Microfluidic chip and single cell culture, cell population screening and derivation method

Abstract

The application provides a microfluidic chip and a single cell culture, cell group screening and derivation method. The chip comprises a substrate layer, a flow channel layer, an inlet flow channel, an outlet flow channel, a plurality of common flow channels and a plurality of functional units. The flow channel layer is located below the substrate layer, and the material of the flow channel layer comprises at least one of silicon dioxide, spin-on glass, non-photosensitive epoxy resin and non-photosensitive polyimide. The two ends of the common flow channel are respectively communicated with the inlet flow channel and the outlet flow channel. The functional unit comprises a single cell derivation inlet, a cell culture and screening cavity, a cell derivation cavity, a cell derivation outlet and a driving element. The driving element is used for providing power for liquid to introduce the single cell entering the common flow channel into the cell culture and screening cavity, and derive the target cell group cultured and screened from the cell derivation outlet. The application can complete the single cell culture, cell group identification and screening and target cell group derivation on the chip, is simple in experimental operation, reduces the risk of cross contamination and avoids the fluorescence interference of the flow channel layer itself.

Description

Technical Field

[0001] This invention belongs to the fields of microfluidics, cell line development, and monoclonal antibody screening, and relates to a microfluidic chip and a method for single-cell culture, cell population screening and export. Background Technology

[0002] Cells are the basic unit of life, and research at the single-cell level can reveal the developmental laws of life activities at a deeper level, with wide applications in monoclonal antibody screening, cell line culture, and other fields. Single-cell isolation is the foundation and key to single-cell research. Currently, single-cell isolation mainly includes microneedle aspiration, limiting dilution, microwell arrays, and microfluidic-based sorting methods. Current isolation methods face problems such as high operational difficulty, low efficiency, and the inability to obtain multiple cells, which are not conducive to subsequent culture and analysis. In monoclonal antibody screening and cell line culture applications, isolated single cells are mostly placed in well plates for culture. After titer and phenotypic analysis, cell populations with good performance are screened for large-scale culture. The entire process is labor-intensive, cumbersome, time-consuming, and inefficient. Therefore, in single-cell research, especially in monoclonal antibody screening and cell line development, there is an urgent need for a simple and efficient research method that integrates single-cell isolation, culture, cell population screening, and export. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a microfluidic chip and a method for single-cell culture, cell population screening and export, to solve the problems of high manpower requirements, cumbersome operation, long time consumption and low efficiency in the processes of cell separation, culture and cell population screening in the prior art.

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

[0005] Substrate layer;

[0006] A flow channel layer is located below the substrate layer, and the material of the flow channel layer includes at least one of silicon dioxide, spin-coated glass, non-photosensitive epoxy resin and non-photosensitive polyimide.

[0007] The inlet and outlet channels are located in the channel layer and are spaced apart.

[0008] Multiple common flow channels are located in the flow channel layer and are spaced apart. The two ends of the common flow channels are respectively connected to the liquid inlet flow channel and the liquid outlet flow channel.

[0009] The system comprises multiple functional units, including a single-cell inlet, a cell culture screening chamber, a cell outlet chamber, a cell outlet, and a driving element. The single-cell inlet is located in the substrate layer and communicates with the common flow channel. The cell culture screening chamber, the cell outlet chamber, and the cell outlet are all located in the flow channel layer, with both ends of the cell culture screening chamber communicating with the common flow channel and the cell outlet chamber, respectively. The cell outlet is located below the cell outlet chamber and communicates with it. The driving element is located in the cell outlet chamber and faces the cell outlet. The driving element provides power to the liquid to introduce single cells introduced into the common flow channel through the single-cell inlet into the cell culture screening chamber, and to export the target cell population cultured and screened in the cell culture screening chamber through the cell outlet.

[0010] Optionally, the inlet channel, the outlet channel, the common channel, the cell culture screening chamber, and the cell export chamber all penetrate the channel surface facing the substrate to expose the substrate, and the driving element is attached to the surface of the substrate.

[0011] Optionally, the substrate layer may be made of silicon.

