An integrated microfluidic chip and single cell culture, screening and export method thereof

Abstract

The present invention provides an integrated microfluidic chip and a single cell culture, screening and export method thereof, the chip includes a base, a liquid inlet flow channel, a liquid outlet flow channel, a plurality of common flow channels and a plurality of functional units, wherein the two ends of the common flow channel are respectively connected to the liquid inlet flow channel and the liquid outlet flow channel, the functional unit includes a single cell introduction port, a cell culture screening chamber, a cell export chamber, a cell export port and a driving element, the driving element is used to provide power for the liquid to introduce the single cell entering the common flow channel into the cell culture screening chamber, and to export the target cell group cultured and screened in the cell culture screening chamber from the cell export port. The integrated microfluidic chip of the present invention can enable single cell culture, cell group identification and screening, and target cell group export to be completed on-chip, thereby improving cell throughput, simplifying experimental operations, reducing reagent usage and potential cross-contamination risks. At the same time, by combining thermal bubble printing technology, the entire process can be made more controllable, convenient and efficient.

Description

Technical Field

[0001] The present invention belongs to the fields of microfluidics, cell line development, monoclonal antibody screening, and the like, and relates to an integrated microfluidics chip and a method for culturing, screening, and exporting single cells thereof. Background Art

[0002] Cells are the fundamental units of life. Research at the single-cell level can reveal the underlying principles of life at a deeper level, and has broad applications in areas such as monoclonal antibody screening and cell line culture. Single-cell isolation is fundamental and crucial to single-cell research. Currently, single-cell isolation methods primarily include microneedle aspiration, limiting dilution, microwell arrays, and microfluidics-based sorting. Current isolation methods suffer from operational difficulty, low efficiency, and the need to obtain multiple cells, hindering subsequent culture and analysis. In monoclonal antibody screening and cell line culture applications, isolated single cells are often cultured in well plates. After titer and phenotypic analysis, the cell populations with the best performance are selected for large-scale culture. This entire process is labor-intensive, cumbersome, time-consuming, and inefficient. Therefore, in single-cell research, especially monoclonal antibody screening and cell line development, there is an urgent need for a simple, efficient method that integrates single-cell isolation, culture, screening, and derivation. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide an integrated microfluidic chip and its single cell culture, screening and export method, which is used to solve the problems in the prior art of cell separation, culture, screening and other processes that require a lot of manpower, are cumbersome to operate, time-consuming and inefficient.

[0004] To achieve the above objectives and other related objectives, the present invention provides an integrated microfluidic chip, comprising:

[0005] A base, comprising a front side and a back side arranged opposite to each other;

[0006] The liquid inlet channel and the liquid outlet channel are buried in the base and spaced apart;

[0007] A plurality of common flow channels are buried in the base and spaced apart, with both ends of the common flow channels being connected to the liquid inlet flow channel and the liquid outlet flow channel respectively;

[0008] Multiple functional units, the functional units include a single cell introduction port, a cell culture screening chamber, a cell export chamber, a cell export port and a driving element, the single cell introduction port is arranged on the front of the base and is connected to the common flow channel, the cell culture screening chamber and the cell export chamber are both buried in the base, and the two ends of the cell culture screening chamber are respectively connected to the common flow channel and the cell export chamber, the cell export port is arranged on the back of the base and is connected to the cell export chamber, the driving element is located at the top of the cell export chamber and faces the cell export port, the driving element is used to provide power for the liquid to introduce the single cell entering the common flow channel into the cell culture screening chamber, and export the target cell group cultured and screened in the cell culture screening chamber through the cell export port.

[0009] Optionally, the extension direction of the liquid inlet channel is parallel to the extension direction of the liquid outlet channel.

[0010] Optionally, the extension direction of the common flow channel is perpendicular to the extension direction of the liquid inlet flow channel.

[0011] Optionally, the thickness of the cell culture screening chamber is set to accommodate only a monolayer of cells.

