CN102071139A - Microfluidic chip-based cell three-dimensional co-culture method - Google Patents
Microfluidic chip-based cell three-dimensional co-culture method Download PDFInfo
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- CN102071139A CN102071139A CN2009102200707A CN200910220070A CN102071139A CN 102071139 A CN102071139 A CN 102071139A CN 2009102200707 A CN2009102200707 A CN 2009102200707A CN 200910220070 A CN200910220070 A CN 200910220070A CN 102071139 A CN102071139 A CN 102071139A
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Abstract
The invention discloses a microfluidic chip-based cell three-dimensional co-culture method. A microfluidic chip consists of 6 cell co-culture units and a culture solution channel which is connected with the 6 cell co-culture units, wherein each co-culture unit comprises two cell culture cells A and B; the culture cell A is positioned between the culture cell B and the culture solution channel, and is lower than the culture cell B and the culture solution channel; and the microfluidic chip is used to provide three-dimensional space support for the growth and interaction of different cells. The invention has the characteristics of simple and quick operation, small sample dosage and the like.
Description
Technical field
The present invention relates to the micro-fluidic chip technology is applied to the biomedical research field, a kind of cell three-dimensional co-culture method based on micro-fluidic chip is provided, utilize micro-fluidic chip to grow and interact the three-dimensional space support is provided for cell.
Background technology
Cultivating altogether is the important means of research cell-cell interaction, and the mode for co-culture of cells research mainly contains following several at present: 1) mixed culture; 2) conditioned medium is cultivated; 3) microcarrier culture method; 4) micropore counterdie cover ware culture method.More than all methods all be suitable for studying the biological behaviour of cell on two dimensional surface, with the residing in vivo three-dimensional space of cell very big difference is arranged, this difference is familiar with by more and more researchers.Therefore, for remedying the existing defective of present co-culture of cells system, we send out the new three-dimensional space co-culture of cells research platform of exhibition, for biomedical research provides an effective instrument.The micro-fluidic chip technology to the biomedical sector infiltration, had shown wide application prospect rapidly in recent years, and increasing sign shows that the micro-fluidic chip technology will become an epochmaking platform of biomedical research.The present invention can be on chip for heterocellular growth and interaction provide the three-dimensional medium support, with the conventional two-dimensional research model more more near internal milieu.And the present invention is simple to operate, quick, is easy to the biomedical researcher and accepts.
Summary of the invention
The purpose of this invention is to provide a kind of cell three-dimensional co-culture method based on micro-fluidic chip.
The invention provides a kind of micro-fluidic chip, this micro-fluidic chip is made up of 6 co-culture of cells unit and 1 nutrient solution passage; Each co-culture of cells unit comprises two cell cultures ponds, i.e. A and B, and cultivation pool A links to each other with waste liquid pool A with cell injection port A; Cultivation pool B links to each other with waste liquid pool B with cell injection port B; The nutrient solution passage links to each other with the nutrient solution waste liquid pool with the nutrient solution injection port.
Micro-fluidic chip provided by the invention, described co-culture of cells unit comprises two cell cultures ponds, i.e. A and B; Cultivation pool A is between cultivation pool B and nutrient solution passage, and it highly is lower than cultivation pool B and nutrient solution passage, therefore between cultivation pool A and nutrient solution passage and cultivation pool A and cultivation pool B, form two fluids and block and be connected, prevent that the interior fluid of cultivation pool A enters in cultivation pool B and the nutrient solution passage in the cell-matrix sample introduction process.
Micro-fluidic chip provided by the invention, the material of described micro-fluidic chip is the PDMS polymkeric substance, with the irreversible sealing-in of glass material, keeps the wetting ability on PDMS surface after the Cement Composite Treated by Plasma.
The invention provides a kind of cell three-dimensional co-culture method based on micro-fluidic chip, procedure is as follows: the mixture with a kind of cell and BME under cold condition adds in the injection port A, this mixture flows in the cultivation pool A under the effect of capillary force, then chip is positioned over room temperature and is frozen into glue to BME; After treating the BME formation gel in the cultivation pool A, mixture with another cell and BME under cold condition adds in the injection port B, this mixture flows in the cultivation pool B under the effect of capillary force, and contact with cell-BME mixture in the cultivation pool A in fluid blocking-up junction, then chip is positioned over room temperature and is frozen into glue to the BME in the cultivation pool B; After treating the BME formation gel in the cultivation pool B, add cell culture fluid in the nutrient solution injection port, nutrient solution is full of the nutrient solution passage under the effect of capillary force, and penetrates in cultivation pool A and the B.
A kind of cell three-dimensional co-culture method based on micro-fluidic chip provided by the invention, described BME is a basement membrane-like material, is liquid during low temperature, is frozen into colloidal state during room temperature.
In a word, the present invention can carry out cell three-dimensional and cultivates altogether on more than one square centimeters chip.With respect to traditional research method, for the growth and the interaction of cell provides more approaching intravital microenvironment.And that the present invention has is simple to operate, fast and characteristics such as amount of samples is few.Have important biomedical researching value and economic worth.
