CN112300929A - Microfluidic experimental plate and double-sided cell culture method - Google Patents
Microfluidic experimental plate and double-sided cell culture method Download PDFInfo
- Publication number
- CN112300929A CN112300929A CN201910698604.0A CN201910698604A CN112300929A CN 112300929 A CN112300929 A CN 112300929A CN 201910698604 A CN201910698604 A CN 201910698604A CN 112300929 A CN112300929 A CN 112300929A
- Authority
- CN
- China
- Prior art keywords
- cell culture
- flow channel
- semi
- culture medium
- permeable membrane
- 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
Images
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
- C12M23/16—Microfluidic devices; Capillary tubes
-
- 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
- C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Clinical Laboratory Science (AREA)
- Dispersion Chemistry (AREA)
- Immunology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a microfluidic experimental plate and a double-sided cell culture method, the microfluidic experimental plate comprises an upper flow channel plate, a lower flow channel plate and at least one cell culture cup, wherein the lower flow channel plate is connected to the back of the upper flow channel plate, the cell culture cup is arranged in a cell culture cup clamping groove in the flow channel plate, a front flow channel and a back flow channel are arranged in the lower flow channel plate, the front flow channel is used for conveying a culture medium into the cell culture cup and conveying waste liquid into a waste liquid discharge hole, the back flow channel is used for conveying the culture medium into the cell culture cup clamping groove and conveying the waste liquid into the waste liquid discharge hole, and a sealing film for covering the culture medium injection hole, the back flow channel, the cell culture cup clamping groove and the waste liquid discharge hole is arranged on the back of the lower flow channel. The microfluidic experimental plate integrates and standardizes microfluidic liquid supply control and cell culture, solves all the structure and processing problems encountered in the integration, can realize the co-culture of various cells, and integrates the shearing force stimulation function.
Description
Technical Field
The invention belongs to the field of microfluidics and biomedicine, and relates to a microfluidic experimental plate and a double-sided cell culture method.
Background
Cell culture is widely used in medical real-time detection and biological and pharmaceutical research in universities and colleges. Most cell cultures are currently limited to monolayer cultures of single cells, however, increasing data indicate that the biological performance of single cells and cells in monolayer cultures in animal tissues is not the same. The culture mode for making the cells more similar to the culture mode in the animal body is that various cells are co-cultured, and even a three-dimensional structure of a plurality of layers of cells is constructed. The current academia refers to the use of more complex structures to simulate organ microenvironments as organ chips. Moreover, most organ chips in academia are temporarily designed and processed according to the research needs of specific organs, and have no universality and stability. Indeed, there is a real need for tools for drug development and production for such multi-cell co-culture.
Therefore, how to provide a general cell co-culture tool to integrate and standardize microfluidic liquid supply control and cell culture becomes an important technical problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides a microfluidic assay plate and a double-sided cell culture method, which are used to solve the problem of the lack of standardized tools for multi-cell co-culture in the prior art.
To achieve the above and other related objects, the present invention provides a microfluidic assay plate, comprising:
the upper flow channel plate is internally provided with at least one first culture medium injection hole, at least one second culture medium injection hole, at least one first waste liquid discharge hole, at least one second waste liquid discharge hole and at least one first cell culture cup clamping groove which penetrate through the front surface and the back surface of the upper flow channel plate;
a lower flow channel plate connected to the back surface of the upper flow channel plate, wherein the lower flow channel plate is provided therein with a third medium injection hole, a fourth medium injection hole, a third waste liquid discharge hole, a fourth waste liquid discharge hole, and a second cell culture cup clamping groove which are vertically aligned with the first medium injection hole, the second medium injection hole, the first waste liquid discharge hole, the second waste liquid discharge hole, and the first cell culture cup clamping groove, respectively, wherein the third medium injection hole, the third waste liquid discharge hole, and the second cell culture cup clamping groove all penetrate through the front surface and the back surface of the upper flow channel plate, and the fourth medium injection hole and the fourth waste liquid discharge hole are opened from the front surface of the lower flow channel plate, extend in the direction of the back surface of the lower flow channel plate, and do not penetrate through the back surface of the lower flow channel plate;
the cell culture cup is arranged in the first cell culture cup clamping groove, the bottom of the cell culture cup extends into the second cell culture cup clamping groove but does not reach the plane where the back surface of the lower-layer runner plate is located, the top and the bottom of the cell culture cup are both open, the bottom opening of the cell culture cup is sealed by a first semi-permeable membrane, and the side wall of the cell culture cup is provided with a culture medium input hole and a waste liquid output hole;
a front flow channel which is opened from the front surface of the lower flow channel plate, extends towards the back surface of the lower flow channel plate, and does not penetrate through the back surface of the lower flow channel plate, and is communicated with the fourth culture medium injection hole, the second cell culture cup clamping groove, the culture medium input hole, the culture medium output port and the fourth waste liquid discharge hole, so that the culture medium from the fourth culture medium injection hole is conveyed into the cell culture cup through the culture medium input hole, and the waste liquid is conveyed to the fourth waste liquid discharge hole through the culture medium output hole;
a back flow channel opened from the back of the lower flow channel plate and extending toward the front of the lower flow channel plate but not penetrating the front of the lower flow channel plate, the back flow channel communicating with the third medium injection hole, the second cell culture cup neck, and the third waste liquid discharge hole, so as to transport the medium from the third medium injection hole to the second cell culture cup neck and transport the waste liquid to the third waste liquid discharge hole;
and a sealing film disposed on the back surface of the lower flow channel plate and covering the third medium injection hole, the back surface flow channel, the second cell culture cup neck, and the third waste discharge hole.
Optionally, the cell culture cup still disposes a bowl cover, the top and the equal opening in bottom of bowl cover, just the bottom opening of bowl cover is sealed through the second semi-permeable membrane, the bottom of bowl cover via the open-top of cell culture cup stretches into in the cell culture cup, just the second semi-permeable membrane is located relatively the culture medium input hole with waste liquid delivery outlet top, the open-top of bowl cover is used for adding the culture medium extremely the second semi-permeable membrane top is in order to prevent culture medium evaporation between first semi-permeable membrane and the second semi-permeable membrane, and prevent that the bubble from passing through the second semi-permeable membrane gets into culture medium between first semi-permeable membrane and the second semi-permeable membrane.
