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  • 专利说明
  • 权利要求
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    • 专利摘要:
    A cell co-culture device of the present invention relates to the technical field of cell cultures, which is composed of a dish body and a dish cover, the dish body is a hollow structure with an upper opening, and the upper opening of the dish body is provided with the dish cover, an interior of the dish body is provided with an isolation ring, the isolation ring divides the interior of the dish body into a first area and a second area, and an interior of the isolation ring is provided with a climbing slide. By using the above-described device, not only the direct contact and co-culture of bovine myocytes and adipocytes can be achieved, but also single-type cells (myocytes or adipocytes) on the climbing slide can be collected for subsequent analysis and processing of transcriptome and proteome, so as to study the effect on myocytes or adipocytes in a co-culture environment, thereby facilitating the further exploration of the interaction between myocytes and adipocytes. It has been proved by many observations that the myocytes and adipocytes cultured by this method have high survival efficiency, good vitality and obvious cell characteristics, and the morphology is consistent with that of myocytes and adipocytes cultured separately, and therefore the method is a good in vitro research model and has certain promotion and research values.
    公开号:NL2026517A
    申请号:NL2026517
    申请日:2020-09-23
    公开日:2021-05-18
    发明作者:Wang Hongbao；Su Xiaotong；Wang Yaning；Li Anqi；Cheng Gong；Zan Linsen
    申请人:Univ Northwest A&F；
    IPC主号:
    专利说明:

    -1- CELL CO-CULTURE DEVICE AND CO-CULTURE METHOD OF BOVINEMYOCYTES AND ADIPOCYTES
    TECHNICAL FIELD The present invention relates to the technical field of cell cultures, and particularly to a cell co-culture device and a co-culture method of bovine myocytes and adipocytes.
    BACKGROUND Compared with monotypic (single cell type) cell culture technology, in order to establish a culture system closer to an /# vivo environment, cell co-culture technology emerged in the late 1980s. Cell co-culture, also known as composite culture or mixed culture, is the co-culture of two or more different types of cells in the same environment. Since the cell co-culture considers the interaction between living cells, simulates the microenvironment iz vivo to the greatest extent, and compensates for the defects of monotypic cell culture, it can be better used to investigate the cell-cell interactions. At present, the cell co-culture techniques mainly include direct contact co-culture method and indirect contact co-culture method. Direct contact co-culture is to mix and culture two or more types of cells in the same culture environment according to a certain inoculation ratio, and the disadvantage is that the subsequent transcriptomic or proteomic analysis cannot be performed on a single cell type. Indirect contact co-culture is to inoculate two or more types of cells on different carriers, and then place the different carriers in the same culture environment. The most typical system used for indirect contact co-culture is Transwell co-culture system, which consists of two parts: an upper compartment and a lower compartment, in which the upper compartment contains an upper culture medium and the lower compartment contains a lower culture medium, and the upper culture medium and lower culture medium are separated by a polycarbonate membrane. Two types of cells are seeded in the upper compartment and lower compartment respectively. Due to the permeability of the polycarbonate membrane, the components in the upper culture medium and the lower culture medium can diffuse to
    -2- each other, but the cells cannot pass through the polycarbonate membrane, so the interaction between the two types of cells could be studied. However, the Transwell culture system also has the following problems:(1) the upper and lower compartments of the Transwell culture system are separated by the polycarbonate membrane, so in order to prevent cells from passing through, a membrane with a pore diameter of 0.4 microns should be used 1n the co-culture, which will affect the passage and action time of cell secretion factors; (2) it is more difficult to observe the cells in the upper and lower compartments intuitively and carry out subsequent operation; and (3) it is expensive.
    There are two main types of fat in beef: intermuscular fat and intramuscular fat. Among them, intramuscular fat is the material basis for the formation of beef marbling, and the content of intramuscular fat can improve the flavor, juiciness and tenderness of beef, which is an important factor determining beef quality. In marbled beef, myocytes (muscle cells) and adipocytes (fat cells) are heterogeneously distributed, and the two cells regulate the life process mutually through direct contact, autocrine or paracrine some cytokines.
    SUMMARY The objective of the present invention is to provide a device for co-culturing bovine myocytes and adipocytes and a culture method thereof, which aims to solve the problem that when myocytes and adipocytes are cultured separately, the interaction between myocytes and adipocytes in vivo is ignored, and the molecular mechanism of character formation of marbled beef cannot be fully analyzed.
