A cell co-culture device and a co-culture method of bovine muscle cells and fat cells

Abstract

The invention discloses a cell co-culture device, which relates to the field of cell culture. The device is composed of a dish body and a dish cover. The dish body is a hollow structure with an upper opening. The dish cover is provided at the opening of the dish body. The dish body is provided with an isolation ring. The isolation ring divides the interior of the dish body into region I and region II. The isolation ring contains a crawling sheet. The device can be used to realize direct contact co-culture of bovine muscle cells and fat cells, and single cells (muscle cells or fat cells) on the crawling sheet can be collected for subsequent transcriptome and proteome analysis and processing to study the effects on muscle cells or fat cells in a co-culture environment. Therefore, it is beneficial to further explore the interaction between muscle cells and fat cells. It has been confirmed through multiple observations that the muscle cells and fat cells cultured in this way have high survival efficiency, good vitality, obvious cell characteristics, and are consistent with the morphology of muscle cells and fat cells cultured alone. It is a good in vitro research model with certain promotion and research value.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a cell co-culture device and a co-culture method of bovine muscle cells and fat cells. Background Art

[0002] Compared with monolayer cell culture technology, in order to establish a culture system closer to the in vivo environment, cell co-culture technology came into being in the late 1980s. Cell co-culture, also known as composite culture or mixed culture, is the co-culture of two or more cells in the same environment. Because it takes into account the interaction between living cells, it simulates the in vivo microenvironment to the greatest extent, making up for the defects of monolayer cell culture, so it can better study the interaction between cells.

[0003] At present, the technologies for studying cell co-culture mainly include direct contact co-culture and indirect contact co-culture. Direct contact co-culture is to place two or more cells in a certain inoculation ratio in the same culture environment for mixed culture. Its disadvantage is that it is impossible to perform subsequent transcriptome or proteome analysis and processing on a single type of cell; indirect contact co-culture is to inoculate two or more cells on different carriers respectively, and then place the two carriers in the same culture environment. The more typical method is the Transwell co-culture system, which includes an upper chamber and a lower chamber. The upper chamber contains the upper culture medium, and the lower chamber contains the lower culture medium. The upper and lower culture medium are separated by a polycarbonate membrane. The two types of cells are inoculated in the upper and lower chambers respectively. Since the polycarbonate membrane is permeable, the components in the culture fluid in the upper and lower layers can diffuse into each other, but the cells themselves cannot pass through. Therefore, it can be used to study the interaction between the two cells. However, there are also problems: (1) The upper and lower chambers of the Transwell culture system are separated by a polycarbonate membrane. To prevent cells from passing through, a membrane with a pore size of 0.4 microns must be used in co-culture, which will affect the passage and action time of cell secretion factors; (2) It is not easy to observe the cells in the upper and lower chambers more intuitively and perform subsequent operations; (3) It is expensive.

[0004] There are two main types of fat in beef: intermuscular fat and intramuscular fat. Intramuscular fat is the material basis for the formation of beef marbling. The intramuscular fat content can improve the flavor, juiciness and tenderness of beef, and is an important factor in measuring beef quality. In marbled beef, muscle cells and fat cells are intertwined. The two cells are in direct contact, and they secrete or paracrine some cytokines to regulate each other's life processes. Summary of the invention

[0005] The purpose of the present invention is to provide a device for co-culturing bovine muscle cells and fat cells and a culturing method thereof, aiming to solve the problem of culturing muscle cells and fat cells separately while ignoring the interaction between muscle cells and fat cells in living bodies and failing to fully analyze the molecular mechanism of the formation of marbled beef traits.

[0006] A cell co-culture device of the present invention is composed of a dish body and a dish cover. The dish body is a hollow structure with an upper opening, and the dish cover is provided at the opening of the dish body. An isolation ring is provided inside the dish body. The isolation ring is a hollow structure with upper and lower openings. The isolation ring divides the interior of the dish body into area I and area II. The interior of the isolation ring is area I, and the area between the isolation ring and the dish body is area II. A climbing sheet is provided inside the isolation ring.

