CN102206583A - Chip for cell co-culture and co-culture method - Google Patents
Chip for cell co-culture and co-culture method Download PDFInfo
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- CN102206583A CN102206583A CN2010101365194A CN201010136519A CN102206583A CN 102206583 A CN102206583 A CN 102206583A CN 2010101365194 A CN2010101365194 A CN 2010101365194A CN 201010136519 A CN201010136519 A CN 201010136519A CN 102206583 A CN102206583 A CN 102206583A
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Abstract
A chip for cell co-culture and a co-culture method. The chip consists of 2-99 same cell culture structures which are separated by separator plate structures. The co-culture method comprises the steps of: adding a cell suspension in the cell culture structures and culturing in a cell incubator; then observing cell adherence and growth state by placing the chip under a microscope. The chip in the invention utilizes physical blocking effects of the separator plates to form different cell culture areas; so that cell co-culture can be realized by only inoculating different cells in different culture areas. The method is easy to operate and has a high repeatability, besides the chip can be used for detecting a plurality of indexes after the cell co-culture.
Description
Technical field
The present invention relates to the co-culture of cells technology, provide a kind of co-culture of cells with chip and co-culture method especially.
Background technology
It is cell, tissue, organ or small individual taking-up of will live that cell in vitro is cultivated, physiological condition in the analogue body, and the aseptic vitro culture of carrying out is kept its existence and growth, is one of important channel of carrying out fundamental research, drug development research and bio-pharmaceuticals.Conventional cultured in monolayer in vitro method can not provide organizes normal growth to grow required envrionment conditions.Under many circumstances, monolayer cell is cultivated and can be lost original biological characteristics gradually because of the change of external environment, and occurs external and the interior diverse experimental result of body.Create and the interior similarly growing environment of body, impelling cell proliferation, break up and present analog inner tissue 26S Proteasome Structure and Function proterties is the development trend that cell in vitro is cultivated.Co-culture of cells just is based on the extracorporeal culturing method that These characteristics is set up, farthest simulated in vivo environment, keep proterties in the body, interact between observation of cell and the cell, these research and development for disease Molecular Study and new drug are significant.
By the interaction between two kinds of dissimilar cells of means observation of co-culture of cells, see the following several method of mainly containing of bibliographical information at present: (1) mixed culture: the different sorts cytomixis of being studied is cultivated in same cultivation vessel, the direct contact of observation of cell, but the different sorts cellular segregation that is difficult to cultivate is together observed.(2) microcarrier culture method: be about to a kind of cell cultures on microcarrier, another kind of cell cultures is in culture dish, also add the microcarrier cell in the culture dish subsequently, cross certain hour and can observe two kinds of cell interactions, but this method generally should migrate to the culture dish from microcarrier to prevent cell less than 4 hours.(3) transwell cell, this cultivation cover ware bottom is made by the microporous membrane of inorganic or organic materials, because soluble substance can freely pass through, the cell of cultivating in the cover ware can be by film and the cell interaction of cultivating in culture dish.The commercialization of this cell, but cost an arm and a leg, only can disposablely use.(4) utilize conditioned medium to carry out, a kind of cell is cultivated under normal operation, get supernatant after centrifugal, stimulate and hatch another cell.Above several method can satisfy the research needs of Routine Test Lab to a certain extent, but equal existing problems: for mixed culture, be difficult to distinguish two kinds of cells, can't the observation of cell metamorphosis; Cultivate for microcarrier, the The limited time of cell interaction only is fit to the research of the special system of small part, does not have universality; Transwell cell and utilize the method for conditioned medium to observe a kind of cell can't be realized the real-time monitored of cellular response; Above several method all exists reagent consumption big simultaneously, shortcomings such as complex operation.
Summary of the invention
The purpose of this invention is to provide a kind of co-culture of cells with chip and co-culture method.
The invention provides a kind of co-culture of cells chip, this chip is made of 2~99 identical cell cultures structures, and these are cultivated between the structure and separate mutually by diaphragm structure.
Co-culture of cells chip provided by the invention, described cell cultures structure be meant and be used for the zone that cell cultures is used, and this structure is circular or fan-shaped, and by chip material around forming; Diaphragm structure is made up of 2~99 platy structures, and dividing plate is connected with the chip periphery.
Co-culture of cells chip provided by the invention, the height of described diaphragm structure are lower than chip periphery height.