    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 upper opening of the dish body is provided with the dish cover; an interior of the dish body is provided with an isolation ring, the isolation ring 1s a hollow structure with an upper opening and a lower opening; the isolation ring divides the interior of the dish body into a first area and a second area, an interior of the isolation ring is the first area, and the area between
    -3- the isolation ring and the dish body is the second area; and the interior of the isolation ring is provided with a climbing slide. As a further improvement of the present invention, both the dish body and the isolation ring are cylinder. As a further improvement of the present invention, a diameter of the dish body is 60 mm, a diameter of the isolation ring is 35 mm, and the climbing slide is a cover slip of 24 mm x 24 mm.
    A method for co-culturing bovine myocytes and adipocytes using the cell co-culture device described above includes the following steps: (1) under aseptic conditions, taking a bovine longissimus dorsi muscle tissue and a perirenal adipose tissue respectively, excising visible blood vessels and connective tissue, rinsing with 5%-10% by weight of double antibody diluted in IxPBS (an abbreviation of Phosphate Buffered Saline), and then cutting remaining tissues into small pieces in a size of 1 mm’ for subsequent use; (2) adding a digestion solution containing 0.25% type II collagenase and 0.1% type II neutral protease to the minced bovine longissimus dorsi muscle tissue obtained in step (1) and a digestion solution containing 0.25% type I collagenase to the minced perirenal adipose tissue obtained in step (1), respectively; and then placing in a oscillating constant temperature water bath at 37°C for digestion until the minced bovine longissimus dorsi muscle tissue and the minced perirenal adipose tissue become a fine slurry respectively; (3) mixing the fine slurry prepared in step (2) with an equal volume of culture medium containing serum to terminate the digestion, then filtering a cell suspension through an 80 um cell sieve, performing a cell centrifugation at 1200 rpm for 10 minutes, then discarding a supernatant to obtain cell pellets, and then adding appropriate amount of serum culture medium to achieve a cell density of 1x10° to 2x10°/mL, pipetting and mixing well to obtain cell suspensions; and
    -4- (4) inoculating the cell suspensions prepared in step (3) in the cell co-culture device, and adding a DMEM/F12 complete medium (a 1:1 mixture of Dulbecco’s Modified Essential Medium (DMEM) and Ham’s F-12 Medium) containing 10% fetal bovine serum and 1% double antibody, wherein in the first area, one of the cell suspensions is inoculated, and in the second area, another cell suspension is inoculated or a mixture of the two cell suspensions in a ratio of 1:1 is inoculated for mixed co-culture; then placing the cell co-culture device in an incubator at 37°C and 5% CO: for culture; and after the cells adhered to the wall, removing the isolation ring to complete the co-culture of bovine myocytes and adipocytes.
    As a further improvement of the present invention, in step (3), the culture medium containing serum contained in the adipocyte cell suspension is a DMEM/F12 medium containing 10% fetal bovine serum and 1% double antibody.
    As a further improvement of the present invention, in step (3), the culture medium containing serum contained in the myocyte cell suspension is a DMEM/F12 medium containing 20% fetal bovine serum and 1% double antibody.
    The advantages of the present invention are as follows.
    (1) The cell co-culture device provided by the present invention has the advantages of simple structure, simple manufacturing process, and easy operation. Bovine myocytes and adipocytes can be inoculated on the climbing slide and a bottom area of the dish simultaneously to achieve the co-culture of the two types of cells and simulate the microenvironment in vivo to the greatest extent, so as to better observe the cell-cell interaction and compensate for the defects of monotypic cell culture.
    (2) By using the cell co-culture device provided by the present invention, not only the direct contact and co-culture of bovine myocytes and adipocytes can be achieved, but also single-type cells (myocytes or adipocytes) on the climbing slide can be collected for subsequent analysis and processing of transcriptome and proteome, so as to study the effect on myocytes or adipocytes in a co-culture environment, thereby facilitating the
    -5- further exploration of the interaction between myocytes and adipocytes.
    (3) In the method for co-culturing bovine myocytes and adipocytes provided by the present invention, bovine myocytes and adipocytes are co-cultured by using the cell co- culture device and using the DMEM/F 12 complete medium containing 10% fetal bovine serum and 1% double antibody (i.e., 10% penicillin/streptomycin). It has been proved by many observations that the myocytes and adipocytes cultured by this method have high survival efficiency, good vitality and obvious cell characteristics, and the morphology is consistent with that of myocytes and adipocytes cultured separately. The method for co-culturing bovine myocytes and adipocytes is a good model for in vitro research and has certain promotion and research values.
    BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of an overall structure of a cell co-culture device for co- culturing bovine myocytes and adipocytes provided by the present invention; FIG. 2 is a side view of the device for co-culturing bovine myocytes and adipocytes provided by the present invention; FIG. 3 is a top view of the device for co-culturing bovine myocytes and adipocytes provided by the present invention (in the figure: FF and MM are adipocytes and myocytes in a separate culture system; FX and MX are co-culture systems; and adipocytes and myocytes are climbed on the climbing slide, respectively); FIG. 4A is a diagram showing cell morphology of the adipocytes in the bovine myocytes and adipocytes co-cultured using the cell co-culture device of the present invention; FIG. 4B is a diagram showing cell morphology of the adipocytes separately cultured using the cell co-culture device of the present invention,
    -6- FIG. 5A is a diagram showing cell morphology of the myocytes in the bovine myocytes and adipocytes co-cultured using the cell co-culture device of the present invention; and FIG. 5B is a diagram showing cell morphology of the myocytes separately cultured using the cell co-culture device of the present invention.
    DETAILED DESCRIPTION OF THE EMBODIMENTS In order to make the objectives, technical solutions and advantages of the present invention clear and definite, the present invention is further described in detail below with reference to the drawings and embodiments. It should be understood that, the specific embodiments described hereinafter are merely intended to explain the present invention rather than limit the present invention. Embodiment 1 As shown in FIG. 1 and FIG. 2, the cell co-culture device of the present invention is composed of the dish body 1 and the dish cover 2; the dish body 1 is a hollow structure with an upper opening, the upper opening of the dish body 1 is provided with the dish cover 2; an interior of the dish body 1 is provided with an isolation ring4, the isolation ring 4 is a hollow structure with an upper opening and a lower opening; the isolation ring 4 divides the interior of the dish body 1 into the first area (I) and the second area (ID, an interior of the isolation ring 4 is the first area (I), and the area between the isolation ring 4 and the dish body 1 is the second area (II); the interior of the isolation ring 4 is provided with the climbing slide 3.
    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 a role of isolation after cell inoculation and before adhesion. The climbing slide 3 1s a cover slip of 24 mm * 24 mm.
    -7- Embodiment 2 The method for co-culturing bovine myocytes and adipocytes using the cell co-culture device described in Embodiment 1 includes the steps as follows.
    (1) Under aseptic conditions, a bovine longissimus dorsi muscle tissue and a perirenal adipose tissue are taken respectively, visible blood vessels and connective tissue are excised, 5%-10% by weight of double antibody (10% penicillin/streptomycin) diluted in 1xPBS is used for rinsing, and then the remaining tissues is cut into small pieces in a size of 1 mm’ for subsequent use.
    (2) A digestion solution containing 0.25% type II collagenase and 0.1% type II neutral protease is added to the minced bovine longissimus dorsi muscle tissue obtained in step (1), and a digestion solution containing 0.25% type I collagenase is added to the minced perirenal adipose tissue obtained in step (1), respectively, and then placed in a oscillating constant temperature water bath at 37°C for digestion until the minced bovine longissimus dorsi muscle tissue and the minced perirenal adipose tissue become a fine slurry respectively.
    (3) The fine slurry prepared in step (2) is mixed with an equal volume of culture medium containing serum to terminate the digestion, then a cell suspension is filtered through an 80 um cell sieve, a cell centrifugation is performed at 1200 rpm for 10 minutes, then a supernatant is discarded to obtain cell pellets, and then appropriate amount of serum culture medium is added to achieve a cell density of 1x105 to 2x105/mL, followed by pipetting and mixing well to obtain cell suspensions (i.e., the adipocyte cell suspension and the myocyte cell suspension prepared respectively). Moreover, the culture medium containing serum contained in the adipocyte cell suspension is a DMEM/F12 medium containing 10% fetal bovine serum and 1% double antibody; and the culture medium containing serum contained in the myocyte cell suspension is a DMEM/F 12 medium containing 20% fetal bovine serum and 1% double antibody.
    (4) The cell suspensions prepared in step (3) is inoculated in the cell co-culture device, and a DMEM/F12 complete medium containing 10% fetal bovine serum and 1% double antibody is added. Specifically, in the first area (I), one of the cell suspensions is inoculated, and in the second area (II), another cell suspension is inoculated or a mixture
    -8- of the two cell suspensions in a ratio of 1:1 is inoculated for mixed co-culture. Subsequently, the cell co-culture device is placed in an incubator at 37°C and 5% CO: for culture; and after the cells adhered to the wall, the isolation ring is removed to complete the co-culture of bovine myocytes and adipocytes.