[0007] As a further improvement of the present invention, the dish body and the isolation ring are both cylindrical.

[0008] As a further improvement of the present invention, the diameter of the dish body is 60 mm, the diameter of the isolation ring is 35 mm, and the climbing sheet is a cover glass of 24 mm*24 mm.

[0009] The method for co-culturing bovine muscle cells and adipocytes using the above cell co-culture device has the following operating steps:

[0010] (1) Under sterile conditions, take the longissimus dorsi muscle and perirenal adipose tissue of the bovine, remove the visible blood vessels and connective tissue, rinse with 1xPBS containing 5% to 10% by weight of double antibody, and then cut the tissue into small pieces of 1 mm3 for later use;

[0011] (2) adding 0.25% type II collagenase / 0.1% type II neutral protease digestion solution to the minced longissimus dorsi muscle tissue of the bovine in step (1), and adding 0.25% type I collagenase digestion solution to the minced perirenal adipose tissue in step (1), and digesting in a constant temperature shaking water bath at 37° C. until a fine slurry is obtained, thereby obtaining a fine slurry;

[0012] (3) The fine slurry prepared in step (2) was mixed with an equal volume of serum culture medium to terminate digestion. The cell suspension was then filtered through an 80 μm cell sieve and centrifuged at 1200 rpm / min for 10 minutes. The supernatant was discarded to obtain a cell pellet. An appropriate amount of serum culture medium was then added to make the cell density 1×10 5 ~2×10 5 / ml, pipette and mix to make cell suspension;

[0013] (4) The cell suspension prepared in step (3) is inoculated into the cell co-culture device according to claim 1, and DMEM / F12 complete medium containing 10% fetal bovine serum and 1% double antibody is added. One type of cell is inoculated in area I, and another type of cell is inoculated in area II, or the two types of cells are mixed and co-cultured in a ratio of 1:1, and the mixture is placed at 37°C and 5% CO. 2 Culture in an incubator, and remove the isolation circle after the cells adhere to the wall, thus completing the co-culture of bovine muscle cells and adipocytes.

[0014] As a further improvement of the present invention, the formula of the adipocyte serum culture medium in step (3) is DMEM / F12 culture medium containing 10% fetal bovine serum and 1% double antibody.

[0015] As a further improvement of the present invention, the serum culture medium for myocytes in step (3) is a DMEM / F12 culture medium containing 20% ​​fetal bovine serum and 1% double antibody.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) The cell co-culture device provided by the present invention has a simple structure, a simple manufacturing method, and is easy to operate. It can simultaneously inoculate bovine muscle cells and fat cells on the slide and the bottom area of ​​the dish to achieve co-culture of the two cells, simulating the in vivo microenvironment to the greatest extent, so as to better observe the interaction between cells and make up for the defects of single-layer cell culture;

[0018] (2) The cell co-culture device provided by the present invention can not only realize the direct contact co-culture of bovine muscle cells and adipocytes, but also collect single cells (muscle cells or adipocytes) on the slide for subsequent transcriptome and proteome analysis and processing, so as to study the effects of the co-culture environment on muscle cells or adipocytes, thus facilitating further exploration of the interaction between muscle cells and adipocytes;

[0019] (3) The present invention provides a method for co-culturing bovine muscle cells and adipocytes. By using the cell co-culture device, bovine muscle cells and adipocytes are co-cultured using DMEM / F12 complete culture medium containing 10% fetal bovine serum and 1% double antibody (i.e., 10% penicillin / streptomycin). Multiple observations have confirmed that the muscle cells and adipocytes cultured in this method have high survival efficiency, good vitality, obvious cell characteristics, and are consistent with the morphology of muscle cells and adipocytes cultured separately. This method is a good in vitro research model with certain promotion and research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the overall structure of the bovine muscle cell and adipocyte co-culture device provided by the present invention;

[0021] Figure 2 A side view of the co-culture device of bovine muscle cells and adipocytes provided by the present invention;

