WO2021056921A1 - 一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法 - Google Patents

一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法 Download PDF

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WO2021056921A1
WO2021056921A1 PCT/CN2020/000101 CN2020000101W WO2021056921A1 WO 2021056921 A1 WO2021056921 A1 WO 2021056921A1 CN 2020000101 W CN2020000101 W CN 2020000101W WO 2021056921 A1 WO2021056921 A1 WO 2021056921A1
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cell
cells
muscle cells
dish body
adipocytes
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French (fr)
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王洪宝
苏晓彤
王亚宁
李安奇
成功
昝林森
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西北农林科技大学
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    • C—CHEMISTRY; METALLURGY
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
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    • C12N2502/00—Coculture with; Conditioned medium produced by

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  • the invention relates to the field of cell culture, in particular to a cell co-cultivation device and a co-cultivation method of bovine muscle cells and fat cells.
  • Cell co-culture is also called compound culture or mixed culture. It is the co-cultivation of two or more cells in the same environment. Because it takes into account the interaction between living cells, it simulates the microenvironment in the body to the greatest extent and makes up for it. The defects of monolayer cell culture, so the interaction between cells can be better studied.
  • the techniques for studying cell co-culture mainly include direct contact co-cultivation and indirect contact co-cultivation.
  • Direct contact co-cultivation is to place two or more kinds of cells in the same culture environment at a certain inoculation ratio.
  • the disadvantage is that the subsequent transcriptome or proteome analysis processing cannot be performed on a single type of cell; indirect; Contact co-cultivation is to inoculate two or more kinds of cells on different carriers, and then place the two carriers in the same culture environment.
  • the more typical method is the Transwell co-cultivation system, which includes the upper chamber and the lower chamber.
  • the chamber There are two parts of the chamber, in which the upper chamber contains the upper layer of culture fluid, the lower chamber contains the lower layer of culture fluid, and the upper and lower layer of culture fluid are separated by a polycarbonate membrane. Inoculate the two kinds of cells in the upper and lower chambers. Due to the permeability of the polycarbonate membrane, the components in the upper and lower layers of the culture fluid can diffuse each other, but the cells themselves cannot pass through, so it can be used to study the difference between the two types of cells. The interaction between the two, but there are also problems: (1) The upper and lower chambers of the Transwell culture system are separated by polycarbonate membranes.
  • a membrane with a pore size of 0.4 micron should be used in the co-culture, which will affect cell secretion.
  • intermuscular 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, and is an important factor in measuring beef quality.
  • 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, and is an important factor in measuring beef quality.
  • muscle cells and fat cells are intermingled and distributed, and the two types of cells are in direct contact, autocrine or paracrine some cytokines, and mutually regulate the process of life.
  • the purpose of the present invention is to provide a device for co-cultivating bovine muscle cells and adipocytes and a culture method thereof, which aims to solve the problem of cultivating muscle cells and adipocytes separately, while ignoring the interaction between muscle cells and adipocytes in the living body. Analyze the molecular mechanism of the formation of marbled beef traits.
  • the cell co-cultivation 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.
  • the opening of the dish body is provided with a dish cover, and the interior of the dish body is provided with an isolation ring.
  • It is a hollow structure with upper and lower openings.
  • the isolation ring divides the interior of the vessel 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 vessel body is area II.
  • the interior of the isolation ring is provided with climbing pieces.
  • both the vessel body and the isolation ring are cylindrical.
  • the diameter of the vessel body is 60mm
  • the diameter of the spacer is 35mm
  • the climbing piece is a cover glass of 24mm*24mm.