Optionally, a first sealing ring is arranged between the inner wall of the first cell culture cup clamping groove and the outer wall of the cell culture cup, a second sealing ring is arranged between the inner wall of the second cell culture cup and the outer wall of the cell culture cup, a first sealing ring fixing groove and a second sealing ring fixing groove are arranged on the outer wall of the cell culture cup, the first sealing ring fixing groove is relatively located above the culture medium input hole and the waste liquid output hole, and the second sealing ring fixing groove is relatively located below the culture medium input hole and the waste liquid output hole.
Optionally, still be equipped with at least one cell culture cup constant head tank in the upper flow channel plate, the constant head tank connect in first cell culture cup draw-in groove one side, the constant head tank certainly upper flow channel plate openly, and toward upper flow channel plate back direction extends, but does not run through the upper flow channel plate back, the side of cell culture cup be equipped with cell culture cup constant head tank matched with location bulge.
Optionally, the number of the first medium injection hole and the third medium injection hole is at least two, the back channel includes N-level sub-channels, N is an integer greater than 1, the first-level channel is connected to the nth-level channel in sequence, the first-level channel includes at least two branches, the number of the branches of the next-level channel is greater than the number of the branches of the previous-level channel, the head end of the first-level channel is connected to the third medium injection hole, the nth-level channel traverses the second cell culture cup clamping groove, and the tail end of the nth-level channel is connected to the third waste liquid discharge hole.
Optionally, the number of the branches of the first-stage sub-channel is equal to the number of the third medium injection holes, and the head end of each branch is connected with one third medium injection hole; the number of branches of the nth stage flow path is equal to the number of the third waste discharge holes, and the end of each branch is connected with one of the third waste discharge holes, respectively.
Optionally, the number of the second medium filling hole, the number of the fourth medium filling hole, the number of the second waste liquid discharging hole and the number of the fourth waste liquid discharging hole are respectively one, the front flow channel includes a sub-flow channel or at least two sub-flow channels which are mutually communicated, and each sub-flow channel passes through at least one second cell culture cup clamping groove.
Optionally, the number of the second medium injection hole, the number of the fourth medium injection hole, the number of the second waste liquid discharge hole, and the number of the fourth waste liquid discharge hole are at least two, the front flow channel includes at least two sub-flow channels that are not communicated with each other, and each sub-flow channel passes through at least one second cell culture cup clamping groove.
The invention also provides a double-sided cell culture method, which comprises the following steps:
providing a microfluidic assay plate according to any one of the above, seeding cells on both sides of the first semi-permeable membrane, respectively;
and supplying a culture medium to the outer surface of the first semi-permeable membrane through the first culture medium injection hole, the third culture medium injection hole, the back flow channel and the second cell culture cup clamping groove, and supplying a culture medium to the inner surface of the first semi-permeable membrane through the second culture medium injection hole, the fourth culture medium injection hole, the front flow channel and the culture medium input hole to culture the cells on both sides of the first semi-permeable membrane.
Optionally, the outer surface of the first semi-permeable membrane is seeded with at least one cell species, the inner surface of the first semi-permeable membrane is seeded with at least one cell species, and the outer surface of the first semi-permeable membrane is at least partially different from the inner surface seeded cells.
Optionally, the method of seeding cells on the outer surface of the first semi-permeable membrane comprises: applying a cell suspension to the outer surface of the first semi-permeable membrane and keeping the outer surface of the first semi-permeable membrane facing upwards for a predetermined time before the cell culture cup is placed in the first cell culture cup channel.
Optionally, the method of seeding cells on the outer surface of the first semi-permeable membrane comprises: after the cell culture cup is arranged in the first cell culture cup clamping groove, introducing cell suspension to the outer surface of the first semi-permeable membrane through the first culture medium injection hole, the third culture medium injection hole, the back flow channel and the second cell culture cup clamping groove, so that cells are inoculated to the outer surface of the first semi-permeable membrane, and the outer surface of the semi-permeable membrane is kept standing upwards for a preset time.
Optionally, the method of seeding cells on the inner surface of the first semi-permeable membrane comprises: and applying a cell suspension to the cell culture cup through the top opening of the cell culture cup, so that the cells are inoculated on the inner surface of the first semi-permeable membrane, and keeping the inner surface of the first semi-permeable membrane upwards and standing for a preset time.
Optionally, the double-sided cell culture method is used for drug stimulation experiments of cells.
Optionally, the microfluidic assay plate includes two first medium injection holes, a drug-containing medium containing a liquid medicine to be tested is introduced into one of the first medium injection holes, a blank medium not containing the liquid medicine to be tested is introduced into the other first medium injection hole, and the drug-containing medium and the blank medium are mixed through the back flow channel to form a drug gradient, so that stimulation of drugs with different concentrations on cells is completed.
Optionally, the two-sided cell culture method is used to test the response of cells to shear force stimulation.
Optionally, the shear force to which the first semi-permeable membrane is subjected is adjusted by adjusting the medium feed rate of the first medium injection hole.
Optionally, the shear force to which the cells on the inner surface of the first semi-permeable membrane are subjected is adjusted by adjusting the medium feed rate of the second medium injection hole.
As described above, the microfluidic experimental plate of the present invention integrates and standardizes microfluidic liquid supply control and cell culture, solving all the structural and processing problems encountered in this integration. The microfluidic experimental plate can realize the co-culture of various cells and realize the integration of the shear force stimulation function on the cell co-culture tool. The microfluidic experimental plate can realize the culture of co-cultured cells on two sides of the semipermeable membrane, so as to simulate intercellular zones among different cells and research the cell interaction passing through the semipermeable membrane. In the bilateral cell culture, the shear force stimulation can be realized on the cells on one side.
Drawings
Fig. 1 is a perspective view of a microfluidic control plate according to the present invention.
Fig. 2 is a view showing a structure of the upper flow channel plate and the lower flow channel plate in a separated state, in which the front and a part of the side surfaces of the upper flow channel plate and the lower flow channel plate are shown.
Fig. 3 is a structural view showing a split structure of the upper flow channel plate and the lower flow channel plate, in which the back and a part of the side surfaces of the upper flow channel plate and the lower flow channel plate are shown.
Fig. 4 is a top view of the upper flow field plate.
Fig. 5 is a bottom view of the upper flow field plate.
