KR20140046690A - Method for cryopreservation of porcine spermatogonial stem cell using cryopreservation medium comprising trehalose - Google Patents
Method for cryopreservation of porcine spermatogonial stem cell using cryopreservation medium comprising trehalose Download PDFInfo
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- KR20140046690A KR20140046690A KR1020120112393A KR20120112393A KR20140046690A KR 20140046690 A KR20140046690 A KR 20140046690A KR 1020120112393 A KR1020120112393 A KR 1020120112393A KR 20120112393 A KR20120112393 A KR 20120112393A KR 20140046690 A KR20140046690 A KR 20140046690A
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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
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Abstract
The present invention relates to a cryopreservation method of pig garden stem cells using a cryopreservation solution containing a specific concentration of trehalose, and to the pig garden stem cells after cryopreservation and thawing of pig garden stem cells by the method of the present invention. The recovery rate is increased, and the survival rate of garden stem cells is excellent after transplanting pig garden stem cells into the testes of awarded pigs. You can expect a breakthrough in development.
Description
The present invention relates to a cryopreservation method of pig garden stem cells using a cryopreservation solution containing a specific concentration of trehalose.
Stem cells are undifferentiated and can proliferate for a long time through self-division and have the ability to differentiate into cells that perform special functions under appropriate conditions. These stem cells are divided into embryonic stem cells and adult stem cells depending on the time of separation. Unlike embryonic stem cells with pluripotency, adult stem cells are undifferentiated cells present in tissues and organs, and maintain proper homeostasis by supplying appropriate cells through self-renewal and differentiation in tissues and organs. To be able.
Spermatogonial stem cells (SSCs), which produce sperm, mediators of male genetic information to the next generation through spermatogenesis, are adult stem cells in testis. Garden stem cells are germline stem cells that form the basis of spermatogenesis. The spermatogenesis process is the process by division and differentiation of garden stem cells in the testis of men and is maintained throughout the male life.
In 1994, Brinster (Proc Natl Acad Sci US A. 1994, 22; 91 (24): 11303-7) developed a technique for transplanting mouse testis cells, demonstrating the presence of garden stem cells in the testes of mice. Electricity was prepared to directly study the functional characteristics of garden stem cells. Since, testicular cell and garden stem cell transplantation techniques have been used as essential and the most essential technology in the development of biological characteristics and applications of garden stem cells. Generally, long-term preservation of animal cells including stem cells includes in vitro culture and cryopreservation. In the case of rodents, an in vitro culture method of garden stem cells has been recently developed to enable long-term culture growth. However, to date, no efficient in vitro culture techniques for garden stem cells have been developed in mammals other than rodents. In 1996, the Brinster (Nature medicine. 1996, 2, 693-696) research team transplanted fused garden stem cells into the testes of donor animals after cryopreservation and derived donor garden stem cells from testes of donor animals. It was confirmed that sperm cells are produced. The cryopreservation method used in this case is the same method as the general somatic cryopreservation method, which increases the possibility of developing an efficient method to replace sperm cryopreservation in terms of long-term preservation of genetic resources. Later, cryopreservation methods developed garden stem cell cryopreservation methods of other species, including Dobrinski (Biology of reproduction, 1999, 61: 1331-1339) in 1999 and Izadyar (Journal of andrology, 2002, 23: 537-545) in 2002. Research has been carried out to increase the efficiency of the system. However, the cryopreservation method of the garden stem cells used in the above-described studies was a cryopreservation method for the somatic cells, which can be easily used. However, since the cell survival rate is relatively low and all cell groups of the testis are frozen, the pure garden stem cells are used. The exact survival rate after freezing and thawing is unknown. Therefore, for long-term preservation of garden stem cells, the development of efficient cryopreservation technology optimized for pure garden stem cells is urgently needed.
In addition, there has not been a study on cryopreservation method of pig garden stem cells so far, therefore, it is urgently needed to develop efficient cryopreservation technology of garden stem cells in order to efficiently preserve long-term pig genotype.
Therefore, the present inventors continued to develop a method for efficiently cryopreserving pig garden stem cells, and when cryopreserving pig garden stem cells in a tissue state using a cryopreservation solution containing trehalose. The recovery and freezing and thawing at the time of transplanting pig garden stem cells into the testes of the donor pigs confirmed that the survival rate is excellent, thereby completing the present invention.
