CN106035315A - Cryopreservation solution of mammary tissue of dairy cow for primary culture of mammary epithelial cells of dairy cow, and application of cryopreservation solution in cryopreservation method for mammary tissue of dairy cow - Google Patents

Abstract

The invention discloses a dairy cow mammary gland tissue cryopreservation solution for primary culture of dairy cow mammary gland epithelial cells, which is composed of fetal bovine serum FBS, DMEM/F12 medium, dimethyl sulfoxide DMSO and HEPES buffer compounded with pyruvic acid The sodium stock solution is prepared according to the volume ratio of 20:12:5:3; and the application of the cryopreservation solution in the cryopreservation method of dairy cow mammary gland tissue is provided. The beneficial effect of the present invention is: a dairy cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells provided by the present invention and its application in the dairy cow mammary gland tissue cryopreservation method can reduce the trouble of frequent material collection, reduce The cost of material collection; the operation process of cutting, cleaning, freezing and resuscitation of tissue blocks is greatly simplified, which is conducive to maintaining the biological activity of tissue blocks and can be effectively preserved for 6 to 8 months.

Description

Dairy cow mammary gland tissue cryopreservation fluid for primary culture of dairy cow mammary gland epithelial cells and its Application in breast tissue cryopreservation method

technical field

The invention relates to the technical field of tissue and cell engineering, in particular to a dairy cow mammary gland tissue cryopreservation solution for primary culture of dairy cow mammary gland epithelial cells and its application in a mammary gland tissue cryopreservation method.

Background technique

Tissue cryopreservation technology has been widely used in the fields of genetic breeding and biomedicine. It can not only preserve scarce tissues, but also reduce the trouble of repeated sampling for some research. However, different uses determine different cryopreservation methods for different tissues. After the tissue samples are isolated, they should be stored at 4°C in time, or they should be collected, fixed or frozen within 30 minutes or within 30 minutes according to different experimental purposes. Wang Xuan (2013) studied the effects of different tissue cryopreservation systems on the expansion of periodontal ligament stem cells in vitro.

Some people use - 70°C absolute ethanol cryopreservation method for frozen tissue samples, that is, put absolute ethanol in the - 70°C refrigerator for pre-cooling, put the tissue into a plastic bag after taking the tissue, seal it with a sealer, and put it in cold absolute ethanol Take it out after 3-4 minutes of medium-quick freezing, absorb the absolute ethanol on the surface of the plastic bag with filter paper, and store it in a low-temperature refrigerator at -70°C for long-term storage. But dairy cow mammary gland tissue is the main material for culturing mammary gland epithelial cells, but the acquisition cost of dairy cow mammary gland tissue with good tissue quality and no pollution is high. In order to carry out the primary culture of dairy cow mammary gland epithelial cells effectively, the cryopreservation technology of dairy cow mammary gland tissue blocks was explored, and a method for separating and culturing dairy cow mammary gland epithelial cells from cryopreserved mammary gland tissue was successfully established.

Contents of the invention

The purpose of the present invention is to address the defects in the above-mentioned prior art, to provide a dairy cow mammary gland tissue cryopreservation solution for primary culture of dairy cow mammary gland epithelial cells and its application in a dairy cow mammary gland tissue cryopreservation method.

In order to achieve the above object, the technical scheme provided by the present invention is: a dairy cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells, which is composed of fetal bovine serum FBS, DMEM/F12 medium, dimethyl sulfoxide The stock solution of DMSO and HEPES buffer mixed with sodium pyruvate was prepared at a volume ratio of 20:12:5:3.

Further, the above-mentioned dairy cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells, the preparation method of the storage solution of the HEPES buffer compounded with sodium pyruvate is: accurately weigh 0.9532g of HEPES, acetone Dissolve 0.1100g of sodium bicarbonate in 30mL of D-Hank's basal medium, fully dissolve and mix evenly, set the volume to 50mL, filter through a 0.22 micron microporous membrane, and store in a sealed place at 4°C in the dark for future use. Preserved for a long time, ready to use immediately; wherein, the content of HEPES buffer solution is 0.8 mol/L, and the content of sodium pyruvate is 0.02 mol/L.