[0012] Optionally, the extension direction of the inlet channel is parallel to the extension direction of the outlet channel.

[0013] Optionally, the extension direction of the common flow channel is perpendicular to the extension direction of the liquid inlet flow channel.

[0014] Optionally, the thickness of the cell culture screening chamber is set to accommodate only a single layer of cells.

[0015] Optionally, in the plane where the flow channel layer is located, and in the direction perpendicular to the cell culture screening cavity pointing to the cell export cavity, the width of the cell culture screening cavity is greater than the width of the cell export cavity.

[0016] Optionally, the single-cell inlet is used to receive single cells ejected from the single-cell printing chip.

[0017] Optionally, the single-cell printing chip includes a thermal bubble printing chip.

[0018] Optionally, the driving element includes one of a heating film, a piezoelectric nozzle, a PDMS microvalve, a solenoid valve, and a peristaltic pump.

[0019] Optionally, the number of the functional units ranges from 10 to 10,000.

[0020] This invention also provides a method for single-cell culture, cell population screening and export, comprising the following steps:

[0021] Provide a microfluidic chip as described in any of the above, and inject a single cell into the common flow channel through the single cell inlet;

[0022] After the cells settle naturally, the driving element is used to drive the liquid flow, introducing the single cells injected into the common flow channel into the cell culture screening chamber.

[0023] After the cells have been cultured in the cell culture screening chamber for a preset time, screening reagents are introduced into the cell culture screening chamber through the liquid inlet channel to identify and screen out the target cell population.

[0024] The target cell population is transferred into a designated container via the cell outlet.

[0025] Optionally, before injecting a single cell into the common channel through the single cell inlet, the cell culture medium is first perfused into the integrated microfluidic chip to remove air bubbles.

[0026] Optionally, after introducing the single cell injected into the common channel into the cell culture screening chamber, cell culture medium is perfused again to culture the cells.

[0027] Optionally, the cell culture medium can be perfused using an injection pump.

[0028] Optionally, after introducing the screening reagent into the cell culture screening chamber, the target cell population is screened out by performing fluorescence image characterization on the cell population in the cell culture screening chamber.

[0029] Optionally, the single-cell culture, cell population screening and export method is used for screening monoclonal antibody cell populations.

[0030] As described above, this invention provides a microfluidic chip for single-cell isolation, culture, screening, and export of target cell populations. This chip enables single-cell culture, cell population identification and screening, and export of target cell populations all to be completed on-chip, increasing cell throughput, simplifying experimental operations, and reducing reagent consumption and potential cross-contamination risks. Furthermore, by incorporating thermal bubble printing technology, the entire process becomes more controllable, convenient, and efficient. In addition, in the microfluidic chip of this invention, the flow channel layer uses a silica layer, spin-coated glass layer, non-photosensitive epoxy resin material layer, or non-photosensitive polyimide material layer instead of a traditional dry film, which avoids fluorescence interference from the flow channel layer itself. Attached Figure Description

[0031] Figure 1 The diagram shows a planar layout of the flow channels in the microfluidic chip of this invention.

[0032] Figure 2The diagram shows the longitudinal layout of the flow channels of the functional units in the microfluidic chip of this invention.

[0033] Figure 3 The flowchart shown is a process for single-cell culture, cell population screening and export of the present invention.

[0034] Component designation explanation

[0035] 1. Microfluidic chip

[0036] 2. Liquid inlet channel

[0037] 3. Liquid outlet channel

[0038] 4. Common flow channel

[0039] 5 Functional Units

[0040] 501 Single-cell inlet

[0041] 502 Cell Culture Screening Chamber

[0042] 503 Cell export cavity

[0043] 504 Cell Export Port

[0044] 505 drive element

[0045] 6 Single-celled organisms

[0046] 7 Single-cell printed chips

[0047] Steps S1 to S4 Detailed Implementation

[0048] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0049] Please see Figures 1 to 3 It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0050] Example 1

[0051] This embodiment provides a microfluidic chip, including a substrate layer, a flow channel layer, an inlet flow channel, an outlet flow channel, multiple common flow channels, and multiple functional units, wherein the flow channel layer is located below the substrate layer.

