CN114196620B

CN114196620B - In-vitro culture method for breast tissue of sow

Info

Publication number: CN114196620B
Application number: CN202210139330.3A
Authority: CN
Inventors: 肖昊; 曹舒婷; 王丽; 蒋宗勇; 胡胜兰; 高开国
Original assignee: Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Priority date: 2022-02-14
Filing date: 2022-02-14
Publication date: 2022-05-20
Anticipated expiration: 2042-02-14
Also published as: CN114196620A

Abstract

The invention belongs to the field of biotechnology, and discloses a method for culturing sow mammary tissue in vitro, which comprises the following steps: (1) sampling and disinfecting the mammary tissue of the sow; (2) carrying out slicing treatment on the mammary tissue of the sow; (3) preparing a solidified glue, placing a sow mammary tissue slice between an upper layer of solidified glue and a lower layer of solidified glue, placing the solidified glue in an upper chamber of a Transwell plate, and placing the solidified glue in an incubator for culturing for 30 minutes to fix the solidified glue; (4) the lower chamber of the Transwell was filled with the medium, and the Transwell plate was placed in a 37-degree incubator for culture. The invention has the characteristics of high in vivo environment simulation degree, low cost and high cell growth speed.

Description

In-vitro culture method for breast tissue of sow

Technical Field

The invention relates to the field of biotechnology, in particular to an in-vitro culture method of sow mammary tissue.

Background

The pig has undeveloped sweat glands, thicker subcutaneous fat and slower heat dissipation, and the temperature is up to more than 30 ℃ in summer for 7-9 months in China, particularly in southern areas, so that the lactating sows are easy to generate heat stress. When the environmental temperature is higher than the temperature of a comfortable area of the lactating sow, the growth performance of the lactating sow and the piglet is affected, so that the feed intake of the sow is reduced, the ingested nutrient is reduced, the lactation amount is reduced, the weight loss in the lactation period is serious, the back fat and the protein loss are increased, and the like, wherein the change of the lactation performance directly affects the growth of the piglet. The lactation performance of the sow has important influence on the birth weight and growth and development of piglets, the heat stress factor easily damages mammary gland tissues, the mammary gland is in a state of vigorous metabolism for a long time, the mammary gland also generates oxidative stress, the function of the mammary gland is damaged, and the lactation performance of a mother body is reduced. Therefore, the construction of the in-vitro culture model of the mammary gland of the sow has important significance for researching the mammary gland injury mechanism of the sow under the stress state, deeply exploring the nutrient regulation and control mechanism to promote the growth performance of the sow and improve the production benefit.

Reference may be made to CN 201811122209. X and CN 202010038062.7 for studies in this respect.

CN 201811122209. X discloses a method for 3D pig mammary gland epithelial cells, and the invention provides an Advanced DMEM/F12 complete culture medium which is combined with a 3D culture method to culture the pig mammary gland cells. According to the invention, EMC protein is used as a growth support, so that cells can be differentiated to generate a certain three-dimensional tissue specific structure, compared with the traditional 2D culture method, the expression quantity of each intracellular milk is obviously improved, but the growth speed of the cells cultured by the method provided by the invention is slow.

CN 202010038062.7 discloses a three-dimensional continuous culture method for porcine mammary epithelial cells, which provides a three-dimensional continuous culture method, combines with a three-dimensional culture medium, can fully and truly simulate in vivo growth modes, and compared with a monolayer cell culture system, the three-dimensional cell culture system can more accurately reflect the actual microenvironment of cells in tissues. However, the three-dimensional cell culture medium used in the invention is a special culture medium prepared according to a single porcine mammary gland epithelial cell, and has high price and overlarge use cost.

Disclosure of Invention

The invention aims to provide an in vitro culture method of a mammary tissue of a sow, which highly simulates an in vivo environment and reduces the preparation cost of a culture medium under the condition of not influencing the growth speed of cells.

Without specific explanation of the present invention: nM for nanomole/liter,. mu.M for micromole/liter,. mu.M for millimole/liter, M for mol/liter;

in order to achieve the purpose, the invention adopts the technical scheme that:

an in vitro culture technology of sow mammary tissue comprises the following steps:

slicing the mammary tissue of the sow, placing the sliced mammary tissue between an upper solidified glue and a lower solidified glue, and contacting the lower solidified glue with a culture medium; changing the culture medium every 2-4 days or changing the culture medium when the culture medium turns yellow;

preferably, the components of the culture medium are as follows:

DMEM-F12：450～550ml；

DMEM basic：350～450 ml；

fetal bovine serum: 80-120 ml;

penicillin-streptomycin: 10-20 ml;

insulin: 8-12 ug/ml;

500 Xgentamicin-amphotericin B mixed solution: 0.8-1.2 times of the volume;

cholera toxin：0.05～0.15 nM；

EGF：5～15ng/ml ；

hydrocortisone: 0.7-13 ug/ml;

ROCK inhibitor Y27632: 6 to 14 μ M.