[0012] Optionally, in a direction perpendicular to the cell culture screening chamber and pointing toward the cell derivation chamber, the width of the cell culture screening chamber is greater than the width of the cell derivation chamber.

[0013] Optionally, the single cell introduction port is used to receive single cells ejected from the single cell printing chip.

[0014] Optionally, the single-cell printing chip includes a thermal bubble printing chip.

[0015] Optionally, the driving element includes one of a heating film, a PDMS microvalve, a solenoid valve and a peristaltic pump.

[0016] Optionally, the number of the functional units ranges from 10 to 10,000.

[0017] The present invention also provides a method for culturing, screening and exporting single cells, comprising the following steps:

[0018] Providing the integrated microfluidic chip as described in any one of the above, and injecting a single cell into the common flow channel from the single cell introduction port;

[0019] After the cells have settled naturally, the driving element is used to drive the liquid to flow, and the single cells injected into the common flow channel are introduced into the cell culture screening chamber;

[0020] After the cells are cultured in the cell culture screening chamber for a preset time, introducing a screening reagent into the cell culture screening chamber through the liquid inlet channel to identify and screen out a target cell population;

[0021] The target cell population is transferred into a designated container via the cell outlet.

[0022] Optionally, before injecting single cells into the common flow channel through the single cell introduction port, cell culture fluid is first perfused into the integrated microfluidic chip to expel bubbles.

[0023] Optionally, after the single cells injected into the common flow channel are introduced into the cell culture screening chamber, cell culture fluid perfusion is performed again to culture the cells.

[0024] Optionally, after the screening reagent is introduced into the cell culture screening chamber, the target cell population is screened out by performing image characterization on the cell population in the cell culture screening chamber.

[0025] Optionally, the single cell culture, screening and derivation method is used to screen monoclonal antibody cell populations.

[0026] As described above, the present invention provides an integrated microfluidic chip for single-cell isolation, culture, screening, and derivation of target cell populations. This chip enables single-cell culture, cell population identification and screening, and derivation of target cell populations to be performed on-chip, improving cell throughput, simplifying experimental procedures, and reducing reagent usage and potential cross-contamination risks. Furthermore, by integrating thermal bubble printing technology, the entire process becomes more controllable, convenient, and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is a schematic top view of the integrated microfluidic chip of the present invention.

[0028] Figure 2 Shown is a side view schematic diagram of the functional unit in the integrated microfluidic chip of the present invention.

[0029] Figure 3 Shown is a flow chart of the single cell culture, screening and derivation method of the present invention.

[0030] Component number description

[0031] 1 base

[0032] 2 Liquid inlet channel

[0033] 3 Liquid outlet channel

[0034] 4 Public flow channels

[0035] 5 Functional Units

[0036] 501 Single cell introduction port

[0037] 502 Cell Culture Screening Chamber

[0038] 503 Cell export cavity

[0039] 504 cell export port

[0040] 505 drive element

[0041] 6 Single Cell

[0042] 7 Single-cell printed chips

[0043] Steps S1 to S4 DETAILED DESCRIPTION

[0044] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0045] See also Figures 1 to 3 It should be noted that the diagrams provided in this embodiment are merely schematic illustrations of the basic concept of the present invention. Therefore, the diagrams only show components relevant to the present invention and are not drawn according to the number, shape, and size of components in actual implementation. In actual implementation, the type, quantity, and proportion of each component may be arbitrarily changed, and the component layout may also be more complex.

[0046] Example 1

[0047] This embodiment provides an integrated microfluidic chip. Figure 1 , which shows a schematic diagram of the top view of the integrated microfluidic chip, including a base 1, a liquid inlet channel 2, a liquid outlet channel 3, multiple common channels 4 and multiple functional units 5. Figure 2 , which is a side structural diagram of the functional unit 5.