[0052] Specifically, the material of the flow channel layer includes at least one of silicon dioxide, spin-on glass (SOG, a polyoxysilane spin-coating material), non-photosensitive epoxy resin, and non-photosensitive polyimide. Because the flow channel layer of the microfluidic chip of this invention uses a non-photosensitive material layer, such as a silicon dioxide layer, a spin-on glass layer, a non-photosensitive epoxy resin layer, or a non-photosensitive polyimide layer, to replace the traditional dry film, the fluorescence interference problem of the flow channel layer itself can be avoided.

[0053] As an example, the substrate layer is made of silicon, in which the required circuit elements and lines can be fabricated. Since the channel layer is made of silicon dioxide, spin-coated glass, non-photosensitive epoxy resin, or non-photosensitive polyimide, not only can the fluorescence interference problem of the channel layer itself be avoided, but it can also bond well with the silicon substrate. Furthermore, it is easy to fabricate the channel layer on the silicon substrate using micro-nano processes to form the required channels. For example, a first channel layer can be formed on the substrate layer first, and the first channel layer can be patterned to obtain the required channels. Then, a second channel layer can be formed on the second channel layer to seal the channels in the first channel layer.

[0054] For example, please refer to Figure 1 and Figure 2 ,in, Figure 1 The diagram shows a planar layout of the flow channels in the microfluidic chip 1. Figure 2 The diagram shows the longitudinal layout of the flow channels of the functional units in the microfluidic chip.

[0055] Specifically, the inlet channel 2 and the outlet channel 3 are both located in the channel layer and are spaced apart; multiple common channels 4 are located in the channel layer and are spaced apart, with both ends of the common channels 4 connected to the inlet channel 2 and the outlet channel 3, respectively; multiple functional units 5 are arranged in an array, each functional unit 5 including a single-cell inlet 501, a cell culture screening chamber 502, a cell outlet chamber 503, a cell outlet 504, and a driving element 505. The single-cell inlet 501 is located in the substrate layer and is connected to the common channels 4. The cell culture screening chamber 502, the cell outlet chamber 503, and the cell outlet 504 are all located in the channel layer and are spaced apart. In the flow channel layer, the two ends of the cell culture screening chamber 502 are respectively connected to the common flow channel 4 and the cell outlet chamber 503. The cell outlet 504 is located below the cell outlet chamber 503 and is connected to the cell outlet chamber 503. The driving element 505 is located at the bottom of the cell outlet chamber 503 and faces the cell outlet 504. The driving element 505 is used to provide power to the liquid to introduce the single cell 6 introduced into the common flow channel 4 through the single cell inlet 501 into the cell culture screening chamber 502, and to export the target cell population cultured and screened in the cell culture screening chamber 502 through the cell outlet 504.

[0056] As an example, the inlet channel 2, the outlet channel 3, the common channel 4, the cell culture screening chamber 502, and the cell export chamber 503 all penetrate the channel surface facing the substrate to expose the substrate, and the driving element 505 is attached to the surface of the substrate.

[0057] Specifically, the functional unit 5 has functions of single-cell isolation, culture, cell population 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, or 5,000.

[0058] As an example, Figure 1 The flow path of the liquid is indicated by dashed arrows. The inlet of the liquid inlet channel 2 and the outlet of the liquid outlet channel 3 can be adjusted as needed, for example, they can be located at a preset position at one end or in the middle of the channel, which is not shown in the figure. The culture medium and reagents can be driven by external power (e.g., an injection pump) to ensure cell culture and screening.

[0059] As an example, such as Figure 1As shown, the extending direction of the inlet channel 2 is parallel to the extending direction of the outlet channel 3, and the extending direction of the common channel 4 is perpendicular to the extending direction of the inlet channel 2. This allows the multiple functional units 5 to be arranged in a regular square array, facilitating cooperation with the printing nozzle of the single-cell printing chip and with the cell receiving device. In other embodiments, the connection method of the inlet channel 2, the outlet channel 3, and the common channel 4 can also be adjusted as needed, and is not limited to this embodiment.

