CN110408536A - Cell migration assay device and method - Google Patents
Cell migration assay device and method Download PDFInfo
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- CN110408536A CN110408536A CN201910503722.1A CN201910503722A CN110408536A CN 110408536 A CN110408536 A CN 110408536A CN 201910503722 A CN201910503722 A CN 201910503722A CN 110408536 A CN110408536 A CN 110408536A
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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- 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
- C12M23/46—Means for fastening
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- 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
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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Abstract
The present invention relates to field of biotechnology, and in particular to a kind of Cell migration assay device and method.The present apparatus is made of PMMA fixture, the PDMS plate with cylindrical-array, hollow PMMA plate, culture dish and two pieces of magnet blocks.PDMS plate both sides have aperture, and injection culture solution can be connect with syringe pump.There are four circular holes for PMMA fixture and culture dish surrounding band, successively can be fixed PMMA fixture, the PDMS plate with cylindrical-array, hollow PMMA plate and culture dish with four groups of screw bolt and nut.Magnet is placed above and below the present apparatus can be such that the cylindrical-array on PDMS is tightly attached on culture dish.
Description
Technical field
The present invention relates to a kind of Cell migration assay device and method, it is to probe into collection body cell to move which, which is a kind of,
The cell culture apparatus for creating cell-free region is moved, i.e. device Applied Physics barrier before cell sowing has blocked cell to exist
Attachment in partial region, to form cell attachment region and both regions of cell-free attachment region in a device.It should
Device can be used for cultivating various cells, and then simulate wound healing, probe into the situation of collective's cell migration, and research is not related to
The collective migration process of cellular damage is significantly such as cancer metastasis, inflammation and angiogenesis etc., is additionally related to be permitted
Mostly normal physiology course, such as tissue repair, angiogenesis and wound healing, it is also possible to which the device carries out simulated experiment.Separately
Outside, influence of the device for detection drug to cell activity is also very convenient suitable.
Background technique
Collective's cell migration is a kind of cellular activity of height adjustment, and the various pathology including wound healing are raw
The important component of reason process.Collective's cell migration plays very important effect in the invasion and transfer of malignant tumour,
Therefore research significance is great.In order to study the dynamic migration process of collection body cell, various ex vivo techniques, such as wound have been developed
Healing assay, the test of the room Boyden and germination test.Wherein, wound healing assay is some most normal of research collective's cell migration
Method because they are simple, and can during cell migration visual cells.
In general, it carries out wound healing assay to need that partial region cell is killed or removed, traditionally using chemistry
Method, optical method, electrical method.For chemical method, chemical reagent, such as sodium hydroxide solution, for dissolving cell need to be used
Wound is generated, the size and location of wound need to be precisely controlled the volume of chemicals and position is added dropwise.Optics rule is to pass through
Laser ablation eliminates scheduled wound area, although can produce the repeatable wound with arbitrary shape, and uses optics
Interface carries out micro- sem observation, but higher cost.And electrical method is then using high-frequency ac voltage electroporation and to kill cell,
Wound area is generated, operation difficulty is larger, and technical requirements are higher.
And in typical wound healing assay, the mode of mechanical scratch is generally used, wound is generated by physical scratch-off, can
To use pipette tip, needle, bladder, shaver, rubber police, cotton bud or teflon scraper to carry out mechanical scraping, for generating list
A scratch or multiple scratches.Although this method is widely used, but have the limitation of several common wound healing assays.Firstly, In
In different experiments, the scraping speed and geometry of damage field can change.These factors may make to be difficult between experiment
It is compared.Second, scraping process is related to the mechanical damage to cell, and intracellular substance can be discharged into environment,
Some influences are generated, and cellular damage degree is difficult to control, and cell migration process may be made to complicate.Third uses cell
It is challenging that epimatrix (ECM) coating carrys out modification of surfaces, because scoring processes may destroy coating.In addition, common wound is cured
Handling capacity, reproducibility and the reusability for closing test are usually limited.Generally, for be used to observe and analyze cell into
The effect of row migration is not especially desirable.
Summary of the invention
The present invention in view of the drawbacks of the prior art, provides a kind of Cell migration assay device that can solve these defects
And method.Compared with traditional method, which is not necessarily to the addition of additional chemical reagent, sets without using valuableness such as laser devices
It is standby, without the operation for using various power equipments to carry out various complexity, just with lower-cost PMMA plate, PDMS plate,
Culture dish, magnet block, bolt and nut make the device, greatly reduce cost, simplify experimentation.
In order to achieve the above objectives, the present invention adopts the following technical solutions:
A kind of Cell migration assay device, including PMMA fixture, PDMS plate, hollow PMMA plate and culture dish, it is characterised in that: by
Screw bolt and nut successively fixes PMMA fixture, PDMS plate, hollow PMMA plate and culture dish, and in the Cell migration assay
The placement magnet up and down of device；Working principle: Applied Physics barrier has blocked cell to train in part cell before cell sowing
The attachment for supporting area, to form cell attachment region and both regions of cell-free attachment region in cell culture area, for into
Row Cell migration assay.
The screw bolt and nut is stainless steel material, which is the socket cap of smooth surface.The PMMA folder
Tool be it is rectangular, centre be it is rectangular hollow, quadrangle nearby there are four hole can be passed through by the bolt.The PDMS plate has spacing
There are aperture in 1550 ± 50 μm of cylindrical-array and two sides, and the section radius of the cylinder is 300 ± 3 μm, the aperture radius
It is 500 ± 5 μm, cell culture fluid can be injected with syringe pump.There is rectangular hollow area among the hollow PMMA plate, for confining
The culture region of the culture dish.There are four holes for the culture dish surrounding, corresponding with the hole of the PMMA fixture, by the bolt
Pass through.The magnet is two pieces of same rectangular strong magnets, and smooth surface makes the cylindrical-array be adjacent to the culture dish.
The present invention compared with prior art, have following obvious prominent substantive distinguishing features and significant technology into
Step:
Compared with common scratch experiment, Cell migration assay device of the invention can be stablized using physical barriers manufactures multiple nothings
Cell compartment blocks cell in some districts using the PDMS plate (size of cylindrical-array is as shown in Fig. 2) with cylindrical-array
Attachment in domain is equivalent to and has made multiple circular scratch marks, also just no longer needs to manufacture scratch using mechanical means in this way, makes thin
Cellular damage minimizes, and not only can simplify experimentation, but also multiple cell-free regions are substantially completely consistent, can pass through sight
The situation for examining each region inner cell migration, obtains more accurately as a result, avoiding fortuitous phenomena in the transition process of cell
Interference.In addition to this, the device preparation cost is low, and simple structure is reusable, can be used for the wound healing examination of various cells
In testing, to observe the collective migration situation of cell.
Detailed description of the invention
Fig. 1 is concrete structure diagram of the invention.
Fig. 2 is the PDMS and its dimensional parameters of the invention with cylindrical-array.
Fig. 3 is physical barriers method of the invention and removes the partial enlargement diagram after the barrier.
Fig. 4 is that device in kind of the invention just sets top view.
Fig. 5 is that device in kind of the invention is inverted top view.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below by way of preferred embodiment combination attached drawing
The present invention is described further.The features in the embodiments and the embodiments of the present application can mutual any combination.
Embodiment one:
Referring to Fig. 1 ~ Fig. 5, this Cell migration assay device includes PMMA fixture, PDMS plate, hollow PMMA plate and culture dish, spy
Sign is: successively being fixed PMMA fixture, PDMS plate, hollow PMMA plate and culture dish by screw bolt and nut, and described thin
The placement magnet up and down of born of the same parents' migration experimental provision；Working principle: Applied Physics barrier has blocked cell to exist before cell sowing
The attachment in part cell culture area, to form cell attachment region and both areas of cell-free attachment region in cell culture area
Domain, for carrying out Cell migration assay.
Embodiment two: the present embodiment is basically the same as the first embodiment, and special feature is as follows:
The screw bolt and nut is stainless steel material, which is the socket cap of smooth surface.The PMMA fixture is
It is rectangular, centre be it is rectangular hollow, quadrangle can nearby be passed through there are four hole by the bolt.The PDMS plate has spacing 1550
There are aperture in ± 50 μm of cylindrical-array and two sides, and the section radius of the cylinder is 300 ± 3 μm, and the aperture radius is 500
± 5 μm, cell culture fluid can be injected with syringe pump.There is rectangular hollow area among the hollow PMMA plate, it is described for confining
The culture region of culture dish.There are four holes for the culture dish surrounding, corresponding with the hole of the PMMA fixture, are led to by the bolt
It crosses.The magnet is two pieces of same rectangular strong magnets, and smooth surface makes the cylindrical-array be adjacent to the culture dish.
Embodiment three:
This Cell migration assay device and method carries out experimental implementation using above-mentioned apparatus, it is characterised in that:
The step of experiment, is as follows:
(1) magnet is placed in Cell migration assay device or more, pastes the cylindrical-array on the PDMS plate tightly
In the culture dish interior bottom wall；
(2) culture solution containing osteocyte is slowly injected into the hollow PMMA by the aperture on the PDMS plate with syringe pump
The fixed culture dish part of sheet frame, injects stationary culture after appropriate culture solution；
(3) it cultivates ± 10 minutes 24 hours, and with micro- sem observation, covers with the fixed culture of the hollow PMMA sheet frame to osteocyte
Behind ware region, the magnet is removed, the cylindrical-array on the PDMS plate is made to leave the culture dish interior bottom wall, is generated many
The cell-free region of circle of the same size；
(4) continue culture 20 ~ after forty minutes, using each border circular areas inner cell of micro- sem observation throughout situation.
Example IV: the cell healing assay of osteocyte.
Firstly, immobilized-cell culture ware, hollow PMMA plate is covered on culture dish, and by ready-made with cylindrical-array
PDMS plate is detained from top, is placed PMMA fixture above, is tightened and fixed with four groups of bolt and nuts.Then magnet is placed in dress
It sets up and down, the cylindrical-array on PDMS plate is made tightly to be attached to culture dish interior bottom wall.Then it will contain osteocyte with syringe pump
Culture solution slowly injects the fixed culture dish part of hollow PMMA sheet frame by the aperture on PDMS plate, injects quiet after appropriate culture solution
Set culture.It cultivates general 24 hours, and with micro- sem observation, covers with the fixed culture dish region of hollow PMMA sheet frame to osteocyte
Afterwards, magnet is removed, the cylindrical-array on PDMS plate is made to leave culture dish interior bottom wall, is generated many of the same size round without thin
Born of the same parents region, as shown in Figure 3.Continue to cultivate cell, in this process, osteocyte can gradually be grown to cell-free border circular areas
Extension.After a period of time, using each border circular areas inner cell of micro- sem observation throughout situation, and the migration of osteocyte is analyzed
Situation and active situation.
In example IV, if wanting to observe effect of certain drug to cell by the device, osteocyte can contained
Drug target is added in culture solution, is then similarly operated, observes the migration situation of osteocyte under the same conditions, passes through
Comparison, it may be determined that the active situation of cell, and then influence of the drug to bone cell activity can be analyzed.
Claims (8)
1. a kind of Cell migration assay device, including PMMA fixture, PDMS plate, hollow PMMA plate and culture dish, it is characterised in that:
Successively PMMA fixture, PDMS plate, hollow PMMA plate and culture dish are fixed by screw bolt and nut, and moved in the cell
Move the placement magnet up and down of experimental provision；Working principle: Applied Physics barrier has blocked cell in part before cell sowing
The attachment in cell culture area, so that cell attachment region and both regions of cell-free attachment region are formd in cell culture area,
For carrying out Cell migration assay.
2. Cell migration assay device as described in claim 1, it is characterised in that:
The screw bolt and nut is stainless steel material, which is the socket cap of smooth surface.