It should be noted that: the 500X gentamicin-amphotericin B mixed solution: the meaning of 0.8-1.2 times volume is as follows: 0.8-1.2 ml of 500 Xgentamicin-amphotericin B mixed solution is added to each 500ml of culture medium.

Preferably, the upper setting adhesive and the lower setting adhesive are both composed of the following components:

collagen I, 10 times of volume of F12 culture medium and buffer solution;

the weight ratio of the collagen I, 10 times of volume of F12 culture medium and buffer solution is 6:1: 1-8: 1: 1.

Preferably, the buffer is prepared from 100mL of sterile water, 0.05M NaOH, 200mm HEPES, 2.2 g NaHCO₃The composition of the mixture.

Preferably, the volume ratio of the upper solidified glue to the lower solidified glue is 1: 1-2.

Preferably, the specific method for the sow mammary tissue section comprises the following steps:

cutting the mammary tissue without connective tissue and fat into small pieces, inserting the mammary tissue into liquid agar of 6% (volume fraction), and placing into refrigerator for solidification;

cutting the agar block containing the mammary tissue into cubes after the agar is solidified;

cutting the cube into slices with thickness of 250-350 μm by using a slicer, and adding PBS (sterile PBS containing KH)₂PO₄，NaCl，Na₂HPO₄pH 7.2-7.4); and stripping off the agar under a stereoscopic microscope to obtain the sliced mammary tissue.

Preferably, the PBS solution contains 3 times the volume of penicillin-streptomycin and 1 time the volume of 500X gentamicin-amphotericin B mixed solution.

Preferably, the cube is 3 x 3cm in size.

Compared with the prior art, the invention has the beneficial effects that:

compared with the prior art, the in vitro culture technology of the sow mammary tissue provided by the invention simulates an in vivo environment better, and can reduce the cost of a culture medium under the condition of not influencing the growth state of cells.

Drawings

Reference numerals 1 to 4 in FIG. 1 are photographs of the cultures of example 1 and comparative example 1 at different times.

FIG. 2 is a photograph of tissue culture after continuous culture for 2 days in comparative example 2.

Reference numerals 1 to 4 in fig. 3 are graphs of HE staining of breast tissue at different magnifications provided in example 1, and the magnifications are 5, 10, 20 and 40 times in sequence.

Reference numerals 1 to 3 in fig. 4 are Tunel staining patterns of mammary tissue at different magnifications provided for example 1, and the magnifications are 10, 20 and 40 times in this order.

FIG. 5 is a photograph of the tissue culture of comparative example 3.

FIG. 6 is a photograph of the tissue culture of comparative example 4.

FIG. 7 is a photograph of the tissue culture of comparative example 5.

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 any inventive step, are within the scope of the present invention.

Description of the reagents

DMEM basic: domestic preparation of Gibco (thermo Fisher scientific)

DMEM F12: domestic preparation of Gibco (thermo Fisher scientific)

Insulin: cat No. I9278, Sigma

Gentamicin/Ampotericin B (500X): item No. HY-B0221, MCE; Gentamicin/Amphotericin B (500X) is named in Chinese: 500 Xgentamicin-amphotericin B mixed solution;

cholera toxin: cargo number HY-129040A, MCE Inc

Epidermal growth factor EGF: commodity number PHG0311, Gibco

Hydrocortisone: cargo number HY-N0583, MCE Co

Y27632 (ROCK inhibitor): cargo number HY-10071, MCE Co

Advanced DMEM/F12: domestic preparation of Gibco (thermo Fisher scientific)

Fetal bovine serum: gibco (thermo Fisher scientific)

Penicillin-streptomycin: gibco (thermo Fisher scientific); (the following examples and comparative examples, where not otherwise specified, "double antibody" refers to penicillin-streptomycin in the present context)

Collagen I: cat # C0130, Sigma

HEPES (high efficiency particulate air): cat No. 15630080, Gibco (thermo Fisher scientific)

NaHCO 3: cat No. 25080094, Gibco (thermo Fisher scientific)

Transwell plate: cat # 3450/3460 Corning

B-27: cat No. 17504044, Gibco (thermo Fisher scientific)

Glutamine (b): cat No. A2916801, Gibco (thermo Fisher scientific)