Fig. 6 is a plan view of the lower flow field plate.
Fig. 7 is a bottom view of the lower flow field plate.
FIG. 8 is a perspective view of the cell culture cup.
FIG. 9 is a schematic view showing an exploded structure of the cell culture cup.
FIG. 10 is a sectional view of the cell culture cup.
FIG. 11 is a side view of the cell culture cup.
Description of the element reference numerals
100 upper flow channel plate
101 first medium injection hole
102 second Medium injection hole
103 first waste liquid discharge hole
104 second waste liquid discharge hole
105 first cell culture cup neck
106 cell culture cup positioning groove
200 lower runner plate
201 third medium injection hole
202 fourth Medium injection hole
203 third waste liquid discharge hole
204 fourth waste liquid discharge hole
205 second cell culture cup neck
206 front flow passage
207 back flow channel
300 cell culture cup
301 first semi-permeable membrane
302 medium inlet port
303 waste liquid output hole
304 locating projection
305 first seal ring
306 second seal ring
307 first seal ring fixing groove
308 second sealing ring fixing groove
400 cup cover
401 second semi-permeable membrane
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Please refer to fig. 1 to 11. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.
Example one
In the present embodiment, a microfluidic experimental plate is provided, please refer to fig. 1, which shows a three-dimensional structure diagram of the microfluidic experimental plate, and includes an upper flow channel plate 100, a lower flow channel plate 200, and at least one cell culture cup 300, wherein the lower flow channel plate 200 is connected to the back surface of the upper flow channel plate 100, and the cell culture cup 300 is installed in the upper flow channel plate 100 and the lower flow channel plate 200.
For example, the upper flow channel plate 100 and the lower flow channel plate 200 are connected by any one of glue, double-sided tape, pressure film, chemical bonding, and thermocompression bonding.
Referring to fig. 2 to 7, fig. 2 and 3 are exploded structural views of the upper flow channel plate 100 and the lower flow channel plate 200, wherein fig. 2 shows the front and partial side surfaces of the upper flow channel plate 100 and the lower flow channel plate 200, fig. 3 shows the back and partial side surfaces of the upper flow channel plate 100 and the lower flow channel plate 200, fig. 4 is a top view of the upper flow channel plate 100, fig. 5 is a bottom view of the upper flow channel plate 100, fig. 6 is a top view of the lower flow channel plate 200, and fig. 7 is a bottom view of the lower flow channel plate 200.
As shown in FIGS. 1, 2, 3, 4 and 5, the upper flow channel plate 100 is provided with at least one first medium inlet 101, at least one second medium inlet 102, at least one first waste liquid outlet 103, at least one second waste liquid outlet 104 and at least one first cell culture cup 300 slot 105 penetrating the front and rear surfaces of the upper flow channel plate 100, wherein the cell culture cup 300 is mounted in the first cell culture cup 300 slot 105.
Specifically, the bottom of the cell culture cup 300 further extends into the slot 205 of the second cell culture cup 300, but does not reach the plane of the back of the lower flow field plate 200.
Illustratively, after the cell culture cup 300 is placed in the first cell culture cup 300 receiving slot 105, the top surface of the cell culture cup 300 is higher than the front surface of the upper flow channel plate 100, thereby facilitating the placement of the cell culture cup 300. In this embodiment, a flange is further disposed at the top opening of the cell culture cup 300, so as to further facilitate taking the cell culture cup 300. The inlet of the first medium inlet 101, the inlet of the second medium inlet 102, the outlet of the first waste liquid outlet 103, and the outlet of the second waste liquid outlet 104 are protruded from the front surface of the upper channel plate 100, so that the input of the medium and the discharge of the waste liquid are facilitated, for example, a hose may be connected to the inlet of the first medium inlet 101 and the inlet of the second medium inlet 102 to supply the medium, and a hose may be connected to the outlet of the first waste liquid outlet 103 and the outlet of the second waste liquid outlet 104 to discharge the waste liquid. .
Illustratively, the upper flow channel plate 100 further comprises at least one positioning groove 106 for the cell culture cup 300, the positioning groove is connected to one side of the groove 105 of the first cell culture cup 300, the positioning groove is opened from the front surface of the upper flow channel plate 100, extends toward the back surface of the upper flow channel plate 100, but does not penetrate through the back surface of the upper flow channel plate 100, and the side surface of the cell culture cup 300 is provided with a positioning protrusion 304 which is engaged with the positioning groove 106 of the cell culture cup 300.
As shown in fig. 1, 2, 3, 6 and 7, the lower channel plate 200 is provided with a third medium inlet 201, a fourth medium inlet 202, a third waste liquid outlet 203, a fourth waste liquid outlet 204 and a second cell culture cup 300 slot 205 which are vertically aligned with the first medium inlet 101, the second medium inlet 102, the first waste liquid outlet 103, the second waste liquid outlet 104 and the first cell culture cup 300 slot 105, respectively, wherein the third medium inlet 201, the third waste liquid outlet 203 and the second cell culture cup 300 slot 205 penetrate the front and back of the upper channel plate 100, and the fourth medium inlet 202 and the fourth waste liquid outlet 204 are opened from the front of the lower channel plate 200 and extend toward the back of the lower channel plate 200, but does not penetrate the back of the lower flow field plate 200.
Specifically, the lower flow channel plate 200 is provided with a front flow channel 206 and a back flow channel 207, wherein the front flow channel 206 is opened from the front of the lower flow channel plate 200, extends toward the back of the lower flow channel plate 200, but does not penetrate through the back of the lower flow channel plate 200, the front flow channel 206 is communicated with the fourth culture medium injection hole 202, the second cell culture cup 300 clamping groove 205, the culture medium input hole 302, the culture medium output port, and the fourth waste liquid discharge hole 204, so that the culture medium from the fourth culture medium injection hole 202 is conveyed into the cell culture cup 300 through the culture medium input hole 302, and the waste liquid is conveyed to the fourth waste liquid discharge hole 204 through the culture medium output hole.
Illustratively, the number of the second medium inlet 102, the fourth medium inlet 202, the second waste liquid outlet 104 and the fourth waste liquid outlet 204 is at least two, and the front flow channel 206 comprises at least two sub-flow channels that are not connected to each other, and each sub-flow channel passes through at least one of the second cell culture cup 300 slots 205. FIG. 6 shows the case where the number of the second medium inlet 102 and the second waste liquid outlet 104 is 3, and each of the sub-channels passes through 8 second cuvette fastening grooves.
