CN104164365A - 一种体外细胞接触式共培养装置及其培养操作方法 - Google Patents
一种体外细胞接触式共培养装置及其培养操作方法 Download PDFInfo
- Publication number
- CN104164365A CN104164365A CN201410386234.4A CN201410386234A CN104164365A CN 104164365 A CN104164365 A CN 104164365A CN 201410386234 A CN201410386234 A CN 201410386234A CN 104164365 A CN104164365 A CN 104164365A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- box body
- barrier film
- cell cultures
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000003501 co-culture Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 11
- 238000000338 in vitro Methods 0.000 title claims abstract description 8
- 238000004113 cell culture Methods 0.000 claims abstract description 37
- 239000012528 membrane Substances 0.000 claims abstract description 10
- 239000004417 polycarbonate Substances 0.000 claims abstract description 9
- 229920000515 polycarbonate Polymers 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 69
- 230000004888 barrier function Effects 0.000 claims description 25
- 239000006285 cell suspension Substances 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 244000309466 calf Species 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 230000001464 adherent effect Effects 0.000 claims description 3
- 239000002356 single layer Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 108090000631 Trypsin Proteins 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 210000004691 chief cell of stomach Anatomy 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims description 2
- 238000005213 imbibition Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000011435 rock Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 239000002699 waste material Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 3
- 239000002207 metabolite Substances 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 239000000969 carrier Substances 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000023402 cell communication Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000001990 heterocyst Anatomy 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
Classifications
-
- 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
- C12M23/02—Form or structure of the vessel
-
- 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
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- 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
- 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
- C12M23/38—Caps; Covers; Plugs; Pouring means
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
-
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
-
- 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
- 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
- C12N5/0656—Adult fibroblasts
-
- 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
