阅读说明：本技术 一种保持人类精子活力的体外培养液及其制备方法 (In-vitro culture solution for maintaining human sperm motility and preparation method thereof ) 是由 余柯达 师帅 毛佳婷 柴娟 包云 高辉 郑群 于 2021-09-22 设计创作，主要内容包括：本发明公开一种保持人类精子活力的体外培养液及制备方法,该体外培养液包含有MenSCs活性上清液和基础培养液,所述MenSCs活性上清液与基础培养液的混合比例为1：9,该MenSCs活性上清液通过无血清培养液培养MenSCs获得。本发明包含有MenSCs活性上清液,其中含有大量活性因子,可有效保护精子在体外不被氧化损伤,保持精子活力,可为ART治疗时提供更有活力的精子,以提高受精率和妊娠率,尤其是逆行射精患者。(The invention discloses an in vitro culture solution for maintaining human sperm motility and a preparation method thereof, the in vitro culture solution comprises MenSCs active supernatant and basic culture solution, the mixing ratio of the MenSCs active supernatant to the basic culture solution is 1: 9, the MenSCs active supernatant was obtained by culturing MenSCs in a serum-free medium. The invention contains MenSCs active supernatant, which contains a large amount of active factors, can effectively protect sperms from being oxidized and damaged in vitro, keep the sperm motility, and provide more viable sperms for ART treatment so as to improve the fertilization rate and pregnancy rate, especially for patients with retrograde ejaculation.)

1. An in vitro culture solution for maintaining the motility of human sperm, which comprises MenSCs active supernatant and a basal culture solution.

2. The in vitro culture solution for maintaining human sperm motility according to claim 1, wherein the mixing ratio of MenSCs active supernatant to basal culture solution is 1: 9, the MenSCs active supernatant is obtained by culturing MenSCs in a serum-free culture solution, contains active factors, and can effectively protect sperms from being oxidized and damaged in vitro.

3. The in vitro culture solution for maintaining human sperm motility according to claim 1, wherein said basal culture solution is a mixture of SSS and mHTF, the SSS and mHTF are mixed at a ratio of 1: 9.

4. the method for preparing an in vitro culture solution for maintaining the motility of human sperm according to any one of claims 1 to 3, comprising the following steps:

1) culturing MenSCs by using a serum-free culture solution, and collecting the cultured serum-free culture solution to obtain active supernatant of MenSCs;

2) SSS and mHTF were mixed as 1: 9 to prepare a basic culture solution;

3) MenSCs active supernatant was mixed with basal medium at 1: 9 to prepare an in vitro culture solution;

4) the prepared in vitro culture solution is filtered by a filter membrane.

5. The method of claim 4, wherein the MenSCs active supernatant is collected from P3-P20 subcultured cells.

6. The method of claim 4, wherein the MenSCs are cultured in a serum-free medium for 72 hours.

7. The method of claim 4, wherein the filter membrane used for filtering the in vitro culture solution has a pore size of 0.22 μm.

Technical Field

The invention relates to the technical field of sperm in-vitro culture, in particular to an in-vitro culture solution containing MenSCs active supernatant and capable of keeping human sperm activity and a preparation method thereof.

Background

Recent survey results in China show that the number of infertility patients in married couples in China accounts for 10%, and the incidence rate is on the rise. In view of this, infertility has become an important factor affecting social stability and family harmony. Human Assisted Reproductive Technology (ART) is a medical treatment for infertility, which allows patients with low fertility or infertility to gain the possibility of having offspring. ART includes both Artificial Insemination (AI) and In vitro fertilization-embryo transfer (IVF-ET) and their derivatives. When artificial insemination and IVF-ET treatment are carried out in the uterine cavity of the husband semen, the male patient needs to take out semen from the body by masturbation, carry out optimization treatment, namely washing seminal plasma, sperms with low activity and dead sperms, and then culture the sperms by using artificially synthesized culture solution including BWW, mHTF, desktop solution and the like. However, the optimization treatment can cause damage to the sperms, active oxygen is released in the culture process, the sperms are further oxidized, damaged and even killed, particularly in a male patient with retrograde ejaculation, the sperms are damaged by urine, although the optimization treatment is rapidly carried out after the sperm is separated, the damage is already caused, and the speed of the sperm losing activity in the culture solution is far higher than that of the sperms of a normal male. Therefore, it is important to develop a culture system for maintaining sperm motility in clinic.