The design that the front flow channel 206 includes at least two sub-flow channels that are not connected to each other can be applied to the situation where each group of inlets corresponds to different kinds of drugs.
In another embodiment, the number of the second medium filling hole 102, the number of the fourth medium filling hole 202, the number of the second waste liquid discharging hole 104 and the number of the fourth waste liquid discharging hole 204 may be one, the front flow channel 206 includes one sub-flow channel or at least two sub-flow channels connected to each other, and each sub-flow channel passes through at least one of the second cell culture cup 300 slots 205.
Specifically, the lower flow channel plate 200 is further provided with a back flow channel 207, the back flow channel 207 is opened from the back of the lower flow channel plate 200, extends toward the front of the lower flow channel plate 200, but does not penetrate the front of the lower flow channel plate 200, and the back flow channel 207 communicates with the third medium injection hole 201, the second cell culture cup 300 clamping groove 205, and the third waste liquid discharge hole 203, so as to convey the medium from the third medium injection hole 201 to the second cell culture cup 300 clamping groove 205, and convey the waste liquid to the third waste liquid discharge hole 203.
Illustratively, the number of the first medium injection hole 101 and the third medium injection hole 201 is at least two, for example, 2 to 10, respectively, so that the medium containing different concentrations of the drug is input through different first medium injection holes 101. In this embodiment, the number of the first medium injection holes 101 is 2, for example.
Illustratively, the back flow channel 207 includes N-stage sub-flow channels, N is an integer greater than 1, wherein the first-stage flow channel to the nth-stage flow channel are connected in sequence, the first-stage flow channel includes at least two branches, the number of branches of the latter-stage flow channel is greater than the number of branches of the former-stage flow channel, the head end of the first-stage flow channel is connected to the third medium injecting hole 201, the nth-stage flow channel traverses the second cell culture cup 300 clamping groove 205, and the tail end of the nth-stage flow channel is connected to the third waste liquid discharging hole 203. The back flow channel 207 is designed to facilitate the formation of a drug gradient.
In this embodiment, the number of the branches of the first-stage sub-channel is equal to the number of the third medium injection holes 201, and the head end of each branch is connected to one third medium injection hole 201; the number of branches of the nth stage flow path is equal to the number of the third waste discharge holes 203, and the end of each branch is connected to one of the third waste discharge holes 203, respectively.
Illustratively, one branch of the nth stage flow channel passes through at least two of the second cell culture cup 300 wells 205. In FIG. 1, a case where one branch of the Nth-stage flow channel passes through at least three culture chambers is shown.
It should be noted that the front channel 206 may also be designed the same as or similar to the back channel 207, so as to be suitable for the situation where the drug needs to be diluted by using a gradient.
Specifically, a sealing film (not shown) is further provided on the back surface of the lower flow channel plate 200, and covers the third medium inlet 201, the back flow channel 207, the second cell culture cup 300 retaining groove 205, and the third waste discharge hole 203.
As an example, the sealing film is selected from any one of a pressure film, a pressure sensitive adhesive film, and a thermocompression bonding sealing film.
Referring to fig. 8 to 11, fig. 8 is a perspective view of the cell culture cup 300, fig. 9 is an exploded view of the cell culture cup 300, fig. 10 is a cross-sectional view of the cell culture cup 300, and fig. 11 is a side view of the cell culture cup 300.
Specifically, the top and the bottom of the cell culture cup 300 are both open, the bottom opening of the cell culture cup 300 is closed by a first semi-permeable membrane 301, and the sidewall of the cell culture cup 300 is provided with a culture medium input hole 302 and a waste liquid output hole 303.
Illustratively, the first semi-permeable membrane 301 is encapsulated on the bottom surface of the cell culture cup 300 by any one of double-sided tape, glue, chemical bonding and thermocompression bonding. The inner surface (the surface facing the inside of the cell culture cup 300) of the first semi-permeable membrane 301 and the outer surface (the surface facing the outside of the cell culture cup 300) of the first semi-permeable membrane 301 are both used as surfaces for adherent growth of cells, and the internal network of the material of the first semi-permeable membrane 301 is a channel for mutual stimulation of cells on both sides through cell secretion.
Specifically, cell culture cup 300 still disposes a bowl cover 400, the top and the equal opening in bottom of bowl cover 400, just the bottom opening of bowl cover 400 seals through second semi-permeable membrane 401, the bottom of bowl cover 400 via the open-top of cell culture cup 300 stretches into in the cell culture cup 300, just second semi-permeable membrane 401 is located relatively culture medium input hole 302 with waste liquid output hole 303 top.
The second semi-permeable membrane 401 is, for example, sealed to the bottom surface of the cap 400 by any one of double-sided tape, glue, chemical bonding and thermocompression bonding. The top opening of the cap 400 is used for adding culture medium to the upper part of the second semi-permeable membrane 401 to prevent the culture medium between the first semi-permeable membrane 301 and the second semi-permeable membrane 401 from evaporating and prevent air bubbles from entering the culture medium between the first semi-permeable membrane 301 and the second semi-permeable membrane 401 through the second semi-permeable membrane 401.
For example, after the cap 400 is installed in the cell culture cup 300, the top surface of the cap 400 is higher than the top surface of the cell culture cup 300, so as to facilitate the installation of the cap 400. In this embodiment, a flange is further disposed at an opening at the top of the cup cover 400, so that the cup cover 400 can be taken conveniently.
For example, a first packing 305 is provided between the inner wall of the locking groove 105 of the first cell culture cup 300 and the outer wall of the cell culture cup 300, a second packing 306 is provided between the inner wall of the second cell culture cup 300 and the outer wall of the cell culture cup 300, a first packing 305 fixing groove 307 and a second packing 306 fixing groove 308 are provided on the outer wall of the cell culture cup 300, the first packing 305 fixing groove is located above the medium inlet hole 302 and the waste liquid outlet hole 303, and the second packing 306 fixing groove is located below the medium inlet hole 302 and the waste liquid outlet hole 303.
For example, the first sealing ring 305 and the second sealing ring 306 may be rubber rings or other materials to prevent liquid from leaking out of the gaps between the upper flow channel plate 100, the lower flow channel plate 200 and the cell culture cup 300.