[0048] Specifically, the base 1 includes a front and a back surface that are relatively arranged; the liquid inlet channel 2 and the liquid outlet channel 3 are buried in the base 1 and are spaced apart; a plurality of common flow channels 4 are buried in the base 1 and are spaced apart, and the two ends of the common flow channel 4 are respectively connected to the liquid inlet channel 2 and the liquid outlet channel 3; a plurality of functional units 5 are arranged in an array, and the functional units 5 include a single cell introduction port 501, a cell culture screening chamber 502, a cell derivation chamber 503, a cell derivation port 504 and a driving element 505, the single cell introduction port 501 is arranged on the front of the base 1 and is connected to the common flow channel 4, the cell culture screening chamber 502 and the cell export chamber 503 are all buried in the base 1, and the two ends of the cell culture screening chamber 502 are respectively connected with the common flow channel 4 and the cell export chamber 503, the cell export port 504 is arranged on the back of the base 1 and is connected with the cell export chamber 503, the driving element 505 is located at the top of the cell export chamber 503 and faces the cell export port 504, the driving element 505 is used to provide power for the liquid to introduce the single cell 6 entering the common flow channel 4 into the cell culture screening chamber 502, and export the target cell group cultured and screened in the cell culture screening chamber 502 through the cell export port 504.

[0049] Specifically, the functional unit 5 has the functions of single-cell isolation, culture, screening, and export. Thousands of these functional units 5 can be integrated into the integrated microfluidic chip as needed to ensure high-throughput single-cell culture and screening. For example, the number of functional units 5 ranges from 10 to 10,000, such as 1,000, 2,000, 5,000, and so on.

[0050] As an example, Figure 1 The dotted arrows in the figure illustrate the liquid flow path. The inlet of the inlet channel 2 and the outlet of the outlet channel 3 can be adjusted as needed, for example, to one end of the channel or a predetermined position in the middle of the channel (not shown). The culture medium and reagents can be driven by external power, providing support for cell culture and screening.

[0051] As an example, Figure 1 As shown, the inlet channel 2 extends parallel to the outlet channel 3, while the common channel 4 extends perpendicular to the inlet channel 2. This allows the functional units 5 to be arranged in a regular square array, facilitating interaction with the print nozzles of the single-cell printing chip and the cell receiving device. In other embodiments, the communication between the inlet channel 2, the outlet channel 3, and the common channel 4 can be adjusted as needed, and is not limited to this embodiment.

[0052] As an example, Figure 2 As shown, the thickness of the cell culture screening chamber 502 is set to accommodate only a single layer of cells. For example, the thickness of the cell culture screening chamber 502 can be set to be equivalent to the size of the cells to be screened, thereby ensuring a single layer of cells during the cell culture process, which is helpful for subsequent cell counting and fluorescence analysis.

[0053] As an example, Figure 1 As shown, in the direction perpendicular to the cell culture screening chamber 502 pointing to the cell export chamber 503, the width of the cell culture screening chamber 502 is greater than the width of the cell export chamber 503 to facilitate the screened cells to enter the cell export chamber 503 in sequence and be exported.

[0054] As an example, the driving element 505 may include one of a heating film, a PDMS (polydimethylsiloxane) microvalve, a solenoid valve and a peristaltic pump. In the present embodiment, the driving element 505 preferably adopts a heating film, thereby forming a hot bubble nozzle together with the cell derivation cavity 503 and the cell derivation port 504. The hot bubble nozzle is made based on micro-nano processing technology and integrated at the bottom of the microchannel. The hot bubble nozzle uses the instantaneous high temperature of the heating film to vaporize the liquid above, generate bubbles to drive the liquid to flow and eject from the nozzle, and then the subsequent liquid is replenished under the action of capillary force, thereby providing power for the continuous flow of the liquid. The hot bubble nozzle is controlled by the underlying circuit.