    (5) The slide 3 1s taken out, and the morphologies of bovine myocytes and adipocytes are observed using a phase contrast microscope. The cell morphology results are shown in FIG. 4 and FIG. 5. FIG. 4A is a diagram showing the cell morphology of the adipocytes in the co-cultured bovine myocytes and adipocytes after 48 hours of culture, represented by FX-F; FIG. 4B is a diagram showing the cell morphology of the separately cultured adipocytes after 48 hours of culture, represented by FF; FIG. SA is a diagram showing the cell morphology of the myocytes in the co-cultured bovine myocytes and adipocytes after 48 hours of culture, represented by MX-M; and FIG. 5B is a diagram showing the cell morphology of the separately cultured myocytes after 48 hours of culture, represented by MM. As can be clearly seen from the figure, the co- cultured adipocytes (or myocytes) and the separately cultured adipocytes (or myocytes) are identical in morphology and the cell morphology is obvious.
    In order to explore the interaction between bovine myocytes and adipocytes, the present invention innovatively established an in vitro research model and disclosed a cell co- culture device, which allows the co-culture of the myocytes and adipocytes in the same environment, simulates the microenvironment iz vivo to the greatest extent, so that not only the direct contact and co-culture of adipocytes and myocytes can be achieved, but also the monotypic myocytes or monotypic adipocytes can be collected for subsequent analysis and processing of transcriptome and proteome. Moreover, the cell co-culture device has the advantages of simple structure, simple manufacturing process, easy operation, and strong practicability, and has certain research and promotion values.
    权利要求:
    Claims (6)
    [1]
    A cell culture device composed of a shell body (1) and a shell cover (2), characterized in that the shell body (1) is a hollow structure with an upper opening, and the upper opening of the shell body ( 1) is provided with the shell cover (2); an inside of the shell body (1) is provided with an insulating ring (4), the insulating ring (4) being a hollow structure having an upper opening and a lower opening; wherein the insulating ring (4) divides the inside of the shell body (1) into a first region and a second region, an inside of the insulating ring (4) being the first region, and the region between the insulating ring (4) and the shell body (1) is the second region; and the inside of the isolation ring (4) is provided with a climbing slide (3).
    [2]
    Device for co-culturing cells according to claim 1, characterized in that both the shell body (1) and the isolation ring (4) are a cylinder.
    [3]
    A cell co-culture device according to claim 2, characterized in that a diameter of the shell body (1) is 60 mm, a diameter of the isolation ring (4) is 35 mm and the climbing slide (3) is a cover glass of 24mm x 24mm.
    [4]
    A method of co-culturing bovine myocytes and adipocytes using the cell co-culturing device of claim 1, wherein the method comprises the steps of: (1) taking a tissue under aseptic conditions, respectively, from a bovine dorsal muscle and bovine perirnal adipose tissue, excising visible blood vessels and connective tissue, rinsing with 5% by weight - 10% by weight of a double antibody diluted in 1x PBS, and then cutting the remaining tissues in small parts in a size of 1 mm3 for subsequent use; (2) adding a degradation solution containing 0.25% collagenase type II and 0.1% neutral protease type II, respectively, to the crushed
    -10 - bovine lung muscle tissue obtained in step (1) and a degradation solution containing 0.25% collagenase type I to the crushed perirenal adipose tissue obtained in step (1); and then placing in an oscillating water bath at a constant temperature at 37°C for degradation until the crushed bovine lumbar muscle tissue and the crushed perirenal adipose tissue, respectively, become a fine suspension; (3) mixing the fine suspension prepared in step (2) with an equal volume of culture medium containing serum to stop the degradation, then filtering a cell suspension through an 80 µm cell strainer, performing cell centrifugation at 1200 rpm for 10 minutes, then discarding a supernatant to obtain cell pellets, then adding an appropriate amount of serum culture medium to give a cell density of 1x105 - 2x105/mL, pipetting and mixing well to obtain cell suspensions; and (4) inoculating the cell suspensions prepared in step (3) into the cell coculture device and adding a DMEM/F12 complete medium containing 10% fetal bovine serum and 1% double antibody, wherein the first region, one of the cell suspensions is inoculated, and in the second region another cell suspension is inoculated or a mixture of the two cell suspensions is inoculated in a ratio of 1:1 for mixed co-culture; then placing the cell co-culture device in an incubator at 37°C and 5% CO 2 for culture; and after the cells are attached to the wall, removing the isolation ring to complete the co-culture of bovine myocytes and adipocytes.
    [5]
    A method for co-culturing bovine myocytes and adipocytes according to claim 4, characterized in that in step (3) the culture medium containing serum contained in the adipocyte cell suspension is a DMEM/F12 medium containing 10% fetal bovine serum and 1% double antibody.
    [6]
    A method for co-culturing bovine myocytes and bovine adipocytes according to claim 4, characterized in that in step (3) the culture medium containing serum contained in the myocyte cell suspension contains a
    “11 - DMEM/F12 medium is containing 20% fetal bovine serum and 1% double antibody.
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    引用文献:
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    法律状态:
    优先权:
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