Description of drawings
Fig. 1 micro-fluidic chip synoptic diagram of the present invention, wherein (1) co-culture of cells unit, (2) nutrient solution passage, (3) cell injection port A, (4) cell injection port B, (5) waste liquid pool A, (6) waste liquid pool B, (7) nutrient solution injection port, (8) nutrient solution waste liquid pool;
Fig. 2 shows that is cultivated a unitary local enlarged diagram altogether, wherein (1) cell cultures pond A, (2) cell cultures pond B, (3) nutrient solution passage;
Fig. 3 shows the cultivation situation of different time ACC-M cell in three-dimensional medium, as the blank of co-culture of cells research;
Fig. 4 shows different time ACC-M cell and the interaction situation of normal fibroblast (HFL1) in three-dimensional medium;
Fig. 5 shows different time ACC-M cell and the interaction situation of cancer associated fibroblast cell (CAF) in three-dimensional medium.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Used micro-fluidic chip designs voluntarily for this laboratory and prepares.Chip material is the PDMS polymkeric substance, is packaged on glass surface by irreversible sealing technology.As shown in Figure 1, this chip comprises 6 co-culture of cells unit, and each unit (Fig. 2) is made up of the cell cultures pond of two circles.Cell cultures pond A is between nutrient solution passage and cell cultures pond B, and it highly is 1/2 of nutrient solution passage and a cell cultures pond B height.Therefore between cultivation pool A and cultivation pool B and nutrient solution passage, form 2 fluids blocking-up and be connected, prevent at cell sample introduction process medium fluid from cultivation pool A inflow cultivation pool B or nutrient solution passage.The mixture that in the A of cell cultures pond, adds adenoid cystocarcinoma of salivary gland cell ACC-M and Basement Membrane Extract (BME) by cell injection port A at low temperatures, after thing to be mixed forms gel, in the B of cell cultures pond, add at low temperatures by cell injection port B and not contain the Basement Membrane Extract (BME) of any cell as blank, after treating that BME forms gel, in the nutrient solution injection port, add cell culture fluid, place 37 ℃ CO
2Cultivated 6 days in the incubator, took pictures every 2 days and determine cell relative position (Fig. 3).
As described in embodiment 1, used micro-fluidic chip designs voluntarily for this laboratory and prepares.The mixture that in the A of cell cultures pond, adds adenoid cystocarcinoma of salivary gland cell ACC-M and Basement Membrane Extract (BME) by cell injection port A at low temperatures, after thing to be mixed forms gel, the mixture that in the B of cell cultures pond, adds human embryonic lung fibroblast strain HFL1 and Basement Membrane Extract (BME) by cell injection port B at low temperatures, after thing to be mixed forms gel, in the nutrient solution injection port, add cell culture fluid, place 37 ℃ CO
2Cultivated 6 days in the incubator, took pictures every 2 days and determine cell relative position (Fig. 4).
As described in embodiment 1, used micro-fluidic chip designs voluntarily for this laboratory and prepares.The mixture that in the A of cell cultures pond, adds adenoid cystocarcinoma of salivary gland cell ACC-M and Basement Membrane Extract (BME) by cell injection port A at low temperatures, after thing to be mixed forms gel, the mixture that in the B of cell cultures pond, adds cancer associated fibroblast cell strain CAF and Basement Membrane Extract (BME) by cell injection port B at low temperatures, after thing to be mixed forms gel, in the nutrient solution injection port, add cell culture fluid, place 37 ℃ CO
2Cultivated 6 days in the incubator, took pictures every 2 days and determine cell relative position (Fig. 5).
Claims (5)
1. micro-fluidic chip, it is characterized in that: this micro-fluidic chip is made up of 6 co-culture of cells unit (1) and 1 nutrient solution passage (2); Each co-culture of cells unit comprises two cell cultures ponds, i.e. A (3) and B (4), and cultivation pool A links to each other with waste liquid pool A (6) with cell injection port A (5); Cultivation pool B links to each other with waste liquid pool B (8) with cell injection port B (7); The nutrient solution passage links to each other with nutrient solution waste liquid pool (10) with nutrient solution injection port (9).
2. according to the described micro-fluidic chip of claim 1, it is characterized in that: described cell cultures pond A is between cultivation pool B and nutrient solution passage, and it highly is cultivation pool B and nutrient solution channel height half.
3. according to the described micro-fluidic chip of claim 1, it is characterized in that: the material of described micro-fluidic chip is the PDMS polymkeric substance.