The present invention also provides the method for co-culture of cells, and procedure is: cell suspension is added in the cell cultures structure, put into cell culture incubator and cultivate, then chip is placed the adherent and growing state of microscopically observation of cell;
Select experiment flow according to detecting the index characteristics:
Flow process one: detect the influence of cultivating altogether for the cell migration behavior
Cell suspension is added in the cell cultures structure, put into cell culture incubator and cultivate, treat that cell attachment and cell density are greater than after 80%, gently chip is thrown off, inhaled and remove substratum, PBS washes once, add public substratum, seek two kinds of intercellular defective regions and take pictures at microscopically immediately; Continue to be positioned in the incubator and cultivate, every 24 hours defective region is taken pictures once thereafter, dynamically take pictures continuously according to requirement of experiment and observe a couple of days; Observe the migration situation of cultivating the back cell altogether.
Flow process two: detect the variation of cultivating each cell phenotype of back altogether respectively
Cell suspension is added in the cell cultures structure, put into cell culture incubator and cultivate, treat that cell attachment and cell density greater than after 60%, inhale and remove substratum, add public substratum, make the substratum liquid level be higher than the dividing plate height; Continue to be positioned in the incubator and cultivate, cultivate a couple of days altogether according to requirement of experiment after, can extract to carry out albumen or nucleic acid equimolecular Biological Detection each cell respectively according to detecting the index characteristics, perhaps respectively each cell is carried out optical imagery and detects.
The present invention also provides the method for co-culture of cells, and described public substratum is the common substratum that uses of common cultured cells, and its composition is difference according to the difference of co-cultured cell kind.
The present invention has following advantage:
1. chip of the present invention has utilized the physical barriers effect of dividing plate, forms different cell cultures zones, only needs can realize at the different different cells of cultivation zone inoculation the common cultivation of cell, and is easy and simple to handle, the experimental repeatability height.
2. chip of the present invention can be used for detecting the multiple index after the co-culture of cells.1. after cell attachment grows to 80% chip is taken off, can be detected the influence (as the tumor cell invasion experiment) of common cultivation for the cell migration behavior.2. inhale behind the cell attachment and remove substratum separately, add public substratum, make the substratum liquid level be higher than the dividing plate height, after continuously a couple of days cultivates altogether, detect the variation of cultivating each cell phenotype of back altogether respectively, can carry out optical imagery and detect, also can extract cell and carry out molecular Biological Detection.
3. the chip material bottom surface viscosity of the present invention's employing is big, is easy to and the reversible sealing-in of culture dish, and the solution ne-leakage has excellent biological compatibility, no fluorescence absorption, and cheap, be fit to scale operation.
Description of drawings
Fig. 1 co-culture of cells chip synoptic diagram (front);
Fig. 2 co-culture of cells chip synoptic diagram (side);
Fig. 3 HFL and ACC2 cultivate the influence to the HFL migratory behaviour altogether;
Fig. 4 HFL and GES-1 cultivate the influence to the HFL migratory behaviour altogether.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Embodiment 1
Experimentize by flow process one described flow process, in a side cell cultures structure of chip, inoculate human fibroblasts (HFL), opposite side inoculation people's adenoid cystic cancer cells (ACC2), cell density about 10
6/ ml treats that cell attachment and cell density greater than after 80%, throw off chip gently, inhales and removes substratum, and PBS wash once, adds public culture medium A CCM, immediately at microscopically two kinds of intercellular defective regions of searching and take pictures; Continue to be positioned in the incubator and cultivate, every 24 hours defective region is taken pictures once thereafter, dynamically take pictures continuously according to requirement of experiment and observe a couple of days; Observe the migration situation of cultivating the back cell altogether.As seen the common cultivation of HFL and ACC2 can promote that HFL moves (see figure 3) to ACC2.
Embodiment 2
Experimentize by flow process one described flow process, in a side cell cultures structure of chip, inoculate human fibroblasts (HFL), opposite side inoculation people's gastric epithelial cell (GES-1), cell density about 10
6/ ml treats that cell attachment and cell density greater than after 80%, throw off chip gently, inhales and removes substratum, and PBS wash once, adds public culture medium A CCM, immediately at microscopically two kinds of intercellular defective regions of searching and take pictures; Continue to be positioned in the incubator and cultivate, every 24 hours defective region is taken pictures once thereafter, dynamically take pictures continuously according to requirement of experiment and observe a couple of days; Observe the migration situation of cultivating the back cell altogether.As seen, compare with the common cultivation of ACC2 with HFL, the migration distance of HFL obviously shortened (see figure 4) when HFL and GES-1 carried out common the cultivation.
Embodiment 3
Experimentize by flow process two described flow processs, in a side cell cultures structure of chip, inoculate human fibroblasts (HFL), opposite side inoculation people's adenoid cystic cancer cells (ACC2), cell density about 10
6/ ml puts into cell culture incubator and cultivates, treat cell attachment after, inhale and to remove substratum, add public culture medium A CCM, make the substratum liquid level be higher than the dividing plate height; Continue to be positioned in the incubator and cultivate, cultivate 7 days altogether after, adopt immunofluorescence technique to detect ACC2 cell transformation growth factor beta (TGF-β) change of Expression.