The microfluidic experimental plate of this example integrates and standardizes microfluidic liquid supply control and cell culture, solving all the structural and processing problems encountered in this integration.
Example two
This example provides a method for performing double-sided cell culture using the microfluidic assay plate described in the first example, including the following steps:
s1: seeding cells on both sides of the first semi-permeable membrane 301, respectively;
s2: the cells on both sides of the first semi-permeable membrane 301 are cultured by supplying the culture medium to the outer surface of the first semi-permeable membrane 301 through the first medium inlet 101, the third medium inlet 201, the back channel 207, the second cell culture cup 300 locking groove 205 and the channels, and supplying the culture medium to the culture chamber on the inner surface of the first semi-permeable membrane 301 through the second medium inlet 102, the fourth medium inlet 202, the front channel 206 and the medium inlet 302. .
Specifically, the cell types seeded on both surfaces of the first semipermeable membrane 301 may be the same or different. In this embodiment, at least one cell type is seeded on the outer surface of the first semi-permeable membrane 301, at least one cell type is seeded on the inner surface of the first semi-permeable membrane 301, and at least a part of the cells seeded on the outer surface and the inner surface of the first semi-permeable membrane 301 are different in type, so that co-culture of a plurality of cells is realized. The microfluidic assay plate is used for culturing co-cultured cells on two sides of the first semi-permeable membrane 301, so that intercellular zones among different cells can be simulated, and the cell interaction through the semi-permeable membrane can be researched.
As an example, the first semi-permeable membrane 301 may be seeded with cells on one side comprising hepatocytes and seeded with cells on the other side comprising antral endothelial cells to partially mimic the in vivo liver environment and facilitate hepatocyte function. During double-sided cell culture, the liver sinusoidal endothelial cells can be activated through hypoxia, the capability of promoting the function of the liver cells is enhanced, no exogenous endothelial cell activator is needed, the introduction of exogenous substances is reduced, and the subsequent cell application is facilitated.
As an example, seeding the outer surface of the first semi-permeable membrane 301 with cells may be performed as follows: before the cell culture cup 300 is placed in the first cell culture cup 300 receiving groove 105, a cell suspension is applied to the outer surface of the first semi-permeable membrane 301, and the outer surface of the first semi-permeable membrane 301 is kept standing for a predetermined time while facing upwards, so as to ensure that cells are attached to the surface of the semi-permeable membrane. For example, a cell suspension is dropped onto the outer surface of the first semipermeable membrane 301, and the outer surface of the first semipermeable membrane 301 is left standing upward for one hour or more.
In another embodiment, seeding the outer surface of the first semi-permeable membrane 301 with cells may also be performed in another way: after the cell culture cup 300 is placed in the first cell culture cup 300 holding groove 105, a cell suspension is introduced into the outer surface of the first semi-permeable membrane 301 of the culture chamber through the first medium inlet 101, the third medium inlet 201, the back flow channel 207 and the second cell culture cup 300 holding groove 205, cells are seeded on the outer surface of the first semi-permeable membrane 301, and the outer surface of the semi-permeable membrane is kept standing upward for a predetermined time.
As an example, the method of seeding cells on the inner surface of the first semi-permeable membrane 301 comprises: after the cells inoculated in the first round are fully attached to the outer surface of the first semi-permeable membrane 301, the assembled microfluidic experiment plate is arranged upright, cell suspension is applied to the cell culture cup 300 through the top opening of the cell culture cup 300, the cells are inoculated to the inner surface of the first semi-permeable membrane 301, and the inner surface of the first semi-permeable membrane 301 is kept upward and kept standing for a preset time.
Illustratively, after the second round of seeded cells is sufficiently attached to the inner surface of the first semi-permeable membrane 301, the cap 400 is inserted into the cell culture cup 300 and an appropriate amount of culture medium is added to the cap 400 to prevent evaporation of the culture medium between the first semi-permeable membrane 301 and the second semi-permeable membrane 401 and to prevent air bubbles from entering between the first semi-permeable membrane 301 and the second semi-permeable membrane 401 through the second semi-permeable membrane 401, as the entry of air bubbles between the first semi-permeable membrane 301 and the second semi-permeable membrane 401 may damage the cells. Hoses are connected to an inlet of the first medium inlet 101, an inlet of the second medium inlet 102, an outlet of the first waste liquid outlet 103, and an outlet of the second waste liquid outlet 104, and then, the introduction of the medium is started to culture the cells. In this example, the culture medium was continuously supplied and the waste liquid was continuously discharged through the waste liquid discharge hole 5 during the cell culture.
It should be noted that both sides of the second semi-permeable membrane 401 may also be seeded with cells, and the scope of the present invention should not be unduly limited herein.
As an example, the two-sided cell culture method is used for drug stimulation experiments of cells. For example, the microfluidic assay plate includes two first medium injection holes 101, a drug-containing medium (e.g., a medium containing a desired highest concentration of a liquid to be assayed) containing a liquid to be assayed is introduced into one of the first medium injection holes 101, a blank medium not containing a liquid to be assayed is introduced into the other first medium injection hole 101, and the drug-containing medium and the blank medium form a drug gradient after being mixed by a branch structure at the downstream of the back flow channel 207, thereby completing stimulation of different concentrations of drugs on cells.
As an example, the culture medium that the cells on the outer surface of the first semi-permeable membrane 301 contact contains a drug, the culture medium that the cells on the inner surface of the first semi-permeable membrane 301 contact does not contain a drug, and there is no concern that the upper and lower culture media will affect each other. Since, in theory, the drug in the lower medium cannot penetrate the cells directly due to the presence of cells on both sides of the first semi-permeable membrane 301, there is no concern that the drug will penetrate the cells directly through the first semi-permeable membrane 301 into the upper medium. In addition, since the medium is continuously fed, even if drug molecules pass through the first semi-permeable membrane 301, they are diluted and washed away.
Of course, in other embodiments, the media contacting the cells on the outer surface and the cells on the inner surface of the first semi-permeable membrane 301 may also contain drugs, and the front flow channel 206 may be designed to form a drug gradient with the same or similar design as the back flow channel 207.
