CN114874909A - Cell culture device and method - Google Patents

Cell culture device and method Download PDF

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CN114874909A
CN114874909A CN202210723360.9A CN202210723360A CN114874909A CN 114874909 A CN114874909 A CN 114874909A CN 202210723360 A CN202210723360 A CN 202210723360A CN 114874909 A CN114874909 A CN 114874909A
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cell
substrate
printing device
tip
droplets
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林金明
谢天择
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Tsinghua University
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Tsinghua University
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
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    • 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
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Abstract

The embodiment of the invention provides a cell culture device and a method, wherein the device comprises: the microfluidic printing device comprises a substrate, a cell sample operating platform and a microfluidic printing device, wherein the substrate is arranged on the cell sample operating platform, and the microfluidic printing device is suspended above the substrate; the microfluidic printing device prints a hydrophilic pattern on the substrate using a patterned material that supports cell adhesion; the substrate and the hydrophilic pattern are covered with an oil phase layer; printing a cell sample on the hydrophilic pattern and below the oil phase layer by a microfluidic printing device to form a cell droplet; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture. In the invention, the hydrophilic pattern can support cell adhesion, allow adherent cells to express morphology and physiological functions, analyze and research the morphology and physiological functions of single cells, and realize batch single cell culture.

Description

Cell culture device and method
Technical Field
The embodiment of the invention relates to the field of cell biology, in particular to a cell culture device and a cell culture method.
Background
Cells are the most fundamental unit for the realization of the functions of living bodies, and therefore, the behavior of individual cells is usually studied in related studies to effectively analyze the functions. In a living body, a large number of cells exist in an adherent form, forming a specific morphology to support their function. In most of the current microfluidic single cell studies, for convenience of processing and analysis, adherent cells are usually in a suspension state and lack normal morphology, and physical properties and adhesion behaviors of the adherent cells are affected.
Therefore, there is a need for a device and method that can culture single adherent cells in batch.
Disclosure of Invention
The embodiment of the invention provides a cell culture device and a cell culture method, which are used for culturing single adherent cells in batch.
In a first aspect, embodiments of the present invention provide a cell culture apparatus, the apparatus comprising: the microfluidic printing device comprises a substrate, a cell sample operating platform and a microfluidic printing device, wherein the substrate is arranged on the cell sample operating platform, and the microfluidic printing device is suspended above the substrate;
the microfluidic printing device prints hydrophilic patterns on the substrate in batch by using a patterned material, wherein the patterned material supports cell adhesion;
an oil phase layer covers the substrate and the hydrophilic pattern;
printing cell samples on the hydrophilic pattern and below the oil phase layer in batches by a micro-fluidic printing device to form a plurality of cell droplets; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the substrate during culture.
Optionally, the device further comprises a handling device suspended above the substrate, the handling device having a liquid channel and a tip with a diameter below 100 microns, the tip contacting the substrate and moving between cell droplets to create aqueous phase microchannels between the cell droplets in the case where the tip is in an infiltrated state as an aqueous phase; and in the case that the infiltration state of the tip is an oil phase, contacting the tip with a substrate and moving to cut off the water phase micro-channel between the cell drops.
Optionally, a microsyringe is connected to the manipulation device, and the extraction of the cell sample is performed by the tip of the manipulation device and the microsyringe.
Optionally, the apparatus further comprises: the heating plate is fixed on the cell operation platform and used for controlling the temperature of the cell operation platform, and an objective lens of the inverted microscope is positioned below the cell operation platform and used for observing the operation device and the cell liquid drops.
Optionally, the apparatus further comprises: an alignment device to which the micro-fluidic printing device and the handling device are fixed, the alignment device being configured to suspend the micro-fluidic printing device above a target site of the culture dish.
Optionally, the microfluidic printing device comprises a signal source, an electrode plate and an inkjet printing chip, wherein the signal source is connected with the electrode plate through a wire, the electrode plate is fixed to each channel of the inkjet printing chip by using a clamp, and the signal source controls the inkjet printing chip to drop liquid drops on a substrate to form a hydrophilic pattern or cell liquid drops;
the cell sample operation platform comprises an electric control objective table, wherein the electric control objective table is used for controlling the substrate to accurately move in two directions xy so as to be matched with a signal source of the micro-fluidic printing device to drop liquid drops on the substrate to form a hydrophilic pattern or cell liquid drops.