  • the method of co-cultivating bovine muscle cells and adipocytes using the above-mentioned cell co-cultivation device has the following steps:
  • step (2) Add 0.25% type II collagenase/0.1% type II neutral protease digestion solution to the muscular tissue of the longissimus dorsi muscle which was cut up in step (1), and then cut up the kidney in step (1) Add 0.25% of type I collagenase digestion solution to the peripheral adipose tissue, and digest it in a constant temperature shaking water bath at 37°C until it becomes a fine slurry to obtain a fine slurry;
  • step (3) Mix the fine slurry prepared in step (2) with an equal volume of serum culture medium, terminate the digestion, then filter the cell suspension through an 80 ⁇ m cell sieve, centrifuge at 1200 rpm/min for 10 minutes, and discard the supernatant to obtain Cell precipitation, then add appropriate amount of serum culture medium to make the cell density 1 ⁇ 10 5 ⁇ 2 ⁇ 10 5 /ml, pipette and mix well to make a cell suspension;
  • step (3) Inoculate the cell suspension prepared in step (3) in the cell co-cultivation device of claim 1, add DMEM/F12 complete medium containing 10% fetal bovine serum and 1% double antibody to the area I inoculate one type of cell, inoculate another type of cell in area II, or inoculate the two types of cells in a ratio of 1:1 for mixed co-cultivation, and place them in a 37°C, 5% CO 2 incubator, and isolate the cells after they adhere to the wall.
  • the circle is taken out to complete the co-cultivation of bovine muscle cells and adipocytes.
  • the adipocyte serum culture solution in step (3) is formulated as DMEM/F12 medium containing 10% fetal bovine serum and 1% double antibody.
  • the muscle cell serum culture solution formula in step (3) is DMEM/F12 medium containing 20% fetal bovine serum and 1% double antibody.
  • the cell co-cultivation device provided by the present invention has simple structure, simple manufacturing method and easy operation. It can simultaneously inoculate bovine muscle cells and adipocytes in the area of the slide and the bottom of the dish to achieve the co-cultivation of the two types of cells to the greatest extent Simulate the microenvironment in vivo to better observe the interaction between cells and make up for the defects of monolayer cell culture;
  • the cell co-cultivation device not only realizes the direct contact and co-cultivation of bovine muscle cells and adipocytes, but also collects single types of cells (muscle cells or adipocytes) on the slide for subsequent transcriptome and protein
  • the analysis and processing of the group is used to study the effect on muscle cells or adipocytes in the co-culture environment, so it is helpful to further explore the interaction between muscle cells and adipocytes;
  • the method for co-cultivating bovine muscle cells and adipocytes uses the cell co-cultivation device containing 10% fetal bovine serum and 1% double antibody (ie 10% penicillin/streptomycin). ) DMEM/F12 complete medium for co-cultivation of bovine muscle cells and adipocytes. Many observations have confirmed that the muscle cells and adipocytes cultured in this way have high survival efficiency, good viability, and obvious cell characteristics. The morphology of the muscle cells and adipocytes are the same, which is a good in vitro research model and has certain promotion research value.
  • Figure 1 is a schematic diagram of the overall structure of the co-cultivation device for bovine muscle cells and adipocytes provided by the present invention
  • Figure 2 is a side view of the device for co-cultivating bovine muscle cells and adipocytes provided by the present invention
  • FIG 3 is a top view of the co-culture device for bovine muscle cells and adipocytes provided by the present invention (in the figure: FF and MM are adipocytes and muscle cells in a separate culture system, respectively; FX and MX are co-culture systems, and the slides are respectively Fat cells and muscle cells);
  • 4a and 4b are respectively cell morphology diagrams of adipocytes in co-cultured bovine muscle cells and adipocytes and adipocytes cultured separately by applying the present invention
  • Figures 5a and 5b are respectively cell morphology diagrams of muscle cells in co-cultured bovine muscle cells and adipocytes and muscle cells cultured separately using the present invention.
  • the present invention provides a device for co-cultivating bovine muscle cells and adipocytes and a culture method thereof.
  • a cell co-cultivation 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, and the opening of the dish body 1 is provided There is a dish cover 2, a spacer ring 4 is arranged inside the dish body 1, and the spacer ring 4 is a hollow structure with upper and lower openings.
