阅读说明：本技术 一种猪种种质资源的快速保存方法 (Method for rapidly preserving pig germplasm resources ) 是由 苗义良 张霞 刘鑫 余智盛 窦成利 徐天 于 2021-09-22 设计创作，主要内容包括：本发明公开了一种猪种种质资源的快速保存方法,属于组织工程技术领域。本发明公开的一种猪种种质资源的快速保存方法,包括一种冻存猪耳组织的冻存液,冻存液以含有胎牛血清的磷酸盐缓冲液为基础液,添加乙二醇和二甲基亚砜；还包括解冻液,解冻液以含有胎牛血清的磷酸盐缓冲液为基础液,添加海藻糖。采用本发明的冻存液对猪耳组织进行低温冻存,可有效实现猪耳组织长期低温保存的效果,配合使用解冻液解冻后可获得用于培养的猪成纤维细胞,达到保存猪种种质资源的目的。(The invention discloses a method for rapidly preserving a swine germplasm resource, belonging to the technical field of tissue engineering. The invention discloses a method for rapidly preserving pig germplasm resources, which comprises a frozen stock solution for freezing and storing pig ear tissues, wherein the frozen stock solution takes phosphate buffer solution containing fetal calf serum as a base solution, and is added with glycol and dimethyl sulfoxide; the anti-freezing solution is prepared by taking phosphate buffer solution containing fetal calf serum as base solution and adding trehalose. The cryopreservation liquid provided by the invention is used for cryopreservation of the pig ear tissues at low temperature, so that the effect of long-term cryopreservation of the pig ear tissues can be effectively realized, and pig fibroblasts for culture can be obtained after unfreezing by using the unfreezing liquid in a matching manner, so that the purpose of storing the germplasm resources of pig breeds is achieved.)

1. A method for rapidly preserving a boar germplasm resource is characterized by comprising the following specific steps:

(1) sterilizing pig ear tissues, and trimming the pig ear tissues into tissue blocks;

(2) putting the tissue block in the step (1) into a pretreatment solution for treatment, and then putting the tissue block into a frozen stock solution for treatment;

the pretreatment solution takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 7.5% of ethylene glycol and 7.5% of dimethyl sulfoxide by volume fraction are added;

the freezing stock solution takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 15% of ethylene glycol, 15% of dimethyl sulfoxide and 0.1M of trehalose by volume fraction are added;

(3) and (3) placing the tissue blocks processed in the step (2) in a freezing storage tube, and adding liquid nitrogen into the freezing storage tube.

2. The method of claim 1, wherein the tissue blocks of step (1) have a size of 5mm x 1 mm.

3. The method for rapidly preserving a porcine germ and germ resource of claim 1, wherein in the step (2), the tissue block of the step (1) is treated in a pretreatment solution for 5min and then in a frozen stock solution for 5 min.

4. The method for rapidly preserving a porcine germplasm resource according to claim 1, further comprising the step (4): placing the frozen and preserved pig ear tissue into the thawing solution I for treatment for 5min, then placing the frozen and preserved pig ear tissue into the thawing solution II for treatment for 5min, and obtaining viable pig ear fibroblasts through tissue mass primary culture;

the thawing solution I takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 0.1M trehalose is added; the thawing solution II takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 0.05M trehalose is added.

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a method for rapidly storing pig germplasm resources.

Background

The germplasm resources of local pigs in China are very rich, but the African swine fever seriously threatens the germplasm resource preservation of local pigs in China as a widely spread high lethal epidemic disease. At present, the germ plasm resources of the pig breeds can be preserved only by freezing and preserving somatic cells and semen, but both the methods have the defects that the preservation of the somatic cells needs to convey fresh tissues to a cell chamber for cell separation culture and then freezing and preserving, the cost is high, the operation on the spot of a pig farm cannot be carried out, and the freezing and the preservation of the semen are suitable for freezing and preserving only part of the semen of the pig due to large individual difference of the pig. How to establish a novel preservation method to effectively protect the germplasm resources of local and newly-cultivated pig breeds becomes the key for coping with major emergency of African swine fever in China at present.

Therefore, the technical personnel in the field need to solve the problem of providing a method for rapidly storing the germplasm resources of the swine species in China under the epidemic situation of the African swine fever.

