluidine blue O) (Sigma, T3260), use the above reagents to stain the differentiated cells for confirmation.

Condition maturation medium production:

When the saturation of P5 cells reaches 80-90% in a 10cm culture dish, wash with DPBS to completely remove the culture medium. Then, add 3ml oocyte maturation medium (M199, Sigma, M4530) containing 10% FBS, 10μL follicle-stimulating hormone (FSH, Sigma, F-2293), 20μL human Human chorionic gonadotropin (hCG, Biovision, 4778-1000), 1% antibiotic-antimycotic (ABAM, Gibco, 15240-062) and 10% porcine follicular fluid ( Porcine follicular fluid (PFF) or porcine zygote medium-3 (PZM-3) was added to each culture plate, and the cells were cultured for 3 hours. The prepared conditioned medium was filtered at 0.22μm and placed in 4 Store at ℃.

Porcine oocyte in vitro maturation (IVM) and in vitro culture (IVC) test:

Keep the obtained slaughterhouse pig ovaries in 1% penicillin/streptomycin saline at 37°C, and after transporting them to the cell laboratory, wash them with 1% penicillin/streptomycin saline 3 times within 1 hour, and then remove the follicles The solution is sucked into a 15mL centrifuge tube with an 18G needle and a 10-20mL syringe, and incubated in a water bath at 37°C for 5 minutes to precipitate the oocytes. Use a pipette to transfer the precipitated liquid to a 10 cm culture dish to select cumulus-oocyte complexes (COCs).

1. IVM test: Wash the cumulus-oocyte complex twice with DPBS, then transfer to (1) normal M199 (normal maturation M199 medium) (C), (2) PFTSCs-treated M199 maturation medium (porcine fallopian) tube stem cell treated M199 medium) (P), (3) Bovine fallopian tube stem cell treated M199 medium (B) treated with PFTSCs, 20~30 cumulus-oocyte complexes/100μL, use Cover culture with mineral oil for 44 hours. After 44 hours, the oocyte complex was treated with 0.1% hyaluronidase to remove the cumulus cells, and the mature oocytes were calculated, which released the first oocytes from each treatment. Polar body (first polar body). Finally, mature oocytes were activated by treatment with calcium ionophore A23187 (Calcium Ionophore, A23187) for 5 minutes and 6-methylaminopurine (6-dimethylaminopurine, 6-DMAP) for 4 hours, and cultured under PZM-3 for 7 days .

2. IVC test: The cumulus-oocyte complex was washed twice through DPBS and transferred to a maturation medium (maturation medium) for 44 hours. Mature and activated oocytes without cumulus cells are in (1) normal PZM-3 (normal PZM medium) (C), (2) PZM-3 (porcine fallopian tube stem cell treated PZM medium) (P ), (3) PZM-3 (bovine fallopian tube stem cell treated PZM medium) (B) treated with BFTSCs was cultured for 7 days.

3. Different concentrations of IVC: Porcine fertilized egg culture medium PZM-3 (conditioned PZM-3) diluted with general PZM-3. Combine mature and activated oocytes with (1) PZM-3 (normal PZM medium) (C), (2) PZM-3 with 25% PFTSCs (25% PFTSCs treated PZM medium) (P25), (3) PZM-3 with 50% PFTSCs (50% PFTSCs treated PZM medium) ( P50), (4) 25% BFTSCs PZM-3 (25% BFTSCs treated PZM medium) (B25), (5) 50% BFTSCs PZM-3 (50% BFTSCs treated PZM medium) (B50) culture for 7 days ( Replace every 2 days). Record the results of each test at the embryonic stage on day 2, day 4, and day 7.

Statistical Analysis:

The data is analyzed and counted through SAS 9.4 (Duncan method), and the conditioned medium is tested for culturing eggs Mother cell, test whether its maturation rate has increased.

Experimental result

Characterization, cell performance and cell differentiation of PFTSC and BFTSC:

When the finely divided tissue of the fallopian tube was cultured for 3 days, please refer to Figure 2. Long stick-like cells were successfully isolated (Figure 2A), which showed proliferation potential (Figure 2B).

Please refer to Figure 3, which shows CD29, but does not show Oct4 and CD34.

Please refer to Figure 2. Short stick-like cells from cattle show strong proliferation potential (Figure 2C and Figure 2D). Please refer to Figure 4, and express CD29 and OCT4 mRNA, which is similar to MSC. It is related to pluripotency.

Please refer to Figure 5, the expression of stem cell (MSCs) markers CD44 and CD105 are 76% and 8.0%, respectively, and the expression of T lymphocyte-related CD4 is 0.4%.

Please refer to Figure 6. In the surface antigen analysis, BFTSCs showed 76.7% CD44, 7.6% CD105 and 3.7% CD4.

Please refer to Fig. 7A and Fig. 8A. After culturing PFTSCs and BFTSCs in osteogenic differentiation medium, calcium deposits gradually formed, and the red complex was displayed by Alizarin Red S staining reagent.

Please refer to Figure 7B and Figure 8B. In adipocyte differentiation, both cell types show the potential to differentiate to produce oil generation (oil generation), which is confirmed by the oil red O staining reagent.

Please refer to Figure 7C and Figure 8C. In the chondrocyte differentiation, cell pellets formed by PFTSCs and BFTSCs can be observed on the third day of culture. On the 21st day, the differentiated cartilage is penetrated through the tissue-embedded section. The toluidine blue O staining reagent showed confirmation of matrix and intercellular space (matrix and intercellular space).

Porcine oocyte maturation and development

Please refer to Table 2 below. After culturing the cumulus-oocyte complex of pigs with the conditioned maturation medium from PFTSCs and BFTSCs, the maturation rate is higher than that of the normal maturation medium, but In blastocyst rate (blastocyst rate), there is no difference between different groups.