  • the spacer ring 4 divides the interior of the dish body 1 into a region I and a region II, and the interior of the spacer ring 4 is a region I.
  • Between the isolation ring 4 and the vessel body 1 is a region II, and a climbing piece 3 is provided inside the isolation ring 4.
  • the dish body 1 is a cylinder with a cross-section of 60mm in diameter
  • the spacer 4 is a cylindrical plastic circle with a cross-section of 35mm in diameter, which is transformed from a 35mm cell culture dish, and is mainly used before the cells are inoculated before they adhere to the wall.
  • the climbing sheet 3 is a 24mm*24mm cover glass.
  • step (2) Add 0.25% type II collagenase/0.1% type II neutral protease digestion solution to the muscular tissue of the longissimus dorsi muscle which was cut up in step (1), and then cut up the kidney in step (1) Add 0.25% of type I collagenase digestion solution to the peripheral adipose tissue, and digest it in a constant temperature shaking water bath at 37°C until it becomes a fine slurry to obtain a fine slurry;
  • step (3) Mix the fine slurry prepared in step (2) with an equal volume of serum culture medium, terminate the digestion, then filter the cell suspension through an 80 ⁇ m cell sieve, centrifuge at 1200 rpm/min for 10 minutes, and discard the supernatant to obtain Cell precipitation, then add appropriate amount of serum culture medium to make the cell density 1 ⁇ 10 5 ⁇ 2 ⁇ 10 5 /ml, pipetting and mixing to make a cell suspension; among them, fat cells with serum culture medium formula contains 10% fetal bovine serum , DMEM/F12 medium with 1% double antibody (); muscle cell serum culture medium formula is DMEM/F12 medium with 20% fetal bovine serum and 1% double antibody.
  • Fig. 4a and Fig. 4b are cell morphology diagrams of adipocytes in co-cultured bovine muscle cells and adipocytes and adipocytes cultured separately after 48 hours of culture, respectively, denoted by FX-F and FF; Fig. 5a and Fig. 5b, respectively
  • FX-F and FF cell morphology of adipocytes in co-cultured bovine muscle cells and adipocytes and the muscle cells cultured separately
  • MX-M and MM the cell morphology of the muscle cells in the co-cultured bovine muscle cells and adipocytes and the muscle cells cultured separately. It can be clearly seen from the figure that the co-cultured adipocytes (or muscle cells) and the adipocytes (or muscle cells) cultured separately have the same morphology, and the cell morphology is obvious.
  • the present invention innovatively establishes an in vitro research model and discloses a cell co-cultivation device.
  • the muscle cells and adipocytes are co-cultured in the same environment. It simulates the microenvironment in the body to a certain extent, not only realizes the direct contact and co-cultivation of adipocytes and muscle cells, but also collects individual muscle cells or adipocytes for subsequent transcriptome and proteome analysis and processing, and its structure is simple and the production method is simple. Simple, easy to operate, strong practicability, and has certain research and promotion value.