[0022] Figure 3 A top view of a co-culture device for bovine myocytes and adipocytes provided by the present invention (in the figure: FF and MM are adipocytes and myocytes in separate culture systems, respectively; FX and MX are co-culture systems, with adipocytes and myocytes on the slides, respectively);

[0023] Figure 4 a. Figure 4 b are cell morphology diagrams of adipocytes in the co-culture of bovine myocytes and adipocytes and adipocytes cultured alone according to the present invention;

[0024] Figure 5 a. Figure 5 b are cell morphology diagrams of the myocytes in the co-culture of bovine myocytes and adipocytes and the myocytes cultured alone using the present invention. DETAILED DESCRIPTION

[0025] The present invention provides a device for co-culturing bovine myocytes and adipocytes and a culturing method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Example 1

[0027] like Figure 1 and Figure 2 As shown, a cell co-culture device provided by the present invention is composed of a dish body 1 and a dish cover 2, wherein the dish body 1 is a hollow structure with an upper opening, a dish cover 2 is provided at the opening of the dish body 1, an isolation ring 4 is provided inside the dish body 1, and the isolation ring 4 is a hollow structure with upper and lower openings. The isolation ring 4 divides the interior of the dish body 1 into area I and area II, the interior of the isolation ring 4 is area I, and the area between the isolation ring 4 and the dish body 1 is area II, and a climbing sheet 3 is provided inside the isolation ring 4.

[0028] The dish body 1 is a cylinder with a cross section of 60 mm in diameter, and the isolation ring 4 is a cylindrical plastic ring with a cross section of 35 mm in diameter, which is modified from a 35 mm cell culture dish and mainly plays an isolation role before the cells are attached to the wall after inoculation. The climbing sheet 3 is a cover glass of 24 mm*24 mm.

[0029] Example 2

[0030] The method for co-culturing bovine muscle cells and adipocytes using the cell co-culture device described in Example 1 has the following operating steps:

[0031] (1) Under sterile conditions, the longissimus dorsi muscle and perirenal adipose tissue were removed, the visible blood vessels and connective tissue were removed, and the tissues were rinsed with 1xPBS containing 5% to 10% double antibody (10% penicillin / streptomycin) by weight, and then cut into 1 mm 3 Small pieces of different sizes, set aside;

[0032] (2) adding 0.25% type II collagenase / 0.1% type II neutral protease digestion solution to the minced longissimus dorsi muscle tissue of the bovine in step (1), and adding 0.25% type I collagenase digestion solution to the minced perirenal adipose tissue in step (1), and digesting in a constant temperature shaking water bath at 37° C. until a fine slurry is obtained, thereby obtaining a fine slurry;

[0033] (3) The fine slurry prepared in step (2) was mixed with an equal volume of serum culture medium to terminate digestion. The cell suspension was then filtered through an 80 μm cell sieve and centrifuged at 1200 rpm / min for 10 minutes. The supernatant was discarded to obtain a cell pellet. An appropriate amount of serum culture medium was then added to make the cell density 1×10 5 ~2×10 5 / ml, blow and mix to make a cell suspension; among them, the serum culture medium formula for adipocytes is DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody (); the serum culture medium formula for muscle cells is DMEM / F12 medium containing 20% ​​fetal bovine serum and 1% double antibody.

[0034] (4) The cell suspension prepared in step (3) is inoculated into the cell co-culture device according to claim 1, and DMEM / F12 complete medium containing 10% fetal bovine serum and 1% double antibody is added. One type of cell is inoculated in area I, and another type of cell is inoculated in area II, or the two types of cells are mixed and co-cultured in a ratio of 1:1, and the mixture is placed at 37°C and 5% CO. 2 Cultivate in an incubator, and remove the isolation circle 4 after the cells adhere to the wall, thus completing the co-culture of bovine muscle cells and adipocytes;

[0035] (5) Take out slide 3 and use phase contrast microscopy to observe the morphology of bovine muscle cells and adipocytes. The cell morphology results are as follows: Figure 4 , Figure 5 shown. Figure 4 a. Figure 4 b Cell morphology of adipocytes in co-cultured bovine myocytes and adipocytes and adipocytes cultured alone after 48 h of culture, indicated by FX-F and FF, respectively; Figure 5 a. Figure 5b are the cell morphology diagrams of the co-cultured bovine myocytes and adipocytes and the myocytes cultured alone after 48 hours of culture, which are represented by MX-M and MM respectively. It can be clearly seen from the figure that the co-cultured adipocytes (or myocytes) and the adipocytes (or myocytes) cultured alone have the same morphology, and the cell morphology is obvious.