Further, the above-mentioned dairy cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells, the DMEM/F12 medium and dimethyl sulfoxide DMSO need to be sterilized by filtration through a 0.22 μm microporous membrane , ready-to-use.

The second object of the present invention is to provide the above-mentioned application of the milk cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells in the dairy cow mammary gland tissue cryopreservation method, which is to cut the mammary gland middle tissue into a volume of 4 ～5mm ³ size tissue block, put into 5mL cryopreservation tube, put 5～6 pieces of tissue block into each cryopreservation tube, then add the cryopreservation liquid described in claim 1, wrap the cryopreservation tube with 20 layers of gauze , frozen at -80°C for 24 h in one step, and then transferred to liquid nitrogen for cryopreservation.

The beneficial effect of the present invention is: a dairy cow mammary gland tissue cryopreservation solution for the primary culture of dairy cow mammary gland epithelial cells provided by the present invention and its application in the dairy cow mammary gland tissue cryopreservation method can reduce the trouble of frequent material collection, Reduce the cost of obtaining materials; greatly simplify the operation process of cutting, cleaning, freezing and resuscitating tissue blocks, which is conducive to maintaining the biological activity of tissue blocks and can be effectively preserved for 6 to 8 months.

Description of drawings

Figure 1 is a comparison of the morphological observations of frozen breast tissue and fresh breast tissue.

Among them, A is the fibroblast that crawled out on the fifth day of frozen tissue culture; B is the fibroblast that crawled out on the fifth day of fresh tissue culture; C is the cobblestone-like epithelial cell that climbed out on the fifth day of frozen tissue culture; D is the cobblestone-like epithelial cells crawled out on the 5th day of fresh tissue culture.

Figure 2 is the immunofluorescence identification of keratin 18 in mammary epithelial cells.

A is fluorescently labeled mammary epithelial cytokeratin-18; B is the result of nuclear staining; C is the merged image of A and B.

detailed description

Example 1:

1. Preparation method of freezing solution:

Freezing solution A: FBS: DMSO: DMEM/F12 is prepared at a volume ratio of 7:2:1, and DMSO and DMEM/F12 are sterilized by filtration through a 0.22 μm microporous membrane, and are ready for use immediately.

Freeze-preservation solution B: FBS: DMEM/F12: DMSO: HEPES buffer mixed with sodium pyruvate storage solution was prepared at a volume ratio of 20:12:5:3. The preparation method of the stock solution of HEPES buffer mixed with sodium pyruvate is as follows: accurately weigh 0.9532g of HEPES and 0.1100g of sodium pyruvate, dissolve them in 30mL of basal medium (D-Hank's), fully dissolve and mix evenly, and constant volume to 50 mL, filtered through a 0.22-micron microporous membrane, and sealed and stored at 4°C in the dark for future use. That is, in the final HEPES buffer solution compounded with sodium pyruvate stock solution, the molar concentration of HEPES is 0.8 mol/L, and the molar concentration of sodium pyruvate is 0.02 mol/L. In addition, DMEM/F12 and DMSO are sterilized by filtration through a 0.22 μm microporous membrane, and the cryopreservation solution is prepared and used immediately.

2. Experimental animals:

The experimental animals were 24-month-old healthy Holstein cows with no lactation history.

3. Collection of whole breast tissue:

After the slaughtered cows were slaughtered, the surface of the udder was fully rinsed with water, sterilized with 75% alcohol, and the whole udder was dissected with a high-pressure sterilized scalpel, placed in a sampling box treated with 75% alcohol, and brought back to the laboratory within 1 hour.

