CN100999724B - Feeder cells using in immature ovocyte in-vitro cultivating maturation - Google Patents

Abstract

The feeder cells used in the in vitro culture of immature oocytes are characterized in that the bone marrow mesenchymal stem cells MSC of female mice are used as feeder cells. The feeder cells are easy to obtain, can secrete a variety of cytokines and growth factors, can promote the development and maturity of more than 90% of immature oocytes cultured in vitro, and promote the development, ovulation and maturation of all levels of follicles in ovarian tissue blocks cultured in vitro ; Overcome the shortcomings of IVM culture medium and feeder cells, improve the culture maturity rate, and better serve the immature oocyte in vitro culture maturation technology for rare and endangered animal preservation, livestock breeding and commercial production, and embryonic stem cells Biological research and the creation of human "egg banks" to provide reproductive insurance open up broader prospects.

Description

Feeder cells used in in vitro maturation of animal immature oocytes

technical field

The invention relates to the fields of reproductive medicine and cell biology, and more specifically relates to a feeder cell used in the culture and maturation of animal immature oocytes in vitro. the

Background technique

Immature oocyte in vitro maturation (IVM) technology emerged with the development of artificial assisted reproductive technology (ART). IVM can shorten the reproductive cycle, maximize the use of good genetic material, and be used for artificial reproduction of rare or endangered animals. And the cultivation and commercial production of livestock breeds; it is convenient to study the development mechanism of oocytes, combined with in vitro fertilization or parthenogenetic activation technology to obtain a large number of embryonic stem cells for scientific research. the

Culture medium is a key content of oocyte IVM technology, but there is no cheap, stable and safe commercial IVM culture medium available. At present, the commonly used basal culture media for IVM include Ham’s F-10, TCM-199, α-MEM, DMEM, HTF, etc., but the maturation rate of oocytes using these culture media alone is very low. The British "Nature" magazine (Nature, 1965, 208: 349) reported that adding high concentrations of serum to these basal media, in vitro cultured mice, sheep, cows, and rhesus monkeys can differentiate towards maturity, but most of them stagnate. During the first meiotic prophase, less than 30% of oocytes reach the MII stage. Currently commonly used added factors include steroid hormones: FSH, Gn, LH, HCG, E2, P, etc., cytokines: transferrin, IL-1, IL-6, etc., growth factors: EGF, FGF, IGF, etc. , but these substances are very expensive and very unstable, prone to inconsistent nucleoplasmic maturation of oocytes. Cambridge, England "Reproduction" magazine (Reproduction, 2001, 121: 925) reported that monkey kidney cells, granulosa cells and oviduct epithelial cells were used as feeder cells, and the secretion of cells was used to reduce the addition of substances and improve the maturation rate of oocytes. The 90% maturation rate of oocytes during ovulation stimulation has not been reached, and there is also the risk of contamination by pathogens and foreign genes. the

The existence of the above reasons greatly limits the play and application of the advantages of IVM technology. the

Contents of the invention

The invention provides a feeder cell used in the in vitro maturation of animal immature oocytes. The feeder cell is easy to obtain and can secrete various cytokines and growth factors to overcome the shortcomings of IVM culture fluid and feeder cells. Improve the culture maturation rate, and better serve the immature oocyte in vitro culture maturation (IVM) technology for the preservation of rare and endangered animals, the breeding and commercial production of livestock breeds, and the cell biology research of embryonic stem cells. the

The technical scheme that the present invention solves technical problem adopts is:

The present invention uses female mouse bone marrow mesenchymal stem cells MSCs as feeder cells. the

The method of obtaining bone marrow mesenchymal stem cells MSCs of female mice in the present invention is as follows: obtaining 8-10 week-old female mice bone marrow cavity flushing liquid, using density gradient centrifugation combined with adherent culture method to cultivate, separate and purify MSCs. the

The culture medium of MSC of the present invention is DMEM, and the fetal calf serum of 10% by volume ratio, 10mmol/LHEPES, 100U/mL penicillin, 100U/mL streptomycin are added therein, culture condition is 37 ℃, volume ratio is 5 % CO ₂ .

The culture medium of the MSC of the present invention can also be RPMI-1640 or Ham's F-12 with the culture condition of 37°C and 5% CO ₂ by volume.