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Abstract

本发明提供一种细胞共培养的装置，其是由皿体和皿盖构成，皿体为上开口的中空结构，皿体开口处有皿盖，皿体内有隔离圈，隔离圈将皿体的内部分割为区域I和区域II，隔离圈内设有爬片。利用该装置在实现牛肌细胞与脂肪细胞直接接触共培养的同时，又可将爬片上的单类细胞(肌细胞或脂肪细胞)收集并进行后续转录组及蛋白组的分析处理，以用来研究共培养环境下对肌细胞或脂肪细胞的作用，因此有利于进一步探究肌细胞与脂肪细胞之间的相互作用；经多次观察证实用这种方法培养的肌细胞和脂肪细胞成活效率高、活力好、细胞特征明显，并与单独培养的肌细胞和脂肪细胞形态一致，是一种很好的体外研究模型，具有一定的推广研究价值。

Description

一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法 技术领域
本发明涉及细胞培养领域，具体涉及一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法。
背景技术
相较于单层细胞培养技术，为了能够建立更接近体内环境的培养体系，在20世纪80年代后期，细胞共培养技术应运而生。细胞共培养又称为复合培养或混合培养，是将两种或两种以上的细胞共同培养于同一环境中，由于它考虑到活体细胞间的相互作用，最大程度地模拟体内微环境，弥补了单层细胞培养的缺陷，所以能更好地研究细胞与细胞之间的相互作用。
目前，研究细胞共培养的技术主要包括直接接触共培养和间接接触共培养两种。直接接触共培养是将将两种或两种以上的细胞按照一定的接种比例放在同一培养环境中共同混合培养，其缺陷是无法对单类细胞进行后续转录组或蛋白组的分析处理；间接接触共培养是将两种或两种以上的细胞分别接种于不同的载体上，然后再将这两种载体置于同一培养环境中，较为典型的方法是Transwell共培养体系，包含上室和下室两部分，其中，上室内盛装上层培养液，下室内盛装下层培养液，上、下层培养液以聚碳酸酯膜相隔。将两种细胞分别接种于上、下室内，由于聚碳酸酯膜具有通透性，上、下层的培养液中的成分可以互相扩散，而细胞本身无法通过，所以可以用来研究两种细胞之间的相互作用情况，但同时也存在的问题是：(1)Transwell培养系统上下室以聚碳酸酯膜相隔，为防止细胞穿过，共培养中要选用0.4微米孔径的膜，会影响细胞分泌因子的通过和作用时间；(2)不易对上下室细胞进行较为直观地观察和后续操作；(3)价格昂贵。
牛肉中的脂肪类型主要包括两种：肌间脂肪和肌内脂肪。其中，肌内脂肪是形成牛肉大理石花纹的物质基础，肌内脂肪含量可以改善牛肉的风味、多汁性和嫩度，是衡量牛肉品质的重要因素。在大理石纹牛肉中，肌细胞与脂肪细胞交杂分布，两种细胞通过直接接触，自分泌或旁分泌一些细胞因子，相互调控生命进程。
发明内容
本发明的目的在于提供一种牛肌细胞与脂肪细胞共培养的装置及其培养方法，旨在解决单独培养肌细胞与脂肪细胞，而忽略了活体中肌细胞与脂肪细胞的相互作用，不能全面解析大理石纹牛肉性状形成的分子机理的问题。
本发明的一种细胞共培养装置，是由皿体和皿盖构成，所述皿体为上开口的中空结构，皿体开口处设有皿盖，皿体的内部设有隔离圈，隔离圈为上下开口的中空结构，隔离圈将皿体的内部分割为区域I和区域II，隔离圈的内部为区域I，隔离圈与皿体之间为区域II，隔 离圈的内部设有爬片。
作为本发明的进一步改进，所述的皿体和隔离圈均为圆柱体。
作为本发明的进一步改进，所述的皿体的直径60mm，隔离圈的直径为35mm，爬片为24mm*24mm的盖玻片。
利用上述细胞共培养装置，共培养牛肌细胞与脂肪细胞的方法，其操作步骤如下：