Mycoplasma remover Plasmocin: punuisan game

N-acetylcysteine: cat # A105422, alatin

0.20 μ M filter: millipore

P38 inhibitor SB 202190: cat No. HY-10295, MCE Inc

Matrigel: cat number 354234, BD Co

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Tissue extraction method

Collecting mammary tissues of sows: after a sow over 90 days old is slaughtered (cleaned), a sampling part is wiped by using 75 percent (volume fraction) of alcohol, the whole mammary gland of a second pair of nipples is cut off, sterile PBS is used for repeatedly washing for 5 times, the two nipples are put into a prepared large beaker with high temperature sterilization, the two nipples are soaked by sterile PBS (containing 3 percent (volume fraction)) of double antibiotics, kraft paper is sealed, the sealed two nipples are put into a foam box with two or three ice bags, and the two nipples are quickly taken back to a laboratory; putting the whole mammary gland into a tray, cutting a 3X 3cm block by taking a nipple as a center, discarding the rest mammary glands, cutting along the nipple, pinching the nipple to touch down to feel a duct, cutting a mammary duct 2-3cm below the nipple into small breast blocks of 1 cubic centimeter, removing obvious connective tissues and fat tissues, cleaning for 3 times by using a solution containing 3% (volume fraction) of double antibody until a culture medium supernatant is clear and free of impurities, and placing the mammary duct into a culture dish containing 1 volume of PBS (phosphate buffer solution) containing 500X gentamicin-amphotericin B mixed solution.

Example 1

(1) Preparing a section: taking out the scissors after high-temperature sterilization, cutting the mammary gland tissue into small pieces, longitudinally inserting the mammary gland tissue into 6% (volume fraction) agar (about 40 ℃, not scalding hands), and putting into a refrigerator at 4 ℃ to solidify the mammary gland tissue; after the agar had solidified, the agar blocks containing mammary tissue were cut into small cubes and adhered to the stage with 520 f strong glue.

(2) Preparing a solidified glue: collagen I, 10 volumes of F12 medium and buffer were mixed at a ratio of 8:1:1 to make a recombinant collagen gel, wherein the buffer was composed of a mixture of 100mL sterile water, 0.05M NaOH, 200mm HEPES, 2.2 g NaHCO 3. If peracid can adjust the pH value to 7.0. After mixing, the mixture was placed on a chamber on a 12-well Transwell plate in an amount of 50 to 100. mu.l per well, and the mixture was allowed to stand in an incubator at 37 ℃ for 15 minutes to coagulate the mixture.

(3) Preparing a slice: cutting into slices of 250-350 μm by a vibrating microtome, and adding PBS (3 times volume of double antibody and 1 time volume of 500 Xgentamycin-amphotericin B mixed solution); the agar is stripped off under a stereomicroscope, the prepared tissue slices are placed on the solidified gel, and 30-50 mu l of the solidified gel is placed on the tissue slices for culture at 37 ℃. 500ul of medium was added below chamber.

Preparing a culture medium: weighing the following substances and mixing: DMEM-F12: 500ml, DMEM basic: 373ml, fetal bovine serum: 100ml, penicillin-streptomycin: 15ml, insulin: 10ug/ml, 500 Xgentamicin-amphotericin B mixed solution: 1-fold volume, cholera toxin: 1 nM, EGF: 10ng/ml, hydrocortisone: 10ug/ml, ROCK inhibitor Y27632: 10 μ M.

Comparing the first day after preparation (reference numeral 1 in FIG. 1) with the fourteenth day after preparation (reference numeral 3 in FIG. 1), it was found that the medium turned yellow (basis for judging the growth state of the cells) one to two days after preparation for fourteen days, and the growth rate of the cells was comparable to that of comparative example 1.

In FIG. 4, reference numerals 1 to 3 indicate the Tunel staining results of the breast tissue on different scales, and in this figure, there are no red areas indicating that the tissue has grown well.

Example 2

The addition amounts of the components of the culture medium of example 1 were changed as follows:

DMEM-F12: 450ml, DMEM basic: 350 ml, fetal bovine serum: 80ml, penicillin-streptomycin: 10ml, insulin: 8ug/ml, 500 Xgentamicin-amphotericin B mixed solution: 0.8 volumes, cholera toxin: 0.05 nM, EGF: 5ng/ml, hydrocortisone: 0.7ug/ml, ROCK inhibitor Y27632: 6 μ M.

The growth condition of the porcine mammary gland epithelial tissue cells cultured by using the culture medium is not greatly different from that of the porcine mammary gland epithelial tissue cells cultured by using the culture medium in the example 1, and the growth speed is slightly slow.