By way of example, the two-sided cell culture method can also be used to test cells for response to shear force stimuli. In the bilateral cell culture, the shear force stimulation can be realized on the cells on one side. For example, when the microfluidic experiment plate is used for carrying out the co-culture of the hepatic cells and the endothelial cells of the hepatic sinus in a standardized way, the stimulation research of the endothelial cells on the fluid shear force can be completed.
Specifically, in the shear force stimulation experiment, the shear force applied to the cells on both sides of the first semipermeable membrane 301 can be adjusted by adjusting the medium introduction speed of the first medium injection hole 101.
Specifically, the shearing force applied to the cells on the inner surface of the first semipermeable membrane 301 can be adjusted by adjusting the medium introduction speed of the second medium injection hole 102.
In conclusion, the microfluidic experimental plate of the present invention integrates and standardizes microfluidic liquid supply control and cell culture, and solves all the structural and processing problems encountered in this integration. The microfluidic experimental plate can realize the co-culture of various cells and realize the integration of the shear force stimulation function on the cell co-culture tool. The microfluidic experimental plate can realize the culture of co-cultured cells on two sides of the semipermeable membrane, so as to simulate intercellular zones among different cells and research the cell interaction passing through the semipermeable membrane. In the bilateral cell culture, the shear force stimulation can be realized on the cells on one side. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (18)
1. A microfluidic assay plate, comprising:
the upper flow channel plate is internally provided with at least one first culture medium injection hole, at least one second culture medium injection hole, at least one first waste liquid discharge hole, at least one second waste liquid discharge hole and at least one first cell culture cup clamping groove which penetrate through the front surface and the back surface of the upper flow channel plate;
a lower flow channel plate connected to the back surface of the upper flow channel plate, wherein the lower flow channel plate is provided therein with a third medium injection hole, a fourth medium injection hole, a third waste liquid discharge hole, a fourth waste liquid discharge hole, and a second cell culture cup clamping groove which are aligned with the first medium injection hole, the second medium injection hole, the first waste liquid discharge hole, the second waste liquid discharge hole, and the first cell culture cup clamping groove, respectively, wherein the third medium injection hole, the third waste liquid discharge hole, and the second cell culture cup clamping groove all penetrate through the front surface and the back surface of the upper flow channel plate, and the fourth medium injection hole and the fourth waste liquid discharge hole are all opened from the front surface of the lower flow channel plate and extend in the direction of the back surface of the lower flow channel plate, but do not penetrate through the back surface of the lower flow channel plate;
the cell culture cup is arranged in the first cell culture cup clamping groove, the bottom of the cell culture cup extends into the second cell culture cup clamping groove but does not reach the plane where the back surface of the lower-layer runner plate is located, the top and the bottom of the cell culture cup are both open, the bottom opening of the cell culture cup is sealed by a first semi-permeable membrane, and the side wall of the cell culture cup is provided with a culture medium input hole and a waste liquid output hole;
a front flow channel which is opened from the front surface of the lower flow channel plate, extends towards the back surface of the lower flow channel plate, and does not penetrate through the back surface of the lower flow channel plate, and is communicated with the fourth culture medium injection hole, the second cell culture cup clamping groove, the culture medium input hole, the culture medium output port and the fourth waste liquid discharge hole, so that the culture medium from the fourth culture medium injection hole is conveyed into the cell culture cup through the culture medium input hole, and the waste liquid is conveyed to the fourth waste liquid discharge hole through the culture medium output hole;
a back flow channel opened from the back of the lower flow channel plate and extending toward the front of the lower flow channel plate but not penetrating the front of the lower flow channel plate, the back flow channel communicating with the third medium injection hole, the second cell culture cup neck, and the third waste liquid discharge hole, so as to transport the medium from the third medium injection hole to the second cell culture cup neck and transport the waste liquid to the third waste liquid discharge hole;
and a sealing film disposed on the back surface of the lower flow channel plate and covering the third medium injection hole, the back surface flow channel, the second cell culture cup neck, and the third waste discharge hole.
2. The microfluidic assay plate of claim 1, wherein: the cell culture cup still disposes a bowl cover, the top and the equal opening in bottom of bowl cover, just the bottom opening of bowl cover is sealed through second semi-permeable membrane, the bottom of bowl cover via the open-top of cell culture cup stretches into in the cell culture cup, just the second semi-permeable membrane is located relatively the culture medium input hole with waste liquid delivery outlet top, the open-top of bowl cover is used for adding the culture medium extremely the second semi-permeable membrane top is in order to prevent first semi-permeable membrane and the evaporation of culture medium between the second semi-permeable membrane, and prevent that the bubble from passing through the second semi-permeable membrane gets into first semi-permeable membrane and the culture medium between the second semi-permeable membrane.
3. The microfluidic assay plate of claim 1, wherein: the cell culture cup is characterized in that a first sealing ring is arranged between the inner wall of the first cell culture cup clamping groove and the outer wall of the cell culture cup, a second sealing ring is arranged between the inner wall of the second cell culture cup and the outer wall of the cell culture cup, a first sealing ring fixing groove and a second sealing ring fixing groove are formed in the outer wall of the cell culture cup, the first sealing ring fixing groove is relatively located above the culture medium input hole and the waste liquid output hole, and the second sealing ring fixing groove is relatively located below the culture medium input hole and the waste liquid output hole.
4. The microfluidic assay plate of claim 1, wherein: still be equipped with an at least cell culture cup constant head tank in the upper flow channel plate, the constant head tank connect in first cell culture cup draw-in groove one side, the constant head tank certainly the openly opening of upper flow channel plate, and toward upper flow channel plate back direction extends, but does not run through the upper flow channel plate back, the side of cell culture cup be equipped with cell culture cup constant head tank matched with location bulge.
5. The microfluidic assay plate of claim 1, wherein: the first culture medium injection hole with the quantity in third culture medium injection hole is at least two respectively, the back runner includes N level sub-runner, and N is for being greater than 1 integer, and wherein, first order runner to nth level runner connect gradually, and first order runner includes two at least branches, and the branch quantity of back level runner is greater than the branch quantity of preceding level runner, the head end of first order runner connect in the third culture medium injection hole, nth level runner crosses second cell culture cup draw-in groove, and the end of nth level runner connect in the third waste liquid discharge hole.