4. Treatment of breast tissue for cell culture:

Then use 75% alcohol to disinfect the surface of the tissue, avoid adipose tissue, connective tissue and blood vessels, cut the mammary gland parenchyma inside the tissue aseptically, and take the middle part of the tissue, which is characterized by yellow, dense tissue and visible small ducts . The excised breast parenchyma tissue was soaked in 75% alcohol for 3 min and then transferred into the intercellular space.

5. Cryopreservation of dairy cow mammary gland tissue:

Soak the tissue with D-Hank's solution 4 times to remove the alcohol, remove the external white tissue, cut the mammary gland parenchyma into small pieces of about 4-5 mm ³ , put 5-6 pieces of tissue into each 5 mL cryopreservation tube, and separate Two methods of freezing solution A and freezing solution B were used for freezing. The tubes were wrapped with 20 layers of gauze, frozen in one step at -80°C for 24 h, and then transferred to liquid nitrogen for storage.

6. Breast tissue recovery and cell culture:

Eight months later, the cryopreserved tubes containing mammary gland tissue were quickly thawed in a 37°C water bath, and the tissue block was fully washed with D-Hank’s solution, and the dairy cow mammary gland epithelial cells were isolated and cultured by the tissue block planting method. When inoculating tissue blocks, each group of 30 pieces was used as a group, and each group was repeated 3 times. Continuous observation was carried out for 10 days. The number of tissue blocks with cells crawling out was counted, and the average value was calculated. The survival rate of tissue blocks is shown in Table 1 (unit : block/day), compare the viability of the cells crawling out of the dairy cow mammary gland tissue preserved in cryopreservation solution A and B. As a result, the survival rate of the tissue blocks preserved by cryopreservation solution A for 8 months was 50%, and that of the tissue blocks preserved by cryopreservation solution B for 8 months was 61.1%. The preservation effect is better. Morphological observation results showed that, as shown in Figure 1, cells crawled out of the frozen tissue on the 4th day, and some frozen tissue blocks could also directly crawl out of cobblestone-like dairy cow mammary epithelial cells, and it was the same as that of fresh tissue. There was no significant difference in cell morphology. As shown in FIG. 2 , the isolated and cultured cells expressed cytokeratin 18 through fluorescence immunoassay detection, indicating that the obtained cells were cow mammary gland epithelial cells.

Table 1

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Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (4)

1. the cow mammary gland tissue freezing solution for cow mammary gland epithelial cells original cuiture, it is characterised in that be by tire Ox blood serum FBS, DMEM/F12 culture medium, dimethyl sulfoxide DMSO and HEPES buffer compound the stock solution of Sodium Pyruvate by body The long-pending proportions than 20:12:5:3.

A kind of cow mammary gland tissue freezing for cow mammary gland epithelial cells original cuiture the most according to claim 1 Liquid, it is characterised in that the compound method of the stock solution that described HEPES buffer compounds Sodium Pyruvate is: accurately weigh HEPES 0.9532g, Sodium Pyruvate 0.1100g, be dissolved in 30mL D-Hank's basal medium, fully dissolves and mix homogeneously, fixed Holding to 50mL, after 0.22 micrometer Millipore membrane filtration, 4 DEG C of sealings of lucifuge save backup, and this stock solution is now with the current.

A kind of cow mammary gland tissue freezing for cow mammary gland epithelial cells original cuiture the most according to claim 1 Liquid, it is characterised in that described DMEM/F12 culture medium and dimethyl sulfoxide DMSO are both needed to remove through 0.22 μm filtering with microporous membrane Bacterium, now with the current.

A kind of cow mammary gland tissue freezing solution for cow mammary gland epithelial cells original cuiture the most according to claim 1 Application in cow mammary gland tissue freezing method, it is characterised in that be that tissue shear in the middle part of mammary gland becomes volume is 4～5mm³Greatly Little piece of tissue, puts into 5mL cryopreservation tube, puts into 5～6 block organization's blocks, add freezing described in claim 1 in every cryopreservation tube Liquid storage, with 20 layers of gauze parcel cryopreservation tube, is transferred in liquid nitrogen frozen after-80 DEG C of one-step freezing 24 h.