A cell culture device and method

Abstract

The embodiment of the present application provides a kind of cell culture device and method, the device includes: substrate, cell sample operation platform, microfluidic printing device, the substrate is placed on the cell sample operation platform, and the microfluidic printing device is suspended above the substrate;The microfluidic printing device prints hydrophilic pattern on the substrate using patterning material, and the patterning material supports cell adhesion;The substrate and its hydrophilic pattern are covered with oil phase layer;Cell sample is printed onto the hydrophilic pattern below the oil phase layer by microfluidic printing device, forming cell droplet;The cell droplet contains cells, the cells are in contact with the hydrophilic pattern, and adhere to the hydrophilic pattern during the culture process.In the present application, the hydrophilic pattern can support cell adhesion, allow adherent cells to exhibit morphological and physiological functions, and analyze and study the morphology and physiological functions of single cells.The present application can also realize batch single cell culture.

Description

TECHNICAL FIELD

[0001] Embodiments of the present application relate to the field of cell biology, and in particular to a cell culture device and method. BACKGROUND

[0002] Cells are the most basic unit to realize the function of living organisms, so in related research, the behavior of a single cell is usually studied to effectively analyze its function. In living organisms, a large number of cells exist in the form of adhesion, forming a specific morphology to support their function. In most current microfluidic single-cell research, in order to facilitate processing and analysis, adherent cells are usually in a suspended state, thus lacking normal morphology, affecting their physical properties and adhesion behavior.

[0003] Therefore, there is an urgent need for a device and method capable of batch culturing single adherent cells. SUMMARY

[0004] Embodiments of the present application provide a cell culture device and method for batch culturing single adherent cells.

[0005] The first aspect of the embodiments of the present application provides a cell culture device, the device comprising: a substrate, a cell sample operation platform, a microfluidic printing device, the substrate being placed on the cell sample operation platform, and the microfluidic printing device being suspended above the substrate;

[0006] The microfluidic printing device uses patterned material to batch print hydrophilic patterns on the substrate, and the patterned material supports cell adhesion.

[0007] The substrate and its hydrophilic patterns are covered with an oil phase layer.

[0008] The microfluidic printing device batch prints cell samples onto the hydrophilic patterns and under the oil phase layer to form a plurality of cell droplets; the cell droplets contain cells, the cells are in contact with the hydrophilic patterns, and adhere to the substrate during the culture process.

[0009] Optionally, the device further comprises an operation device suspended above the substrate, the operation device having a liquid channel and a tip with a diameter less than 100 microns, in the case that the tip is in a water phase wetting state, the tip is in contact with the substrate and moves between the cell droplets to generate water phase microchannels between the cell droplets; in the case that the tip is in an oil phase wetting state, the tip is in contact with the substrate and moves to cut off the water phase microchannels between the cell droplets.

[0010] Optionally, the operation device is further connected with a micro-syringe, and the extraction of the cell sample is completed by the tip of the operation device and the micro-syringe.

[0011] Optionally, the device further comprises a heating plate fixed on the cell sample operation platform for controlling the temperature of the cell sample operation platform, and an inverted microscope with an objective lens below the cell sample operation platform for observing the operation device and the cell droplet.

[0012] Optionally, the device further comprises an alignment device, and the microfluidic printing device and the operation device are fixed on the alignment device, and the alignment device is used for suspending the microfluidic printing device above the target site of the culture dish.

[0013] Optionally, the microfluidic printing device comprises a signal source, an electrode sheet and an inkjet printing chip, the signal source is connected to the electrode sheet through a wire, the electrode sheet is fixed to each channel of the inkjet printing chip using a clamp, and the signal source controls the inkjet printing chip to drop droplets on the substrate to form a hydrophilic pattern or a cell droplet.

[0014] The cell sample operation platform comprises an electrically controlled stage for controlling the accurate movement of the substrate in two xy directions to cooperate with the signal source of the microfluidic printing device to drop droplets on the substrate to form a hydrophilic pattern or a cell droplet.

[0015] Optionally, the microfluidic printing device comprises a signal source, an electrode sheet and an inkjet printing chip, the signal source is connected to the electrode sheet through a wire, the electrode sheet is fixed to each channel of the inkjet printing chip using a clamp, and the microfluidic printing device further comprises a position control platform for controlling the accurate movement of the microfluidic printing device in two xy directions to drop droplets on the substrate to form a hydrophilic pattern or a cell droplet.