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1335—Skeletal muscle cells, myocytes, myoblasts, myotubes
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Clinical Laboratory Science (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种体外细胞接触式共培养装置及其培养操作方法,其是在培养盒体的底端设有盒底,盒体的上端设有盒盖,培养盒体内设有细胞培养槽,所述的培养盒体设为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底直径相同,培养槽的中部设有隔离膜,隔离膜固定于培养槽的中部将培养槽分隔为上下两个培养槽,本发明巧妙的运用了聚酸酯膜将培养槽分隔成上下培养槽,并在其上下的两侧分别接种,翻转后再置于细胞培养盒中,实现了细胞实质性的接触式共培养,便于研究一种细胞代谢物对另一种细胞的影响以及直接接触而导致的胞间作用。
Description
技术领域
本发明涉及一种细胞培养装置,确切的说是一种体外细胞接触式共培养装置及其培养操作方法。
背景技术
在动物体内,细胞处于一个高度信息化的微环境中,其间存在各种复杂的物理化学信号,细胞对各种信号刺激做出响应,以实现其特定的生物学功能。细胞与这种微环境间的相互作用包括来自周围细胞、细胞外基质和可溶性因子的各种复杂的生物化学、生物力学和生物电学信号。其中,细胞与细胞之间是通过直接接触或旁分泌可溶性因子而发生相互作用。20世纪80年代后期,为了建立更类似于体内环境的培养体系,尽可能使体外环境与体内环境相吻合,从而使细胞间能相互沟通信息,相互支撑生长增殖,人们在细胞培养技术的基础上发展出了细胞共培养技术。细胞共培养技术是将2种或2种以上的细胞共同培养于同一环境中,由于其具有更好地反映体内环境的优点,所以这种方法被广泛应用于现代细胞研究中。
目前,现有的细胞共培养体系主要通过两种方法建立:
一是直接共培养体系,是将两种或两种以上的细胞按照一定比例同时或分别接种于细胞培养皿或板孔中,在同一培养环境中进行共同培养,不同种类的细胞之间直接接触。常用的是细胞培养皿和不同孔径的细胞培养板,或者把细胞培养皿或板孔分割成不同空间,把不同细胞接种在底部。
二是间接共培养体系,是将两种或两种以上的细胞分别接种于不同的载体上,然后将这两种载体置于同一培养环境之中,使不同种类的细胞共用同一种培养体系而不直接接触,从而达到研究细胞的分泌或代谢产物对目的细胞生长、迁移、增殖、分化等生物学行为影响的目的。
然而,现有的细胞共培养装置不能满足不同形式和不同目的的培养。传统接触式共培养有的是不同细胞混养在一起,很难调控体系中同型与异型细胞间相互作用比例,且后期深入研究时也很难有效的把不同种细胞分离开,所以不好检测两类细胞增殖分化以及相关基因的表达。有的细胞是生长在分割出不同空间的同一细胞培养皿或板孔中,由于细胞都是贴底壁生长,这种情况下只能实现不同细胞间分泌物质的交流,但并不能实现不同细胞间的实质性接触。就象非接触式共培养体系仅能部分模拟体内细胞间的相互作用,缺乏由异种细胞间直接接触而导致的胞间作用。所以,在细胞体外培养过程中,如何有效模拟体内微环境,调控不同细胞之间、细胞与细胞外微环境间的相互作用,对于细胞形态与功能的维持以及细胞间的相互影响至关重要。
发明内容
本发明目的就是克服上述缺陷,提供一种细胞交流更加充分,可以实现细胞实质性接触式共培养,细胞换液更加方便的体外细胞接触式共培养装置。
本发明的另一个目的就是提供该培养装置的使用操作方法。
本发明的方案是由培养盒体、盒底及盒盖组成,培养盒体的底端设有盒底,盒体的上端设有盒盖,培养盒体内设有细胞培养槽,所述的培养盒体设为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底直径相同,培养槽的中部设有隔离膜,隔离膜为一具有通透性能的聚碳酸酯膜,固定于培养槽的中部将培养槽分隔为上下两个培养槽。
所述的隔离膜为聚碳酸酯膜,其孔径为3-8μm,能够使细胞的分泌物及各种营养因子及其他分子物质通过,而阻止细胞穿过。
本发明的有益效果:首先一是巧妙的运用了聚酸酯膜将培养槽分隔成上下培养槽,并在其上下的两侧分别接种,翻转后再置于细胞培养盒中,实现了细胞实质性的接触式共培养,便于研究一种细胞代谢物对另一种细胞的影响以及直接接触而导致的胞间作用;二是容易控制体系中不同细胞接种比例和细胞间相互作用比例,且后期深入研究时也很容易有效地把不同种细胞分离开,易于检测两类细胞增殖分化以及相关基因的表达;三是两种细胞生长在细胞培养槽的聚碳酸酯膜两侧,取拿细胞培养槽方便易于细胞的换液工作,不易污染。
下面结合附图作进一步详细说明。
附图说明
图1为培养装置结构示意图;
图2为细胞培养槽结构示意图;
图3为细胞培养盒外形示意图;
图4为细胞共培养装置使用状态参考图。
具体实施方式
图1-4中示出的培养盒体2的底端设有盒底1,盒体的上端设有盒盖3,培养盒体内设有细胞培养槽5,所述的培养盒体为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底相同,细胞培养槽的中部设有隔离膜4,隔离膜为一具有通透性能的聚碳酸酯膜,隔离膜固定于培养槽的中部,将培养槽分隔为上下两个培养槽。
图4中,先将C2C12成肌细胞悬液6接种在隔离膜的上侧,盖上盒盖,放置在培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,贴附在隔离膜上壁上,将细胞培养槽反转过来,使贴壁的C2C12细胞翻转在隔离膜的下侧面,再将3T3-L成纤维细胞7接种在隔离膜的上侧面。
共培养装置的操作方法步骤如下
1、向C2C12细胞培养瓶中加入1ml浓度为0.25%胰蛋白酶液消化细胞,再加入浓度为10%胎牛血清的高糖DMEM液体培养基5ml,用胶头滴管搅拌混合均匀,然后将混合液移入10ml离心管中,在离心机上离心5min,弃上清液,离心机转速为1500转/min,使其配制成浓度为3×104个/ml细胞悬液;
2、将配制好的细胞悬液置入细胞培养槽内的隔离膜的上侧,在隔离膜上侧的细胞悬液中接种C2C12成肌细胞悬液,将细胞培养槽置于细胞培养盒中,盖上细胞培养盒盖,放在超净工作台上缓慢摇晃以混匀细胞,然后将细胞培养盒放在37℃的恒温培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,粘附在隔离膜上壁上;
