KR20170050930A - Device for ovarian tissue vitrification - Google Patents
Device for ovarian tissue vitrification Download PDFInfo
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- KR20170050930A KR20170050930A KR1020150153087A KR20150153087A KR20170050930A KR 20170050930 A KR20170050930 A KR 20170050930A KR 1020150153087 A KR1020150153087 A KR 1020150153087A KR 20150153087 A KR20150153087 A KR 20150153087A KR 20170050930 A KR20170050930 A KR 20170050930A
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- germ cell
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
- A01N1/0257—Stationary or portable vessels generating cryogenic temperatures
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- Biomedical Technology (AREA)
- Mechanical Engineering (AREA)
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- Dentistry (AREA)
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- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Reproductive Health (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to a germ cell freezing apparatus, and more particularly, to a germ cell freezing apparatus including a germ cell holding unit, a storage unit, a refrigerant inflow / outflow unit, a protrusion, and a tagging unit. The frozen germ cells according to the present invention can provide germ cell retention which is remarkably superior to that of the conventional apparatus with a simple structure and a low cost.
Description
The present invention relates to a germ cell freezing apparatus. More specifically, the present invention relates to a germ cell freezing apparatus including a germ cell holding section, a storage section, a refrigerant inflow / outflow section, a protrusion section, and a tagging section.
Germ cells develop in the fetus after fertilization in the mother's womb and migrate into the embryo. In the female, they are differentiated into ovaries through the constitutive process. The oocyte produced at this time is kept in the ovary while stopped in the first meiosis shear cycle. After birth, some of the oocytes in the body undergo a series of processes through primordial follicles, primary follicles, secondary follicles and tertiary follicles through the maturation process, The oocyte is released and plays a role as a reproductive cell. At 20 weeks of gestation, there are about 7 million oocytes in the fetal ovary, but the number of oocytes after birth is reduced to 2 million, and the number of oocytes after birth is steadily decreasing to about 500,000 And the number of oocytes released during a woman's lifetime is generally limited to 300 to 500.
Freezing methods to preserve fertility in female cancer patients include embryo freezing, oocyte freezing, and ovarian freezing. The choice of the most appropriate method depends on the timing of chemotherapy, the type of cancer, the age of the patient and the presence of the spouse. Currently, clinically established methods are cryopreservation of embryos and oocytes, which is not possible in women who can not delay treatment for induction of ovulation. On the other hand, cryopreservation of the ovaries is possible in all pre-adolescent pediatricians and women who can not delay chemotherapy. The main goal of ovarian tissue freezing is to transplant ovarian tissue that is cryopreserved to a heterotopic site other than the pelvis, such as the orthotopic site or the upper arm, abdominal wall, when the patient is cured or the disease is cured.
In cryopreservation of ovarian tissue, adequate permeation of cryoprotective agent (CPA) to oocytes as well as granulosa cells and stromal cells is necessary to prevent the formation of ice crystals. Together, we must consider the toxicity of high concentrations of CPA. So far, slow-freezing using propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol as a CPA has been used as a standardized method in cryopreservation of human ovaries. However, recent studies have shown that vitrification, which is a rapid freezing method, can prevent damage caused by ice crystals during ovarian tissue freezing. Vitrification requires the tools most appropriate for this procedure because tissue must be put into liquid nitrogen in a very short time. When the oocyte or embryo is frozen, tools such as cryotop, cryoleaf, and cryoloop can be used. However, in order to freeze the ovary, specialized tools are limited.
Accordingly, the present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, the present inventors have found that the germ cell freezing device manufactured by including the germ cell holding part, the storage part, the refrigerant inflow / outflow part, the protruding part and the tagging part has remarkably superior germ cell preservation ability And the present invention has been completed.
It is an object of the present invention to provide a germ cell freezing apparatus having a simple constitution and an excellent germ cell preservation ability at low cost.