[0016] The second aspect of the embodiment of the present application provides a cell culture method, which comprises:

[0017] The microfluidic printing device is used to batch print a hydrophilic pattern on the substrate.

[0018] An oil phase layer is overlaid on the substrate.

[0019] The microfluidic printing device is used to batch print a cell sample on the hydrophilic pattern and under the oil phase layer to form a plurality of cell droplets, the cell droplets contain cells, the cells are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during the culture process.

[0020] Optionally, the method further comprises:

[0021] In the case that the wetting state of the tip of the operating device is water phase, contacting the tip with the substrate and moving between the cell droplets generates water phase microchannels between the cell droplets; in the case that the wetting state of the tip is oil phase, contacting the tip with the substrate and moving cuts off the water phase microchannels between the cell droplets,

[0022] The diameter of the tip of the operating device is less than 100 microns.

[0023] Optionally, the method further comprises:

[0024] Extraction of the cell sample is completed by the tip of the operating device and the microsyringe connected thereto.

[0025] In the embodiment of the present application, a hydrophilic pattern is printed on the substrate by microfluidic printing, which can achieve a precision of ten microns and meet the requirements of single cell culture. In the embodiment of the present application, the hydrophilic pattern can support cell adhesion and allow adherent cells to exhibit morphological and physiological functions, and the morphology and physiological functions of single cells can be analyzed and studied. In addition, the microfluidic printing device can print an array of hydrophilic patterns on the substrate, and thus obtain an array of cell droplets, thereby simply and conveniently realizing batch single cell culture.

[0026] In the embodiment of the present application, the substrate is covered with an oil phase layer, so that the cells can be injected by the microfluidic printing device and extracted by the operating device, without the need for integrating a corresponding extraction device, and the culture and extraction of cells can be simply and conveniently realized. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the present application. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0028] Figure 1 is a schematic diagram of a cell culture device according to an embodiment of the present application;

[0029] Figure 2 is a schematic diagram of an operating device in a cell culture device according to an embodiment of the present application;

[0030] Figure 3 is a schematic diagram of an operating process for generating water phase microchannels between cell droplets by an operating device according to an embodiment of the present application;

[0031] Figure 4 is a flowchart of a cell culture method according to an embodiment of the present application. DETAILED DESCRIPTION

[0032] In order to make the above objectives, features and advantages of the present application more obvious and comprehensible, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0033] The first aspect of the present embodiment provides a cell culture device, as shown in the following figure: Figure 1

[0034] The device comprises a substrate (2), a cell sample operation platform (8), and a microfluidic printing device, wherein the substrate (2) is placed on the cell sample operation platform (8), and the microfluidic printing device is suspended above the substrate.

[0035] The microfluidic printing device prints a hydrophilic pattern (6) on the substrate in batches by using a patterned material, and the patterned material supports cell adhesion.

[0036] The substrate (2) and the hydrophilic pattern (6) thereon are covered with an oil phase layer (9).

[0037] The cell sample is printed on the hydrophilic pattern and under the oil phase layer in batches by the microfluidic printing device to form a plurality of cell droplets (7), wherein the cell droplets contain cells, the cells are in contact with the hydrophilic pattern, and the cells adhere to the hydrophilic pattern during the culture process.

[0038] In the present embodiment, the substrate (2) can be a common culture dish, which serves as a batch single-cell culture container.

[0039] In the present embodiment, the patterned material can be a poly-D-lysine solution, and the microfluidic printing device delivers droplets of the solution to the substrate, and the hydrophilic pattern is formed after the water in the solution evaporates.

[0040] In the present embodiment, the patterned material supports cell adhesion and maintains cell activity. Therefore, the cell culture solution commonly used in the art can be used as the patterned material, and the present embodiment does not make specific limitations thereon.

[0041] In the present embodiment, after the hydrophilic pattern is formed, an oil phase layer can be entirely covered on the substrate, and the oil phase layer can be paraffin oil. In the present embodiment, the oil phase layer covers the subsequent cell droplets to prevent the water in the cell droplets from evaporating. Therefore, the oil phase material commonly used in the art can be used as the oil phase layer, and the present embodiment does not make specific limitations thereon.

[0042] In the present embodiment, the oil phase layer can also be quickly covered after the cell droplets are added.

[0043] ​In the embodiment of the present application, after the microfluidic printing device drops the cell droplet on the hydrophilic pattern and under the oil phase layer, the cells in the cell droplet can adhere to the hydrophilic pattern during the culture process, and then adhere to the substrate to exhibit the normal morphology and physiological function of the adherent cells.