[0055] As an example, Figure 2 As shown, the single-cell inlet 501 is used to receive single cells 6 ejected from the single-cell printing chip 7. The position of the single-cell inlet 501 is configured such that, after the single cells enter the common flow channel through the single-cell inlet 501, they settle at the entrance area of ​​the cell culture screening chamber 502, allowing them to accurately enter the cell culture screening chamber 502 under the drive of the drive element 505. The single-cell printing chip 7 can adopt the single-cell printing chip disclosed in Chinese Patent CN108330065A or other suitable single-cell printing chip to achieve single-cell separation and introduction into the single-cell inlet 501. In this embodiment, a single-cell printing chip including a thermal bubble printing chip is preferably used, as it causes minimal damage to cells and allows for efficient and gentle introduction of single cells into the single-cell inlet 501.

[0056] The integrated microfluidic chip of the present invention can be used for single-cell isolation, culture, screening, and extraction of target cell populations. This allows single-cell culture, cell population identification and screening, and target cell population extraction to be completed on-chip, increasing cell throughput, simplifying experimental procedures, and reducing reagent usage and potential cross-contamination risks. Furthermore, by integrating thermal bubble printing technology, the entire process can be made more controllable, convenient, and efficient.

[0057] Example 2

[0058] This example provides a method for culturing, screening, and exporting single cells. Figure 3 , shown as a flow chart of the method, comprising the following steps:

[0059] S1: providing the integrated microfluidic chip described in Example 1, and injecting a single cell into the common flow channel from the single cell introduction port;

[0060] S2: After the cells have settled naturally, the driving element is used to drive the liquid flow, and the single cells injected into the common flow channel are introduced into the cell culture screening chamber;

[0061] S3: After the cells have been cultured in the cell culture screening chamber for a preset time, introducing a screening reagent into the cell culture screening chamber through the liquid inlet channel to identify and screen out a target cell population;

[0062] S4: Transferring the target cell population into a designated container via the cell outlet.

[0063] As an example, in step S1, before injecting single cells into the common flow channel from the single cell introduction port, cell culture fluid is first perfused into the integrated microfluidic chip to expel bubbles.

[0064] As an example, in step S2, after the single cell injected into the common flow channel is introduced into the cell culture screening chamber, cell culture fluid perfusion is performed again to culture the cells.

[0065] As an example, in step S3, after introducing a screening reagent into the cell culture screening chamber, the target cell population is screened by imaging the cell population in the cell culture screening chamber. The screening reagent can be a phenotypic identification antibody or other target, depending on the cells to be screened.

[0066] As an example, in step S4, thermal bubble printing is preferably used to transfer the target cell population to a designated container for subsequent culture or analysis. Thermal bubble printing offers the advantages of fast response, strong driving force, ease of control, ease of integration, and miniaturization, ensuring a convenient and efficient process.

[0067] As an example, the single cell culture, screening, and derivation method of this embodiment can be used to screen monoclonal antibody cell populations or other types of cell populations.

[0068] Example 3

[0069] This embodiment uses the microfluidic chip in Example 1 to screen monoclonal cell lines. First, the cell culture fluid is perfused into the chip to expel bubbles. Then the perfusion of the culture fluid is stopped, and the transfected cells are placed in the single cell introduction port of the chip in the form of single cells through the single cell printing chip. The operation of the hot bubble nozzle is controlled by the underlying circuit to bring the single cell into the cell culture screening chamber. In order to obtain a cell group with a single layer of cells, the height of the cell culture screening chamber can be adjusted according to the size of the cells used. The culture fluid is re-perfused, and after the cells are cultured to a certain group, the relevant fluorescent antibodies are injected into the reagent injection port to characterize the antibody titer secreted by each cell group. The target cell group is then screened out as needed. At this time, the target cell group is again transferred to the designated container through the hot bubble nozzle of the cell lead-out port to complete the screening of the monoclonal antibody cell group.