(1)在无菌条件下，分别取牛背最长肌和肾周脂肪组织，切除可见的血管和结缔组织，用5％～10％质量百分比双抗的1xPBS冲洗，然后将组织剪成1mm3大小的小块，备用；
(2)分别在(1)步骤中剪碎的牛背最长肌肌肉组织加入0.25％的II型胶原酶/0.1％的II型中性蛋白酶消化液，在(1)步骤中剪碎的肾周脂肪组织中加入0.25％的I型胶原酶消化液，恒温振荡水浴锅37℃消化直至细浆状，得到细浆液；
(3)将(2)步骤制得的细浆液与等体积有血清培养液混合，终止消化，然后将细胞悬液通过80μm细胞筛过滤，经1200rpm/min离心10分钟，弃上清液以获得细胞沉淀，再加入适量血清培养液使细胞密度1×10 5～2×10 5/ml，吹打混匀，制成细胞悬液；
(4)将(3)步骤制得的细胞悬液接种于权利要求1所述的细胞共培养装置中，加入含10％胎牛血清、1％双抗的DMEM/F12完全培养基，在区域I接种一种细胞，在区域II接种另一种细胞或按照l∶1的比例接种两种细胞混合共培养，并置于37℃、5％CO 2培养箱培养，待细胞贴壁后将隔离圈取出，即完成牛肌细胞与脂肪细胞的共培养。
作为本发明的进一步改进，步骤(3)中的脂肪细胞有血清培养液配方为含10％胎牛血清、1％双抗的DMEM/F12培养基。
作为本发明的进一步改进，步骤(3)中的肌细胞有血清培养液配方为含20％胎牛血清、1％双抗的DMEM/F12培养基。
本发明的有益效果如下：
(1)本发明所提供的细胞共培养装置，结构简单、制作方法简单、易操作，可同时将牛肌细胞与脂肪细胞接种于爬片及皿底区域，实现两种细胞共培养，最大程度地模拟体内微环境，以便于更好地观察细胞与细胞之间的相互作用，弥补了单层细胞培养的缺陷；
(2)本发明提供的细胞共培养装置在实现牛肌细胞与脂肪细胞直接接触共培养的同时，又可将爬片上的单类细胞(肌细胞或脂肪细胞)进行收集进行后续转录组及蛋白组的分析处理，以用来研究共培养环境下对肌细胞或脂肪细胞的作用，因此有利于进一步探究肌细胞与脂肪细胞之间的相互作用；
(3)本发明提供的牛肌细胞与脂肪细胞共培养的培养方法，通过应用所述的细胞共培养装置，用含10％胎牛血清、1％双抗(即10％青霉素/链霉素)的DMEM/F12完全培养基对牛肌细 胞与脂肪细胞进行共培养，经多次观察证实用这种方法培养的肌细胞和脂肪细胞成活效率高、活力好、细胞特征明显，并与单独培养的肌细胞和脂肪细胞形态一致，是一种很好的体外研究模型，具有一定的推广研究价值。
附图说明
图1为本发明提供的牛肌细胞与脂肪细胞共培养装置的整体结构示意图；
图2为本发明提供的牛肌细胞与脂肪细胞共培养装置的侧视图；
图3为本发明提供的牛肌细胞与脂肪细胞共培养装置的俯视图(图中：FF和MM分别为单独培养体系中的脂肪细胞和肌细胞；FX和MX为共培养体系，爬片上分别为脂肪细胞和肌细胞)；
图4a、图4b分别为应用本发明共培养牛肌细胞与脂肪细胞中的脂肪细胞和单独培养的脂肪细胞的细胞形态图；
图5a、图5b分别为应用本发明共培养牛肌细胞与脂肪细胞中的肌细胞和单独培养的肌细胞的细胞形态图。
具体实施方式
本发明提供一种牛肌细胞与脂肪细胞共培养的装置及其培养方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下结合附图和实施例，对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1
如图1和图2所示，本发明所提供的一种细胞共培养装置，是由皿体1和皿盖2构成，所述皿体1为上开口的中空结构，皿体1开口处设有皿盖2，皿体1的内部设有隔离圈4，隔离圈4为上下开口的中空结构，隔离圈4将皿体1的内部分割为区域I和区域II，隔离圈4的内部为区域I，隔离圈4与皿体1之间为区域II，隔离圈4的内部设有爬片3。
所述的皿体1是横截面为直径60mm的圆柱体，隔离圈4是横截面直径为35mm的圆柱形塑料圈，由35mm细胞培养皿改造而成，主要在细胞接种后未贴壁前起到一个隔离的作用。所述的爬片3为24mm*24mm的盖玻片。