According to one aspect,
(a) a reproductive cell holding part;
(b) a storage part for storing the germ cell holding part;
(c) a plurality of outflow portions for circulating the refrigerant formed in the accommodating portion;
(d) a protrusion extending in the longitudinal direction at one end of the accommodating portion; And
(e) a tagging portion attached to one end of the storage portion,
Wherein the storage portion includes one or two or more reproductive cell holding portions. The shape of the germ cell freezing apparatus according to the present invention is shown in Figs.
In the germ cell freezing apparatus according to the present invention, the germ cells may be a primordial germ cell, an oogonia cell, a primordial follicle, a primary follicle, a secondary follicle secondary follicle, and tertiary follicle.
In the germ cell freezing apparatus according to the present invention, the germ-cell holding section may include a thermally conductive material. The thermally conductive material may be at least one selected from the group consisting of metals such as silver, copper, aluminum and stainless steel, thermally conductive ceramics such as aluminum nitride, silicon nitride and alumina, metal powder and thermally conductive ceramics such as aluminum nitride, , And the like, but the present invention is not limited thereto.
In the germ cell freezing apparatus according to the present invention, the germ cell holding unit may be formed while maintaining a plurality of dot-shaped or net-shaped gaps at regular intervals, but the present invention is not limited thereto.
In the germ cell freezing apparatus according to the present invention, the containing portion may include a plurality of outflow portions for circulating the coolant. For example, the receiving portion may include one or more, two or more, three or four or more refrigerant outlet ports on one or both sides.
In the germ cell freezing apparatus according to the present invention, the protrusions may be used for handling the germ cell freezing apparatus and for mounting in the cooling apparatus.
In the gonad cell freezing apparatus according to the present invention, the tagging unit may be a cell and tissue information to be stored.
In the germ cell freezing apparatus according to the present invention, the germ cells may be 50% or more.
In accordance with the present invention, the germ cell cell apoptosis may be less than 20%.
1 is a front view of a germ cell freezing apparatus according to the present invention.
2 is a perspective view of a germ cell freezing device according to the present invention.
3 is a plan view of the germ cell freezing apparatus according to the present invention.
Figure 4 is the ratio of good follicles in tissues preserved by the gonad cell freezing device according to the present invention.
Figure 5 is the ratio of native follicles in tissues preserved by the gonad cell freezing device according to the present invention.
FIG. 6 is the ratio of follicles that caused apoptosis in tissues preserved by the gonad cell freezing apparatus according to the present invention.
The present invention provides a germ cell freezing apparatus having a simple constitution and an excellent germ cell preservation ability at low cost. The germ cell freezing apparatus comprises:
(a) a reproductive cell holding part;
(b) a storage part for storing the germ cell holding part;
(c) a plurality of outflow portions for circulating the refrigerant formed in the accommodating portion;
(d) a protrusion extending in the longitudinal direction at one end of the accommodating portion; And
(e) a tagging portion attached to one end of the storage portion.
Hereinafter, the present invention will be described in detail with reference to the drawings.
The germ
The germ-
The
The
According to the germ cell freezing apparatus of the present invention, the germ cell can be preserved by 50% or more. Further, according to the germ cell freezing apparatus of the present invention, the cell apoptosis rate of the germ cells can be less than 20%.
Hereinafter, various embodiments are provided to facilitate understanding of the present invention. The following examples are provided to facilitate understanding of the invention and are not intended to limit the scope of the invention.
< Example >
Example 1. Collection of ovarian tissue
The ovaries of cattle slaughtered at the slaughterhouse were collected and the collected ovaries were stored in a 4 ° C L-15 (Leibovitz, Sigma-Aldrich) culture medium and transferred to a clean-vet in the laboratory within 2 hours from the slaughterhouse. The tissues other than the surface lipids and other ovaries were removed from the ovaries and washed with phosphate buffer saline (PBS) containing 0.4% bovine serum albumin (BSA) The surface of the ovaries was sterilized with alcohol. The treated ovaries were cut in the center on a Petri dish containing L-15 culture medium and divided into two equal parts. The medullary part of the tissue was removed, and the cortex part was cut into 1 mm thickness.