[0070] In summary, the present invention provides an integrated microfluidic chip for single-cell isolation, culture, screening, and derivation of target cell populations. This chip enables single-cell culture, cell population identification and screening, and derivation of target cell populations to be performed on-chip, improving cell throughput, simplifying experimental procedures, and reducing reagent usage and potential cross-contamination risks. Furthermore, by incorporating thermal bubble printing technology, the entire process can be made more controllable, convenient, and efficient. Therefore, the present invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0071] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. An integrated microfluidic chip, characterized in that: include: A base, comprising a front side and a back side arranged opposite to each other; The liquid inlet channel and the liquid outlet channel are buried in the base and spaced apart; A plurality of common flow channels are buried in the base and spaced apart, with both ends of the common flow channels being connected to the liquid inlet flow channel and the liquid outlet flow channel respectively; a plurality of functional units, each comprising a single cell introduction port, a cell culture screening chamber, a cell export chamber, a cell export port, and a driving element, wherein the single cell introduction port is disposed on the front of the base and communicates with the common flow channel, the cell culture screening chamber and the cell export chamber are both embedded in the base, and both ends of the cell culture screening chamber are respectively communicated with the common flow channel and the cell export chamber, the cell export port is disposed on the back of the base and communicates with the cell export chamber, the driving element is located at the top of the cell export chamber and faces the cell export port, the driving element is used to provide power for a liquid to introduce a single cell entering the common flow channel into the cell culture screening chamber, and to export a target cell population cultured and screened in the cell culture screening chamber through the cell export port; The number of the functional units ranges from 10 to 10,000, and the single-cell introduction port is used to receive the single cells ejected from the single-cell printing chip; The thickness of the cell culture screening chamber is set to only accommodate a single layer of cells; In a direction perpendicular to the cell culture screening chamber and pointing toward the cell derivation chamber, the width of the cell culture screening chamber is greater than the width of the cell derivation chamber.

2. The integrated microfluidic chip according to claim 1, characterized in that: The extension direction of the liquid inlet channel is parallel to the extension direction of the liquid outlet channel.

3. The integrated microfluidic chip according to claim 2, characterized in that: An extending direction of the common flow channel is perpendicular to an extending direction of the liquid inlet flow channel.

4. The integrated microfluidic chip according to claim 1, characterized in that: The single-cell printing chip includes a thermal bubble printing chip.

5. The integrated microfluidic chip according to claim 1, characterized in that: The driving element includes one of a heating film, a PDMS microvalve, a solenoid valve and a peristaltic pump.

6. A method for culturing, screening and deriving single cells, characterized in that: The following steps are involved: Providing the integrated microfluidic chip according to any one of claims 1 to 5, injecting a single cell into the common flow channel from the single cell introduction port; After the cells have settled naturally, the driving element is used to drive the liquid to flow, and the single cells injected into the common flow channel are introduced into the cell culture screening chamber; After the cells are cultured in the cell culture screening chamber for a preset time, introducing a screening reagent into the cell culture screening chamber through the liquid inlet channel to identify and screen out a target cell population; The target cell population is transferred into a designated container via the cell outlet.

7. The method for culturing, screening and deriving single cells according to claim 6, wherein: Before injecting single cells into the common flow channel from the single cell introduction port, cell culture fluid is first perfused into the integrated microfluidic chip to expel air bubbles.

8. The method for culturing, screening and deriving single cells according to claim 7, wherein: After the single cells injected into the common flow channel are introduced into the cell culture screening chamber, cell culture fluid perfusion is performed again to culture the cells.

9. The method for culturing, screening and deriving single cells according to claim 6, wherein: After the screening reagent is introduced into the cell culture screening chamber, the target cell population is screened out by performing image characterization on the cell population in the cell culture screening chamber.

10. The method for culturing, screening and deriving single cells according to claim 6, wherein: The single cell culture, screening and derivation method is used for screening monoclonal antibody cell populations.

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