An object of the present invention is to provide a method for cryopreservation of pig garden stem cells using a cryopreservation solution containing trehalose.
In order to achieve the above object, the present invention provides a cryopreservation method of pig garden stem cells containing trehalose.
By using the pig garden stem cell cryopreservation method of the present invention, the recovery rate of pig garden stem cells after cryopreservation and thawing is increased, and the survival rate of garden stem cells is excellent after transplanting pig garden stem cells into the testes of awarded pigs. In addition to the development of the field of transgenic stocks of breeders, we can expect significant developments in the development of transgenic animal production methods using frozen pig garden stem cells.
1 is a diagram showing the number of garden stem cells recovered after cryopreservation and thawing for one month (S: freezing donor cells in a single cell state upon freezing;
T: freeze donor cells into tissue state upon freezing;
1: basic cryopreservative (MEM-α with 10% DMSO, 10% fetal bovine serum)
2: cryopreservation solution containing 50 mM trehalose;
3: cryopreservation solution containing 100 mM trehalose;
4: cryopreservation solution containing 200 mM trehalose;
5: cryopreservation solution containing 50 mM sucrose;
6: cryopreservation solution containing 100 mM sucrose;
7: cryopreservation solution containing 200 mM sucrose;
Number (X 10 4 ) of PGP 9.5 positive cells per gram: number of cells stained in PGP 9.5 (pig spermatogonia-specific marker) after freezing and thawing per gram of testis tissue;
a ~ g: Displays grouped results using statistics (one-way analysis of variance and tukey`s HSD test), p <0.05
2 is a diagram showing the results of immunocytochemistry using PGP 9.5 after freezing and thawing. [A: Immune cells after freezing and in vitro culture using a cryopreservation solution containing 200 mM trehalose. Photographs of porcine garden stem cells observed under a microscope after chemistry;
B: fluorescence micrograph of A;
C: Cell photographs observed under fluorescence microscopy after immunocytochemistry using frozen cryopreservation solution containing no glycosylated substance and recovering as a single cell after in vitro culture in tissue state;
D: cell photographs observed under fluorescence microscopy after immunocytochemistry using frozen cryopreservation solution containing 200 mM trehalose and recovered as a single cell after in vitro culture in a tissue state;
Each arrow represents porcine garden stem cells expressing PGP 9.5, bar: 50 μm)].
Figure 3 is a diagram showing the appearance of animal testes after freezing and thawing porcine garden stem cells 1.5 months after transplantation [A: frozen and in vitro culture in a tissue state using a cryopreservation solution containing 200 mM trehalose Stained garden stem cells with red fluorescent staining reagent (PKH26), transplanted into the recipient testis and 1.5 months after testis;
B: Pictures of single colonies derived from donor cells when observed in testes of A
4 is a diagram showing the number of colonies after 1.5 months cryopreservation using the cryopreservation solution containing 200 mM trehalose of the present invention, and transplanted into a recipient animal using the same [DMSO only: added sugar substance Garden stem cells frozen using unfrozen cryopreservative;
200 mM trehalose: Garden stem cells frozen using cryopreservation solution containing 200 mM trehalose;
Colonies / 10 5 cells transplated: colonies formed per number transplanted 10 5 cells;
*: Indicates a statistically significant difference compared to the group frozen with the base stock solution (p <0.05)].
The present invention is a freeze containing 10% -20% fetal calf serum, Alpha Modification of Eagle's Minimum Essential Media (MEM-α) containing 10% -20% DMSO (dimethylsulfoxide) and 0.15 M ~ 0.25M trehalose It provides a cryopreservation method of pig garden stem cells, including; preserving the pig garden stem cells isolated from the testis of pigs in a tissue state using a preservative solution.
The tissue may be removed to remove the testicles of the testis and cut the testis tissue using a surgical scissors, scalpel, tweezers and the like can be used for cryopreservation, the size of the tissue is preferably 3 × 3 × 3 mm.