Table 2. The effects of PFTSCs and BFTSCs treated media on in vitro maturation (IVM) of porcine oocytes

C: In vitro culture-in the general M199 maturation medium (IVM with normal maturation M199 medium).

P: In vitro culture-PFTSCs treated M199 maturation medium (IVM with porcine fallopian tube stem cell treated M199 medium).

B: In vitro culture-BFTSCs treated M199 maturation medium (IVM with bovine fallopian tube stem cell treated M199 medium).

Repeat the experiment 5 times.

^A and ^b in Table 2 represent significant differences between groups C, P and B.

Please refer to Table 3 below. In the in vitro culture test, compared with the general PZM-3 group, the morula rate of PZM-3 treated with PFTSCs is lower than that of the general PZM-3 group, which is derived from the PZM-3 treatment The blastocyst rate of PZM-3 treated with BFTSCs decreased.

Table 3. Effects of PFTSCs and BFTSCs treated media on in vitro culture (IVC) pig oocytes

C: In vitro cultured in general PZM-3 medium (IVC with normal PZM medium).

P: In vitro cultured in PZM-3 medium (IVC with porcine fallopian tube stem cell treated PZM medium) treated with PFTSCs.

B: In vitro cultured in PZM-3 medium (IVC with bovine fallopian tube stem cell treated PZM medium) treated with BFTSCs.

Repeat the experiment 6 times.

^A , ^b , and ^{ab in} Table 3 represent significant differences between groups C, P and B.

Please refer to Table 4 below. When the pig fertilized egg culture medium PZM-3 (conditioned PZM-3) diluted with general PZM-3 is used, the morula rate of P50 group decreases on the 4th day of culture, while the P25 group is The morula rate and blastocyst rate of the P50 group on the last day were lower than those of the C group. in addition, There was no difference between the B25 group and the B50 group in the 7-day duration compared with the C group.

Table 4. The effect of adjusted medium treated with different concentrations of PFTSCs and BFTSCs on IVC

C: In vitro culture-in general PZM-3 medium (IVC with normal PZM medium).

P25: In vitro cultured in PZM-3 medium (IVC with 25% PFTSCs treated PZM medium) treated with 25% PFTSCs.

P50: In vitro cultured in PZM-3 medium (IVC with 50% PFTSCs treated PZM medium) treated with 50% PFTSCs.

B25: In vitro cultured in 25% BFTSCs treated PZM-3 medium (IVC with 25% BFTSCs treated PZM medium).

B50: In vitro cultured in 50% BFTSCs treated PZM-3 medium (IVC with 50% BFTSCs treated PZM medium).

Repeat the experiment 5 times.

^A , ^b , and ^{ab in} Table 4 represent significant differences between C, P25, P50, B25, and B25.

It is proved by the above that the different concentrations of IVC of the present invention: among them, the porcine fertilized egg medium PZM-3 diluted and treated with general PZM-3, preferably in vitro cultured in the PZM-3 medium treated with 25% PFTSCs, can still significantly reduce the cyst Embryo development rate.

According to our experimental results, PFTSCs and BFTSCs can increase the maturation rate of porcine oocytes during IVM and contribute to the maturation of oocytes.

The present invention is to present the preferred embodiments or examples of the technical means used to solve the problem, and is not used to limit the scope of implementation of the patent of the present invention, that is, everything that is consistent with the scope of the patent application of the present invention or made in accordance with the scope of the patent of the present invention Equal changes and modifications are all covered by the invention patent.

S101~S102: Porcine fallopian tube stem cells promote oocyte maturation in vitro and embryo development steps

S201~S202: Yellow cattle fallopian tube stem cells promote oocyte maturation and embryo development in vitro

Claims (10)

A preparation method of isolated porcine oviduct stem cells, which comprises the following steps: Porcine oocytes were matured in vitro and cultured in M199 medium treated with porcine oviduct stem cells, and cultured in vitro in PZM-3 medium treated with porcine oviduct stem cells. The method for preparing porcine oviduct stem cells as described in claim 1, wherein the porcine oviduct stem cells express CD44 at a high level and CD105 at a moderate level. The method for preparing porcine oviduct stem cells isolated from claim 1, wherein the porcine oviduct stem cells have the potential to induce differentiation into sclerocytes, adipocytes and chondrocytes. A porcine oviduct stem cell preparation, which is prepared by the preparation method described in claim 1 which is isolated from porcine oviduct stem cells. A preparation method of porcine fallopian tube stem cells as described in claim 1, the use of the porcine fallopian tube stem cells for preparing preparations for promoting oocyte maturation and embryo development in vitro and for establishing a therapeutic experimental platform system. A preparation method of oviduct stem cells isolated from cattle, which comprises the following steps: Porcine oocytes were matured in vitro in M199 culture medium treated with cattle fallopian tube stem cells, and in vitro matured in PZM-3 medium treated with cattle fallopian tube stem cells. The method for preparing oviduct stem cells isolated from cattle as described in claim 6, wherein the oviduct stem cells of cattle highly express CD44. The method for preparing oviduct stem cells isolated from cattle as described in claim 6, which has the potential to induce differentiation into sclerocytes, adipocytes and chondrocytes. A porcine oviduct stem cell preparation, which is isolated from the oviduct of yellow cattle as described in claim 6 Prepared by the preparation method of stem cells. A method for preparing oviduct stem cells isolated from cattle as described in claim 6, the use of which cattle oviduct stem cells are used to prepare preparations that promote oocyte maturation and embryo development in vitro and to establish a therapeutic experimental platform system.

Images