CN115287185A - Cell sphere preparation device and preparation method - Google Patents
Cell sphere preparation device and preparation method Download PDFInfo
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- CN115287185A CN115287185A CN202210874295.XA CN202210874295A CN115287185A CN 115287185 A CN115287185 A CN 115287185A CN 202210874295 A CN202210874295 A CN 202210874295A CN 115287185 A CN115287185 A CN 115287185A
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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
- C12M23/48—Holding appliances; Racks; Supports
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- 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
- B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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- 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
- B01L3/502738—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 characterised by integrated valves
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- 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
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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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/01—Drops
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/20—Heat exchange systems, e.g. heat jackets or outer envelopes the heat transfer medium being a gas
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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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The invention provides a cell sphere preparation device and a preparation method. The preparation method of the cell sphere comprises the following steps: the water phase liquid storage device, the liquid storage pipe and the first pump are communicated in sequence, and the first pump sucks the cell suspension into the liquid storage pipe; the second pump, the liquid storage tube and the first inlet of the microfluidic chip are communicated in sequence, and the cell suspension in the liquid storage tube is conveyed to the first inlet by the second pump; flowing the oil phase material to a second inlet of the microfluidic chip to form droplets of the cell suspension inside the microfluidic chip; the droplets are solidified to form cell spheres. The cell sphere preparation device and the preparation method provided by the invention can improve the sample introduction efficiency of the microfluidic chip, so that the preparation efficiency of the cell spheres is improved; in addition, the liquid inlet quantity of the cell suspension of the microfluidic chip can be accurately controlled, the quality of the cell spheres is ensured, and the quality stability of the cell spheres is improved.
Description
Technical Field
The invention relates to the technical field of organoid preparation, in particular to a cell sphere preparation device and a preparation method.
Background
Organoid spheres can be cultured from cell spheres. Briefly, a cell suspension may be prepared, and then the cell suspension and an oil phase material may be mixed in a T-shaped channel, the cell suspension may be sheared by the oil phase material to form droplets, the droplets may be solidified to form cell spheres, and the cell spheres may be subsequently cultured to form organoid spheres.
However, in the prior art, the preparation efficiency of the cell spheres is low, and the quality and quality stability of the cell spheres are poor due to the low control precision of the liquid inlet amount of the cell suspension.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the cell sphere preparation device, which has high efficiency of preparing the cell spheres and high quality and quality stability of the prepared cell spheres.
The invention also provides a preparation method of the cell spheres.
A cell sphere preparation apparatus according to an embodiment of the first aspect of the invention includes: the device comprises a rack, and a feeding module, a micro-fluidic chip and a curing module which are arranged on the rack; the feeding module comprises an upper computer, a switching valve, a water phase liquid storage device, an oil phase liquid storage device, a first pump, a second pump and a liquid storage pipe, wherein the water phase liquid storage device is connected with the switching valve, the oil phase liquid storage device is used for storing cell suspension, the first pump, the second pump and the switching valve are all in communication connection with the upper computer, and therefore the upper computer can drive the first pump, the second pump and the switching valve to operate; the microfluidic chip is provided with a three-way flow channel, the three-way flow channel is provided with a first inlet, a second inlet and an outlet, the outlet is connected with the solidification module, the outlet is used for allowing liquid drops of the cell suspension to flow out, and the oil phase liquid storage device is connected with the second inlet so that the oil phase material in the oil phase liquid storage device can flow to the second inlet; the solidification module is used for solidifying the liquid drops of the cell suspension; the switching valve has a sample feeding state and a preparation state; when the switching valve is in a sample introduction state: the water phase liquid storage device, the liquid storage pipe and the first pump are sequentially communicated, and the first pump can suck the cell suspension in the water phase liquid storage device so that the cell suspension is arranged in the liquid storage pipe; when the switching valve is in the preparation state: the second pump, the liquid storage pipe and the first inlet are communicated in sequence, and the cell suspension in the liquid storage pipe can be conveyed to the first inlet by the second pump.
The cell sphere preparation device provided by the embodiment of the invention has at least the following beneficial effects: according to the preparation device, the upper computer drives the first pump, the second pump and the switching valve, so that automatic sample introduction of the cell suspension and oil phase materials is realized, and a user does not need to manually drive the pump or adjust the state of the valve in the sample introduction process, so that the sample introduction efficiency is improved, and the preparation efficiency of cell spheres is improved. In addition, the liquid amount of the cell suspension in the liquid storage tube can be accurately controlled by adjusting the state switching time interval of the switching valve, the running time of the first pump and the running time of the second pump, so that the liquid amount of the cell suspension entering the microfluidic chip in the preparation state is controlled, the accuracy of the liquid inlet amount of the cell suspension of the microfluidic chip is ensured, the quality of cell spheres is improved, and the quality stability of the cell spheres is improved.
According to some embodiments of the invention, the switching valve has a first interface, a second interface, a third interface, a fourth interface, a fifth interface, and a sixth interface; the water phase liquid storage device is connected with the first interface, the first pump is connected with the second interface, two ends of the liquid storage tube are respectively connected with the third interface and the sixth interface, the second pump is connected with the fourth interface, and a first inlet of the microfluidic chip is connected with the fifth interface; when the switching valve is in a sample injection state: the first port is communicated with the sixth port, the second port is communicated with the third port, and the fourth port is communicated with the fifth port; when the switching valve is in the preparation state: the first port is communicated with the second port, the third port is communicated with the fourth port, and the fifth port is communicated with the sixth port.
According to some embodiments of the invention, the second pump is connected to the oil phase reservoir; when the switching valve is in the preparation state, the second pump can inject the oil phase material in the oil phase reservoir into the reservoir tube so that the oil phase material pushes the cell suspension in the reservoir tube towards the first inlet.
According to some embodiments of the invention, further comprising a printing module, the printing module comprising: the printing head is connected with the curing module and is used for ejecting the cell spheres; and the multi-axis displacement mechanism is mounted at the top of the frame, and the printing head is mounted on the multi-axis displacement mechanism which can drive the printing head to move.
According to some embodiments of the invention, the rack comprises a preparation chamber and a curing chamber arranged at a distance from each other, the feeding module and the microfluidic chip are both arranged in the preparation chamber, and the curing module is arranged in the curing chamber.
According to some embodiments of the invention, the apparatus further comprises a temperature regulation module, the temperature regulation module is mounted inside the rack and used for regulating the temperature of the preparation chamber.
A method for preparing a cell sphere according to an embodiment of the second aspect of the present invention comprises: sequentially communicating an aqueous phase reservoir, a reservoir tube and a first pump, wherein the first pump sucks the cell suspension in the aqueous phase reservoir so as to enable the interior of the reservoir tube to have the cell suspension; communicating a second pump, the liquid storage tube and a first inlet of the microfluidic chip in sequence, wherein the second pump conveys the cell suspension in the liquid storage tube to the first inlet; flowing the oil phase material in the oil phase reservoir to a second inlet of the microfluidic chip, causing the oil phase material to shear the cell suspension in the microfluidic chip and form droplets of the cell suspension; solidifying the droplets to form cell spheres.
The cell sphere preparation method provided by the embodiment of the invention has at least the following beneficial effects: the sample introduction efficiency of the microfluidic chip can be improved, and the preparation efficiency of the cell spheres is improved; moreover, the method can accurately control the liquid inlet quantity of the cell suspension of the microfluidic chip, ensure the quality of the cell spheres and improve the quality stability of the cell spheres.
According to some embodiments of the invention, the cell suspension is prepared by obtaining a cell sample by lancing a sample of the cell.
The cell sphere preparation method provided by the embodiment of the invention has at least the following beneficial effects: the sampling is convenient, and the demand of the sample volume is less, which is beneficial to reducing the material cost of the cell microsphere or the organoid sphere.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a schematic diagram of a cell sphere preparation apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic system diagram of a cell sphere preparation apparatus according to an embodiment of the present invention;
FIG. 3 is a schematic view of a flow channel of a microfluidic chip of the cell sphere preparation apparatus according to the present invention;
FIG. 4 is a schematic diagram of the switching valve of the cell sphere preparation apparatus according to the present invention in a sample injection state;
FIG. 5 is a schematic view of a switching valve of the cell sphere preparation apparatus according to the present invention in a preparation state;
FIG. 6 is a schematic diagram of a method for preparing a cell sphere according to an embodiment of the present invention.
Reference numerals: 100-preparation device, 101-frame, 102-cell culture plate, 103-multi-axis displacement mechanism, 104-preparation cavity, 201-feeding module, 202-water phase reservoir, 203-oil phase reservoir, 204-reservoir, 205-second pump, 206-switching valve, 207-first pump, 208-microfluidic chip, 209-solidification module, 210-printing module, 211-host computer, 300-three-way flow channel, 301-first inlet, 302-second inlet, 303-outlet, 401-first interface, 402-second interface, 403-third interface, 404-fourth interface, 405-fifth interface and 406-sixth interface.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it should be understood that the orientation or positional relationship referred to in the description of the orientation, such as the upper, lower, front, rear, left, right, etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the description of the present invention, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present numbers, and the above, below, within, etc. are understood as including the present numbers. If there is a description of first and second for the purpose of distinguishing technical features only, this is not to be understood as indicating or implying a relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of technical features indicated.
In the description of the present invention, unless otherwise specifically limited, terms such as set, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention by combining the specific contents of the technical solutions.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The present invention provides a cell sphere preparation apparatus (hereinafter referred to as an apparatus for short, corresponding to the reference numeral "100"), in which the preparation apparatus 100 can prepare droplets of a cell suspension by using a shearing force of an oil phase material against the cell suspension (the cell suspension corresponds to an aqueous phase material), and then solidify the droplets to form cell spheres. The cell type contained in the cell suspension depends on the type of organoid to be prepared, and the cell may be specifically a muscle cell, a liver cell, etc. After the user obtains the cell sample, the cells may be added to a solution such as matrigel, hydrogel, or the like to obtain a cell suspension. The oil phase material may be a fluorine oil.
The preparation device 100 comprises a rack 101, a feeding module 201, a microfluidic chip 208 and a curing module 209, wherein the feeding module 201, the microfluidic chip 208 and the curing module 209 are all installed in the rack 101.
Referring to fig. 3, the microfluidic chip 208 has a three-way flow channel 300, and the three-way flow channel 300 has a first inlet 301, a second inlet 302, and an outlet 303. If the oil phase material enters the three-way flow channel 300 through the second inlet 302 and the cell suspension enters the three-way flow channel 300 through the first inlet 301, the cell suspension and the oil phase material are converged at the three-way position, and then the cell suspension is dispersed into uniform micro droplets wrapping the cells. Droplets of the cell suspension will exit the microfluidic channel from outlet 303 and the oil phase material will also exit the microfluidic chip 208 from outlet 303.
Referring to fig. 2, the outlet 303 of the microfluidic chip 208 is connected to the solidifying module 209, and the droplet leaving the microfluidic chip 208 enters the solidifying module 209, and the solidifying module 209 can solidify the droplet and form a cell sphere including a cell. The cell spheres comprise a solidified hydrogel or matrigel, and cells encapsulated in the hydrogel or matrigel. Specifically, the curing module 209 is capable of heating the droplets to cure the droplets at a temperature range; for example, the solidifying module 209 includes a solidifying channel (not specifically shown), the droplet exiting the microfluidic chip 208 enters the solidifying channel, and the solidifying module 209 heats the droplet in the solidifying channel to solidify the droplet into a cell sphere.
The curing module 209 may heat the curing tube in a water bath. Accordingly, the curing module 209 may further include a water bath container capable of containing water, and a heating member (e.g., an electric heating wire) for heating the water in the water bath container, and the curing pipe may be soaked in the water bath container. Still alternatively, the curing module 209 includes electric heating wires directly arranged outside the curing pipe through which the liquid droplets flow, and the electric heating wires directly heat the curing pipe. There are many ways to configure the curing module 209, as long as it can heat the liquid drop, which is not listed here.
The feed module 201 will be described further below.
Referring to fig. 2, the supply module 201 includes an upper computer 211, a switching valve 206, and a water phase reservoir 202, an oil phase reservoir 203, a first pump 207, a second pump 205, and a reservoir pipe 204 connected to the switching valve 206. The aqueous phase reservoir 202 is used to store the cell suspension and the oil phase reservoir 203 is used to store the oil phase material. The host computer 211 can be a computer capable of sending out control commands, and the first pump 207 and the second pump 205 are both in communication connection with the host computer 211. The first pump 207, the second pump 205 and the switching valve 206 can act according to instructions sent by the upper computer 211, so that sample introduction is realized; the sample injection in the present invention refers to inputting the cell suspension and the oil phase material into the microfluidic chip 208.
It should be noted that, in some embodiments, the curing module 209 and the printing module 210 may also be in communication connection with the upper computer 211, and the operations of the curing module 209 and the printing module 210 may be controlled by the upper computer 211.
The switching valve 206 has a sample injection state and a preparation state. Referring to fig. 4, when the switching valve 206 is in the sample injection state, the aqueous phase reservoir 202, the reservoir pipe 204 and the first pump 207 are sequentially communicated, and the first pump 207 sucks the cell suspension in the aqueous phase reservoir 202, and accordingly, the cell suspension enters the interior of the reservoir pipe 204. Referring to fig. 5, when the switching valve 206 is in the preparation state, the second pump 205, the reservoir 204 and the first inlet 301 are sequentially communicated, and the second pump 205 delivers the cell suspension in the reservoir 204 to the first inlet 301.
Referring to fig. 2, the oil phase reservoir 203 may be connected (may be connected by a pipe) to the second inlet 302 of the microfluidic chip 208, so that the oil phase material in the oil phase reservoir 203 may flow into the interior of the microfluidic chip 208 from the second inlet 302. Also, the oil phase material may be caused to flow from the oil phase reservoir 203 to the second inlet 302 while the switching valve 206 is in the preparation state. After the cell suspension entering from the first inlet 301 and the oil phase material entering from the second inlet 302 are merged, the cell suspension is dispersed into droplets.
In some embodiments, it may be that feed module 201 further comprises a third pump (not shown) for driving the flow of oil phase material in oil phase reservoir 203 towards second inlet 302. Of course, the third pump is not essential, and in other embodiments, oil phase reservoir 203 may be disposed at a higher position, and the oil phase material may flow toward second inlet 302 by gravity; accordingly, a valve for controlling the flow of the oil phase material may be installed on the pipe between oil phase reservoir 203 and second inlet 302.
According to the preparation device 100, the upper computer 211 drives the first pump 207, the second pump 205 and the switching valve 206, so that automatic sample introduction of cell suspension and oil phase materials is realized, and a user does not need to manually drive the pumps or adjust the states of valves in the sample introduction process, so that the sample introduction efficiency is improved, and the preparation efficiency of cell spheres is improved. In addition, the liquid amount of the cell suspension in the liquid storage tube 204 can be accurately controlled by adjusting the state switching time interval of the switching valve 206, the operation time of the first pump 207 and the operation time of the second pump 205, so that the liquid amount of the cell suspension entering the microfluidic chip 208 in the preparation state is controlled, the accuracy of the liquid inlet amount of the cell suspension of the microfluidic chip 208 is ensured, the quality of cell spheres is improved, and the quality stability of the cell spheres is improved.
Referring to fig. 2, in some embodiments, the preparation device 100 further comprises a printing module 210, the printing module 210 being connected to the curing module 209, the printing module 210 being configured to eject the cell spheres. The print module 210 includes a print head (print head not shown) and a multi-axis displacement mechanism 103, referring to fig. 1, the multi-axis displacement mechanism 103 is mounted on top of the frame 101, and the multi-axis displacement mechanism 103 is used to drive the print head to move. The inlet end of the print head is connected with the outlet end of the curing module 209, the cell spheres flowing out of the outlet 303 end of the curing module 209 enter the print head, and the print head ejects the cell spheres through air pressure. Referring to fig. 1, cell spheres ejected from a print head may be ejected into a cell culture plate 102 or petri dish. The multi-axis displacement mechanism 103 is used to drive the movement of the print head to eject a plurality of cell spheres into different wells of the cell culture plate 102. When the cell spheres are ejected from the print head, the oil phase material is also ejected from the print head and volatilizes.
In some embodiments, the switching valve 206 is provided as a six-channel switching valve. Referring to fig. 4 or 5, in particular, the switching valve 206 includes a first port 401, a second port 402, a third port 403, a fourth port 404, a fifth port 405, and a sixth port 406. The water phase reservoir 202 is connected with a first interface 401, the first pump 207 is connected with a second interface 402, two ends of the reservoir 204 are respectively connected with a third interface 403 and a sixth interface 406, the second pump 205 is connected with a fourth interface 404, and the first inlet 301 of the microfluidic chip 208 is connected with a fifth interface 405.
Referring to fig. 4, when the switching valve 206 is in the sample injection state, the first port 401 communicates with the sixth port 406, the second port 402 communicates with the third port 403, and the fourth port 404 communicates with the fifth port 405. In this state, the first pump 207 is activated to operate, and the cell suspension in the aqueous phase reservoir 202 can flow to the first pump 207, and during this flow, the cell suspension enters the reservoir 204.
Referring to fig. 5, when the switching valve 206 is in the preparation state, the first port 401 is in communication with the second port 402, the third port 403 is in communication with the fourth port 404, and the fifth port 405 is in communication with the sixth port 406. In this state, the second pump 205 is activated, and the second pump 205 can pump the cell suspension in the reservoir 204 to the first inlet 301.
To enable the second pump 205 to pump the cell suspension in the reservoir pipe 204 to the first inlet 301, in some embodiments, the second pump 205 may be configured to deliver an oil phase material and push the cell suspension in the reservoir pipe 204 with the oil phase material to the first inlet 301, since the gas does not tend to push the cell suspension to the first inlet 301.
Specifically, referring to fig. 2 and 5, the second pump 205 is connected to the oil phase reservoir 203, and when the switching valve 206 is in the preparation state, the second pump 205 can inject the oil phase material in the oil phase reservoir 203 into the reservoir pipe 204, and make the oil phase material that enters the reservoir pipe 204 later push the cell suspension that was present in the reservoir pipe 204 to the first inlet 301. The oil phase reservoir 203 may have two separated oil storage cavities, wherein the oil phase material in one oil storage cavity is used to flow to the second inlet 302, and the oil phase material in the other oil storage cavity flows to the reservoir pipe 204 (the oil phase materials in the two oil storage cavities are the same) under the driving of the second pump 205. Of course, oil phase reservoir 203 may have only one oil reservoir cavity therein, but oil phase reservoir 203 still needs to have two outlets 303 for oil phase material to flow out.
It should be noted that, after the oil phase material enters the liquid storage tube 204, the oil phase material and the cell suspension do not join in the flow channel having the three-way shape, the oil phase material does not generate enough shearing force on the cell suspension to disperse the cell suspension into droplets, the cell suspension is not dispersed into droplets in the liquid storage tube 204, and the droplets of the cell suspension are formed in the three-way flow channel 300 of the microfluidic chip 208. In addition, the cell suspension and oil phase materials are immiscible.
In the preparation process of the cell spheres, the cell suspension needs to be stored at a proper temperature, and the cell suspension and the oil phase material need to be converged at a proper temperature, so that the preparation quality of the cell spheres is ensured. For example, certain cell suspensions require storage at 4 ℃.
To maintain the cell suspension at a suitable temperature, the housing 101 includes a preparation chamber 104 and a curing chamber, the preparation chamber 104 and the curing chamber being spaced apart from each other. The curing chamber is not specifically shown, and the position of the curing chamber is flexibly selected under the condition that the preparation chamber 104 and the curing chamber are arranged at intervals.
The curing module 209 is installed in the curing chamber, and the feeding module 201 and the microfluidic chip 208 are installed in the preparation chamber 104. Since higher temperatures are required for the curing of the droplets (e.g. the droplets need to be cured at 37 ℃), mounting the curing module 209 in a further cavity (curing chamber) may reduce the effect of the curing module 209 on the temperature at the supply module 201 and the microfluidic chip 208.
In addition, in some embodiments, the preparation apparatus 100 further comprises a temperature regulation module, which is installed inside the frame 101 (not shown) and is used for regulating the temperature in the preparation chamber 104, thereby regulating the temperature of the cell suspension in the supply module 201 and the microfluidic chip 208.
The temperature regulation module may be configured as a vapor compression refrigeration unit, that is, the temperature regulation module includes a compressor, a condenser, a throttle valve, an evaporator, and a refrigerant circulating through the foregoing components, which are connected in sequence. The evaporator may be disposed inside a wall surface of the preparation chamber 104, and after the refrigerant is evaporated in the evaporator, the refrigerant absorbs air in the preparation chamber 104 and heat of the module, thereby cooling the preparation chamber 104. Or the temperature adjusting module further comprises a fan, the fan is used for driving air to flow through the evaporator and the preparation cavity 104 in sequence, the temperature of the air is reduced after the air flows through the surface of the evaporator, and low-temperature air is blown into the preparation cavity 104, so that the temperature of the module in the preparation cavity 104 is reduced.
It should be noted that the temperature adjustment module may also be in communication connection with the upper computer 211, and the operation of the temperature adjustment module may also be controlled by the upper computer 211.
Referring to fig. 6, based on the above inventive concept, the present invention further provides a method for preparing a cell sphere (hereinafter referred to as a preparation method), the preparation method comprising the following steps:
s1: the aqueous phase reservoir 202, the reservoir pipe 204 and the first pump 207 are sequentially communicated, and the first pump 207 sucks the cell suspension in the aqueous phase reservoir 202, so that the cell suspension is arranged in the reservoir pipe 204;
s2: the second pump 205, the liquid storage tube 204 and the first inlet 301 of the microfluidic chip 208 are sequentially communicated, and the second pump 205 conveys the cell suspension in the liquid storage tube 204 to the first inlet 301;
s3: flowing the oil phase material in the oil phase reservoir 203 to the second inlet 302 of the microfluidic chip 208 to form the cell suspension into droplets in the three-way flow channel 300 in the microfluidic chip 208;
s4: the droplets are solidified to form cell spheres.
By using the method, the sample introduction efficiency of the microfluidic chip 208 can be improved, and the preparation efficiency of the cell spheres can be improved; moreover, the method can accurately control the liquid inlet amount of the cell suspension of the microfluidic chip 208, ensure the quality of cell spheres and improve the quality stability of the cell spheres. After obtaining the cell spheres, the cell spheres may be cultured and organoid spheres obtained.
Further, referring to fig. 6, in some embodiments, the preparation method further includes the following step S0: cell samples were obtained by needle biopsy sampling and used to prepare cell suspensions. In the prior art, the organoid modeling is mostly carried out by using surgical excision samples, the sampling is inconvenient, and the sample size needs more. The invention can utilize the sample of the puncture biopsy to prepare the cell spheres and model the organoid, the sampling is convenient, and the demand of the sample volume is small, which is beneficial to reducing the material cost of the cell microspheres or the organoid spheres.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (8)
1. The cell sphere preparation device is characterized by comprising a rack, and a feeding module, a microfluidic chip and a curing module which are arranged on the rack;
the feeding module comprises an upper computer, a switching valve, a water phase liquid storage device, an oil phase liquid storage device, a first pump, a second pump and a liquid storage pipe, wherein the water phase liquid storage device is connected with the switching valve, the oil phase liquid storage device is used for storing cell suspension, the first pump, the second pump and the switching valve are all in communication connection with the upper computer, and therefore the upper computer can drive the first pump, the second pump and the switching valve to operate;
the microfluidic chip is provided with a three-way flow channel, the three-way flow channel is provided with a first inlet, a second inlet and an outlet, the outlet is connected with the solidification module, the outlet is used for allowing liquid drops of the cell suspension to flow out, and the oil phase liquid storage device is connected with the second inlet so that the oil phase material in the oil phase liquid storage device can flow to the second inlet;
the solidification module is used for solidifying the liquid drops of the cell suspension;
the switching valve has a sample feeding state and a preparation state;
when the switching valve is in a sample injection state: the water phase liquid storage device, the liquid storage pipe and the first pump are sequentially communicated, and the first pump can suck the cell suspension in the water phase liquid storage device so that the cell suspension is arranged in the liquid storage pipe;
when the switching valve is in the preparation state: the second pump, the liquid storage pipe and the first inlet are communicated in sequence, and the second pump can convey the cell suspension in the liquid storage pipe to the first inlet.
2. The device for preparing cell spheres of claim 1, wherein the switching valve has a first port, a second port, a third port, a fourth port, a fifth port, and a sixth port;
the water phase liquid storage device is connected with the first interface, the first pump is connected with the second interface, two ends of the liquid storage tube are respectively connected with the third interface and the sixth interface, the second pump is connected with the fourth interface, and the first inlet of the microfluidic chip is connected with the fifth interface;
when the switching valve is in a sample injection state: the first port is communicated with the sixth port, the second port is communicated with the third port, and the fourth port is communicated with the fifth port;
when the switching valve is in the preparation state: the first port is communicated with the second port, the third port is communicated with the fourth port, and the fifth port is communicated with the sixth port.
3. The cell sphere preparation apparatus according to claim 1 or 2, wherein the second pump is connected to the oil phase reservoir;
when the switching valve is in the preparation state, the second pump can inject the oil phase material in the oil phase reservoir into the reservoir tube so that the oil phase material pushes the cell suspension in the reservoir tube towards the first inlet.
4. The cell sphere preparation apparatus of claim 1, further comprising a printing module comprising:
the printing head is connected with the curing module and is used for ejecting the cell spheres;
and the multi-axis displacement mechanism is mounted at the top of the frame, and the printing head is mounted on the multi-axis displacement mechanism which can drive the printing head to move.
5. The device for preparing cell spheres, according to claim 1, wherein the frame comprises a preparation chamber and a curing chamber which are arranged at a distance from each other, the feeding module and the microfluidic chip are both arranged in the preparation chamber, and the curing module is arranged in the curing chamber.
6. The device for preparing cell spheres, as claimed in claim 5, further comprising a temperature regulating module mounted inside the frame for regulating the temperature of the preparation chamber.
7. The preparation method of the cell sphere is characterized by comprising the following steps:
sequentially communicating an aqueous phase reservoir, a reservoir tube and a first pump, wherein the first pump sucks the cell suspension in the aqueous phase reservoir so as to enable the interior of the reservoir tube to have the cell suspension;
communicating a second pump, the liquid storage tube and a first inlet of the microfluidic chip in sequence, wherein the second pump conveys the cell suspension in the liquid storage tube to the first inlet;
flowing the oil phase material in the oil phase reservoir toward a second inlet of the microfluidic chip, causing the oil phase material to shear the cell suspension in the microfluidic chip and form droplets of the cell suspension;
solidifying the droplets to form cell spheres.
8. The method for preparing a cell sphere according to claim 7, further comprising the steps of:
a cell sample is obtained by sampling by puncturing, and the cell suspension is prepared using the cell sample.
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| CN202210874295.XA CN115287185A (en) | 2022-07-21 | 2022-07-21 | Cell sphere preparation device and preparation method |
| PCT/CN2023/106991 WO2024017118A1 (en) | 2022-07-21 | 2023-07-12 | Cell sphere preparation device and preparation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540305A (en) * | 2022-01-07 | 2022-05-27 | 杭州海兰时生物科技有限责任公司 | Preparation method of organoid structure based on microfluidic technology high-flux culture |
| WO2024017118A1 (en) * | 2022-07-21 | 2024-01-25 | 清华大学深圳国际研究生院 | Cell sphere preparation device and preparation method |
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| JPH0623743B2 (en) * | 1989-06-29 | 1994-03-30 | 株式会社島津製作所 | Sample introduction device |
| US6558946B1 (en) * | 2000-08-29 | 2003-05-06 | The United States Of America As Represented By The Secretary Of The Army | Automated sample processing for identification of microorganisms and proteins |
| JP3109378U (en) * | 2004-12-14 | 2005-05-19 | 出光興産株式会社 | Polymeric substance analysis equipment |
| CN107418872A (en) * | 2016-08-31 | 2017-12-01 | 四川蓝光英诺生物科技股份有限公司 | Prepare the device of microballoon and prepare the method for microballoon |
| US11166920B2 (en) * | 2017-10-05 | 2021-11-09 | Auburn University | Microfluidics device for fabrication of large, uniform, injectable hydrogel microparticles for cell encapsulation |
| CN110711608B (en) * | 2019-09-23 | 2022-03-15 | 中国科学院上海微系统与信息技术研究所 | Microfluidic chip for cell detection and preparation method thereof |
| JP7331328B2 (en) * | 2019-10-17 | 2023-08-23 | 株式会社アイスティサイエンス | Liquid chromatograph equipped with flow switching valve |
| CN113583960B (en) * | 2021-06-02 | 2023-07-07 | 清华大学 | Method and device for constructing personalized tumor assembly based on droplet microfluidic technology |
| CN115287185A (en) * | 2022-07-21 | 2022-11-04 | 清华大学深圳国际研究生院 | Cell sphere preparation device and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114540305A (en) * | 2022-01-07 | 2022-05-27 | 杭州海兰时生物科技有限责任公司 | Preparation method of organoid structure based on microfluidic technology high-flux culture |
| WO2024017118A1 (en) * | 2022-07-21 | 2024-01-25 | 清华大学深圳国际研究生院 | Cell sphere preparation device and preparation method |
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