[0044] In the embodiment of the present application, in the printing process of the microfluidic printing device, when the diameter of the plurality of cell droplets obtained by printing is small, the plurality of cell droplets include a small amount of empty droplets (without cells), a large amount of single-cell droplets (one cell droplet contains one cell), and a very small amount of multi-cell droplets (one cell droplet contains multiple cells); when the diameter of the plurality of cell droplets obtained by printing is large, the plurality of cell droplets include a very small amount of empty droplets, a large amount of single-cell droplets, and a small amount of multi-cell droplets.

[0045] Specifically, the diameter of the hydrophilic pattern and the diameter of the cell droplet obtained by printing can be controlled by adjusting the related parameters of the microfluidic printing device, and then the distribution of single-cell droplets in the cell droplet is controlled, so that single-cell culture is realized.

[0046] Specifically, in the embodiment of the present application, when the diameter of the droplet printed by the microfluidic printing device is less than 200 microns, the plurality of cell droplets obtained by printing contain a large amount of single-cell droplets, and single-cell culture can be realized.

[0047] In the embodiment of the present application, the microfluidic printing device can also be used to print an array of hydrophilic patterns on the substrate, and then the microfluidic printing device is used to print an array of cell droplets on the substrate, so that batch single-cell culture is realized.

[0048] In an alternative embodiment, the device further comprises an alignment device (1), the microfluidic printing device is fixed on the alignment device (1), and the alignment device is used to suspend the microfluidic printing device above the target site of the culture dish.

[0049] In the embodiment of the present application, the alignment device can be used to control the movement of the microfluidic printing device in XYZ three dimensions, so as to suspend the microfluidic printing device above the target site of the culture dish.

[0050] In an alternative embodiment, the microfluidic printing device contains a signal source (3), an electrode sheet (4) and an inkjet printing chip (5), the signal source (3) is connected to the electrode sheet (4) through a wire, the electrode sheet (4) is fixed to each channel of the inkjet printing chip (5) using a clamp, and the signal source (3) controls the inkjet printing chip (5) to drop droplets on the substrate (2) to form a hydrophilic pattern or a cell droplet.

[0051] In an alternative embodiment, the cell sample operation platform comprises an electrically controlled stage for controlling the accurate movement of the substrate in two directions of x and y to cooperate with the signal source of the microfluidic printing device to drop droplets on the substrate to form a hydrophilic pattern or cell droplets.

[0052] In an alternative embodiment, the microfluidic printing device comprises a signal source, an electrode sheet and an inkjet printing chip, the signal source is connected to the electrode sheet through a wire, and the electrode sheet is fixed to each channel of the inkjet printing chip using a clamp; the microfluidic printing device further comprises a position control platform for controlling the accurate movement of the microfluidic printing device in two directions of x and y to drop droplets on the substrate to form a hydrophilic pattern or cell droplets.

[0053] In the embodiments of the present application, the electrically controlled stage can be used to control the accurate movement of the substrate to cooperate with the signal source of the microfluidic printing device to drop droplets on the substrate to form corresponding hydrophilic patterns or cell droplets. The position control platform can also be used to control the accurate movement of the microfluidic printing device to cooperate with the signal source to drop droplets on the substrate to form corresponding hydrophilic patterns or cell droplets.

[0054] The microenvironment of cells largely determines their behavior. Since cells communicate with each other in the form of chemical factors, in the common culture system, cells are subjected to uncontrollable chemical stimulation, making it difficult to accurately control their microenvironment. In the related art, solid materials are used to separate the chambers where cells are located, making it difficult to dynamically adjust the microenvironment of cells. At the same time, since the cells are enclosed, it is very difficult to load or in-situ detect single cells.

[0055] Based on this, in an alternative embodiment of the present application, the cell culture device further comprises an operation device, such as Figure 2 As shown, the operation device (10) is suspended above the substrate, and the operation device (10) has a liquid channel and a tip with a diameter less than 100 microns.

[0056] The device further comprises a heating plate (11) and an inverted microscope (12), the heating plate (11) is fixed on the cell sample operation platform (8) for controlling the temperature of the cell sample operation platform (8), and the objective lens of the inverted microscope (12) is located below the cell sample operation platform (8) for observing the operation device (10) and the cell droplets (7).

[0057] In the embodiments of the present application, the operation device (10) can also be fixed on the alignment device (1), and the alignment device is used to suspend the operation device above the target site of the culture dish.

[0058] The operation device in the embodiment of the present application can be a capillary.

[0059] In the embodiment of the present application, the operation device and the cell droplets can be observed by using an inverted microscope, so that the connection and disconnection between the cell droplets can be operated by changing the wetting state of the tip and sliding on the substrate, as shown in the following figure. Figure 3 As shown in the figure, in the case that the wetting state of the tip is water phase, the tip is in contact with the substrate and moves between the cell droplets to generate the water phase microchannel (13) between the cell droplets; in the case that the wetting state of the tip is oil phase, the tip is in contact with the substrate and moves to cut off the water phase microchannel between the cell droplets.

[0060] In the embodiment of the present application, after the cell droplets are formed by using the substrate in the cell culture device, the cell sample operation platform and the microfluidic printing device, the connection and disconnection between the cell droplets can be operated by using the tip of the operation device. In the embodiment of the present application, since the cell droplets are separated to form a plurality of single cell culture systems on the hydrophilic pattern and under the oil phase layer. When the tip of the operation device in the water phase wetting state is used to draw from one cell droplet to another, a water phase microchannel can be formed on the substrate to connect two cell droplets, so that two single cell culture systems are connected, and when the tip in the oil phase wetting state is used to draw the water phase microchannel in the direction intersecting the water phase microchannel, the water phase microchannel can be disconnected. Therefore, the embodiment of the present application can dynamically regulate the microenvironment of single cells, and further accurately explore and manipulate the behavior and physiological function of specific single cells.

[0061] In the embodiment of the present application, when the tip of the control device is immersed in the cell droplet, the wetting state of the tip is water phase, and when the tip of the control device is immersed in the oil phase layer and does not contact the cell droplet, the wetting state of the tip is oil phase.

[0062] In an alternative embodiment of the present application, the operation device is further connected with a micro-syringe, and the extraction of the cell sample is completed by the tip of the operation device and the micro-syringe.

[0063] Specifically, the liquid channel of the operation device can be connected with the micro-syringe through a hose.

[0064] In the embodiment of the present application, the oil phase is used to replace the solid phase in the traditional system, and the cells can be injected by the microfluidic printing device and extracted by the operation device, without the need to integrate corresponding devices in the system, so that batch single cell culture and extraction can be simply and conveniently realized.

[0065] In the embodiment of the present application, the extraction device commonly used in the field can also be additionally collected to extract the cell droplets. In the embodiment of the present application, the cell detection device commonly used in the field can also be additionally collected to stimulate and analyze the cells in the cell droplets in situ.

[0066] Based on the same inventive concept, the second aspect of the embodiment provides a cell culture method. The cell culture method provided in the embodiment of the present application can be applied to the cell culture device provided in any one of the first aspect of the embodiment. As shown in the figure, the method comprises: Figure 4

[0067] S401, using a microfluidic printing device to batch print a hydrophilic pattern on the substrate;

[0068] S402, covering the substrate with an oil phase layer;

[0069] S403, using the microfluidic printing device to batch print a cell sample on the hydrophilic pattern and under the oil phase layer to form a plurality of cell droplets; the cell droplets contain cells, the cells are in contact with the hydrophilic pattern, and adhere to the hydrophilic pattern during the culture process.

[0070] In an alternative embodiment of the present application, the method further comprises:

[0071] S404, in the case that the wet state of the tip of the operation device is water phase, contacting the tip with the substrate and moving between the cell droplets to generate water phase microchannels between the cell droplets; in the case that the wet state of the tip is oil phase, contacting the tip with the substrate and moving to cut off the water phase microchannels between the cell droplets;

[0072] Wherein, the diameter of the tip of the operation device is less than 100 microns.

[0073] In an alternative embodiment of the present application, the method further comprises:

[0074] The extraction of the cell sample is completed by the tip of the operation device and the microsyringe connected thereto.

[0075] The method is specifically described, which is similar to the above-mentioned device related description, and will not be repeated here.

[0076] This embodiment is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0077] ​While the preferred embodiments of the application have been described above, it will be recognized and understood that various modifications and changes can be made to these embodiments by those skilled in the art having the benefit of this detailed description without departing from the spirit and scope of the application. It is therefore intended that the appended claims cover all such modifications and changes as fall within the scope of the application.

[0078] Finally, it is to be understood that the phraseology or terminology employed herein, such as "first" and "second", etc., are for descriptive purposes only and should not be construed to connote or otherwise imply any kind of ordering, precedence or relationships between or among the elements or acts described. Moreover, the terms "comprising", "including", or "having" and variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that comprises, includes, or has an element or a plurality of elements does not include only those listed elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. In addition, the word "comprising" does not exclude the presence of elements or steps other than those listed in a claim.

[0079] The above provides a cell culture device and method, the principle and implementation of the application are described by using specific examples, the above embodiment is only used to help understand the method and core idea of the application; meanwhile, for the person skilled in the art, according to the idea of the application, the specific implementation and application range can be changed, and the above description should not be understood as the limitation of the application.

Claims

1. A cell culture device, characterized in that, The device includes: a substrate, a cell sample handling platform, and a microfluidic printing device, wherein the substrate is placed on the cell sample handling platform and the microfluidic printing device is suspended above the substrate; The microfluidic printing device uses a patterning material to print hydrophilic patterns in batches on the substrate, and the patterning material supports cell adhesion. The substrate and its hydrophilic pattern are covered with an oil phase layer; Cell samples are printed in batches onto the hydrophilic pattern and below the oil phase using a microfluidic printing device, forming multiple cell droplets; the cell droplets contain cells, which are in contact with the hydrophilic pattern and adhere to the hydrophilic pattern during culture; The device further includes an operating device suspended above the substrate. The operating device has a liquid channel and a tip with a diameter of less than 100 micrometers. When the tip is in an aqueous phase, the tip contacts the substrate and moves between cell droplets to create aqueous microchannels between the cell droplets. When the tip is in an oil phase, the tip contacts the substrate and moves to cut off the aqueous microchannels between the cell droplets.

2. The cell culture apparatus according to claim 1, characterized in that, The operating device is also connected to a micro-syringe, through which cell samples are extracted.

3. The cell culture apparatus according to claim 2, characterized in that, The device further includes a heating plate and an inverted microscope. The heating plate is fixed to the cell sample operation platform and is used to control the temperature of the cell sample operation platform. The objective lens of the inverted microscope is located below the cell sample operation platform for observing the operation device and the cell droplets.

4. The cell culture apparatus according to claim 1, characterized in that, The device further includes an alignment device, on which the microfluidic printing device and the operating device are fixed, and the alignment device is used to suspend the microfluidic printing device above the target site in the culture dish.

5. The cell culture apparatus according to any one of claims 1-4, characterized in that, The microfluidic printing device includes a signal source, an electrode sheet, and an inkjet printing chip. The signal source is connected to the electrode sheet via wires. The electrode sheet is fixed to each channel of the inkjet printing chip using a clamp. The signal source controls the inkjet printing chip to droplets onto the substrate to form hydrophilic patterns or cell droplets. The cell sample handling platform includes an electrically controlled stage, which is used to control the precise movement of the substrate in both the x and y directions to cooperate with the signal source of the microfluidic printing device to droplets onto the substrate to form hydrophilic patterns or cell droplets.

6. The cell culture apparatus according to any one of claims 1-4, characterized in that, The microfluidic printing device includes a signal source, electrode sheets, and an inkjet printing chip. The signal source is connected to the electrode sheets via wires, and the electrode sheets are fixed to the channels of the inkjet printing chip using a fixture. The microfluidic printing device also includes a position control platform, which is used to control the microfluidic printing device to move precisely in the x and y directions to droplets onto the substrate to form hydrophilic patterns or cell droplets.

7. A cell culture method, characterized in that, The method, applied to the cell culture apparatus according to any one of claims 1 to 6, comprises: Hydrophilic patterns were mass-printed on a substrate using a microfluidic printing device. An oil phase layer is coated on the substrate; The microfluidic printing device is used to batch print cell samples on the hydrophilic pattern and below the oil phase layer to form multiple cell droplets; the cell droplets contain cells, the cells are in contact with the hydrophilic pattern, and adhere to the hydrophilic pattern during the culture process; The method further includes: When the tip of the operating device is wetted in an aqueous phase, the tip is brought into contact with the substrate and moved between the cell droplets to create an aqueous microchannel between the cell droplets; when the tip is wetted in an oil phase, the tip is brought into contact with the substrate and moved to cut off the aqueous microchannel between the cell droplets. The diameter of the tip of the operating device is less than 100 micrometers.

8. The cell culture method according to claim 7, characterized in that, The method further includes: Cell sample extraction is accomplished through the tip of the operating device and the micro-syringe connected to it.

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