4. the described a kind of cell three-dimensional co-culture method of claim 1 based on micro-fluidic chip, it is characterized in that: procedure is as follows:
On ice the mixture of a kind of cell and BME is being added in the injection port A, this mixture flows in cultivation pool A under the effect of capillary force, then chip is positioned over room temperature and is frozen into glue to BME;
After treating the BME formation gel in the cultivation pool A, mixture at another cell and BME on ice adds in the injection port B, this mixture flows in the cultivation pool B under the effect of capillary force, and contact with cell-BME mixture in the cultivation pool A in fluid blocking-up junction, then chip is positioned over room temperature and is frozen into glue to the BME in the cultivation pool B;
After treating the BME formation gel in the cultivation pool B, add cell culture fluid in the nutrient solution injection port, nutrient solution is full of the nutrient solution passage under the effect of capillary force, and penetrates in cultivation pool A and the B.
5. according to the described a kind of cell three-dimensional co-culture method based on micro-fluidic chip of claim 4, it is characterized in that: described BME is a basement membrane-like material, is liquid during low temperature, is frozen into colloidal state during room temperature.
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| CN102746986A (en) * | 2012-07-13 | 2012-10-24 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103041878A (en) * | 2012-12-31 | 2013-04-17 | 苏州汶颢芯片科技有限公司 | Novel drug screening microfluidic chip and preparation method thereof |
| CN103045476A (en) * | 2012-12-31 | 2013-04-17 | 苏州汶颢芯片科技有限公司 | Microfluidic chip for researching interaction between cells and preparation method thereof |
| CN103060197A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Reagent concentration responsive microfluidic cell culture chip and preparation method thereof |
| CN103055974A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Micro-fluidic chip for high-efficiency medicine screening and preparation method of micro-fluidic chip |
| CN103060193A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Novel temperature response micro-fluidic cell culturing chip and preparation method thereof |
| CN103060195A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Microfluidic chip for multi-cell cocultivation and preparation method thereof |
| CN103060194A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Micro-fluidic cell culturing chip and preparation method thereof |
| CN103060196A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Novel efficient micro-fluidic multi-cell co-culturing chip and preparation method thereof |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN103103121A (en) * | 2013-01-17 | 2013-05-15 | 中国科学院深圳先进技术研究院 | Cell-culture microfluidic chip |
| CN102071138B (en) * | 2009-11-23 | 2013-12-04 | 中国科学院大连化学物理研究所 | Microfluidic chip and application thereof |
| TWI509067B (en) * | 2014-04-10 | 2015-11-21 | Nat Univ Tsing Hua | In vitro cell automatic positioning co-cultivation platform systems |
| CN106811415A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of transwell micro-fluidic chips being combined with dimensional culture and preparation method thereof |
| CN107354094A (en) * | 2017-08-07 | 2017-11-17 | 江苏大学 | The equally distributed micro-current controlled cell culture chip of cell and cell sample injection method |
| CN108795755A (en) * | 2018-05-25 | 2018-11-13 | 辽宁工业大学 | One kind being based on micro-fluidic chip cell culture apparatus |
| CN109957597A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | Detect the method and its application and micro-fluidic chip of biofilm formation |
| CN113862151A (en) * | 2021-09-29 | 2021-12-31 | 清华大学 | Microfluidic chip device for cell co-culture and cell co-culture method |
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| CN102071138B (en) * | 2009-11-23 | 2013-12-04 | 中国科学院大连化学物理研究所 | Microfluidic chip and application thereof |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN102746986A (en) * | 2012-07-13 | 2012-10-24 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102746986B (en) * | 2012-07-13 | 2014-07-16 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103055974A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Micro-fluidic chip for high-efficiency medicine screening and preparation method of micro-fluidic chip |
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| CN103045476A (en) * | 2012-12-31 | 2013-04-17 | 苏州汶颢芯片科技有限公司 | Microfluidic chip for researching interaction between cells and preparation method thereof |
| CN103103121B (en) * | 2013-01-17 | 2014-10-08 | 中国科学院深圳先进技术研究院 | Cell-culture microfluidic chip |
| CN103103121A (en) * | 2013-01-17 | 2013-05-15 | 中国科学院深圳先进技术研究院 | Cell-culture microfluidic chip |
| TWI509067B (en) * | 2014-04-10 | 2015-11-21 | Nat Univ Tsing Hua | In vitro cell automatic positioning co-cultivation platform systems |
| CN106811415A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of transwell micro-fluidic chips being combined with dimensional culture and preparation method thereof |
| CN107354094A (en) * | 2017-08-07 | 2017-11-17 | 江苏大学 | The equally distributed micro-current controlled cell culture chip of cell and cell sample injection method |
| CN109957597A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | Detect the method and its application and micro-fluidic chip of biofilm formation |
| CN108795755A (en) * | 2018-05-25 | 2018-11-13 | 辽宁工业大学 | One kind being based on micro-fluidic chip cell culture apparatus |
| CN113862151A (en) * | 2021-09-29 | 2021-12-31 | 清华大学 | Microfluidic chip device for cell co-culture and cell co-culture method |
| CN113862151B (en) * | 2021-09-29 | 2024-03-08 | 清华大学 | Microfluidic chip device for cell co-culture and cell co-culture method |
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Application publication date: 20110525 |