CN114196539B - In-vitro pump-free culture chip based on microfluidic technology - Google Patents
In-vitro pump-free culture chip based on microfluidic technology Download PDFInfo
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- CN114196539B CN114196539B CN202111540096.7A CN202111540096A CN114196539B CN 114196539 B CN114196539 B CN 114196539B CN 202111540096 A CN202111540096 A CN 202111540096A CN 114196539 B CN114196539 B CN 114196539B
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- 238000000338 in vitro Methods 0.000 title claims abstract description 12
- 238000005516 engineering process Methods 0.000 title claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 40
- 239000012528 membrane Substances 0.000 claims abstract description 13
- 239000002609 medium Substances 0.000 claims description 19
- -1 polyethylene terephthalate Polymers 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 8
- 239000011148 porous material Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 5
- 239000004698 Polyethylene Substances 0.000 claims description 3
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 3
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 3
- 229920000515 polycarbonate Polymers 0.000 claims description 3
- 239000004417 polycarbonate Substances 0.000 claims description 3
- 229920000728 polyester Polymers 0.000 claims description 3
- 229920000573 polyethylene Polymers 0.000 claims description 3
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 3
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 3
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 210000001519 tissue Anatomy 0.000 description 37
- 210000001550 testis Anatomy 0.000 description 10
- 230000021595 spermatogenesis Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000005452 bending Methods 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 210000004336 spermatogonium Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
Classifications
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- 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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- 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
- C12M23/16—Microfluidic devices; Capillary tubes
Abstract
The application discloses an in-vitro pump-free tissue culture chip based on a microfluidic technology, and belongs to the technical field of tissue culture. The application comprises an upper chip and a lower chip, wherein the upper chip is arranged above the lower chip; the surface of the lower chip is provided with a cavity channel and a flow resistance channel, one end of the cavity channel is connected with the inlet, the other end of the cavity channel is connected with one end of the groove-shaped flow resistance channel, and the other end of the flow resistance channel is connected with the outlet; the upper chip is provided with a tissue cavity for placing a sample, a sample loading port is arranged in the tissue cavity, the tissue cavity is arranged above the cavity channel, and a porous membrane is arranged between the tissue cavity and the cavity channel to support the sample; and a culture medium collecting tank, a culture medium storage tank and a culture medium balancing tank are arranged above the upper chip. The chip provided by the application can realize dynamic flow of the culture medium under the condition of no pump valve and external pipeline, and is convenient to prepare and simple to operate.
Description
Technical Field
The application belongs to the technical field of tissue culture, and particularly relates to an in-vitro pump-free tissue culture chip based on a microfluidic technology.
Background
From drug design, discovery and safety analysis to clinical personalized medicine, appropriate in vitro models are required as detection tools in order to more accurately understand the biological basis of the disease and to improve the accuracy of drug screening. Although the structure of the isolated primary tissue is more complex and the function is closer to that of the in vivo, compared with the cell line, the primary tissue is difficult to maintain in vitro for a longer culture time because of being out of the steady-state microenvironment in vivo. In vivo tissue is exposed to physiological fluid conditions, whereas conventional static culture is difficult to provide a similar microenvironment.
In recent years, microfluidic organ chip technology has been developed, and dynamic circulation of culture medium in the chip provides a more physiologically relevant environment for tissue culture and solves many problems of static organ tissue culture systems. Organ-chips have been reported to maintain various types of tissue, including liver, intestinal tract, retina, artery, lymph, tumor xenografts, testes, and the like.
Although microfluidic chips provide a powerful tool for tissue dynamic culture, the fluids of conventional chips require external pump valve actuation, which undoubtedly increases the use threshold and cost.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides an in-vitro pump-free culture chip based on a microfluidic technology, and the chip can realize dynamic flow of culture medium under the conditions of no pump valve and external pipeline, and provides a novel in-vitro tissue culture platform which is simple and easy to use.