Disclosure of Invention

In view of the above, the present invention provides a method for rapidly preserving a porcine germ plasm resource, wherein a frozen stock solution is used to freeze and preserve porcine ear tissues, trehalose is added to the frozen stock solution to effectively freeze and preserve the porcine ear tissues, and viable porcine fibroblasts can be obtained by primary culture after thawing.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for rapidly preserving a boar germplasm resource comprises the following specific steps:

(1) freezing and storing the pig ear tissues by using a liquid nitrogen quick freezing method, sterilizing the pig ear tissues on site in a pig farm, and trimming the pig ear tissues into tissue blocks;

(2) putting the tissue block in the step (1) into a pretreatment solution for treatment, and then putting the tissue block into a frozen stock solution for treatment;

the pretreatment solution takes Phosphate Buffered Saline (PBS) containing 20% Fetal Bovine Serum (FBS) by volume fraction as a base solution, and 7.5% of glycol and 7.5% of dimethyl sulfoxide by volume fraction are added;

the freezing stock solution takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 15% of ethylene glycol, 15% of dimethyl sulfoxide and 0.1M of trehalose by volume fraction are added;

(3) and (3) placing the tissue blocks processed in the step (2) in a freezing storage tube, and adding liquid nitrogen into the freezing storage tube.

Further, the tissue block size in step (1) is 5mm × 1mm × 1 mm.

Further, the tissue block in the step (1) is placed in a pretreatment solution for treatment for 5min, and then is placed in a freezing storage solution for treatment for 5min in the step (2).

Further, the method for rapidly preserving the pig germplasm resources further comprises the following steps (4): placing the frozen and preserved pig ear tissue into the thawing solution I for treatment for 5min, then placing the frozen and preserved pig ear tissue into the thawing solution II for treatment for 5min, and obtaining viable pig ear fibroblasts through tissue mass primary culture;

the thawing solution I takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 0.1M trehalose is added; the thawing solution II takes phosphate buffer solution containing 20% fetal calf serum by volume fraction as base solution, and 0.05M trehalose is added.

Ethylene Glycol (EG) and dimethyl sulfoxide (DMSO) are osmotic cryoprotectants, have low molecular weights, and can permeate into the interior of cells through the lipid bilayer of the cell membrane; the protection mechanism is that the frozen cell suspension permeates into cells before being completely solidified, generates a certain molar concentration inside and outside the cells, and reduces the concentration of electrolyte in unfrozen solution inside and outside the cells, thereby protecting the cells from being damaged by high-concentration electrolyte.

Trehalose is an impermeable cryoprotectant, has a large molecular weight, cannot permeate into cells, can promote the formation of a solution glassy state, and can improve the cell viability in the cryopreservation process of cell suspensions and whole organs. In addition, studies have demonstrated that trehalose has the ability to scavenge free radicals.

According to the technical scheme, compared with the prior art, the invention discloses the method for rapidly storing the pig germplasm resources, the pig ear tissue cryopreservation liquid can effectively perform low-temperature storage on the pig ear tissue, and cultured fibroblasts can be successfully separated after the tissue is thawed; the method has simple steps, and pig ear tissues can be directly frozen and preserved by using the method in a pig farm.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the cell clone number at the 7 th day of culture after thawing frozen pig ear tissues with different freezing media according to the present invention; *: p < 0.05;

FIG. 2 is a graph showing the cell clone number at the 7 th day of culture after thawing the minced pig ear tissue of the present invention;

FIG. 3 is a drawing showing the primary fibroblasts cultured after thawing frozen and stored porcine ear tissues for different periods of time according to the present invention;

wherein ab c is unfrozen after being frozen and stored for one day; a migration of cells from the tissue mass; b, culturing on day 7, and carrying out cell growth after passage c;

d e f is unfreezing after being frozen for one week; d cells migrate out of the tissue mass; e, culturing on the 7 th day, and f, carrying out cell growth after passage;

g h i is unfreezing after being frozen for one month; g cells migrate out of the tissue mass; h, culturing for 7 days; i cell growth after passage;

panel j k l is thawed after three months of cryopreservation; j cells migrate out of the tissue mass; k culture for day 7; l cell growth after passage;

all the pictures are taken under a 100-fold microscope.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

PBS is HyClone, FBS is VISTECH, trehalose is Michelin, DMEM is Seimer Feishel technology, EG and DMSO are Sigma, and pancreatin and streptomycin are Gibco respectively.

Example 1

(1) Preparing a treating fluid

A. Pretreatment liquid: PBS 7.5% EG + 7.5% DMSO + 20% FBS;

B. freezing and storing liquid: PBS 15% EG + 15% DMSO + 20% FBS +0.1M trehalose;

C. thawing solution I: adding 20% FBS and 0.1M trehalose into PBS;

D. and (3) thawing solution II: adding 20% FBS and 0.05M trehalose into PBS;

E. cell culture solution: DMEM was supplemented with 15% FBS and 100IU/mL streptomycin.

The above percentages are volume fractions, and after preparation, the mixture is filtered through a 0.22 μm filter into a sterile 50mL centrifuge tube, 10mL liquid per tube, stored in a refrigerator at 4 ℃ and allowed to stand at room temperature for 15min before use.