实施例2
利用实施例1所述的一种细胞共培养装置，共培养牛肌细胞与脂肪细胞的方法，其操作步骤如下：
(1)在无菌条件下，分别取牛背最长肌和肾周脂肪组织，切除可见的血管和结缔组织，用5％～10％质量百分比双抗(10％青霉素/链霉素)的1xPBS冲洗，然后将组织剪成1mm 3大小的小块，备用；
(2)分别在(1)步骤中剪碎的牛背最长肌肌肉组织加入0.25％的II型胶原酶/0.1％的II型中性蛋白酶消化液，在(1)步骤中剪碎的肾周脂肪组织中加入0.25％的I型胶原酶消化液，恒温振荡水浴锅37℃消化直至细浆状，得到细浆液；
(3)将(2)步骤制得的细浆液与等体积有血清培养液混合，终止消化，然后将细胞悬液通过80μm细胞筛过滤，经1200rpm/min离心10分钟，弃上清液以获得细胞沉淀，再加入适量血清培养液使细胞密度1×10 5～2×10 5/ml，吹打混匀，制成细胞悬液；其中，脂肪细胞有血清培养液配方为含10％胎牛血清、1％双抗()的DMEM/F12培养基；肌细胞有血清培养液配方为含20％胎牛血清、1％双抗的DMEM/F12培养基。
(4)将(3)步骤制得的细胞悬液接种于权利要求1所述的细胞共培养装置中，加入含10％胎牛血清、1％双抗的DMEM/F12完全培养基，在区域I接种一种细胞，在区域II接种另一种细胞或按照1∶1的比例接种两种细胞混合共培养，并置于37℃、5％CO 2培养箱培养，待细胞贴壁后将隔离圈4取出，即完成牛肌细胞与脂肪细胞的共培养；
(5)取出爬片3，使用相差显微镜观察牛肌细胞与脂肪细胞形态，细胞形态结果如图4、图5所示。图4a、图4b分别为培养48小时后，共培养牛肌细胞与脂肪细胞中的脂肪细胞和单独培养的脂肪细胞的细胞形态图，分别用FX-F和FF表示；图5a、图5b分别为培养48小时后，共培养牛肌细胞与脂肪细胞中的肌细胞和单独培养的肌细胞的细胞形态图，分别用MX-M和MM表示。从图中可以明显看出，共培养的脂肪细胞(或肌细胞)和单独培养的脂肪细胞(或肌细胞)形态一致，而且细胞形态明显。
为了探究牛肌细胞与脂肪细胞之间的相互作用，本发明创新的建立起了一种体外研究模型，公开了一种细胞共培养装置，将肌细胞与脂肪细胞在同一环境下共培养，最大程度地模拟体内微环境，不仅实现了脂肪细胞与肌细胞的直接接触共培养，还可对单独的肌细胞或脂肪细胞进行收集进行后续转录组及蛋白组的分析处理，并且其结构简单制作方法简单、易操作，实用性较强，具有一定的研究推广价值。

Claims (6)

  1. 一种细胞共培养装置，是由皿体(1)和皿盖(2)构成，其特征在于：所述皿体(1)为上开口的中空结构，皿体(1)开口处设有皿盖(2)，皿体(1)的内部设有隔离圈(4)，隔离圈(4)为上下开口的中空结构，隔离圈(4)将皿体(1)的内部分割为区域I和区域II，隔离圈(4)的内部为区域I，隔离圈(4)与皿体(1)之间为区域II，隔离圈(4)的内部设有爬片(3)。
  2. 根据权利要求1所述的一种细胞共培养装置，其特征在于所述的皿体(1)和隔离圈(4)均为圆柱体。
  3. 根据权利要求2所述的一种细胞共培养装置，其特征在于所述的皿体(1)的直径60mm，隔离圈(4)的直径为35mm，爬片(3)为24mm*24mm的盖玻片。
  4. 利用权利要求1所述的装置共培养牛肌细胞与脂肪细胞的方法，其操作步骤如下：
    (1)在无菌条件下，分别取牛背最长肌和肾周脂肪组织，切除可见的血管和结缔组织，用5％～10％质量百分比双抗的1xPBS冲洗，然后将组织剪成1mm 3大小的小块，备用；
    (2)分别在(1)步骤中剪碎的牛背最长肌肌肉组织加入0.25％的II型胶原酶/0.1％的II型中性蛋白酶消化液，在(1)步骤中剪碎的肾周脂肪组织中加入0.25％的I型胶原酶消化液，恒温振荡水浴锅37℃消化直至细浆状，得到细浆液；