MSC is mouse bone marrow mesenchymal stem cell, namely mesenchymal stem cell, referred to as MSC. the

DMEM is a modified MEM by Dulbecco, namely Dulbecco’s modified Eagle’s medium

Compared with prior art, beneficial effect of the present invention is reflected in:

1. The feeder cell MSC of the present invention not only has multi-directional differentiation potential, but also has very important nutritional effect on surrounding cells and inhibition of cell apoptosis. MSC can synthesize and secrete IL-1, IL-6, EGF, FGF, IGF-1, HGF, LIF, TGF-β and other biologically active substances, many of which are necessary for oocyte maturation in vitro. In addition, MSC can also promote the synthesis and secretion of various hormones in granulosa cells, such as E2, P, LH, follicular fluid meiosis-activating sterol (FF-MAS), enhance the interaction between its synapse and oocyte cytoplasm, and promote Oocyte nucleoplasm matures synchronously. the

2. The feeder cell MSC of the present invention has a high maturation rate for immature oocytes, good repeatability and stable results. the

3. The feeder cell MSC of the present invention is easy to obtain. When used in larger animals such as cattle and sheep, MSCs can be obtained by acupuncture with minimal trauma; oocytes can also be obtained by needle aspiration under the guidance of B-ultrasound. the

Description of drawings

Fig. 1 is the primary MSC × 200 after the suspended cells are washed away in the present invention. the

Figure 2 shows the mature oocytes obtained by co-culturing ovulation-induced oocytes with MSCs for 40 minutes × 200. the

Figure 3 is the immature oocytes obtained from ovarian tissue suction and co-cultured with MSCs for 7 days, the oocytes after the preantral follicles were digested to remove granulosa cells×200. the

Figure 4 shows the antral follicles on the surface of the tissue block after 4 days of co-culture of immature oocytes and MSCs in the ovarian tissue block×100. the

Figure 5 shows the immature oocytes discharged from the tissue block after the immature oocytes in the ovarian tissue block were co-cultured with MSCs×100. the

Figure 6 shows the development of immature oocytes discharged from the tissue block to M II stage after the co-culture of immature oocytes and MSCs in the ovarian tissue block ×100. the

The present invention will be further described below in combination with specific embodiments. the

Detailed ways

Isolation and culture of MSC: Obtain 8-10-week-old female mouse bone marrow cavity flushing solution, add an equal proportion to the sterile Percol solution with a density of 1.077 in the centrifuge tube, centrifuge at 2000 rpm for 20 minutes, and collect the liquid between the two liquid layers For the mononuclear cell layer, centrifuge at 10 times the volume of 1×PBS at 1200 rpm for 10 minutes and wash twice. Cells were resuspended in DMEM containing 10% fetal bovine serum by volume, inoculated into a culture flask at a cell density of 2×10 ⁶ /ml, and cultured at 37°C and 5% CO ₂ by volume. After 3 days, gently shake the culture flask, pour off the non-adherent cells, and wash 2 times with 1×PBS to obtain the purified primary MSC; add DMEM containing 10% fetal bovine serum by volume and continue to culture until 80% of the bottom of the flask is covered ( As shown in Figure 1), the weight volume ratio was 0.25% trypsin digestion and passage, 6 × 10 ⁵ /ml cell density seeded 6-well plate was the second-generation MSC, and the culture supernatant was collected after 3 days of culture to be the conditioned medium.

For larger animals, bone marrow fluid can be obtained directly by acupuncture. the

Example 1:

Using MSC as a feeder cell for the culture of immature oocytes separated by stimulation

8-10 weeks old female Kunming mice, intraperitoneal injection of 10IU PMSG, 48 hours later intraperitoneal injection of 10IU HCG, 16-20 hours later cut oviduct, at 37 ℃, volume ratio of 5% _CO2 to balance overnight BSS Pick eggs under a stereomicroscope. Oocytes below the MII stage were cultured for 40 minutes with second-generation MSCs and conditioned medium at 37°C and 5% _CO by volume to obtain mature oocytes (Figure 2). Show).

Experiments have shown that the oocytes below the MII stage are cultured with second-generation MSCs or cultured with conditioned medium at 37 ° C and 5% CO ₂ for 40 minutes, and the maturation rate reaches 95%. Cells continue to divide in MSC or conditioned medium, and mature oocytes are digested with 0.5mg/ml hyaluronidase to remove granulosa cells, and oocytes are transferred to M16 containing 0.4% BSA by weight and volume medium.