3、将余下的培养液用吸液枪吸出打到废液缸后,把细胞培养槽反转过来,倒置于细胞培养盒内,使贴壁的C2C12细胞翻转在隔离膜的下侧面;
4、按照上述同样的方法将1.5g碳酸氢钠、4.5g葡萄糖混合离心后,用浓度10%胎牛血清的高糖DMEM液体培养基5ml,将其配制成浓度为4×105个/ml细胞悬液;
5、将配制好的细胞悬液置入细胞培养槽隔离膜的上侧,在隔离膜上侧的细胞悬液中接种3T3-L成纤维细胞,这样就形成了在细胞培养槽的聚碳酸脂膜下侧接种C2C12细胞,上侧接种3T3-L细胞的细胞直接接触式共培养状态;
6、将此装置放入37℃的恒温培养箱内孵育,每隔一天为两种细胞换液1次。
由于生长在聚碳酸酯膜的两侧,两种细胞能直接接触,细胞产物也可以充分交流,可以在培养的不同阶段,观察两种细胞的生长情况,并分析相互影响的结果。
本装置实验时可用于两种细胞的接触式共培养,作为载体的通透性膜有利于相邻细胞间的直接交流,很好的模拟了体内细胞共存的情况。
所述的C2C12细胞为一种商业化小鼠成肌细胞系,3T3-L细胞是一种商业化细胞,是从小鼠胚胎克隆的特种细胞系,市场均有售,也可从中国科学院上海细胞生物学研究所;高糖DMEM液体培养基:市场有售,货号:12491-015,或购自Gibco公司;所述的超净工作台,市场有售或购于苏州净化有限责任公司。
Claims (3)
1.一种体外细胞接触式共培养装置,由培养盒体、盒底及盒盖组成,其特征在于:所述培养盒体(2)的底端设有盒底(1),盒体的上端设有盒盖(3),培养盒体内设有细胞培养槽(5)。
2.根据权利要求1所述的体外细胞接触式共培养装置,其特征在于:所述的培养盒体为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底相同,细胞培养槽的中部设有隔离膜(4),隔离膜为一具有通透性能的聚碳酸酯膜,隔离膜固定于培养槽的中部,将培养槽分隔为上下两个培养槽。
3.根据权利要求1或2所述的共培养装置的培养操作方法,其特征在于:它包括下列步骤:
(1)向C2C12细胞培养瓶中加入1ml浓度为0.25%胰蛋白酶液消化细胞,再加入浓度为10%胎牛血清的高糖DMEM液体培养基5ml,用胶头滴管搅拌混合均匀,然后将混合液移入10ml离心管中,在离心机上离心5min,弃上清液,离心机转速为1500转/min,使其配制成浓度为3×104个/ml细胞悬液;
(2)将配制好的细胞悬液置入细胞培养槽内的隔离膜的上侧,在隔离膜上侧的细胞悬液中接种C2C12成肌细胞悬液,将细胞培养槽置于细胞培养盒中,盖上细胞培养盒盖,放在超净工作台上缓慢摇晃以混匀细胞,然后将细胞培养盒放在37℃的恒温培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,粘附在隔离膜上壁上;
(3)将余下的培养液用吸液枪吸出打到废液缸后,把细胞培养槽反转过来,倒置于细胞培养盒内,使贴壁的C2C12细胞翻转在隔离膜的下侧面;
(4)按照上述同样的方法将1.5g碳酸氢钠、4.5g葡萄糖混合离心后,用浓度10%胎牛血清的高糖DMEM液体培养基5ml,将其配制成浓度为4×105个/ml细胞悬液;
(5)将配制好的细胞悬液置入细胞培养槽隔离膜的上侧,在隔离膜上侧的细胞悬液中接种3T3-L成纤维细胞,这样就形成了在细胞培养槽的聚碳酸脂膜下侧接种C2C12细胞,上侧接种3T3-L细胞的细胞直接接触式共培养状态;
(6)将此装置放入37℃的恒温培养箱内孵育,每隔一天为两种细胞换液1次。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410386234.4A CN104164365B (zh) | 2014-08-08 | 2014-08-08 | 一种体外细胞接触式共培养装置及其培养操作方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410386234.4A CN104164365B (zh) | 2014-08-08 | 2014-08-08 | 一种体外细胞接触式共培养装置及其培养操作方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104164365A true CN104164365A (zh) | 2014-11-26 |
CN104164365B CN104164365B (zh) | 2016-06-29 |
Family
ID=51908349
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201410386234.4A Expired - Fee Related CN104164365B (zh) | 2014-08-08 | 2014-08-08 | 一种体外细胞接触式共培养装置及其培养操作方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104164365B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106119108A (zh) * | 2016-06-28 | 2016-11-16 | 潍坊医学院 | 双层流动腔***力学微环境下HSCs与EPCs分层共培养工具和方法 |
CN107345205A (zh) * | 2017-09-06 | 2017-11-14 | 阜阳师范学院 | 一种成熟脂肪细胞去分化培养的装置及方法 |
CN108148759A (zh) * | 2018-03-02 | 2018-06-12 | 河南科技大学 | 一种用于贴壁细胞培养/共培养的多用途透气容器 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811256A (en) * | 1995-10-10 | 1998-09-22 | Monell Chemical Senses Center | Method for measuring sensory irritation in vitro |