[0060] As an example, such as 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 comparable to the size of the cells to be screened, thereby ensuring the arrangement of a single layer of cells during the cell culture process, which is helpful for subsequent cell counting and fluorescence analysis.

[0061] As an example, such as Figure 1 As shown, in the plane of the flow channel layer, and in the direction perpendicular to the cell culture screening cavity 502 pointing to the cell export cavity 503, the width of the cell culture screening cavity 502 is greater than the width of the cell export cavity 503 so that the screened cells can sequentially enter the cell export cavity 503 and be exported.

[0062] As an example, the driving element 503 may include one of a heating film, a piezoelectric nozzle, a PDMS (polydimethylsiloxane) microvalve, a solenoid valve, and a peristaltic pump. In this embodiment, the driving element 503 is preferably a heating film, thereby forming a thermal bubble nozzle with the cell outlet cavity 503 and the cell outlet 504. The thermal bubble nozzle is fabricated using micro-nano processing technology and integrated into the bottom of the microchannel. The thermal bubble nozzle utilizes the instantaneous high temperature of the heating film to vaporize the liquid above, generating bubbles that propel the liquid flow and eject it from the nozzle. Subsequently, subsequent liquid replenishes the flow under capillary action, thus providing power for the continuous flow of liquid. The thermal bubble nozzle is controlled by the underlying circuitry.

[0063] As an example, such as Figure 2As 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 cell enters the common channel through the single-cell inlet 501, the sedimentation position of the single cell is located in the entrance region of the cell culture screening chamber 502, so that it can accurately enter the cell culture screening chamber 502 under the driving action of the driving element 505. The single-cell printing chip 7 can be the single-cell printing chip disclosed in Chinese Patent CN108330065A or other suitable single-cell printing chips to achieve the separation of single cells and their introduction into the cell inlet 501. In this embodiment, a single-cell printing chip including a thermal bubble printing chip is preferably used, which causes less damage to the cells and can efficiently and gently introduce single cells into the single-cell inlet 501.

[0064] The integrated microfluidic chip of this invention can be used for single-cell isolation, culture, screening and export of target cell populations, enabling single-cell culture, cell population identification and screening, and export of target cell populations to all be completed on-chip. This increases cell throughput, simplifies experimental operations, and reduces reagent consumption and potential cross-contamination risks. Furthermore, by combining it with thermal bubble printing technology, the entire process becomes more controllable, convenient, and efficient.

[0065] Example 2

[0066] This embodiment provides a method for single-cell culture, cell population screening, and export. Please refer to [link to relevant documentation]. Figure 3 The flowchart of this method is shown below, including the following steps:

[0067] S1: Provide the integrated microfluidic chip as described in Embodiment 1, and inject a single cell into the common flow channel through the single cell inlet;

[0068] S2: After the cells settle naturally, the driving element is used to drive the liquid flow and introduce the single cell injected into the common flow channel into the cell culture screening chamber.

[0069] S3: After the cells have been cultured in the cell culture screening chamber for a preset time, screening reagents are introduced into the cell culture screening chamber through the liquid inlet channel to identify and screen out the target cell population.

[0070] S4: Transfer the target cell population into the designated container via the cell outlet.

[0071] As an example, in step S1, before injecting a single cell into the common channel through the single cell inlet, the cell culture medium is first perfused into the integrated microfluidic chip to remove air bubbles.

[0072] As an example, in step S2, after the single cells injected into the common channel are introduced into the cell culture screening chamber, cell culture medium is perfused again, allowing nutrients to diffuse into the cell culture screening chamber to culture the cells. In this embodiment, the cell culture medium is perfused using an injection pump after all single cells have entered their respective cell culture screening chambers.

[0073] As an example, in step S3, after introducing the screening reagent into the cell culture screening chamber, the target cell population is screened by performing fluorescence image characterization on the cell population within the cell culture screening chamber. The screening reagent can be a phenotypic identification antibody or other targets, depending on the cells to be screened. In this embodiment, after the cells are cultured in the cell culture screening chamber for several days, phenotypic identification antibodies or other targets are injected into the chip, and the target cell population with good performance is screened based on the fluorescence characteristics of the cell population.