The cost of example 1 and example 2 is similar.

Example 3

DMEM-F12: 550ml, DMEM basic: 450ml, fetal bovine serum: 120ml, penicillin-streptomycin: 20ml, insulin: 12ug/ml, 500X gentamicin-amphotericin B mixed solution: 1.2 volumes, cholera toxin: 0.15 nM, EGF: 15ng/ml, hydrocortisone: 1.3ug/ml, ROCK inhibitor Y27632: 14 μ M.

The growth condition of the porcine mammary gland epithelial tissue cells cultured by the culture medium of the comparative example is slightly worse than that of the porcine mammary gland epithelial tissue cells cultured by the culture medium of the comparative example 1, and the growth speed is equivalent.

The cost of example 1 and example 3 is similar.

Comparative example 1

(2) Preparing a solidified glue: collagen I, 10 times volume of F12 culture medium and buffer solution are mixed according to the mass ratio of 8:1:1 to prepare recombinant collagen gel, wherein the buffer solution is composed of 100mL of sterile water, 0.05M NaOH, 200mm HEPES and 2.2 g of NaHCO 3. If peracid can adjust the pH value to 7.0. After mixing, the mixture was placed on a chamber on a 12-well Transwell plate in an amount of 50 to 100. mu.l per well, and the mixture was allowed to stand in an incubator at 37 ℃ for 15 minutes to coagulate the mixture.

(3) Preparing a slice: cutting into slices of 250-350 μm by a vibrating microtome, and adding PBS (3 times volume of double antibody and 1 time volume of 500 Xgentamycin-amphotericin B mixed solution); stripping off agar under a stereoscopic microscope, placing the prepared tissue section on the coagulating glue, placing 30-50 mu l of the coagulating glue on the tissue section, and culturing at 37 ℃. 500ul of control medium was added below chamber.

(4) Preparation of a contrast medium:

1 mL Advanced DMEM/F12 medium: 40-60% (volume fraction) of L-WRN cells culture supernatant, wherein the supernatant is a conditioned medium rich in Wnt3 alpha, R-spondins and Noggin protein factors, 8-12% (volume fraction) of fetal calf serum, 1-2% (volume fraction) of penicillin-streptomycin, 8-12 mu M of ROCK inhibitor Y27632 and 8-12 mu M P38 of inhibitor SB 202190; 8-12 ng/mL epidermal growth factor; hydrocortisone of 0.4-0.6 μ g/mL; 1 × B-27; 1 × glutamine; 0.01 to 0.02% (volume fraction) ITS; 1 amphotericin B & gentamicin; 25 μ M mycoplasma remover Plasmocin; 1.00-1.50 mM N-acetylcysteine, and the medium was filtered through a 0.20 μ M filter.

Comparing the first day after the preparation (reference numeral 1 in FIG. 1) with the fourteenth day after the preparation (reference numeral 3 in FIG. 1), it was found that the medium was significantly yellowed (basis for judging the growth state of the cells) fourteen days after the preparation.

The cell growth rate was comparable to that of example 1.

The cost of the culture medium of comparative example 1 is about 5 times the cost of the culture medium of example 1.

Comparative example 2

(2) Preparing a slice: cutting into slices of 250-350 μm by a vibrating microtome, and adding PBS (3 times volume of double antibody and 1 time volume of 500 Xgentamycin-amphotericin B mixed solution); the agar was peeled off under a stereomicroscope, and the sliced tissue was placed in 500. mu.l of a liquid medium and cultured at 37 ℃.

(3) The composition of the liquid medium was as follows: weighing the following substances and mixing: MEM-F12500 ml, DMEM basic 373ml, 10ug/ml insulin, 1 volume of 500 Xgentamycin-amphotericin B cocktail, cholera toxin 0.1 nM, 10ng/ml EGF, 1ug/ml hydrocortisone, 10 μ M Y27632. It is identical to the medium composition of example 1.

Fig. 2 shows the results of culturing mammary gland tissue with a liquid culture medium, and it can be found that mammary gland tissue cannot be cultured smoothly with the liquid culture medium, which is characterized by easy drying of tissue and very slow culture speed.

Comparative example 3

Porcine mammary epithelial tissue was cultured using the method and medium described in example 1 in CN 202010038062.7, and the tissue was digested and cultured in 3D according to the protease digestion method described in this reference.

Referring to fig. 5, the porcine mammary epithelial tissue in fig. 5 grew well.