In particular,
High purity garden stem cells are recovered from the testicular cell group recovered after cryopreservation and thawing in a tissue state including the garden stem cells through high purity. Recovered garden stem cells can be cultured in vitro to recover a single cell group of garden stem cells. It is preferable to use the differential plating technique for the high purity process for the recovery of high purity garden stem cells. Differential plating of testicular cells centrifuged with a single cell group was performed by incubating 10 × 10 6 single cells in a 60 mm culture dish in an incubator at 5% CO 2 , 37 ° C for 12 hours, and then removing the cells that did not adhere to the bottom of the culture dish. The high purity garden stem cell group can be recovered by recovering.
The in vitro culture is preferably cultured in a serum-free medium containing growth factors and an in vitro incubator coated with a support cell layer. The growth factor is preferably GDNF (Glial cell line derived neurotrophic factor, neurotrophic factor), GFRα1 (GDNF family co-receptor α1), bFGF (Basic fibroblast growth factor, fibroblast growth factor) is preferred. It is preferable to include replacing at intervals of one day, subculture at intervals of 7-9 days.
In addition, after cryopreservation using trehalose, thawing and survival and centrifugation, recovery and proliferation can be measured, and trehalose can be used for long-term cryopreservation. Trehalose (D-glucopyranosyl- (1↔1) -D-glucopyranoside, see Manaught, AD, (1997) Adv. Carbohydr. Chem. Biochem., 52, 43-177) "Is a non-reducing disaccharide having two kinds of isomers in which two glucoses are bonded to each carbon position as shown in Chemical Formula 1 below. Most of the isomers in nature are alpha, alpha-trehalose. It is a type of storage carbohydrate for many kinds of bacteria, fungi, yeasts, insects, animals and plants, and serves as a power source for flying insects, and for external physical effects on yeasts and E. coli. Acts as a resist (Elbein, AD (1974) Adv. Carbohydr. Chem. Biochem., 30, 227-256; Yoshida, M. et al. (1995) Oyo Toshitsu Kagaku, 42, 19-25; Van Laere, A. (1989) FEMS Microbiol. Rev., 63, 201-210; Kassen, I. et al. (1994) Gene, 145, 9-15).
Trehalose is a sweetener with about half the sweetness of sugar in the same concentration, as well as the stabilizing effect of biomolecules in the fields of basic science, medicine, agriculture and bio-electronics. For various applications (Colaco, C. et al. (1992) Bio / Technology., 10, 1007-1011). Trehalose can be used in medicine as a stabilizer in dry or frozen foods and as a diagnostic reagent and pharmaceutical pharmaceutical additive, and as a stabilizer to prevent thermal coagulation and thermal denaturation of bioactive proteins, including enzymes, as well as in people's cosmetics. As a moisturizer, it is very useful in various fields.
[Chemical Formula 1]
In Formula 1, a) is alpha, alpha-trehalose, b) is alpha, beta-trehalose, c) is beta, beta-trehalose.
In the cryopreservation method, the cells of 0.5 x 10 6 cells / ml concentration are placed in a cryopreservation solution to which various concentrations of trehalose are added and stored in 1 ml of one freezing container. The freezing vessel is stored in a cryocontainer containing isopropanol (cryocontainer) to lower the temperature at a rate of about -1 ℃ / min to -80 ℃ (hereinafter referred to as gentle freezing method) and stored for one day. The freezing vessel can then be transferred back to -196 ℃ liquid nitrogen and stored for 30 days.
When cryopreservation is performed using cryopreservation solution containing 0.2 M of trehalose in tissue state, cryopreservation efficiency is lower than that of cryopreservation of single cell group using cryopreservation solution containing 0.2 M of trehalose. great.
In addition, the present invention, after cryopreservation of adult livestock species garden stem cells, by applying recovery and purity enhancement techniques, pure garden stem cells are collected from the testes of excellent pigs that have excellent genetic ability but are not able to semen due to disease or aging. And efficient preservation can improve germ cell preservation efficiency of good pigs.
In addition, it can be applied to the transplantation method of the garden stem cells to improve the utilization of the superior species sow by producing sperm derived from the garden stem cells of the seed sows transplanted from the recipient testis after transplanting to the young recipient testis.
Hereinafter, the present invention will be described in detail through examples. The following examples are merely examples for describing the present invention, and the scope of the present invention is not limited thereto.
Example
One:
Comparison of Survival Efficiency of Garden Stem Cells according to Donor Cell Status and Cryopreservation Conditions of Porcine Garden Stem Cells.