Claims (8)
1. co-culture of cells chip, it is characterized in that: this chip is made of 2~99 identical cell cultures structures, and these are cultivated between structures and separate mutually by diaphragm structure.
2. according to the described co-culture of cells chip of claim 1, it is characterized in that: described cell cultures structure is for circular or fan-shaped, and this cultivation structure is centered on by chip material and forms.
3. according to the described co-culture of cells chip of claim 1, it is characterized in that: described diaphragm structure is made up of 2~99 platy structures, and dividing plate is connected with the chip periphery.
4. according to the described co-culture of cells chip of claim 1, it is characterized in that: the aspect ratio chip periphery height of described diaphragm structure hangs down 0~2cm.
5. method of utilizing the described chip of claim 1 to carry out co-culture of cells, it is characterized in that: procedure is:
Cell suspension is added in the cell cultures structure, put into cell culture incubator and cultivate, then chip is placed the adherent and growing state of microscopically observation of cell.
6. according to the method for the described co-culture of cells of claim 5, it is characterized in that: cell suspension is added in the cell cultures structure, putting into cell culture incubator cultivates, treat that cell attachment and cell density are greater than after 80%, gently chip is thrown off, inhaled and remove substratum, phosphate buffered saline buffer washes once, add public substratum, seek two kinds of intercellular defective regions and take pictures at microscopically immediately; Continue to be positioned in the incubator and cultivate, every 24 hours defective region is taken pictures once thereafter, dynamically take pictures continuously according to requirement of experiment and observe a couple of days; Observe the migration situation of cultivating the back cell altogether.
7. according to the method for the described co-culture of cells of claim 5, it is characterized in that: cell suspension is added in the cell cultures structure, putting into cell culture incubator cultivates, treat that cell attachment and cell density are greater than after 60%, substratum is removed in suction, add public substratum, make the substratum liquid level be higher than the dividing plate height; Continue to be positioned in the incubator and cultivate, cultivate a couple of days altogether according to requirement of experiment after, can extract to carry out albumen or nucleic acid equimolecular Biological Detection each cell respectively according to detecting the index characteristics, perhaps respectively each cell is carried out optical imagery and detects.
8. according to the method for claim 6,7 described co-culture of cells, it is characterized in that: described public substratum is the common substratum that uses of common cultured cells, and its composition is difference according to the difference of co-cultured cell kind.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103710263A (en) * | 2012-09-28 | 2014-04-09 | 江苏吉锐生物技术有限公司 | Cell culture apparatus |
CN103805513A (en) * | 2014-03-12 | 2014-05-21 | 山东大学 | Cell culture plate for cell immunofluorescence and immunochemistry research |
CN106811410A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | External extremely low oxygen inducing function nerve unit forming method based on micro-fluidic chip |
Citations (3)
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CN2322972Y (en) * | 1997-12-04 | 1999-06-09 | 中国科学院新疆化学研究所 | Common culture utensil for plurality of cultures |
CN201071371Y (en) * | 2007-06-01 | 2008-06-11 | 郑州安图绿科生物工程有限公司 | Combined culture medium flat plate |
CN101586075A (en) * | 2009-07-08 | 2009-11-25 | 中国科学院大连化学物理研究所 | Method for detecting cell injury repair ability, and special chip thereof |
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2010
- 2010-03-31 CN CN2010101365194A patent/CN102206583A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN2322972Y (en) * | 1997-12-04 | 1999-06-09 | 中国科学院新疆化学研究所 | Common culture utensil for plurality of cultures |
CN201071371Y (en) * | 2007-06-01 | 2008-06-11 | 郑州安图绿科生物工程有限公司 | Combined culture medium flat plate |
CN101586075A (en) * | 2009-07-08 | 2009-11-25 | 中国科学院大连化学物理研究所 | Method for detecting cell injury repair ability, and special chip thereof |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103710263A (en) * | 2012-09-28 | 2014-04-09 | 江苏吉锐生物技术有限公司 | Cell culture apparatus |
CN103710263B (en) * | 2012-09-28 | 2015-05-27 | 江苏吉锐生物技术有限公司 | Cell culture apparatus |
CN103805513A (en) * | 2014-03-12 | 2014-05-21 | 山东大学 | Cell culture plate for cell immunofluorescence and immunochemistry research |
CN106811410A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | External extremely low oxygen inducing function nerve unit forming method based on micro-fluidic chip |
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Application publication date: 20111005 |