Comparative example 4

Porcine mammary gland epithelial tissue was cultured by the method described in example 1 in CN 202010038062.7 and the medium mentioned in example 1 of the present invention, and the tissue was digested and cultured in 3D by protease digestion referred to in this reference.

Referring to fig. 6, in fig. 6, after the culture medium of the present invention is cultured on porcine mammary gland epithelial tissue for 8 days by using a 3D model, slow cell growth, even cell death, etc. are observed.

Through the tests of comparative example 3 and comparative example 4, it can be found that:

the medium of the present invention is not applicable to all culture systems, and is applicable only to the gas-liquid culture system of the present invention.

But it cannot be ignored that the cost of the contrast medium is too high, with major limitations in large-scale industrial applications.

Comparative example 5

This comparative example was conducted to test the adaptation of different low-cost media to the culture model of the present invention.

The specific culture method is described in example 1.

CN 201811122209. X the medium used in example 1 is as follows:

the composition of the Advanced DMEM/F12 complete medium is as follows: advanced DMEM/F12 basal medium, fetal bovine serum, penicillin, streptomycin, insulin, hydrocortisone and epithelial growth factor, wherein the volume ratio of the Advanced DMEM/F12 basal medium to the fetal bovine serum is 9:1, and the concentrations of the penicillin, the streptomycin, the insulin, the hydrocortisone and the epithelial growth factor in the Advanced DMEM/F12 complete medium are 50 mu g/ml, 4 mu g/ml, 10ug/ml, 1ug/ml and 10ng/ml respectively.

The culture result of the culture medium of the comparative example on the mammary tissue cells is as follows:

the cost of the medium in this comparative example was also not high, but it is apparent from reference to FIG. 7 that the growth of porcine mammary epithelial cells cultured using the medium in this comparative example was poor.

In summary, the following steps:

1. the gas-liquid culture mode of the invention is combined with the culture medium of the invention, so that the aim of culturing the mammary tissue quickly and with low cost can be achieved.

2. The culture medium of the invention of the prior application patent is either too costly or too slow in culture speed after being applied to the culture system of the invention, both of which increase the cost of industrial scale-up production.

3. The culture medium of the present invention is not suitable for liquid culture methods, in other words, the traditional liquid culture methods have too high requirements for nutrient components of the culture medium, and low-cost culture media cannot be suitable.

Claims

1. A method for culturing sow mammary tissue in vitro is characterized by comprising the following steps: slicing the mammary gland tissue of the sow, placing the sliced mammary gland tissue between an upper solidified glue and a lower solidified glue, and contacting the lower solidified glue with a culture medium; changing the culture medium every 2-4 days or changing the culture medium when the culture medium turns yellow;

the 1L of the culture medium comprises the following components:

DMEM-F12：500ml；

DMEM basic：373 ml；

fetal bovine serum: 100 ml;

penicillin-streptomycin: 15 ml;

insulin: 10 ug/ml;

500 Xgentamicin-amphotericin B mixed solution: 1 volume;

cholera toxin：1 nM；

EGF：10ng/ml ；

hydrocortisone: 10 ug/ml;

ROCK inhibitor Y27632: 10 μ M.

2. The method for in vitro culture of the mammary tissue of the sow as claimed in claim 1, wherein the upper and lower coagulating glues are both composed of the following components:

collagen I, 10 times of volume of F12 culture medium and buffer solution;

3. The method for in vitro culture of mammary tissue of sow as claimed in claim 2, wherein the buffer is prepared from 100mL of sterile water, 0.05M NaOH, 200mm HEPES, 2.2 g NaHCO₃Is prepared from the mixture of (A) and (B).

4. The in vitro culture method of the mammary gland tissue of the sow as claimed in claim 1, wherein the volume ratio of the upper solidified glue to the lower solidified glue is 1: 1-2.

5. The method for culturing the breast tissue of the sow in vitro as claimed in claim 1, wherein the specific method for the breast tissue section of the sow is as follows:

cutting the mammary tissue without connective tissue and fat into small pieces, inserting the mammary tissue into liquid agar with volume fraction of 6%, and placing into refrigerator for solidification;

cutting the agar blocks containing the mammary tissue into cubes after the agar is solidified;

cutting the cube into slices with the thickness of 250-350 mu m by using a slicer, and putting the slices into a PBS solution; and stripping off the agar under a stereoscopic microscope to obtain the sliced mammary tissue.

6. The method for in vitro culture of mammary tissue of sow as claimed in claim 5, wherein the PBS solution contains 3 times the volume of penicillin-streptomycin and 1 time the volume of 500 Xgentamicin-amphotericin B mixed solution.

7. The method of claim 5, wherein the cube is 3 x 3cm in size.