Optionally, the microfluidic printing device comprises a signal source, an electrode plate and an inkjet printing chip, wherein the signal source is connected with the electrode plate through a lead, and the electrode plate is fixed on each channel of the inkjet printing chip by using a clamp; the microfluidic printing device further comprises a position control platform, and the position control platform is used for controlling the microfluidic printing device to accurately move in two directions xy so as to drop droplets on the substrate to form a hydrophilic pattern or cell droplets.
In a second aspect, embodiments of the present invention provide a cell culture method, including:
printing hydrophilic patterns on the substrate in batch by using a microfluidic printing device;
covering the substrate with an oil phase layer;
printing a cell sample in batch above the hydrophilic pattern and below the oil phase layer by using the microfluidic printing device to form a plurality of cell droplets; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture.
Optionally, the method further comprises:
contacting the tip with the substrate and moving between the cell droplets to create aqueous phase microchannels between the cell droplets in the case where the tip of the manipulation device is in a wet state in an aqueous phase; contacting the tip with a substrate and moving to cut off the aqueous phase microchannel between the cell droplets in the case where the wet state of the tip is an oil phase,
wherein the tip of the manipulation device has a diameter of less than 100 microns.
Optionally, the method further comprises:
the extraction of the cell sample is accomplished by the tip of the manipulator and its attached microsyringe.
In the embodiment of the invention, the hydrophilic pattern is printed on the substrate in a microfluidic printing mode, so that the precision of ten microns can be achieved, and the requirement of single cell culture is met. In addition, in the embodiment of the invention, the hydrophilic pattern can support cell adhesion, allow adherent cells to express morphology and physiological functions, and analyze and research the morphology and physiological functions of single cells. And the microfluidic printing device can print a hydrophilic pattern array on the substrate, so that a cell drop array is obtained, and batch single cell culture is simply and conveniently realized.
In the embodiment of the invention, the substrate is covered by the oil phase layer, so that cells can be injected through the microfluidic printing device and can be extracted through the operating device, a corresponding extraction device is not required to be additionally integrated, and the culture and extraction of the cells can be simply and conveniently realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments of the present invention will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive labor.
FIG. 1 is a schematic view of a cell culture apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic view of an operating device in the cell culture apparatus according to the embodiment of the present invention;
FIG. 3 is a schematic diagram of the operation of the device for generating aqueous phase micro-channels between cell droplets in the practice of the present invention;
FIG. 4 is a flow chart of a cell culture method according to an embodiment of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
In a first aspect, embodiments of the present invention provide a cell culture apparatus, as shown in fig. 1:
the device comprises: the cell sample printing device comprises a substrate (2), a cell sample operating platform (8) and a micro-flow control printing device, wherein the substrate (2) is arranged on the cell sample operating platform (8), and the micro-flow control printing device is suspended above the substrate.
The microfluidic printing device is printed with hydrophilic patterns (6) on the substrate in batches using patterned material that supports cell adhesion;
the substrate (2) and the hydrophilic patterns (6) thereof are covered with an oil phase layer (9);
printing a cell sample on the hydrophilic pattern and below the oil phase layer in batches by a micro-fluidic printing device to form a plurality of cell droplets (7); the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture.
In the embodiment of the invention, the substrate (2) can be a common culture dish and is used as a batch single cell culture container.
In the embodiment of the invention, the patterning material can be poly-D-lysine solution, the micro-fluidic printing device delivers the solution droplets to the substrate, and the hydrophilic pattern is formed when the moisture in the solution is volatilized.
In embodiments of the invention, the patterned material functions to support cell adhesion and maintain cell viability. Therefore, a cell culture solution commonly used in the art may be used as the patterning material, and the embodiment of the present invention is not particularly limited thereto.
In the embodiment of the invention, after the hydrophilic pattern is formed, the substrate can be covered with an oil phase layer, and the oil phase layer can be paraffin oil. In the embodiment of the present invention, the oil phase layer is used to cover the subsequent cell droplets and prevent the water in the cell droplets from volatilizing, so that all oil phase materials commonly used in the art can be used as the oil phase layer, which is not specifically limited in the embodiment of the present invention.
In the embodiment of the invention, the oil phase layer can be quickly covered after the cell drops are dripped.
In the embodiment of the invention, after the microfluidic printing device is used for dropwise adding the cell droplets above the hydrophilic pattern and below the oil phase layer, cells in the cell droplets can be adhered to the hydrophilic pattern in the culture process and further adhered to the substrate so as to show the normal morphology and physiological functions of the adherent cells.
In an embodiment of the present invention, in a printing process of a microfluidic printing device, when a plurality of cell droplets obtained by printing have a small diameter, the plurality of cell droplets include: a small number of empty droplets (containing no cells), a large number of single-cell droplets (one cell droplet containing one cell), and a very small number of multi-cell droplets (one cell droplet containing a plurality of cells), and in the case where the diameter of the plurality of printed cell droplets is large, the plurality of cell droplets include: a very small number of empty droplets, a large number of single-cell droplets and a small number of multi-cell droplets.
Specifically, the diameter of the hydrophilic pattern and the diameter of the cell droplets obtained by printing can be controlled by adjusting relevant parameters of the microfluidic printing device, and then the distribution condition of the single-cell droplets in the cell droplets is controlled, so that single-cell culture is realized.
Specifically, in the embodiment of the invention, under the condition that the diameter of the droplet printed by the microfluidic printing device is less than 200 microns, a plurality of single-cell droplets are contained in the plurality of printed cell droplets, so that single-cell culture can be realized.
In the embodiment of the invention, the microfluidic printing device can be used for printing the hydrophilic pattern array on the substrate, and then the microfluidic printing device is used for printing the cell droplet array on the substrate, so that the batch culture of single cells is realized.
In an alternative embodiment, the apparatus further comprises: an alignment device (1) secured to the alignment device (1) for suspending the microfluidic printing device above a target site of a culture dish.
In an embodiment of the invention, the alignment device may be configured to control the microfluidic printing device to move in three XYZ dimensions to suspend the microfluidic printing device above the target site of the culture dish.
In an alternative embodiment, the microfluidic printing device comprises a signal source (3), an electrode plate (4) and an inkjet printing chip (5), wherein the signal source (3) is connected with the electrode plate (4) through a lead, the electrode plate (4) is fixed on each channel of the inkjet printing chip (5) by using a clamp, and the signal source (3) controls the inkjet printing chip (5) to drop liquid drops on a substrate (2) to form a hydrophilic pattern or cell liquid drops.
In an alternative embodiment, the cell sample operation platform includes an electronic control stage, and the electronic control stage is configured to control the substrate to precisely move in two directions xy to drop droplets on the substrate in cooperation with a signal source of the micro-fluidic printing device to form a hydrophilic pattern or a cell droplet.
In an alternative embodiment, the microfluidic printing device comprises a signal source, an electrode plate and an inkjet printing chip, wherein the signal source is connected with the electrode plate through a lead, and the electrode plate is fixed on each channel of the inkjet printing chip by using a clamp; the microfluidic printing device further comprises a position control platform, and the position control platform is used for controlling the microfluidic printing device to accurately move in two directions xy so as to drop droplets on the substrate to form a hydrophilic pattern or cell droplets.
In the embodiment of the invention, the electric control stage can be used for controlling the substrate to accurately move so as to be matched with a signal source of the micro-flow control printing device, and liquid drops are dripped on the substrate to form a corresponding hydrophilic pattern or cell liquid drops. The position control platform can also be used for controlling the micro-fluidic printing device to move accurately so as to be matched with a signal source, and liquid drops are dripped on the substrate to form corresponding hydrophilic patterns or cell liquid drops.
The microenvironment in which the cells reside largely determines their behavior. Since the cells communicate with each other through chemical factors, the cells are stimulated by chemical stimuli that are difficult to control in the conventional culture system, so that the microenvironment of the cells is difficult to control accurately. At present, in the related art, the chambers of the cells are separated by using solid materials, so that dynamic adjustment of the microenvironment of the cells is difficult to realize, and meanwhile, because the cells are sealed, the loading or in-situ detection of single cells becomes quite difficult.
In this regard, in an alternative embodiment of the invention, the cell culture apparatus further comprises a handling device, as shown in fig. 2, the handling device (10) is suspended above the substrate, the handling device (10) having a liquid channel and a tip with a diameter below 100 microns.
The device further comprises: the heating plate (11) is fixed on the cell operation platform (8) and used for controlling the temperature of the cell operation platform (8), and an objective lens of the inverted microscope (12) is positioned below the cell operation platform (8) and used for observing the operation device (10) and the cell liquid drops (7).
In an embodiment of the invention, the handling device (10) may also be fixed to the alignment device (1) for suspending the handling device above a target site of the culture dish.
In an embodiment of the present invention, the operation device may be a capillary tube.
In the embodiment of the invention, the operation device and the cell drops can be observed by using an inverted microscope, so that the connection and disconnection between the cell drops can be operated by changing the infiltration state of the tip and sliding on the substrate, as shown in figure 3, in the case that the infiltration state of the tip is an aqueous phase, the tip is contacted with the substrate and moves between the cell drops to generate an aqueous phase microchannel (13) between the cell drops; and in the case that the infiltration state of the tip is an oil phase, contacting the tip with a substrate and moving to cut off the water phase micro-channel between the cell drops.
In the embodiment of the invention, after the cell droplets are formed by the substrate, the cell sample operation platform and the microfluidic printing device in the cell culture device, the connection and disconnection between the cell droplets can be operated by the tip of the operation device. In the embodiment of the invention, the cell drops are arranged above the hydrophilic pattern, and the oil phase layer is separated to form a plurality of single cell culture systems. When the tip of the operation device in the aqueous phase-infiltrated state is used to scribe one cell droplet to another cell droplet, an aqueous phase microchannel may be formed on the substrate to connect the two cell droplets so that the two single cell culture systems communicate, and when the tip in the oil phase-infiltrated state is used to scribe the aqueous phase microchannel in a direction intersecting the aqueous phase microchannel, the aqueous phase microchannel may be disconnected. Therefore, the embodiment of the invention can dynamically regulate and control the microenvironment of the single cells, and further accurately explore and control the behavior and physiological functions of the specific single cells.
In the embodiment of the invention, when the tip of the control device is immersed in the cell drop, the infiltration state of the tip is a water phase, and when the tip of the control device is immersed in the oil phase layer and does not contact the cell drop, the infiltration state of the tip is an oil phase.
In an alternative embodiment of the present invention, the manipulation device is further connected to a microsyringe, and the extraction of the cell sample is performed by the tip of the manipulation device and the microsyringe.
Specifically, the liquid channel of the operation device may be connected to the microsyringe through a hose.
In the embodiment of the invention, the oil phase is used for replacing a solid phase in a traditional system, cells can be injected through the microfluidic printing device and can also be extracted through the operation device, a corresponding device is not required to be additionally integrated in the system, and batch single cell culture and extraction can be simply and conveniently realized.
In the embodiment of the present invention, an extraction device commonly used in the art may be additionally integrated to extract the cell droplets. In the embodiment of the present invention, a cell detection device commonly used in the art may be additionally integrated to perform in situ stimulation and analysis on cells in the cell droplet.
Based on the same inventive concept, the second aspect of the embodiments provides a cell culture method, and the cell culture method provided in the embodiments of the present invention can be applied to the cell culture apparatus provided in any one of the embodiments of the first aspect; as shown in fig. 4, the method includes:
s401, printing hydrophilic patterns on the substrate in batch by using a microfluidic printing device;
s402, covering an oil phase layer on the substrate;
s403, printing cell samples on the hydrophilic patterns and below the oil phase layer in batch by using the microfluidic printing device to form a plurality of cell droplets; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture.
In an alternative embodiment of the invention, the method further comprises:
s404, under the condition that the wetting state of the tip of the operating device is an aqueous phase, enabling the tip to be in contact with the substrate and moving among the cell drops to generate an aqueous phase micro-channel among the cell drops; contacting the tip with a substrate and moving to cut off the aqueous phase microchannel between the cell droplets in the case where the wet state of the tip is an oil phase;
wherein the tip of the manipulation device has a diameter of less than 100 microns.
In an alternative embodiment of the invention, the method further comprises:
the extraction of the cell sample is accomplished by the tip of the manipulator and its attached microsyringe.
The detailed description of the method is similar to that of the above-mentioned apparatus, and is not repeated herein.
The present invention is not limited to the above embodiments, and any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
While preferred embodiments of the present invention have been described, additional variations and modifications of these embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.
Finally, it should also be noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or terminal that comprises the element.
The cell culture apparatus and method provided by the present invention are described in detail above, and the principle and the embodiment of the present invention are explained in detail herein by using specific examples, and the description of the above examples is only used to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims (10)

1. A cell culture apparatus, the apparatus comprising: the microfluidic printing device comprises a substrate, a cell sample operating platform and a microfluidic printing device, wherein the substrate is arranged on the cell sample operating platform, and the microfluidic printing device is suspended above the substrate;
the microfluidic printing device prints hydrophilic patterns on the substrate in batch by using a patterned material, wherein the patterned material supports cell adhesion;
an oil phase layer covers the substrate and the hydrophilic pattern;
printing cell samples on the hydrophilic pattern and below the oil phase layer in batches by a micro-fluidic printing device to form a plurality of cell droplets; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture.
2. The cell culture device of claim 1, further comprising a manipulator suspended above the substrate, the manipulator having a liquid channel and a tip with a diameter of less than 100 microns, the tip contacting the substrate and moving between the cell droplets to create aqueous phase microchannels between the cell droplets in the event the tip is in an aqueous phase as an infiltrated state; and in the case that the infiltration state of the tip is an oil phase, contacting the tip with a substrate and moving to cut off the water phase micro-channel between the cell drops.
3. The cell culture apparatus of claim 2, wherein the manipulator is further coupled to a microsyringe, and wherein the extraction of the cell sample is accomplished by the tip of the manipulator and the microsyringe.
4. The cell culture apparatus of claim 3, further comprising: the heating plate is fixed on the cell operation platform and used for controlling the temperature of the cell operation platform, and an objective lens of the inverted microscope is positioned below the cell operation platform and used for observing the operation device and the cell liquid drops.
5. The cell culture apparatus of claim 1, further comprising: an alignment device to which the micro-fluidic printing device and the handling device are fixed, the alignment device being configured to suspend the micro-fluidic printing device above a target site of the culture dish.
6. The cell culture device according to any one of claims 1 to 5, wherein the microfluidic printing device comprises a signal source, an electrode plate and an inkjet printing chip, the signal source is connected with the electrode plate through a lead, the electrode plate is fixed on each channel of the inkjet printing chip by using a clamp, and the signal source controls the inkjet printing chip to drop liquid drops on a substrate to form a hydrophilic pattern or cell liquid drops;
the cell sample operation platform comprises an electric control objective table, wherein the electric control objective table is used for controlling the substrate to accurately move in two directions xy so as to be matched with a signal source of the micro-fluidic printing device to drop liquid drops on the substrate to form a hydrophilic pattern or cell liquid drops.
7. The cell culture apparatus according to any one of claims 1 to 5, wherein the microfluidic printing apparatus comprises a signal source, an electrode plate, and an inkjet printing chip, the signal source is connected to the electrode plate via a wire, and the electrode plate is fixed to each channel of the inkjet printing chip by using a clamp; the microfluidic printing device further comprises a position control platform, and the position control platform is used for controlling the microfluidic printing device to accurately move in two directions xy so as to drop droplets on the substrate to form a hydrophilic pattern or cell droplets.
8. A method of cell culture, the method comprising:
printing hydrophilic patterns on the substrate in batch by using a microfluidic printing device;
covering the substrate with an oil phase layer;
printing a cell sample in batch above the hydrophilic pattern and below the oil phase layer by using the microfluidic printing device to form a plurality of cell droplets; the cell droplets contain cells therein, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture.
9. The cell culture method of claim 7, further comprising:
contacting the tip with the substrate and moving between the cell droplets to create aqueous phase microchannels between the cell droplets, in the case where the wetted state of the tip of the manipulation device is aqueous; contacting the tip with a substrate and moving to cut off the aqueous phase microchannel between the cell droplets in the case where the wet state of the tip is an oil phase;
wherein the tip of the manipulation device has a diameter of less than 100 microns.
10. The cell culture method of claim 7, further comprising:
the extraction of the cell sample is accomplished by the tip of the manipulator and its attached microsyringe.
CN202210723360.9A 2022-06-24 2022-06-24 Cell culture device and method Pending CN114874909A (en)

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