The chip adopted for solving the technical problem comprises an upper chip and a lower chip; the surface of the lower chip is provided with a groove-shaped cavity channel, one end of the cavity channel is connected with the inlet, culture solution enters the cavity channel from the inlet, the other end of the cavity channel is connected with one end of a groove-shaped flow resistance channel, and the other end of the flow resistance channel is connected with the outlet; the upper chip is provided with a tissue cavity for placing a sample, a sample loading port is arranged in the tissue cavity, the tissue cavity is arranged above the cavity channel, and a porous membrane is arranged between the tissue cavity and the cavity channel to support the sample; the culture medium collection device is characterized in that a culture medium collection tank, a culture medium storage tank and a culture medium balancing tank are arranged above the upper chip, the bottom of the culture medium collection tank is connected with the outlet, the culture medium storage tank is connected with the inlet, and the culture medium balancing tank is connected with the sample loading port.
Optionally, the flow resistance channels are grooves which are narrow in width, long in length and arranged in a zigzag manner, and the number of the grooves for bending the flow resistance channels is 1-200.
Optionally, the total length of the flow resistance channel is 1-500mm, the width is 10-500 μm, and the depth is 10-500 μm.
Optionally, the culture medium collecting tank, the culture medium storage tank and the culture medium balancing tank are all cylindrical, the bottom of the culture medium collecting tank penetrates through the upper chip to be connected with the outlet, and the bottom of the culture medium storage tank penetrates through the upper chip to be connected with the inlet.
Optionally, the pore diameter of the porous membrane is micro-nano scale, and the pore diameter is 0.01-100 μm.
Optionally, the porous membrane material is selected from one of polymer materials such as polycarbonate, polyethylene terephthalate, polytetrafluoroethylene, polydimethylsiloxane, polyester, polyethylene, polypropylene and the like.
Optionally, the fluid velocity in the flow resistance channel is 0.01-10 μl/min.
Optionally, the number of tissue chambers is at least one.
Optionally, the upper chip, the porous membrane and the lower chip are manufactured by one of an ultrasonic method, a plasma sealing method and a clamp assembling method.
The beneficial effects are that:
(1) The application provides a structure-oriented gravity-driven micro-fluidic tissue culture chip, which can realize dynamic flow of culture medium under the condition of no pump valve and external pipeline, and provides a novel simple and easy-to-use in-vitro tissue culture platform.
(2) The microfluidic nonwoven tissue culture chip provided by the application can be used for detecting tissues by adopting a common biological detection means, including morphological tracking observation, cell dead-living marker staining, cell immunofluorescence staining, gene expression detection, protein detection, flow cytometry, HE staining and the like. The chip is convenient to prepare and simple to operate, and can provide a novel in-vitro platform for microfluidic tissue culture.
Drawings
FIG. 1 is a schematic diagram of the structure of the in vitro pump-free culture chip.
FIG. 2 is a bright field plot of mouse testis tissue growth at different times on-chip in example 2, wherein: (a) is a bright field map of a mouse testis tissue just placed in a chip, (b) is a bright field map of a mouse testis tissue placed in a chip for 40 days, (c) is a bright field map of a mouse testis tissue placed in a chip for 80 days, and (d) is a bright field map of a mouse testis tissue placed in a chip for 160 days.
In the figure, 1, an inlet, 2, a loading port, 3, an upper chip, 4, a lower chip, 5, a porous membrane, 6, a tissue chamber, 7, a sample, 8, a flow resistance channel, 9, a chamber channel, 10, an outlet, 11, a culture medium collecting tank, 12, a culture medium storage tank and 13, a culture medium balancing tank.
Detailed Description
The following examples further illustrate the application, but are not intended to limit it.
Example 1
As shown in fig. 1, the chip of the present embodiment includes an upper chip 3 and a lower chip 4, where the upper chip 3 is disposed above the lower chip 4; the surface of the lower chip 4 is provided with a groove-shaped cavity channel 9, one end of the cavity channel 9 is connected with the inlet 1, culture solution enters the cavity channel 9 from the inlet 1, the other end of the cavity channel 9 is connected with one end of a groove-shaped flow resistance channel 8, and the other end of the flow resistance channel 8 is connected with the outlet 10; the upper chip 3 is provided with a tissue chamber 6 for placing a sample 7, a sample loading port 2 is arranged in the tissue chamber 6, the tissue chamber 6 is arranged above the chamber channel 9, and a porous membrane 5 is arranged between the tissue chamber 6 and the chamber channel 9 to support the sample 7; the culture medium collection tank 11, the culture medium liquid storage tank 12 and the culture medium balancing tank 13 are arranged above the upper chip 3, the bottom of the culture medium collection tank 11 is connected with the outlet 10, the culture medium liquid storage tank 12 is connected with the inlet 1, and the culture medium balancing tank 13 is connected with the sample loading port 2.