Mesenchymal Stem Cells (MSCs) are a member of adult stem cells, are derived from the mesoderm of embryonic development, have the characteristics of self-renewal, multidirectional differentiation potential and the like, and have very important application value in regenerative medicine. MSCs were first discovered from bone marrow in 1957, and were subsequently discovered from tissues such as umbilical cord, adipose tissue, and placenta. In recent years, a novel MSCs (menstrualbood-derived MSCs) derived from menstrual blood is discovered, compared with MSCs derived from other sources, MenSCs have the advantages of wide source, easily obtained materials, high content, quick replication, noninvasive acquisition, and human waste, and the ethical problem is avoided. The mechanism of MSCs in tissue repair was originally thought to be by homing to damaged tissues, differentiating and replacing damaged cells, and subsequent studies showed that engraftment and differentiation of MSCs in damaged tissues was very rare and transient, and it is now generally believed that MSCs exert their repair primarily through paracrine action. A large number of active factors and exosomes are secreted in the MSCs culture process, and the exosomes contain functional proteins, mRNAs, microRNAs and other substances, so that the damage to sperms can be effectively repaired, and the vitality and the fertilization capability of the sperms are maintained.

Disclosure of Invention

The invention aims to provide an in vitro culture solution for maintaining human sperm motility and a preparation method thereof, the invention comprises MenSCs active supernatant, wherein the MenSCs active supernatant contains a large amount of active factors, can effectively protect sperms from being oxidized and damaged in vitro, maintains the sperm motility, can provide more viable sperms for ART treatment, and can improve the fertilization rate and pregnancy rate, especially for patients with retrograde ejaculation.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

an in vitro culture solution for maintaining human sperm motility, the in vitro culture solution comprising MenSCs activity supernatant and basal culture solution.

Preferably, the mixing ratio of the MenSCs activity supernatant to the basic culture solution is 1: 9, the MenSCs active supernatant is obtained by culturing MenSCs in a serum-free culture solution, contains active factors, and can effectively protect sperms from being oxidized and damaged in vitro.

Preferably, the basal culture solution is a mixed solution of SSS and mHTF, and the mixing ratio of the SSS to the mHTF is 1: 9.

a preparation method of an in vitro culture solution for maintaining the activity of human sperms is characterized by comprising the following steps:

1) culturing MenSCs by using a serum-free culture solution, and collecting the cultured serum-free culture solution to obtain active supernatant of MenSCs;

2) SSS and mHTF were mixed as 1: 9 to prepare a basic culture solution;

3) MenSCs active supernatant was mixed with basal medium at 1: 9 to prepare an in vitro culture solution.

4) The prepared in vitro culture solution is filtered by a filter membrane.

Preferably, the MenSCs active supernatant is collected from P3-P20 subcultured cells.

Preferably, MenSCs are cultured in a serum-free medium for 72 hours.

Preferably, the pore size of the filter used for filtering the in vitro culture solution is 0.22. mu.m.

The invention has the following advantages:

the invention contains MenSCs active supernatant, which contains a large amount of active factors, can effectively protect sperms from being oxidized and damaged in vitro, keep the sperm motility, and provide more viable sperms for ART treatment so as to improve the fertilization rate and pregnancy rate, especially for patients with retrograde ejaculation. Meanwhile, MenSCs can be derived from female patients among infertility patients, cells derived from a third party are not generated in the whole treatment process, and the risk of biological infection can be effectively avoided.

Drawings

FIG. 1 is a morphological observation of MenSCs (scale bar: 100 μm);

FIG. 2 shows the identification of molecular markers on the surface of MenSCs;

FIG. 3 shows the maintenance of sperm motility in normal males (P < 0.05);

fig. 4 shows the maintenance of sperm motility in retrograde ejaculation patients (P < 0.05).

Detailed Description

In order to better understand the objects, compositions and method steps of the present invention, an in vitro culture solution for maintaining human sperm motility and a preparation method thereof are further described in detail with reference to the examples.