6. The microfluidic assay plate of claim 5, wherein: the number of the branches of the first-stage sub-channel is equal to the number of the third culture medium injection holes, and the head end of each branch is connected with one third culture medium injection hole; the number of branches of the nth stage flow path is equal to the number of the third waste discharge holes, and the end of each branch is connected with one of the third waste discharge holes, respectively.
7. The microfluidic assay plate of claim 1, wherein: the number of the second culture medium injection hole, the number of the fourth culture medium injection hole, the number of the second waste liquid discharge hole and the number of the fourth waste liquid discharge hole are respectively one, the front flow channel comprises a sub-flow channel or at least two sub-flow channels which are mutually communicated, and each sub-flow channel at least passes through one second cell culture cup clamping groove.
8. The microfluidic assay plate of claim 1, wherein: the number of the second culture medium injection hole, the number of the fourth culture medium injection hole, the number of the second waste liquid discharge hole and the number of the fourth waste liquid discharge hole are at least two, the front flow channel comprises at least two sub-flow channels which are not communicated with each other, and each sub-flow channel at least passes through one second cell culture cup clamping groove.
9. A double-sided cell culture method is characterized by comprising the following steps:
providing a microfluidic assay plate according to any one of claims 1 to 8, seeding cells on both sides of the first semi-permeable membrane, respectively;
and supplying a culture medium to the outer surface of the first semi-permeable membrane through the first culture medium injection hole, the third culture medium injection hole, the back flow channel and the second cell culture cup clamping groove, and supplying a culture medium to the inner surface of the first semi-permeable membrane through the second culture medium injection hole, the fourth culture medium injection hole, the front flow channel and the culture medium input hole to culture the cells on both sides of the first semi-permeable membrane.
10. The bifacial cell culture method of claim 9, wherein: the outer surface of the first semi-permeable membrane is seeded with at least one cell species, the inner surface of the first semi-permeable membrane is seeded with at least one cell species, and the outer surface of the first semi-permeable membrane is at least partially different from the inner surface seeded cells.
11. The bifacial cell culture method of claim 9, wherein: a method of seeding cells on the outer surface of the first semi-permeable membrane comprises: applying a cell suspension to the outer surface of the first semi-permeable membrane and keeping the outer surface of the first semi-permeable membrane facing upwards for a predetermined time before the cell culture cup is placed in the first cell culture cup channel.
12. The bifacial cell culture method of claim 9, wherein: a method of seeding cells on the outer surface of the first semi-permeable membrane comprises: after the cell culture cup is arranged in the first cell culture cup clamping groove, introducing cell suspension to the outer surface of the first semi-permeable membrane through the first culture medium injection hole, the third culture medium injection hole, the back flow channel and the second cell culture cup clamping groove, so that cells are inoculated to the outer surface of the first semi-permeable membrane, and the outer surface of the semi-permeable membrane is kept standing upwards for a preset time.
13. The bifacial cell culture method of claim 9, wherein: a method of seeding cells on the inner surface of the first semi-permeable membrane comprises: and applying a cell suspension to the cell culture cup through the top opening of the cell culture cup, so that the cells are inoculated on the inner surface of the first semi-permeable membrane, and keeping the inner surface of the first semi-permeable membrane upwards and standing for a preset time.
14. The bifacial cell culture method of claim 9, wherein: the double-sided cell culture method is used for drug stimulation experiments of cells.
15. The bifacial cell culture method of claim 14, wherein: the micro-fluidic experiment plate comprises two first culture medium injection holes, a drug-containing culture medium containing a liquid medicine to be detected is introduced into one of the first culture medium injection holes, a blank culture medium not containing the liquid medicine to be detected is introduced into the other first culture medium injection hole, and the drug-containing culture medium and the blank culture medium form a drug gradient after being mixed through the back flow channel, so that stimulation of drugs with different concentrations on cells is completed.
16. The bifacial cell culture method of claim 9, wherein: the two-sided cell culture method is used to test the response of cells to shear force stimulation.
17. The bifacial cell culture method of claim 16, wherein: the shearing force applied to the cells on both sides of the first semipermeable membrane is adjusted by adjusting the medium introduction speed of the first medium injection hole.
18. The bifacial cell culture method of claim 16, wherein: and adjusting the culture medium feeding speed of the second culture medium injection hole to adjust the shearing force applied to the cells on the inner surface of the first semi-permeable membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910698604.0A CN112300929B (en) | 2019-07-31 | 2019-07-31 | Microfluidic experiment plate and double-sided cell culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910698604.0A CN112300929B (en) | 2019-07-31 | 2019-07-31 | Microfluidic experiment plate and double-sided cell culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112300929A true CN112300929A (en) | 2021-02-02 |
| CN112300929B CN112300929B (en) | 2022-12-02 |
Family
ID=74485805
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910698604.0A Active CN112300929B (en) | 2019-07-31 | 2019-07-31 | Microfluidic experiment plate and double-sided cell culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112300929B (en) |
Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3924089A (en) * | 1988-08-10 | 1990-02-15 | Endotronics, Inc. | Cell culture apparatus |
| CA2106918A1 (en) * | 1992-09-28 | 1994-03-29 | Susan Barker | Cell Culture Insert |