The flow resistance channels 8 are grooves which are narrow in width, long in length and are arranged in a zigzag manner, and 1-200 grooves for bending the flow resistance channels 8 are formed.
The total length of the flow resistance channels 8 is 1-500mm, the width is 10-500 μm, and the depth is 10-500 μm.
The culture medium collecting tank 11, the culture medium liquid storage tank 12 and the culture medium balancing tank 13 are all cylindrical, the bottom of the culture medium collecting tank 11 penetrates through the upper chip 3 to be connected with the outlet 10, and the bottom of the culture medium liquid storage tank 12 penetrates through the upper chip 3 to be connected with the inlet 1.
The pore diameter of the porous membrane 5 is micro-nano scale, and the pore diameter is 0.01-100 mu m.
The porous membrane 5 is made of one of polymer materials such as polycarbonate, polyethylene terephthalate, polytetrafluoroethylene, polydimethylsiloxane, polyester, polyethylene, polypropylene and the like.
The fluid velocity in the flow resistance channel 8 is 0.01-10 μl/min.
The number of tissue chambers 6 is at least one.
The upper chip 3, the porous membrane 5 and the lower chip 4 are manufactured by one of an ultrasonic method, a plasma sealing method and a clamp assembling method.
Example 2
In this example, sample 7 was a mouse testis tissue, and the procedure was as follows: the mouse testis tissue is placed in the tissue chamber 6, the medium reservoir 12 and the medium collecting tank 11 are connected to the inlet 1 and the outlet 10, and the medium balancing tank 13 is connected to the loading port 2 of the tissue chamber 6. An appropriate amount of medium is added to the medium reservoir 12, and the medium can flow into the medium balance tank 13 and the medium collection tank 11 under the driving of gravity. The level of the medium in the medium equalizing tank 13 will soon coincide with the medium reservoir 12 and then the medium will slowly flow into the medium collecting tank 11. The method requires the timed addition of medium to the medium reservoir 12 and the removal of medium from the medium reservoir 11.
In 2011, japanese student takehiko ogawa cultured mouse testis tissue by using a gas-liquid interface culture system, successfully replicated the whole process of mouse spermatogonium to spermatogenesis, and proved the fertility of sperms by an in vitro fertilization technology, however, the spermatogenesis efficiency is lower and the maintenance time is short by adopting the method.
The microfluidic tissue culture chip adopted in the embodiment can prolong the testis tissue culture time to 160 days, and the spermatogenesis efficiency is greatly improved compared with that of a gas-liquid interface culture method. The method can provide a scientific tool for researching the spermatogenesis mechanism and developing the regenerative medical technology.
While the application has been described in terms of preferred embodiments, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the application, and it is intended that the application is not limited to the specific embodiments disclosed.
Claims (9)
1. An in-vitro pump-free culture chip based on a microfluidic technology is characterized by comprising an upper chip (3) and a lower chip (4), wherein a groove-shaped cavity channel (9) is arranged on the surface of the lower chip (4), one end of the cavity channel (9) is connected with an inlet (1), culture solution enters the cavity channel (9) from the inlet (1), the other end of the cavity channel (9) is connected with one end of a groove-shaped flow resistance channel (8), and the other end of the flow resistance channel (8) is connected with an outlet (10); the upper chip (3) is provided with a tissue chamber (6) for placing a sample (7), a sample loading port (2) is arranged in the tissue chamber (6), the tissue chamber (6) is arranged above the chamber channel (9), and a porous membrane (5) is arranged between the tissue chamber (6) and the chamber channel (9) to support the sample (7); the culture medium collection device is characterized in that a culture medium collection tank (11), a culture medium liquid storage tank (12) and a culture medium balancing tank (13) are arranged above the upper chip (3), the bottom of the culture medium collection tank (11) is connected with the outlet (10), the culture medium liquid storage tank (12) is connected with the inlet (1), and the culture medium balancing tank (13) is connected with the sample loading port (2).
2. The chip according to claim 1, wherein the flow resistance channels (8) are narrow, long and zigzag grooves, and the number of the bent grooves of the flow resistance channels (8) is 1-200.
3. Chip according to claim 1, characterized in that the flow resistance channel (8) has a total length of 1-500mm, a width of 10-500 μm and a depth of 10-500 μm.
4. The chip according to claim 1, characterized in that the bottom of the medium reservoir (11) is connected to the outlet (10) through the upper chip (3), and the bottom of the medium reservoir (12) is connected to the inlet (1) through the upper chip (3).
5. Chip according to claim 1, characterized in that the pore size of the porous membrane (5) is micro-nano-scale, the pore size being 0.01-100 μm.
6. The chip of claim 1, wherein the porous film (5) is made of one selected from the group consisting of polycarbonate, polyethylene terephthalate, polytetrafluoroethylene, polydimethylsiloxane, polyester, polyethylene, and polypropylene.
7. The chip according to claim 1, characterized in that the fluid velocity in the flow resistance channel (8) is 0.01-10 μl/min.
8. Chip according to claim 1, characterized in that the number of tissue chambers (6) is at least one.
9. The chip according to claim 1, wherein the upper chip (3), the porous membrane (5) and the lower chip (4) are manufactured by one of an ultrasonic method, a plasma sealing method and a jig assembling method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111540096.7A CN114196539B (en) | 2021-12-15 | 2021-12-15 | In-vitro pump-free culture chip based on microfluidic technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111540096.7A CN114196539B (en) | 2021-12-15 | 2021-12-15 | In-vitro pump-free culture chip based on microfluidic technology |
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| Publication Number | Publication Date |
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| CN114196539A CN114196539A (en) | 2022-03-18 |
| CN114196539B true CN114196539B (en) | 2023-11-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202111540096.7A Active CN114196539B (en) | 2021-12-15 | 2021-12-15 | In-vitro pump-free culture chip based on microfluidic technology |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006058280A (en) * | 2004-03-16 | 2006-03-02 | Fuji Photo Film Co Ltd | Assay chip |
| JP2007071711A (en) * | 2005-09-07 | 2007-03-22 | Fujifilm Corp | Analytic chip |
| CN101773861A (en) * | 2010-04-02 | 2010-07-14 | 华中科技大学 | Microfluidic sample feeding method, device and application thereof |
| CN102827769A (en) * | 2012-08-14 | 2012-12-19 | 中国科学院广州生物医药与健康研究院 | Automatic stem cell culture and amplification device based on microfluidics |
| CN106222088A (en) * | 2016-09-21 | 2016-12-14 | 东南大学 | A kind of for animal tissue's micro-fluidic chip that comparison is cultivated in situ |
| WO2020177088A1 (en) * | 2019-03-01 | 2020-09-10 | 深圳市博瑞生物科技有限公司 | Microfluidic chip |
| CN112300940A (en) * | 2020-10-30 | 2021-02-02 | 大连医科大学 | Periodontal soft tissue bionic chip constructed based on microfluidic technology and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080064088A1 (en) * | 2006-09-08 | 2008-03-13 | Michael Shuler | Devices and methods for pharmacokinetic-based cell culture system |
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2021
- 2021-12-15 CN CN202111540096.7A patent/CN114196539B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006058280A (en) * | 2004-03-16 | 2006-03-02 | Fuji Photo Film Co Ltd | Assay chip |
| JP2007071711A (en) * | 2005-09-07 | 2007-03-22 | Fujifilm Corp | Analytic chip |
| CN101773861A (en) * | 2010-04-02 | 2010-07-14 | 华中科技大学 | Microfluidic sample feeding method, device and application thereof |
| CN102827769A (en) * | 2012-08-14 | 2012-12-19 | 中国科学院广州生物医药与健康研究院 | Automatic stem cell culture and amplification device based on microfluidics |
| CN106222088A (en) * | 2016-09-21 | 2016-12-14 | 东南大学 | A kind of for animal tissue's micro-fluidic chip that comparison is cultivated in situ |
| WO2020177088A1 (en) * | 2019-03-01 | 2020-09-10 | 深圳市博瑞生物科技有限公司 | Microfluidic chip |
| CN112300940A (en) * | 2020-10-30 | 2021-02-02 | 大连医科大学 | Periodontal soft tissue bionic chip constructed based on microfluidic technology and application thereof |
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| CN114196539A (en) | 2022-03-18 |
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