1. Cryopreservation of garden stem cells in single cell state
Testis were surgically extracted from newborn pigs at 16 weeks of age and transported to a laboratory in a container containing 4 ° C cell culture fluid. Then, the testicle is removed from the testes, and the testis tissue is chopped with surgical scissors, scalpels, and tweezers, and then the enzymes such as collagenase, trypsin, and Dnase I are mixed. Single cells containing stem cells were isolated. The isolated single cells were stored in the cryopreservation solution containing the basic cryopreservation solution (MEM-α containing 10% DMSO, 10% fetal bovine serum), 50 mM, 100 mM, and 200 mM trehalose in the basic cryopreservation solution. , 100 mM, 200 mM sucrose (sucrose) was cryopreserved using the cryopreservation solution additionally included in the basic cryopreservation solution.
In the specific cryopreservation method, cells of 0.5 x 10 6 cells / ml concentration were added to each cryopreservation solution and stored in one freezing container in 1 ml. The freezing vessel was stored in a cryocontainer containing isopropanol (cryocontainer) and then stored at a temperature of about −1 ° C./min down to −80 ° C. (hereinafter referred to as a gentle freezing method) for one day. Afterwards, the freezing vessel was again transferred to -196 ℃ liquid nitrogen and stored for 30 days.
2. Cryopreservation of garden stem cells in tissue state
After extracting the testis in the same manner as in the above 1, the testis tissue was chopped into 3 × 3 × 3 mm, and then the basic cryopreservation solution (MEM-α containing 10% DMSO, 10% fetal calf serum), 50 mM, 100 Cryopreservation was carried out using cryopreservation solution containing 50 mM, 200 mM trehalose in addition to the basic cryopreservation solution, and cryopreservation solution containing 50 mM, 100 mM and 200 mM sucrose in addition to the basic cryopreservation solution. Cryopreservation was carried out in the same manner as described above.
3. Recovery rate of garden stem cells according to cryopreservation and donor test
After thawing the tissue or single cell group containing the freeze-preserved garden stem cells in 1, 2, and put in a serum-free culture medium containing growth factors in vitro cultured in a culture plate coated with the support cells, PGP 9.5 (pig garden) Stem cell specific markers) to perform immunocytochemistry to measure the number of stained cells, which are shown in FIGS. 1 and 2.
As shown in FIG. 1, when freezing was performed in a tissue state using a cryopreservation solution containing 200 mM trehalose, the recovery rate of garden stem cells after thawing and in vitro culture was 18.7 ± 0.6 X 10 The highest recovery was 4 / gram compared to the other freezing groups. In addition, when frozen in a single cell state using cryopreservation solution containing 200 mM trehalose, the recovery rate of garden stem cells after thawing and in vitro culture was 13.8 ± 1.0 X 10 4 / gram on average. The recovery rate was higher than that of, but the recovery rate was significantly lower than that of freezing in tissue state using cryopreservation solution containing 200 mM trehalose. This suggests that tissue state freezing is more advantageous in stem cell survival efficiency than cryopreservation of pig cell stem cells rather than single cell state freezing.
Example 2. Freeze and Thawed Comparison of Stem Cell Activity of Garden Stem Cells
The testicular cells of the basic cryopreservative test group of Example 1 and the cryopreservative test group to which 200 mM trehalose was added were transplanted into the receiving testes after freezing and thawing in the tissue state, and 1.5 months later, the garden stems. Cells were stained with red fluorescent staining reagent (PKH26) and the number of colonies formed (one colony formed by autologous division of one garden stem cell) was measured. The results are shown in FIGS. 3 and 4.
As shown in Figures 3 and 4, the number of garden stem cells in the basal cryopreservation experimental group and the cryopreservation experimental group to which 200 mM trehalose was added were 28 ± 2.2 / 10 5 and 79 ± 4.8 / 10 5 . This means that the addition of 200 mM of trehalose to freeze the testis into a tissue state increases the survival rate of stem cells compared to not adding trehalose.
Claims (7)
Culture the tissue in vitro, recovering a single cell group of pig garden stem cells; further comprising, cryopreservation method of pig garden stem cells.
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| US10172347B2 (en) | 2014-07-08 | 2019-01-08 | Jeong Chan Ra | Composition for improving stability of stem cells |
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