US20100273200A1 (en) * | 2005-12-19 | 2010-10-28 | Masami Niwa | In-Vitro Model of Blood-Brain Barrier, In-Vitro Model of Diseased Blood-Brain Barrier, and Drug Screening Method, Analysis Method for Functions of Diseased Blood-Brain Barrier, and Analysis Method for Pathogenesis Using the Same |
CN203065491U (zh) * | 2012-09-28 | 2013-07-17 | 江苏吉锐生物技术有限公司 | 细胞培养装置 |
-
2014
- 2014-08-08 CN CN201410386234.4A patent/CN104164365B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811256A (en) * | 1995-10-10 | 1998-09-22 | Monell Chemical Senses Center | Method for measuring sensory irritation in vitro |
US20100273200A1 (en) * | 2005-12-19 | 2010-10-28 | Masami Niwa | In-Vitro Model of Blood-Brain Barrier, In-Vitro Model of Diseased Blood-Brain Barrier, and Drug Screening Method, Analysis Method for Functions of Diseased Blood-Brain Barrier, and Analysis Method for Pathogenesis Using the Same |
CN203065491U (zh) * | 2012-09-28 | 2013-07-17 | 江苏吉锐生物技术有限公司 | 细胞培养装置 |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106119108A (zh) * | 2016-06-28 | 2016-11-16 | 潍坊医学院 | 双层流动腔***力学微环境下HSCs与EPCs分层共培养工具和方法 |
CN106119108B (zh) * | 2016-06-28 | 2018-12-28 | 潍坊医学院 | 双层流动腔***力学微环境下HSCs与EPCs分层共培养工具和方法 |
CN107345205A (zh) * | 2017-09-06 | 2017-11-14 | 阜阳师范学院 | 一种成熟脂肪细胞去分化培养的装置及方法 |
CN108148759A (zh) * | 2018-03-02 | 2018-06-12 | 河南科技大学 | 一种用于贴壁细胞培养/共培养的多用途透气容器 |
Also Published As
Publication number | Publication date |
---|---|
CN104164365B (zh) | 2016-06-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106544270B (zh) | 一种用于细胞共培养的微流控芯片及其细胞培养方法 | |
CN103146572B (zh) | 一种实现细胞集落均质性生长的装置和方法 | |
CN203048951U (zh) | 嵌入式多细胞共培养装置 | |
CN205188305U (zh) | 细胞共培养的培养皿 | |
CN102399693B (zh) | 一种仿真三维细胞培养器及培养方法 | |
CN204644371U (zh) | 一种集成式多细胞非接触共培养装置 | |
CN104312976A (zh) | 一种肿瘤干细胞的分离方法 | |
CN104164365B (zh) | 一种体外细胞接触式共培养装置及其培养操作方法 | |
CN101711890A (zh) | 一种用于研究胚胎干细胞发育分化的细胞外基质凝胶模型 | |
CN205088253U (zh) | 一种生物实验用培养皿 | |
CN103396995B (zh) | 一种筛选乳腺癌干细胞的三维培养方法 | |
CN217025936U (zh) | 一种细胞培养瓶 | |
CN205676484U (zh) | ***及胚胎运输装置 | |
CN201501859U (zh) | 能在显微镜下直接观察的真菌小培养装置 | |
CN207130275U (zh) | 一种共培养装置 | |
CN209923357U (zh) | 一种用于细胞三维培养的接种装置 | |
CN204198747U (zh) | 一种便于操作的细胞培养袋 | |
CN102363764A (zh) | 一种培养胰腺干/祖细胞的方法 | |
CN102206583A (zh) | 一种细胞共培养用芯片及共培养方法 | |
CN206396253U (zh) | 一种细胞共培养装置 | |
CN101948798B (zh) | 一种羊水来源的间充质干细胞的分离纯化方法 | |
CN204138673U (zh) | 一种培养皿及培养瓶两用装置 | |
CN103589637A (zh) | 细胞体外立体培养*** | |
CN203833942U (zh) | 细胞培养器皿 | |
CN203846033U (zh) | 组合式双面固体培养基的制备装置 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20220217 Address after: 510000 No. 231, Gaotang Road, Tianhe District, Guangzhou City, Guangdong Province (part 0601, 0602, 0603, building 05) (office use only) Patentee after: Guangzhou Chen Yunxian Life Technology Co.,Ltd. Address before: 236037 Qinghe West Road, Fuyang, Anhui Province, No. 100 Patentee before: FUYANG NORMAL University |
|
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160629 |