[0036] In order to explore the interaction between bovine muscle cells and adipocytes, the present invention innovatively establishes an in vitro research model and discloses a cell co-culture device, in which muscle cells and adipocytes are co-cultured in the same environment to simulate the in vivo microenvironment to the greatest extent, which not only realizes the direct contact co-culture of adipocytes and muscle cells, but also can collect individual muscle cells or adipocytes for subsequent transcriptome and proteome analysis and processing. In addition, the device has a simple structure, a simple production method, is easy to operate, has strong practicality, and has certain research and promotion value.

Claims

1. A method for co-culturing bovine muscle cells and adipocytes, characterized in that: The steps are as follows: (1) Under sterile conditions, the longissimus dorsi muscle and perirenal adipose tissue were removed, the visible blood vessels and connective tissue were removed, and the tissues were rinsed with 1xPBS containing 5% to 10% double antibody by weight, and then cut into 1 mm 3 Small pieces of different sizes, set aside; (2) adding 0.25% type II collagenase / 0.1% type II neutral protease digestion solution to the minced longissimus dorsi muscle tissue of the bovine in step (1), and adding 0.25% type I collagenase digestion solution to the minced perirenal adipose tissue in step (1), and digesting in a constant temperature shaking water bath at 37° C. until a fine slurry is obtained, thereby obtaining a fine slurry; (3) The fine slurry prepared in step (2) was mixed with an equal volume of serum-containing culture medium to terminate digestion. The cell suspension was then filtered through an 80 μm cell sieve and centrifuged at 1200 rpm / min for 10 minutes. The supernatant was discarded to obtain a cell pellet. Serum-containing culture medium was then added to make the cell density 1×10 5 ~2×10 5 / ml, pipetting and mixing to prepare cell suspension; the formula of serum culture medium for adipocytes is DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody; the formula of serum culture medium for muscle cells is DMEM / F12 medium containing 20% ​​fetal bovine serum and 1% double antibody; (4) Inoculate the cell suspension prepared in step (3) into a cell co-culture device, add DMEM / F12 complete medium containing 10% fetal bovine serum and 1% double antibody, inoculate one type of cell in area I, inoculate another type of cell in area II, or inoculate the two types of cells in a 1:1 ratio for mixed co-culture, and place in a 37°C, 5% CO2 incubator for culture. After the cells adhere to the wall, remove the isolation circle (4), thereby completing the co-culture of bovine muscle cells and adipocytes; The cell co-culture device is composed of a dish body (1) and a dish cover (2); the dish body (1) is a hollow structure with an upper opening; the dish cover (2) is provided at the opening of the dish body (1); an isolation ring (4) is provided inside the dish body (1); the isolation ring (4) is a hollow structure with upper and lower openings; the isolation ring (4) divides the inside of the dish body (1) into region I and region II; the inside of the isolation ring (4) is region I, and the area between the isolation ring (4) and the dish body (1) is region II; and a climbing sheet (3) is provided inside the isolation ring (4).

2. The method for co-culturing bovine muscle cells and adipocytes according to claim 1, characterized in that: The vessel body (1) and the isolation ring (4) are both cylindrical.

3. The method for co-culturing bovine muscle cells and adipocytes according to claim 2, characterized in that: The diameter of the dish body (1) is 60 mm, the diameter of the isolation ring (4) is 35 mm, and the climbing sheet (3) is a cover glass of 24 mm×24 mm.

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