Example 2. Ovarian tissue Vitrification
The ovarian tissue of Example 1 was inoculated into the gonad cell freezing apparatus of the present invention and then cultured in a culture medium containing 20% fetal bovine serum (FBS) containing 7.5% ethylene glycol (EG) and 7.5% DMSO (20% FBS-PBS) containing 20% ethylene glycol, 20% DMSO and 0.5 M sucrose for 10 min at room temperature for 2 min. Were rapidly frozen in liquid nitrogen and vitrified. The frozen tissue was stored in a normal tissue preservation liquid nitrogen tank.
Example 3. Thawing of ovarian tissue
The frozen ovary tissues of Example 2 were transferred to the atmosphere in the state of being immobilized by the gonad cell freezing apparatus of the present invention, exposed for 2 minutes at room temperature, and treated for 3 minutes in a constant temperature water bath maintained at 30 to 37 ° C. The tissues recovered in the constant temperature water bath were treated for 5 minutes in a culture medium containing 1 M of sucrose, 5 minutes in a culture medium containing 0.5 M of sucrose, 5 minutes in a culture medium containing 0.25 M of sucrose, Rehydration.
< Experimental Example >
Experimental Example One. Vitrification Comparison of gonadal cell structure of ovary
In order to observe the effect of the present invention, ovarian tissue slices (hereinafter, referred to as 'fresh') which were not frozen in ovarian tissue slices (hereinafter referred to as 'SNUBH device') thawed by the method of Example 3 and conventional equipment used for vitrification of ovarian tissues The ratio of good follicles, primordial follicles, and apoptotic follicles were compared with vitrification of Example 3 and ovarian tissue sections (hereinafter referred to as 'Kitazato' and 'Cryovial') of Example 4, which were observed under a microscope.
As can be seen from FIG. 4, the SNUBH device showed superior fecal ratio in the state of good preservation compared with the conventional equipment Kitazato and cryovial.
As can be seen from FIG. 5, the SNUBH device was found to retain more primordial follicles, which were in the state before egg production, as compared with the conventional equipment Kitazato and Cryovial.
As can be seen from FIG. 6, the SNUBH device did not show any significant difference in apoptosis as compared with the conventional equipment Kitazato and Cryovial.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (6)
A storage part for storing the germ cell holding part;
A plurality of outflow portions for circulating the refrigerant formed in the accommodating portion;
A protrusion extending in the longitudinal direction at one end of the accommodating portion; And
And a tagging portion attached to one end of the accommodating portion,
Wherein the storage section includes one or two or more reproductive cell holding sections.
The germ cells include primordial germ cells, oogonia cells, primordial follicles, primary follicles, secondary follicles, and tertiary follicles. ≪ / RTI > wherein the cell comprises one or more cells selected from the group comprising < RTI ID = 0.0 >
Wherein the reproductive cell holding part is made of a thermally conductive material selected from the group consisting of silver, copper, aluminum, stainless steel, aluminum nitride, silicon nitride, alumina, metal powder, aluminum nitride and carbon fiber.
Wherein the reproductive cell holding portion is formed with a plurality of dot-shaped or net-shaped portions while maintaining a constant gap.
Wherein said germ cells are preserved by 50% or more.
Wherein the germ cell cell apoptosis rate is less than 20%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150153087A KR20170050930A (en) | 2015-11-02 | 2015-11-02 | Device for ovarian tissue vitrification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150153087A KR20170050930A (en) | 2015-11-02 | 2015-11-02 | Device for ovarian tissue vitrification |
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| KR20170050930A true KR20170050930A (en) | 2017-05-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| KR1020150153087A KR20170050930A (en) | 2015-11-02 | 2015-11-02 | Device for ovarian tissue vitrification |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115462370A (en) * | 2022-11-03 | 2022-12-13 | 能科达(上海)干细胞研究中心有限公司 | Gel solution for preserving umbilical cord near totipotent stem cells and application thereof |
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2015
- 2015-11-02 KR KR1020150153087A patent/KR20170050930A/en active Search and Examination
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115462370A (en) * | 2022-11-03 | 2022-12-13 | 能科达(上海)干细胞研究中心有限公司 | Gel solution for preserving umbilical cord near totipotent stem cells and application thereof |
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