(2) Cryopreservation method of pig ear tissues

The pig ear tissue is from the pig farm of the pig determination center of university of agriculture in Huazhong. Sterilizing in vitro pig ear tissue with iodine tincture, clamping one ear edge tissue with two sterilized hemostatic forceps, cutting off fan-shaped pig ear tissue, soaking in 75% alcohol for sterilization for 1min, and washing with 500IU/mL streptomycin-containing PBS for several times. Then, the pig ear tissue is trimmed into tissue blocks of 5mm × 1mm × 1mm by using an ophthalmic scissors, the tissue blocks are soaked in a pretreatment solution for 5min at room temperature, then soaked in a freezing solution for 5min at room temperature, the treated tissue blocks are transferred to a freezing tube, liquid nitrogen is added into the freezing tube, a cover is screwed, and the tissue blocks are put into a liquid nitrogen tank for storage.

(3) Thawing method for frozen pig ear tissue

Taking out the frozen tube from the liquid nitrogen tank, opening the cover, standing for 20-30s, transferring the frozen tissue into thawing solution I for soaking at room temperature for 5min, transferring into thawing solution II for soaking at room temperature for 5min, and transferring into freezing solution IITransferring to PBS with 20% FBS, soaking at room temperature for 5min, and washing with PBS. Then placing the pig ear tissue block into 1.5mL EP tube, cutting with ophthalmic scissors, adding 0.05% pancreatin, digesting at 38.5 deg.C for 30min, adding cell culture solution containing serum to stop digestion, centrifuging at 1200 rpm for 5min, discarding supernatant, adding cell culture solution, inoculating into 6-well plate, inoculating at 38.5 deg.C and 5% CO₂And culturing in an incubator with saturated humidity. The culture plate is not shaken as much as possible within 3-5 days. And replacing the solution every other day after the cells adhere to the wall.

Test examples

(1) Freezing pig ear tissue with different freezing medium, thawing, and culturing on the seventh day

And (3) putting the pig ear tissue blocks into different freezing solutions for treatment, freezing for one day and then thawing. The remaining operating steps and parameters were in accordance with example 1. The frozen stock solutions were: PBS was added 15% EG + 15% DMSO + 20% FBS +0.1M trehalose (0.1M trehalose, experimental group); PBS was added 15% EG + 15% DMSO + 20% FBS +0.5M trehalose (0.5M trehalose, control 1); PBS was added 15% EG + 15% DMSO + 20% FBS +0.5M sucrose (0.5M sucrose, control 2); to PBS was added 15% EG + 15% DMSO + 20% FBS +0.5M sucrose +0.1M trehalose (0.5M trehalose, control 3).

The number of cell clones (number) at the 7 th day of culture after thawing the pig ear tissue frozen with different freezing media was counted, and the results are shown in FIG. 1. The results in fig. 1 show that after thawing, at day 7 of culture, the number of cell clones in the 0.1M trehalose-supplemented group was significantly higher than that in the other three groups, and the difference between the other three groups was not significant. It was demonstrated that the 0.1M trehalose group had better cryoprotective effect than the 0.5M sucrose group and the 0.5M trehalose group, and the 0.5M sucrose and 0.1M trehalose group.

(2) Freezing and thawing the minced pig ear tissue, culturing on day 7

After the tissue is cut into pieces (<1mm³) Freezing (cutting and freezing) and cutting the tissue into strips of 5mm multiplied by 1mm (strip freezing), thawing the frozen strips one day, and culturing the frozen strips for 7 days. The remaining operating steps and parameters were in accordance with example 1.

The number (number) of cell clones at the 7 th day of culture after freezing and thawing the minced pig ear tissue was counted, and the results are shown in FIG. 2. The results in FIG. 2 show that there were an average of 49 clones in the cut-to-fragment group and an average of 58 clones in the cut-to-stripe group, with no significant difference between the two groups. The method shows that the cell clone generation rate after unfreezing is not influenced after the frozen stock of the cut strip group, the method is easier to operate in a pig farm, and the freezing procedure is simplified.

Under the condition of spreading the African swine fever epidemic situation, all pig species in a pig farm can be frozen and preserved by using the method, and are marked with numbers. After sampling and detecting whether all the pigs have the African swine fever, the ear tissues which are positive in the African swine fever detection can be abandoned, and the healthy pig species germplasm resources are further subjected to targeted preservation.

(3) Primary fibroblast cultured after thawing frozen pig ear tissues stored for different time

The frozen pig ear tissue blocks after one day, one week, one month and three months are thawed respectively, and primary culture operation is carried out. The specific operating steps and parameters were in accordance with example 1. As a result, as shown in FIG. 3, it was found that the frozen pig ear tissue blocks frozen for one day, one week, one month and three months were able to migrate primary fibroblasts which were highly proliferative and could be passaged further, indicating that the pig ear tissue could be preserved for a long period of time by the method of the present invention.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.