The same experiment also included co-cultivation of oocytes below the MII stage with DMEM containing 10% fetal bovine serum by volume, the maturation rate after 12 hours was only 18%, and the oocytes could not be well developed . the

Example 2:

Using MSC as feeder cells, for the culture of immature oocytes obtained by aspirating from ovarian tissue

Under sterile conditions, remove both ovaries of female Kunming mice aged 8-10 weeks, and place them in DMEM containing 10% fetal bovine serum by volume at 37°C and equilibrated overnight with 5% _CO2 by volume. Use 1ml 25G syringe needle suction under the microscope to obtain free antral follicles and preantral follicles. Under the condition of 37°C and 5% CO ₂ by volume, part of them are mixed with second-generation MSCs, and part of them are mixed with conditioned medium for 24 hours to 7 days. cultured to obtain mature oocytes (shown in Figure 3).

Experiments have shown that, depending on the developmental stage of the follicles, in order to obtain mature oocytes, the culture time varies from 24 hours to 7 days. The maturation rate of mother cells is 93%; the maturation rate of oocytes is 85% after being cultured with conditioned medium for 7 days. In order to prevent mature oocytes from continuing to divide in MSC or conditioned medium, the mature oocytes were digested with 0.5 mg/ml hyaluronidase to remove granulosa cells, and the oocytes were transferred to a medium containing 0.4% BSA in M16 medium. the

In the same experiment, a part of immature oocytes obtained by aspirating from ovarian tissue were cultured in DMEM containing 10% fetal bovine serum by volume for 7 days, and the oocyte maturation rate was only 10%. the

For larger animals, oocytes can be obtained by aspiration with a puncture needle under the guidance of B-ultrasound. the

Example 3:

Using MSCs as feeder cells for the cultivation of ovarian tissue blocks

Prepare an agar scaffold with a weight-to-volume ratio of 0.4%: inoculate a 6-well plate with second-generation MSCs at a cell density of 1×10 ⁵ /ml, pour off the culture medium after the cells adhere to the wall; prepare 0.4% agar with conditioned medium, and wait until Aspirate 3ml at 37°C-42°C and spread on MSCs, equilibrate overnight at 37°C with 5% CO ₂ by volume, and then use it for ovarian tissue block culture.

Under aseptic conditions, take out both ovaries of 8-10 weeks old female Kunming mice, place them in 4°C pre-cooled DMEM containing 10% fetal bovine serum by volume, and quickly make 1 -2mm thin slices, paste on agar with 0.4% weight volume ratio of MSC at 37°C and 5% volume ratio of _CO2 overnight, press lightly to embed the tissue block into the agar, and add 3 international units per ml at the same time HCG; part of the ovarian tissue slices were placed on the agar with a weight volume ratio of 0.4% prepared in the conditional solution without MSC; part of the ovarian tissue slices were placed on the weight volume of DMEM containing 10% fetal bovine serum without MSC Ratio of 0.4% on agar; both also added 3IU/mlHCG at the same time, 37 ° C, volume ratio of 5% CO ₂ conditions.

On the 4th day of the ovarian tissue pieces co-cultured with MSCs, many antral follicles formed on the surface of the ovarian tissue (shown in Figure 4), and 100% of the discharged oocytes developed to the MII stage (shown in Figures 5 and 6); Ovarian tissues cultured on 0.4% agar by weight volume ratio prepared by condition solution and 0.4% by weight volume ratio by weight volume ratio prepared by MSC-free DMEM containing 10% fetal bovine serum were also visible on day 4 Follicles form, but no oocytes are expelled, and no oocytes are able to develop to the MII stage. the

Ovarian tissues were cultured on agar with a weight-to-volume ratio of 0.4% with MSC or on agar with a weight-to-volume ratio of 0.4% prepared by conditioned medium without MSC. DMEM containing 10% fetal bovine serum by volume was cultured on agar with a weight volume ratio of 0.4%, and the aforementioned three hormones did not increase significantly. the

For each of the above examples, observe the morphological changes of the oocyte under a stereo microscope: the cytoplasm is uniform, the color is light, the particles are uniform, and the first polar body can be seen in the perivitelline space to determine that the oocyte is mature, that is, the MII stage oocyte . the

The levels of E2, P, and LH in the culture fluid after 3 days of co-culture of follicles or ovarian tissue pieces and MSCs were several to ten times higher than those of follicles or ovarian tissue pieces and MSCs cultured alone (Table 1). the

Table 1 Hormone levels in supernatants of follicles and ovarian tissue pieces co-cultured with MSCs and cultured alone

Claims (1)

1. One kind with female mice mesenchymal stem cells MSCs MSC as the purposes that is applied in the feeder cell in the animal immature oocyte vitro culture maturation.

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