3. Cell migration assay device as described in claim 1, it is characterised in that:
The PMMA fixture be it is rectangular, centre be it is rectangular hollow, quadrangle nearby there are four hole can be passed through by the bolt.
4. Cell migration assay device as described in claim 1, it is characterised in that:
The PDMS plate has 1550 ± 50 μm of spacing of cylindrical-array and there are aperture in two sides, and the section radius of the cylinder is
300 ± 3 μm, the aperture radius is 500 ± 5 μm, can inject cell culture fluid with syringe pump.
5. Cell migration assay device as described in claim 1, it is characterised in that:
There is rectangular hollow area among the hollow PMMA plate, for confining the culture region of the culture dish.
6. Cell migration assay device as described in claim 1, it is characterised in that:
There are four holes for the culture dish surrounding, corresponding with the hole of the PMMA fixture, are passed through by the bolt.
7. Cell migration assay device as described in claim 1, it is characterised in that:
The magnet is two pieces of same rectangular strong magnets, and smooth surface makes the cylindrical-array be adjacent to the culture dish.
8. a kind of Cell migration assay method carries out experiment behaviour using Cell migration assay device according to claim 1
Make, it is characterised in that:
The step of experiment, is as follows:
(1) magnet is placed in Cell migration assay device or more, pastes the cylindrical-array on the PDMS plate tightly
In the culture dish interior bottom wall；
(2) culture solution containing osteocyte is slowly injected into the hollow PMMA by the aperture on the PDMS plate with syringe pump
The fixed culture dish part of sheet frame, injects stationary culture after appropriate culture solution；
(3) it cultivates ± 10 minutes 24 hours, and with micro- sem observation, covers with the fixed culture of the hollow PMMA sheet frame to osteocyte
Behind ware region, the magnet is removed, the cylindrical-array on the PDMS plate is made to leave the culture dish interior bottom wall, is generated many
The cell-free region of circle of the same size；
(4) continue culture 20 ~ after forty minutes, using each border circular areas inner cell of micro- sem observation throughout situation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910503722.1A CN110408536A (en) | 2019-06-12 | 2019-06-12 | Cell migration assay device and method |
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| CN201910503722.1A CN110408536A (en) | 2019-06-12 | 2019-06-12 | Cell migration assay device and method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203048919U (en) * | 2013-01-08 | 2013-07-10 | 复旦大学 | Stamp type experimental device for cell migration experiments |
| CN104774747A (en) * | 2015-04-14 | 2015-07-15 | 浙江大学 | Microfluidic liquid drop chip device for cell migration analysis experiment and method |
| CN105838607A (en) * | 2016-05-27 | 2016-08-10 | 上海臻和生物科技有限公司 | Cell cultivation insertion component for scratch experiments and application of cell cultivation insertion component |
| CN106047665A (en) * | 2016-07-29 | 2016-10-26 | 北京大学 | Method for high-throughput cell migration research and cell migration research system |
| CN106281964A (en) * | 2016-08-10 | 2017-01-04 | 中国科学院电子学研究所 | A kind of three-dimensional cell cut chip and preparation method and application |
-
2019
- 2019-06-12 CN CN201910503722.1A patent/CN110408536A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203048919U (en) * | 2013-01-08 | 2013-07-10 | 复旦大学 | Stamp type experimental device for cell migration experiments |
| CN104774747A (en) * | 2015-04-14 | 2015-07-15 | 浙江大学 | Microfluidic liquid drop chip device for cell migration analysis experiment and method |
| CN105838607A (en) * | 2016-05-27 | 2016-08-10 | 上海臻和生物科技有限公司 | Cell cultivation insertion component for scratch experiments and application of cell cultivation insertion component |
| CN106047665A (en) * | 2016-07-29 | 2016-10-26 | 北京大学 | Method for high-throughput cell migration research and cell migration research system |
| CN106281964A (en) * | 2016-08-10 | 2017-01-04 | 中国科学院电子学研究所 | A kind of three-dimensional cell cut chip and preparation method and application |
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Application publication date: 20191105 |