CN113774016B - Oocyte in-vitro maturation 3D culture system - Google Patents
Oocyte in-vitro maturation 3D culture system Download PDFInfo
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
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- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
Abstract
The invention belongs to the field of biological medicine, and particularly relates to establishment of a 3D culture system based on oocyte in-vitro maturation of novel hydrogel. The invention establishes a 3D IVM culture system with interaction of oocytes and granulosa cells based on the novel laminin functional peptide modified hydrogel, adds the effective components in the ECM, provides the biophysical environment required by the oocyte development, reduces the external stimulus and oxidative stress level of the oocyte, maintains the interaction of the oocyte and the granulosa cells, improves the oocyte maturation rate and embryo development potential after fertilization, and has wide clinical application panorama.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to establishment of a 3D culture system for oocyte in-vitro maturation based on laminin peptide hydrogel.
Background
The follicle consists of an oocyte, a plurality of layers of granulosa cells around the oocyte, a basement membrane consisting of extracellular matrix (ECM) and outer membrane cells, and the components within the follicle all have different functions. In the long-term development process of the oocyte, the oocyte and surrounding granulosa cells are connected through gaps and regulated in a paracrine bidirectional manner, and the bidirectional signal is continuously changed along with the time so as to synchronize the follicular development and the oocyte maturation, and play an important role in the processes of metabolism of the oocyte, recovery of meiosis, fertilization and the like.
In recent years, the incidence of infertility is gradually increased, and the auxiliary reproduction technology is gradually a powerful helper for solving the fertility requirements of people. Oocyte In Vitro Maturation (IVM) is an assisted reproductive technology based on the modification of traditional In Vitro Fertilisation (IVF), i.e. the process of spontaneous maturation in vitro into MII-stage oocytes by removing GV-stage immature oocytes from sinus follicles with little or no stimulation by exogenous gonadotrophin. The IVM is suitable for various clinical diagnosis and treatment processes, such as clinical ovum donation, FSH resisting in vitro fertilization of patients, in vitro fertilization of thrombus formation high-risk patients, fertility preservation of ovarian cancer patients and prevention of excessive stimulation of gonadotrophin by polycystic ovary syndrome patients.
Oocytes treated by the existing IVM technology are mechanically separated from premature immature follicles and then directly placed in culture droplets in a planar culture dish for in vitro culture. The oocyte is extremely fragile, is easy to be stimulated by the outside when lacking the protection of extracellular structures, increases the level of oxidative stress, is extremely easy to cause the death or dysfunction of the oocyte, and causes the low success rate of IVM. The existing IVM culture system has huge difference with the in-vivo living and developing environment of the oocyte, the temperature, humidity, ion balance and gas balance conditions of the culture system are unstable, and the requirements on operators and the whole operation environment are high, so that the oocyte has low development potential, implantation rate, pregnancy rate and living yield, and the clinical application of the IVM is limited.
Disclosure of Invention
Aiming at the problems, the invention establishes a 3D culture system with interaction of oocytes and granulosa cells based on the laminin functional peptide modified hydrogel, adds the active ingredients in the ECM, provides the physical environment required by the oocyte development, reduces the external stimulus and oxidative stress level of the oocyte, maintains the interaction of the oocyte and the granulosa cells, and improves the oocyte maturation rate and embryo development potential after fertilization.
In order to achieve the above purpose, the present invention provides the following technical solutions.
The invention provides an oocyte in-vitro maturation 3D culture system which is characterized by comprising in-vitro maturation culture solution, granulosa cells and ionic hydrogel.
Further, the ionic hydrogel modifies two laminin complex peptide fragments.
Preferably, the two laminin composite peptide fragments are Laminin subunit alpha-1 and Laminin subunit beta-1, respectively.
Further, the ionic hydrogel modified two laminin composite peptide segments have the effect of providing extracellular matrix functional components to promote the meiosis of oocytes.
Further, the ionic hydrogel has the effects of reducing external stimulus to the oocyte, reducing the level of oxidative stress, improving the mitochondrial function of the oocyte and further improving the developmental potential of the oocyte.
Further, the granulosa cells are derived from a cumulus complex of an autologous mature oocyte, and have the function of providing an in-vitro oocyte microenvironment similar to that of an in-vivo immature oocyte for the immature oocyte, so that the oocyte maturation rate is increased, and an in-vitro maturation bionic culture system of the oocyte is established.
Further, the culture system comprises the following steps of in vitro culturing oocytes:
(1) Collecting the cumulus complexes and immature oocytes of the mature oocytes obtained after ovulation stimulation;
(2) Digesting the collected cumulus complexes of mature oocytes obtained after ovulation promotion by using hyaluronidase, collecting granular cells, centrifuging, and placing into in vitro maturation culture solution to suspend for standby;
(3) Mixing the laminin peptide hydrogel and the in vitro maturation culture solution containing granulosa cells according to a certain volume ratio to prepare hydrogel mixed solution microdroplets, and placing the hydrogel mixed solution microdroplets into a 37 ℃ incubator for standing until gel is formed;
(4) Placing the collected immature oocytes into hydrogel-granulosa cell microdroplets, placing 1-2 oocytes in each microdroplet, capping the in vitro maturation medium, and culturing at 37deg.C with 5% CO 2 And culturing for 22-24h under 100% humidity;
(5) Observing the maturation condition of the oocyte in the step (4), wherein the oocyte reaching the second meiosis metaphase is the mature oocyte.
Preferably, the ratio of the laminin peptide hydrogel of step (3) to the in vitro maturation medium containing granulosa cells is 4:1.
Preferably, the culture conditions in step (4) are those under conditions containing 5% CO 2 And culturing at 37deg.C under 100% humidity; the culture time is 22-24h.
Further, the culture system is used for preparing a reagent and/or a kit for treating infertility.
Compared with the prior art, the invention has the beneficial effects.
1. The invention firstly proposes to use laminin peptide ion hydrogel to establish an oocyte 3D in vitro culture system, simulate the physical environment and biological environment of the oocyte in vivo, reduce the influence of external stimulus on the oocyte, and maximally improve the development potential of the oocyte.
2. The invention provides a 3D culture system for oocytes loaded with granulosa cells, which maintains the interaction among cells, improves the development microenvironment of the oocytes through the oocyte maturation-promoting factors secreted by the granulosa cells of the mature oocytes, and improves the oocyte maturation capacity.
Drawings
Figure 1 shows that laminin in normal rat ovarian tissue is mainly expressed in the cumulus oophorus complex, and the content of laminin is significantly higher than that of a model rat with premature ovarian failure, which proves that laminin can be an important extracellular matrix component for regulating oocyte development.
FIG. 2 shows the physical pressure developed after setting of hydrogels at different concentrations and the viability and maturation rate of oocytes in 3D culture systems formed from hydrogels at different concentrations. The comparison of bionic analogy, survival rate and maturation rate proves that the concentration is 1: the 3D culture system composed of 5 sets of hydrogels was most beneficial for oocyte survival and meiosis.
FIG. 3 shows the viability and maturation rate of oocytes cultured in gelatin hydrogels, alginate hydrogels, hyaluronic acid hydrogels, collagen peptide fragment ion hydrogels and laminin peptide fragment hydrogels. The laminin peptide ion hydrogels proved to be most beneficial to the survival and meiosis of oocytes by comparison with other common types of hydrogels.
FIG. 4 shows the expression of granulosa cell oocytes Bmp15 and Oas1d at different concentrations.
FIG. 5 shows an image of mature oocytes in a 3D culture system with no granulosa cells and with granulosa cells in a previous culture.
FIG. 6 shows the maturation rate of mature oocytes in previous culture, granulosa-free and granulosa-cell-containing 3D culture systems.
FIG. 7 shows the effect of the 3D culture system of the present invention on oocyte maturation rate, fertilization rate, cleavage rate, high quality embryo formation rate, blastocyst formation rate, high quality blastocyst formation rate and litter size, as compared to prior IVM culture techniques. The invention obviously improves the oocyte maturation rate, the high-quality embryo formation rate, the blastula formation rate, the high-quality blastula formation rate and the litter size.
FIG. 8 shows oocytes, fertilization procedures, blastomeres and blastocysts developed in the 3D culture system of the present invention.
FIG. 9 shows that in the 3D culture system of the present invention, the level of oxidative stress of oocytes containing granulosa cells is lower than that of oocytes without granulosa cells and oocytes after culture by the prior IVM technique.
FIG. 10 shows that in the 3D culture system of the present invention, the mitochondrial membrane potential of oocytes containing granulosa cells is higher than that of oocytes without granulosa cells and oocytes after culture by the prior IVM technique.
FIG. 11 shows the effect of the 3D culture system of the present invention on oocyte maturation rate, fertilization rate, cleavage rate, high quality embryo formation rate, blastocyst formation rate and high quality blastocyst formation rate compared to prior IVM techniques. The invention obviously improves the oocyte maturation rate, the blastula formation rate and the high-quality blastula formation rate.
Detailed Description
The following examples will aid in the understanding of the present invention, but are merely illustrative of the invention and the invention is not limited thereto. The methods of operation in the examples are all conventional in the art.
The formulation of the in vitro maturation medium in the examples is: FBS, glutamine, FSH, E2, HCG, EGF, penicillin and streptomycin were dissolved in M16 to prepare M16 culture broth (sigma) containing 10% FBS,0.029mmol/L glutamine, 0.075IU/mL FSH,10ug/mL E2,0.15IU/mL HCG,10ng/mL EGF,100IU/mL penicillin and 100IU/mL streptomycin, i.e. in vitro maturation culture broth.
Example 1 this example provides a 3D culture system and a method of preparing the 3D culture system while culturing oocytes using the 3D culture system.
The oocyte in-vitro maturation culture method of the 3D culture system comprises the following steps:
(1) Collecting the cumulus complexes and immature oocytes of the mature oocytes obtained after ovulation stimulation;
(2) Digesting the collected cumulus complexes of mature oocytes obtained after ovulation promotion by using hyaluronidase, collecting granular cells, centrifuging, and placing into in vitro maturation culture solution to suspend for standby;
(3) Mixing laminin peptide hydrogel and in-vitro maturation culture solution containing granulosa cells according to a certain volume ratio to prepare a hydrogel mixed solution, and placing the mixed solution into a 37 ℃ incubator for standing until gel is formed;
(4) Placing the collected immature oocytes into hydrogel-granulosa cell microdroplets, placing 1-2 oocytes in each microdroplet, capping the in vitro maturation medium, and culturing at 37deg.C with 5% CO 2 Culturing for 22-24h under the condition of air and 100% humidity;
(5) Observing the maturation condition of the oocyte in the step (4), and placing the oocyte reaching the second meiosis metaphase under a micromanipulator for the next treatment.
2. Dilution ratio optimization test of laminin peptide fragment hydrogel:
oocytes were matured in vitro according to the above culture method, wherein laminin peptide fragment hydrogel was cultured according to 1: 3. 1: 5. 1:7, comparing the survival rate and the maturation rate of the oocytes, and the result is shown in figure 2, wherein the dilution ratio of the laminin peptide hydrogel is 1: at 5, the survival and maturation rate of oocytes were higher than other dilution concentrations. .
3. Comparative experiments of gelatin hydrogels, alginate hydrogels, hyaluronic acid hydrogels, collagen peptide fragment ion hydrogels and laminin peptide fragment ion hydrogels:
comparative experiments were performed using gelatin hydrogel, alginate hydrogel, hyaluronic acid hydrogel, collagen peptide ion hydrogel and laminin peptide ion hydrogel according to the above-described culture methods, and the survival rate and maturation rate of oocytes were compared, and the results are shown in fig. 3, in which the survival rate and maturation rate of oocytes cultured in the laminin peptide ion hydrogel were higher than those of other groups.
4. Comparative experiments with granulosa cells and granulosa cells free:
after the oocyte containing the granulosa cells and the oocyte without the granulosa cells are matured in vitro according to the culture method, the maturing rate of the oocyte containing the granulosa cells is high, the oxidative stress level of the oocyte is low, and the mitochondrial membrane potential is high. As a result, referring to FIGS. 6, 9 and 10, it can be seen from FIG. 6 that the maturation rate of the oocytes in the method of the present invention is higher than that in the control group, that the active oxygen level of the oocytes in the method of the present invention is lower than that in the control group, and that the mitochondrial membrane potential of the oocytes in the method of the present invention is higher than that in the control group, as shown in FIG. 10.
5. Experiments for optimizing the ratio of oocytes to granulosa cells:
the ratio of oocyte to granulosa cells was 1: 1000. 1: 2000. 1: 5000. 1:10000, comparing the expression of the oocyte quality related gene, the result is shown in figure 4, the ratio of oocyte to granulosa cell reaches 1: at 5000, the expression of oocyte Bmp15 and Oas1d is obviously increased, and the increase of the number of granulosa cells has no obvious influence on the expression of genes above the oocyte.
Example 2 in vitro culture of mouse oocytes.
1. The mice were injected with 10IU PMSG intraperitoneally to promote ovulation, and after 46-48 hours, the ovaries of the mice were excised and the immature cumulus complexes of the mice were collected.
2. Extraction and culture of granulosa cells: and digesting the cumulus complexes of the mature oocytes obtained after ovulation promotion by using hyaluronidase, collecting granular cells, centrifuging, and placing into in vitro maturation culture solution for suspension for later use.
3. Mixing the laminin peptide hydrogel with the in vitro maturation culture solution containing granulosa cells according to the volume of 4:1 to prepare a hydrogel mixed solution, and placing the hydrogel mixed solution into a 37 ℃ incubator for standing until gel is formed.
4. The oocytes identified as immature oocytes obtained after ovulation stimulation were collected and placed in hydrogel-granulosa cell microdroplets, each of which was placed in 1-2 oocytes, capped with in vitro maturation medium. The culture time is 22-24h, the culture condition is 37 ℃ and the culture medium contains 5% of CO 2 Is 100% of the humidity of the air.
5. Mature oocytes are fertilized by intracytoplasmic sperm injection (ICSI) technique: after the immature mouse oocyte is cultured in vitro for 22-24 hours, the maturation condition of the immature mouse oocyte is observed. Oocytes that reached the second meiosis stage (MII stage) were placed under a micromanipulator and ICSI treatment was performed.
6. Embryo culture: g-1PLUS (Vitrolife) embryo culture medium was placed in 35mm petri dishes and made into 50. Mu.L droplets covered with mineral oil. The fertilized egg is cultured to 8-cell blastomere embryo by using the culture solution.
7. Culturing blastula: g-2PLUS (Vitrolife) embryo culture medium was placed in 35mm petri dishes and made into 50. Mu.L droplets covered with mineral oil. The culture solution is used for culturing embryo at the cleavage stage after 8 cells develop until the blastocyst stage.
The maturation rate, embryo formation rate, high quality embryo formation rate, blastocyst formation rate, high quality blastocyst formation rate and litter size of the mouse oocytes cultured using the methods of the prior art and the present invention were compared. The results are shown in FIGS. 5 and 7. Experimental results show that the oocyte maturation rate, the high-quality embryo formation rate, the blastula formation rate and the litter size of the mice are higher than those of a control group in the experiment.
Example 3 in vitro culturing of oocytes in sterile line patients.
1. The hospital reproductive medicine center collects granulosa cells and immature oocytes from patients who are sterile for IVF.
2. The procedure was followed as in examples 2-8.
The maturation rate, embryo formation rate, high quality embryo formation rate, blastocyst formation rate and high quality blastocyst formation rate of oocytes cultured using the methods of the prior art and the present invention were compared. The results are shown in FIG. 11. Experimental results show that the oocyte maturation rate, the blastula formation rate and the high-quality blastula formation rate of the method are higher than those of a control group.
From the results, the novel hydrogel-based IVM 3D culture system can obviously increase the maturation rate of the immature oocyte, simultaneously improve the cleavage rate and blastula formation rate of the fertilized oocyte and the litter size of mice, and improve the development potential and clinical outcome of the immature oocyte.
Claims (7)
1. An oocyte in-vitro maturation 3D culture system, which is characterized by comprising an in-vitro maturation culture solution, granulosa cells and ionic hydrogel; the granulosa cells are derived from a cumulus complex of an autologous mature oocyte, and have the function of providing an in-vitro oocyte microenvironment similar to that of an in-vivo for an immature oocyte, so that the oocyte maturation rate is increased, and an in-vitro maturation bionic culture system of the oocyte is established; the ionic hydrogel modifies two laminin composite peptide fragments; the two laminin composite peptide fragments are Laminin subunit alpha-1 and Laminin subunit beta-1 respectively.
2. An oocyte in vitro maturation 3D culture system according to claim 1, wherein the ionic hydrogel modification of the two laminin complex peptide fragments has the effect of providing extracellular matrix functional components to promote meiosis of the oocyte.
3. The in vitro maturation 3D culture system of claim 1 wherein the ionic hydrogel has the effect of reducing the external stimulus to the oocyte, reducing the level of oxidative stress, improving the mitochondrial function of the oocyte and thus improving the developmental potential of the oocyte.
4. The in vitro maturation 3D culture system of claim 1, wherein the culture system comprises the following steps:
(1) Collecting the cumulus complexes and immature oocytes of the mature oocytes obtained after ovulation stimulation;
(2) Digesting the collected cumulus complexes of mature oocytes obtained after ovulation promotion by using hyaluronidase, collecting granular cells, centrifuging, and placing into in vitro maturation culture solution to suspend for standby;
(3) Mixing the laminin peptide hydrogel and the in vitro maturation culture solution containing granulosa cells according to a certain volume ratio to prepare hydrogel mixed solution microdroplets, and placing the hydrogel mixed solution microdroplets into a 37 ℃ incubator for standing until gel is formed;
(4) Placing the collected immature oocytes into hydrogel-granulosa cell microdroplets, placing 1-2 oocytes in each microdroplet, capping the in vitro maturation medium, and culturing at 37deg.C with 5% CO 2 And culturing for 22-24h under 100% humidity;
(5) Observing the maturation condition of the oocyte in the step (4), wherein the oocyte reaching the second meiosis metaphase is the mature oocyte.
5. The culture system according to claim 4, wherein the ratio of the laminin peptide hydrogel to the in vitro maturation medium containing granulosa cells in step (3) of the culture system for in vitro culturing of oocytes is 4:1.
6. The culture system according to claim 4, wherein the culture conditions in step (4) of culturing the oocyte in vitro in the culture system are under conditions of 5% CO 2 And culturing at 37deg.C under 100% humidity; the culture time is 22-24h.
7. Use of an oocyte in vitro maturation 3D culture system according to claim 1 for the preparation of a reagent and/or kit for the treatment of infertility.
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| CN105039245A (en) * | 2015-07-01 | 2015-11-11 | 浙江大学 | Method for promoting in-vitro maturing of human immature oocyte by utilizing 3D printing technology |
| CN106566800A (en) * | 2016-11-15 | 2017-04-19 | 广西医科大学 | Method for facilitating in-vitro maturation of human immature oocytes by utilizing 3D printing technology |
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| CN105039245A (en) * | 2015-07-01 | 2015-11-11 | 浙江大学 | Method for promoting in-vitro maturing of human immature oocyte by utilizing 3D printing technology |
| CN106566800A (en) * | 2016-11-15 | 2017-04-19 | 广西医科大学 | Method for facilitating in-vitro maturation of human immature oocytes by utilizing 3D printing technology |
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