    (3)将(2)步骤制得的细浆液与等体积有血清培养液混合，终止消化，然后将细胞悬液通过80μm细胞筛过滤，经1200rpm/min离心10分钟，弃上清液以获得细胞沉淀，再加入血清培养液使细胞密度1×10 5～2×10 5/ml，吹打混匀，制成细胞悬液；
    (4)将(3)步骤制得的细胞悬液接种于权利要求1所述的细胞共培养装置中，加入含10％胎牛血清、1％双抗的DMEM/F12完全培养基，在区域I接种一种细胞，在区域II接种另一种细胞或按照1∶1的比例接种两种细胞混合共培养，并置于37℃、5％CO 2培养箱培养，待细胞贴壁后将隔离圈(4)取出，即完成牛肌细胞与脂肪细胞的共培养。
  5. 根据权利要求1所述的共培养牛肌细胞与脂肪细胞的方法，其特征在于步骤(3)中的脂肪细胞有血清培养液配方为含10％胎牛血清、1％双抗的DMEM/F12培养基。
  6. 根据权利要求1所述的共培养牛肌细胞与脂肪细胞的方法，其特征在于步骤(3)中的肌细胞有血清培养液配方为含20％胎牛血清、1％双抗的DMEM/F12培养基。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN203923211U (zh) * 2014-06-24 2014-11-05 谢一泓 一种细胞共培养孔板
CN205188305U (zh) * 2015-10-22 2016-04-27 深圳爱生再生医学科技有限公司 细胞共培养的培养皿
CN107723238A (zh) * 2017-09-29 2018-02-23 东北大学 一种细胞培养装置及其应用
CN107794223A (zh) * 2017-11-07 2018-03-13 广东药科大学附属第医院 细胞共培养装置及模拟体内厌氧菌与需氧细胞相互作用的体外研究模型及方法
CN109486752A (zh) * 2018-12-18 2019-03-19 西北农林科技大学 一种秦川肉牛肌内脂肪细胞分离的方法
CN110511871A (zh) * 2019-09-25 2019-11-29 西北农林科技大学 一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法
CN210736788U (zh) * 2019-09-25 2020-06-12 西北农林科技大学 一种细胞共培养的装置

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN203923211U (zh) * 2014-06-24 2014-11-05 谢一泓 一种细胞共培养孔板
CN205188305U (zh) * 2015-10-22 2016-04-27 深圳爱生再生医学科技有限公司 细胞共培养的培养皿
CN107723238A (zh) * 2017-09-29 2018-02-23 东北大学 一种细胞培养装置及其应用
CN107794223A (zh) * 2017-11-07 2018-03-13 广东药科大学附属第医院 细胞共培养装置及模拟体内厌氧菌与需氧细胞相互作用的体外研究模型及方法
CN109486752A (zh) * 2018-12-18 2019-03-19 西北农林科技大学 一种秦川肉牛肌内脂肪细胞分离的方法
CN110511871A (zh) * 2019-09-25 2019-11-29 西北农林科技大学 一种细胞共培养的装置及牛肌细胞与脂肪细胞的共培养方法
CN210736788U (zh) * 2019-09-25 2020-06-12 西北农林科技大学 一种细胞共培养的装置

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