Example 1

1. Obtaining of MenSCs Activity supernatant

(1) Cell isolation culture

Menstrual blood samples were collected from 20-35 year old healthy female volunteers during menstrual periods using sterile menstrual cups. Collecting the collected warpBlood was mixed with an equal volume of PBS (containing 1% penicillin-streptomycin) and stored at 2-8 ℃ under refrigeration. Separating MenSCs from menstrual blood by Ficoll density gradient centrifugation, mixing menstrual blood samples, placing on Ficoll separating medium (SIGMA corporation, USA), centrifuging for 25min at 1800r/min, collecting the centrifuged intermediate cell layer, washing with PBS for 2 times, removing lymphocyte separating medium, resuspending cells with 10% FBS-containing DMEM/F-12 (GIBCO corporation, USA), inoculating into six-well culture plate, placing at 37 deg.C and 5% CO₂Conventionally culturing under the conditions of concentration and saturation humidity, wherein the cell density reaches 90%, and digesting and passaging. A small amount of irregular, polygonal or polygonal adherent cells can be observed after 24 hours of primary separated cells, the liquid is changed in a full amount after 72 hours, and the impurity cells are discarded. After 12-14d, the primary cells (P0) attached to a monolayer and were passaged to generate P1 cells. With increasing passage times, the cell population was progressively homologous, and most cells were spindle-shaped, arranged in a vortex and clustered, exhibiting typical MSCs characteristics (fig. 1).

(2) Cell surface marker detection

Taking well-grown MenSCs, digesting with 0.25% pancreatin, rinsing with PBS 2 times, and making into 1 × 10 with PBS⁶Each of the samples to be tested was added with 1. mu.L of each of CD29 PE, CD34 FITC, CD45FITC, CD73 PE, CD90 FITC, CD105 PE, CD117 PE, and HLA-DR FITC antibody (purchased from BD Co., USA) in a single cell suspension, while PBS was used as a blank. Incubating at room temperature in dark for 30min, washing with PBS for 2 times, centrifuging at 1300r/min for 5min, resuspending cells with PBS, and detecting with flow cytometry. FIG. 2 shows surface molecular markers of stem cells derived from menstrual blood, indicating that the cells highly express CD29, CD73, CD90 and CD105, and lowly express CD34, CD45, CD117 and HLA-DR, and meet the expression standards of the surface molecular markers of MSCs.

(3) Obtaining of MenSCs Activity supernatant

Culturing identified MenSCs with DMEM/F-12 culture solution containing 10% FBS for passage, discarding the culture solution when the cells are fused to 70% -80% confluency, washing the cells with PBS for three times, adding serum-free culture solution (Lonza company in America) for continuous culture for 72h, recovering the supernatant, collecting the serum-free culture solution after the culture of P3-P20 MenSCs, namely MenSCs active supernatant, wherein the active supernatant contains a large amount of active factors, can effectively protect sperms from being oxidized and damaged in vitro, keeps the sperms active, and is frozen at-80 ℃ for later use.

2. In vitro culture of normal male sperm

(1) Preparation of in vitro culture solution

Firstly, taking out the collected MenSCs active supernatant from a refrigerator at the temperature of-80 ℃ and unfreezing; serum replacement (SSS, irvinesercific, usa) and gamete preparation (Modified HTF Medium, mHTF, irvinesercific, usa) were mixed in a ratio of 1: 9 to prepare a basic culture solution; thawed MenSCs active supernatant was then mixed with basal medium at a ratio of 1: 9 to prepare an in vitro culture solution; and finally, filtering the prepared in-vitro culture solution through a filter membrane with the aperture of 0.22 mu m, wherein the filtered liquid is the usable in-vitro culture solution.

(2) Treatment of sperm cells

Collecting sperm samples with normal activity (standard according to WHO five edition), and placing the sperm taking cup with the collected semen into a water bath with 37 ℃ for liquefaction. The liquefied semen is optimized by adopting a density gradient centrifugation method, 1.0mL of upper layer separating medium in Isolate sperm separating medium (Irvine scientific company, USA) is added into a sterile centrifuge tube, then equal lower layer separating medium is added into the bottom of the upper layer separating medium, and the completely liquefied semen is slowly added above the separating medium along the tube wall, so that the interface is kept clear and visible; placing in a centrifuge, centrifuging at 220g for 16 min; the centrifuged sperm pellet was aspirated and added to a fresh centrifuge tube containing 1mL of 10% SSS mHTF and mixed well.

(3) Culture of sperm

Equally dividing the sperm suspension into two groups, one group is a control group, the other group is an experimental group, and adjusting the optimized sperm density to 10 × 10⁶and/mL, taking sperm for microscopic examination, and recording sperm motility. Centrifuging the two groups at 220g for 5min, discarding the supernatant, adding a certain volume of mixed solution of serum-free culture solution and basal culture solution into the control group, wherein the ratio of the two is 1: 9, the experimental group added the same volume of the above in vitro culture. Culturing control group and experimental group in 35 deg.C incubator for 24 hr, taking sperm, microscopic examination, and recording spermThe activity of the seed.

(4) Results of the culture

As shown in FIG. 3, there is no significant difference in the forward motile sperm rate between the control group and the experimental group (P >0.05) when the sperm is just optimized (0h), after culturing at 35 ℃ for 24h, the forward motile sperm rate of the experimental group is 91.33 + -1.53%, the forward motile sperm rate of the control group is only 81.67 + -2.52%, and the experimental group is significantly higher than the control group (P < 0.05).

Example 2

1. Obtaining of MenSCs Activity supernatant

The procedure is as in example 1.

2. In vitro culture of retrograde ejaculation male sperm

(1) Preparation of in vitro culture solution

Firstly, taking out the collected MenSCs active supernatant from a refrigerator at the temperature of-80 ℃ and unfreezing; serum replacement (SSS, irvinesercific, usa) and gamete preparation (Modified HTF Medium, mHTF, irvinesercific, usa) were mixed in a ratio of 1: 9 to prepare a basic culture solution; thawed MenSCs active supernatant was then mixed with basal medium at a ratio of 1: 9 to prepare an in vitro culture solution; and finally, filtering the prepared in-vitro culture solution through a filter membrane with the aperture of 0.22 mu m, wherein the filtered liquid is the usable in-vitro culture solution.

(2) Treatment of sperm cells

Collecting urine specimen after emptying bladder and masturbating ejaculation of retrograde ejaculation patient, centrifuging collected urine containing sperm for 5min at 220g, discarding supernatant, resuspending sperm precipitate with 2mL of 10% SSS-containing mHTF, performing optimization treatment by density gradient centrifugation, adding 1.0mL of upper layer separating medium in sperm separating medium (Irvine scientific company, USA) into a sterile centrifuge tube, adding equal amount of lower layer separating medium to the bottom of the upper layer separating medium, slowly adding resuspended sperm above the separating medium along the tube wall, and keeping the interface clear and visible; placing in a centrifuge, centrifuging at 220g for 16 min; the centrifuged sperm pellet was aspirated and added to a fresh centrifuge tube containing 1mL of 10% SSS mHTF and mixed well.

(3) Culture of sperm

Mixing the aboveThe sperm suspension is equally divided into two groups, one group is a control group, the other group is an experimental group, and the optimized sperm density is adjusted to 10 multiplied by 10⁶and/mL, taking sperm for microscopic examination, and recording sperm motility. Centrifuging the two groups at 220g for 5min, discarding the supernatant, adding a certain volume of mixed solution of serum-free culture solution and basal culture solution into the control group, wherein the ratio of the two is 1: 9, the experimental group added the same volume of the above in vitro culture. And (3) placing the control group and the experimental group in a 35 ℃ incubator for 24h, taking sperm for microscopic examination, and recording the sperm activity.

(4) Results of the culture

As shown in FIG. 4, there is no significant difference in the forward motile sperm rates between the control group and the experimental group (P >0.05) just after optimization of sperm (0h), after 24h of incubation at 35 ℃, the forward motile sperm rate of the experimental group is 67.33 + -4.16%, the forward motile sperm rate of the control group is only 46.33 + -4.04%, and the experimental group is significantly higher than the control group (P < 0.05).

The invention has the beneficial effects that:

the invention contains MenSCs active supernatant, which contains a large amount of active factors, can effectively protect sperms from being oxidized and damaged in vitro, keep the sperm motility, and provide more viable sperms for ART treatment so as to improve the fertilization rate and pregnancy rate, especially for patients with retrograde ejaculation. Meanwhile, MenSCs can be derived from female patients among infertility patients, cells derived from a third party are not generated in the whole treatment process, and the risk of biological infection can be effectively avoided.

It is to be understood that the present invention has been described with reference to certain embodiments, and that various changes in the features and embodiments, or equivalent substitutions may be made therein by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.