| US5707869A (en) * | 1994-06-28 | 1998-01-13 | Wolf; Martin L. | Compartmentalized multiple well tissue culture plate |
| AU2006335679A1 (en) * | 2006-01-16 | 2007-07-19 | Reliance Life Sciences Pvt Ltd | Device for culturing and transporting cells |
| CN101629143A (en) * | 2008-12-02 | 2010-01-20 | 中国科学院上海微系统与信息技术研究所 | Microfluidic cell array chip for high-throughput medicament screening, method and use |
| US20130143230A1 (en) * | 2011-12-02 | 2013-06-06 | The Trustees Of The Stevens Institute Of Technology | Microfluidic-based cell-culturing platform and method |
| CN103981096A (en) * | 2014-05-27 | 2014-08-13 | 东南大学 | Two-layer cell culture system organ chip and preparation method thereof |
| WO2014175710A1 (en) * | 2013-04-26 | 2014-10-30 | 나노바이오시스 주식회사 | Minute fluid cell chip, method for culturing cell using same, and apparatus for analyzing cell image using same |
| CN104877905A (en) * | 2015-05-04 | 2015-09-02 | 清华大学深圳研究生院 | Cell in-vitro co-culture microfluidic chip, system and method |
| CN105754855A (en) * | 2016-04-12 | 2016-07-13 | 中国航天员科研训练中心 | Fed-batch type two-layer cell co-culture chip |
| WO2016117281A1 (en) * | 2015-01-21 | 2016-07-28 | 東洋製罐グループホールディングス株式会社 | Cell culture container, cell culture method, and method for using cell culture container |
| CN106238112A (en) * | 2016-08-27 | 2016-12-21 | 宋波 | A kind of micro-fluidic chip and the application in the qualification and drug sensitive experiment of pathogen thereof |
| CN106399076A (en) * | 2016-09-14 | 2017-02-15 | 南京大学 | Micro-fluidic gel gas-liquid interface flue gas exposing device |
| KR101965076B1 (en) * | 2017-12-27 | 2019-04-02 | 고려대학교 산학협력단 | Three-dimensional cell culture chip |
| CN208844091U (en) * | 2018-09-05 | 2019-05-10 | 上海新微技术研发中心有限公司 | dPCR in-situ chip based on through hole structure and microfluidics |
| CN109789406A (en) * | 2016-08-10 | 2019-05-21 | 新加坡科技研究局 | Integrated micro flow control system for cultivating and testing |
-
2019
- 2019-07-31 CN CN201910698604.0A patent/CN112300929B/en active Active
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3924089A (en) * | 1988-08-10 | 1990-02-15 | Endotronics, Inc. | Cell culture apparatus |
| CA2106918A1 (en) * | 1992-09-28 | 1994-03-29 | Susan Barker | Cell Culture Insert |
| US5707869A (en) * | 1994-06-28 | 1998-01-13 | Wolf; Martin L. | Compartmentalized multiple well tissue culture plate |
| AU2006335679A1 (en) * | 2006-01-16 | 2007-07-19 | Reliance Life Sciences Pvt Ltd | Device for culturing and transporting cells |
| CN101629143A (en) * | 2008-12-02 | 2010-01-20 | 中国科学院上海微系统与信息技术研究所 | Microfluidic cell array chip for high-throughput medicament screening, method and use |
| US20130143230A1 (en) * | 2011-12-02 | 2013-06-06 | The Trustees Of The Stevens Institute Of Technology | Microfluidic-based cell-culturing platform and method |
| WO2014175710A1 (en) * | 2013-04-26 | 2014-10-30 | 나노바이오시스 주식회사 | Minute fluid cell chip, method for culturing cell using same, and apparatus for analyzing cell image using same |
| CN103981096A (en) * | 2014-05-27 | 2014-08-13 | 东南大学 | Two-layer cell culture system organ chip and preparation method thereof |
| WO2016117281A1 (en) * | 2015-01-21 | 2016-07-28 | 東洋製罐グループホールディングス株式会社 | Cell culture container, cell culture method, and method for using cell culture container |
| CN104877905A (en) * | 2015-05-04 | 2015-09-02 | 清华大学深圳研究生院 | Cell in-vitro co-culture microfluidic chip, system and method |
| CN105754855A (en) * | 2016-04-12 | 2016-07-13 | 中国航天员科研训练中心 | Fed-batch type two-layer cell co-culture chip |
| CN109789406A (en) * | 2016-08-10 | 2019-05-21 | 新加坡科技研究局 | Integrated micro flow control system for cultivating and testing |
| CN106238112A (en) * | 2016-08-27 | 2016-12-21 | 宋波 | A kind of micro-fluidic chip and the application in the qualification and drug sensitive experiment of pathogen thereof |
| CN106399076A (en) * | 2016-09-14 | 2017-02-15 | 南京大学 | Micro-fluidic gel gas-liquid interface flue gas exposing device |
| KR101965076B1 (en) * | 2017-12-27 | 2019-04-02 | 고려대학교 산학협력단 | Three-dimensional cell culture chip |
| CN208844091U (en) * | 2018-09-05 | 2019-05-10 | 上海新微技术研发中心有限公司 | dPCR in-situ chip based on through hole structure and microfluidics |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112300929B (en) | 2022-12-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9388374B2 (en) | Microfluidic cell culture systems | |
| Tehranirokh et al. | Microfluidic devices for cell cultivation and proliferation | |
| JP7398955B2 (en) | Air bubble removal in microfluidic devices | |
| CN106497771B (en) | A kind of multifunctional microflow control chip screened simultaneously for a variety of drugs and cell | |
| US20070036690A1 (en) | Inlet channel volume in a reactor | |
| JP2017093467A (en) | Cell culture and gradient migration assay method and device | |
| CN104877905A (en) | Cell in-vitro co-culture microfluidic chip, system and method | |
| US20210284944A1 (en) | High-throughput multi-organ perfusion models | |
| CN113388517B (en) | Biological culture micro-fluidic chip suitable for assembling microgravity gyroscope and cell culture method thereof | |
| EP3428266A1 (en) | Cell culture device and cell culture method | |
| US20190062686A1 (en) | Microfluidic device for culturing cells | |
| US20210238523A1 (en) | Stacked Recirculating Bioreactor | |
| CN105842324A (en) | Integrated micro-fluidic chip and working method thereof | |
| JP2008501365A (en) | Generation of shear force in the reactor | |
| CN112300929B (en) | Microfluidic experiment plate and double-sided cell culture method | |
| US10053663B2 (en) | Centrifugation chamber with gas permeable membrane layers for cell cultivation | |
| CN112300930A (en) | Microfluidic experimental plate and double-sided cell culture method | |
| US20190316071A1 (en) | Perfusion device | |
| CN111607519A (en) | Culture hole plate for drug concentration screening multi-cell co-culture and use method thereof | |
| US11643632B2 (en) | Method for gas enrichment and simultaneously for displacement of a fluid, and system for controlling the cell environment on a corresponding multi-well cell culture plate | |
| CN107236668A (en) | Micro-fluidic chip for breast carcinoma stem cell culture and Pharmaceutical Analysis | |
| CN115926980A (en) | Chip device and method for cell culture | |
| US11591555B2 (en) | Method for cultivating cells | |
| CN114231397A (en) | High-throughput drug testing centrifugal microfluidic system and use method thereof | |
| EP2821145B1 (en) | Centrifugation chamber with gas-permeable membranes layers for cell cultivation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |