CN112293407A

CN112293407A - Method for programmed cryopreservation of ovarian tissues

Info

Publication number: CN112293407A
Application number: CN201910686041.3A
Authority: CN
Inventors: 符晓倩
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-28
Filing date: 2019-07-28
Publication date: 2021-02-02

Abstract

The invention relates to a technical system for programmed cryopreservation of mammalian ovarian tissues, belonging to the technical field of tissue cryopreservation. The ovary tissue frozen and revived by the invention can keep good physiological activity. The refrigeration of the ovary tissue not only can preserve the function of generating gametes of the ovary, but also can maintain the function of endocrine, and has incomparable advantages in the aspect of fertility preservation. After the ovarian tissue is obtained and processed, the ovarian tissue is preserved in liquid nitrogen through cryoprotectant balance and programmed freezing instrument temperature reduction. By improving the components and the proportion of the cryoprotectant, the balance time and the freezing and cooling procedures, the cryoprotectant can well protect ovarian follicles in ovarian tissues from being damaged by freezing-resuscitation stimulation, and a feasible method is provided for protecting and preserving the fertility of mammals.

Description

Method for programmed cryopreservation of ovarian tissues

Technical Field

The invention relates to a technical system for programmed cryopreservation of mammalian ovarian tissues, which can ensure that the mammalian ovarian tissues can keep good physiological activity after freezing-resuscitation and belongs to the technical field of tissue cryopreservation.

Background

Ovarian tissue freezing has its unique advantages in preserving ovarian function. First, the ovarian cortex contains a large number of primordial germ cells, located in the cortical region 1mm from the ovarian surface, and this part of the follicle is the primary target of protection for ovarian tissue freezing. Secondly, primordial follicles in ovarian tissue are small in size, in a very low metabolic state, and have a very low content of intracellular lipids that are sensitive to low temperatures, so primordial follicles are more tolerant to the cryogenic freezing process than mature follicles. Third, the human ovarian cortex can be obtained laparoscopically or by open surgery, and is not affected by the menstrual cycle, nor delays the time for tumor treatment. Fourth, ovarian cortical freezing can preserve not only the fertility of the patient, but also its endocrine function. Fifth, ovarian tissue freezing can be used to preserve the fertility function of prepubertal girls. Ovarian tissue cryopreservation has the advantages of no alternatives as described above.

However, ovarian tissue freezing is much more complex than gamete or embryo freezing because it requires the preservation of a tissue system that has a certain volume and contains multiple cell types. Ovarian tissue freezing is closer to organ freezing than gamete freezing. The majority of follicles in the ovarian cortex are primordial follicles in a quiescent state, surrounded by a monolayer of flattened promyelocytic cells, which contain a primary oocyte, the volume of which is only 1/10 of the mature ovum. Compared to the growing follicle, the primordial follicle is small in size, allows rapid penetration of the cryoprotectant solution into the inner oocyte, and is slowly metabolized, has a low number of supporting cells, and is more resistant to toxicity caused by freezing. Furthermore, although primordial follicles can be technically isolated, the surrounding tissues have a major impact on the further maturation of the follicles, and the only practical way to preserve primordial follicles is to preserve them in the cortex at present.

The ovary tissue programmed freezing mainly adopts low-concentration cryoprotectant to carry out gradient dehydration on the ovary tissue, and the temperature is slowly reduced by a programmed freezing instrument. The slow freezing technique has been widely used in many research fields, and has been widely used for embryo freezing and embryo freezing in assisted reproduction, and has achieved surprising performance in ovarian tissue freezing in many species including humans. Currently, over 130 healthy infants have been successfully delivered worldwide by ovarian tissue cryopreservation technology, and not only the fertility of patients is preserved but also the reproductive secretion function is reestablished by autograft technology. While programmable freezing has achieved good performance at present, there are a number of challenges to optimizing programmable freezing protocols. Because the ovarian tissue contains many kinds of cells, such as interstitial cells, oocytes, granulosa cells and theca cells, and also many blood vessels and nerves penetrate through the ovarian tissue, the structures are closely related to the functional recovery after the transplantation of the ovarian tissue. Factors which can affect the viability of the tissues after freeze thawing, such as permeability to cryoprotectants, toxicity tolerance, hydraulic conductivity and the like, are different among different cells, so that the concentration of the cryoprotectant and the cooling requirements of the complex components of the ovarian tissues are different, and the freezing scheme of the ovarian tissues needs to achieve compromise among the optimal dehydration time, cooling and rewarming speed of different types of cells.

Disclosure of Invention

The invention aims to provide an antifreeze agent for low-damage cryopreservation of mammalian ovarian tissues and a programmed cooling scheme for freezing the mammalian ovarian tissues by adopting the antifreeze agent. And (3) observing the follicular morphology of each group by using fresh ovarian tissues as a control through conventional HE (high-grade hematoxylin) staining, counting primordial and primary follicles with normal and abnormal morphologies, and calculating the morphologic normal rate of the primordial and primary follicles of each group of ovarian tissues. PROH cryoprotectant compatibility and freeze-thaw technology systems were evaluated to preserve the physiological activity of mammalian ovarian tissue after freeze-thaw.

According to the technical scheme provided by the invention, the method for freezing the mammalian ovarian tissue by using the PROH as the main component of the cryoprotectant and adopting the programmed cooling scheme of the cryoprotectant is characterized by comprising the following steps of:

1. preparation of ovary tissue refrigerating fluid, thawing fluid and culture fluid

The solutions for freezing and unfreezing the ovarian tissues are prepared by adding different types of cryoprotectants with different concentrations into a freezing and thawing base solution. All solutions were filter sterilized through a 0.22 μm pore size filter.

1.1 freezing and thawing base liquid: namely, HEPES buffered human fallopian tube fluid (HTF) containing 5mg/ml Human Serum Albumin (HSA): 5ml of HSA was added to 95ml of HEPES-HTF culture medium.

1.2 thawing recovery liquid: 3.4g of sucrose was added to 10ml of freeze-thaw base solution to a final sucrose concentration of 1M.

1.3 first cryoprotectant solution: mu.l of PROH was added to 890. mu.l of the freeze-thaw base solution to a final concentration of 1.5M.

1.4 second cryoprotectant: to 790. mu.l of the freeze-thaw base solution were added 110. mu.l of PROH and 100. mu.l of the thawing recovery solution, and the final concentration of PROH was 1.5M.

1.5 first resuscitation fluid: freezing and thawing the base solution: thawing recovery liquid (v/v) ═ 2: 1 (sucrose 0.28M)

1.6 second resuscitation fluid: freezing and thawing the base solution: thawing recovery liquid (v/v) ═ 4: 1 (sucrose 0.17M)

2. Process for freezing and thawing ovarian tissue

2.1 at room temperature (22-25 ℃), soaking the ovary tissue in the first freezing protection solution for 5 minutes, then transferring the tissue strips into a 1.8ml freezing tube filled with 1ml of the second freezing protection solution, balancing for 30 minutes at 4 ℃, and transferring to a programmed freezing instrument for programmed freezing.

2.2 program freezer operating program as follows:

2.2.1 starting from 4 ℃ and reducing the temperature from-2 ℃/min to-8 ℃;

2.2.2 maintaining at-8 deg.C for 10 min; then, manually planting ice by using forceps precooled in liquid nitrogen in advance; then maintaining the temperature at-8 ℃ for 10 minutes;

2.2.3, cooling to-40 ℃ at the speed of-0.3 ℃/min;

2.2.4, cooling to-150 ℃ at the speed of-30 ℃/min;

2.2.5 quickly put into liquid nitrogen for preservation.

And 2.3, after freezing for 48 hours, performing resuscitation. The cryovial was removed from the liquid nitrogen and allowed to stand in air for 10 seconds. Then the freezing tube is placed in a water bath at 37 ℃ and is continuously shaken, the ice crystals can be completely dissolved by naked eyes within 2-3 minutes, and the ovary tissue is taken out. Sequentially re-absorbing water in the first resuscitation solution and the second resuscitation solution gradually, balancing for 5 minutes each time, rinsing in a freeze-thaw basic solution for several times, placing at 37 deg.C and 5% CO₂And carrying out subsequent treatment in a wet incubator.

Drawings

Figures 1A and B present images of typical primordial and primary follicles in fresh ovarian tissue (HE staining x 400): a is a typical primordial follicle, in which a layer of flat granular cells surrounds the periphery of an ovum; b a typical primary follicle, the outer edge of the ovum surrounds a layer of cuboidal granulosa cells; c and D show images of typical primordial and primary follicles in ovarian tissue after programmed freezing and resuscitation (HE staining x 400): c, a typical primordial follicle, wherein a layer of flat granular cells surrounds the periphery of an ovum; d typically primary follicles, with a layer of cuboidal granulosa cells surrounding the outer edge of the ovum. It can be seen that the morphology of each stage of ovarian follicles in the ovarian tissues after programmed freezing and revival has not changed obviously.

FIG. 2 temperature lowering curve of programmed freezer temperature lowering program

Detailed Description

The invention is further illustrated but is not to be construed as being limited by the following examples. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Examples of the experiments

1.1 freezing and thawing base liquid: namely, HEPES buffered human fallopian tube fluid (HTF) containing 5mg/ml Human Serum Albumin (HSA): 5ml HAS was added to 95ml HEPES-HTF medium.

2. Acquisition of ovarian tissue

After the ovaries were harvested, they were washed 10 times in HEPES buffered-HTF medium at room temperature, and the ovarian tissues were then soaked in HEPES buffered-HTF medium tubes, clamped between ice cubes, and returned to the laboratory.

3. Pretreatment of ovarian tissue

Ovaries were dissected under a microscope with a scalpel and eye cut into small strips of ovarian tissue approximately 2-3mm by 1mm in size. All the medulla is scraped off with a surgical blade. Pre-frozen ovarian tissue cutting and treatment were performed in HEPES-buffered-HTF culture at room temperature, and all manipulations were completed in l hours at room temperature in a sterile ultra clean bench.

4. Process for freezing and thawing ovarian tissue

4.1 at room temperature (22-25 ℃), transferring the ovary tissues into a first freezing protection solution to be soaked for 5 minutes, then transferring the tissue strips into a 1.8ml freezing tube filled with 1ml of a second freezing protection solution to be balanced for 30 minutes at 4 ℃, and transferring the tissue strips into a programmed freezing instrument to be subjected to programmed freezing.

4.2 program freezer operating program as follows:

4.2.1 starting from 4 ℃ and reducing the temperature from-2 ℃/min to-8 ℃;

4.2.2 maintaining at-8 deg.C for 10 min; then, manually planting ice by using forceps precooled in liquid nitrogen in advance; then maintaining the temperature at-8 ℃ for 10 minutes;

4.2.3, cooling to-40 ℃ at the speed of-0.3 ℃/min;

4.2.4, cooling to-150 ℃ at the speed of-30 ℃/min;

4.2.5 quickly put into liquid nitrogen for preservation.

4.3 after freezing for 48h, resuscitation was performed. The cryovial was removed from the liquid nitrogen and allowed to stand in air for 10 seconds. Then the freezing tube is placed in a water bath at 37 ℃ and is continuously shaken, the ice crystals can be completely dissolved by naked eyes within 2-3 minutes, and the ovary tissue is taken out. Sequentially re-absorbing water in the first resuscitation solution and the second resuscitation solution gradually, balancing for 5 minutes each time, rinsing in a freeze-thaw basic solution for several times, placing at 37 deg.C and 5% CO₂And carrying out subsequent treatment in a wet incubator.

5. Histological analysis

Before and after freezing, each ovarian tissue block was randomly sampled and histologically analyzed by 4% paraformaldehyde fixation. Tissues were embedded in paraffin, 4 μm serial sections, and stained with hematoxylin-eosin (HE). Each 10 sections of tissue were analyzed 1 time. The morphology of primordial and primary follicles was observed by a single blind method. Follicular morphology was observed as follows: morphologically normal follicles: the shape of the oocyte is regular, the zona pellucida is complete, the arrangement of follicular cells/granulosa cells is regular, no drop occurs, and the radiation crown is regular; morphologically altered follicles: the oocyte shrinks, the nucleus shrinks, the zona pellucida is incomplete or disappears, the granular cells are irregularly arranged, and parts of the granular cells fall off. Primordial and primary follicle counts the proportion of morphologically normal primordial and primary follicles in each ovarian tissue piece was calculated using the oocyte nucleus as a marker. The number of follicles at other developmental stages was very small and not counted.

6. Statistical analysis

The data were processed with SPSS 22.0 statistical software, and the percentage of follicular morphology normality and follicular apoptosis between groups was compared using χ²And (5) checking and analyzing. P < 0.05 considered the difference to be significant.

7. The experimental results are as follows:

the normal form rate of primordial follicles in the frozen tissues is 89.37 +/-3.12 percent, and the normal form rate of primary follicles is 79.64 +/-4.00 percent; the normal morphology rate of primordial follicles in fresh ovarian tissue is 91.64 +/-3.38%, the normal morphology rate of primary follicles is 83.29 +/-7.06%, and the difference is not statistically significant (P > 0.05). The invention can well protect the ovarian follicle in the ovarian tissue from being damaged by the freeze-resuscitation stimulation.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be considered as within the protection scope of the present invention.

Claims