[0074] As an example, in step S4, thermal bubble printing is preferably used to transfer the target cell population into a designated container for subsequent culture or analysis. Thermal bubble printing has the advantages of minimal cell damage, rapid response, strong driving force, easy control, easy integration, and miniaturization, ensuring the convenience and efficiency of the entire process.

[0075] As an example, the single-cell culture, cell population screening and export method of this embodiment can be used to screen monoclonal antibody cell populations or other types of cell populations.

[0076] Example 3

[0077] This embodiment uses the microfluidic chip from Embodiment 1 for monoclonal cell line screening. First, cell culture medium is perfused into the chip, and air bubbles are expelled. Then, perfusion is stopped, and transfected cells are placed individually at the single-cell inlet of the chip using a single-cell printing chip. The underlying circuitry controls the operation of the thermal bubble nozzle to introduce the single cells into the cell culture screening chamber. To obtain a monolayer of cells, the height of the cell culture screening chamber can be adjusted according to the size of the cells used. The culture medium is re-perfused, and after the cells have reached a certain population size, relevant fluorescent antibodies are injected into the reagent inlet to characterize the antibody titer secreted by each cell population. Then, the target cell population is screened as needed. At this point, the target cell population is again transferred to a designated container through the thermal bubble nozzle at the cell outlet, completing the screening of the monoclonal antibody cell population.

[0078] In summary, this invention provides an integrated microfluidic chip for single-cell isolation, culture, screening, and export of target cell populations. This chip enables on-chip completion of single-cell culture, cell population identification and screening, and export of target cell populations, increasing cell throughput, simplifying experimental operations, and reducing reagent consumption and potential cross-contamination risks. Furthermore, by incorporating thermal bubble printing technology, the entire process becomes more controllable, convenient, and efficient. In addition, in the microfluidic chip of this invention, the flow channel layer uses a silica layer, spin-coated glass layer, non-photosensitive epoxy resin material layer, or non-photosensitive polyimide material layer instead of a traditional dry film, avoiding fluorescence interference from the flow channel layer itself. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0079] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A microfluidic chip, characterized in that, include: Substrate layer; A flow channel layer is located below the substrate layer, and the material of the flow channel layer includes at least one of silicon dioxide, spin-coated glass, non-photosensitive epoxy resin and non-photosensitive polyimide. The inlet and outlet channels are located in the channel layer and are spaced apart. Multiple common flow channels are located in the flow channel layer and are spaced apart. The two ends of the common flow channels are respectively connected to the liquid inlet flow channel and the liquid outlet flow channel. The system comprises multiple functional units, including a single-cell inlet, a cell culture screening chamber, a cell outlet, a cell export port, and a driving element. The single-cell inlet is located in the substrate layer and communicates with the common flow channel. The cell culture screening chamber, the cell export chamber, and the cell export port are all located in the flow channel layer, with both ends of the cell culture screening chamber communicating with the common flow channel and the cell export chamber, respectively. The cell export port is located below the cell export chamber and communicates with it. The driving element is located in the cell export chamber and faces the cell export port. The driving element is used to provide power to the liquid to introduce the single cell introduced into the common flow channel through the single-cell inlet into the cell culture screening chamber, and to export the target cell population cultured and screened in the cell culture screening chamber through the cell export port. The inlet channel, the outlet channel, the common channel, the cell culture screening chamber, and the cell export chamber all penetrate the channel surface facing the substrate to expose the substrate. The driving element is attached to the surface of the substrate. The extension direction of the inlet channel is parallel to the extension direction of the outlet channel. The extension direction of the common channel is perpendicular to the extension direction of the inlet channel.

2. The microfluidic chip according to claim 1, characterized in that: The substrate layer is made of silicon.

3. The microfluidic chip according to claim 1, characterized in that: The thickness of the cell culture screening chamber is set to accommodate only a single layer of cells.

4. The microfluidic chip according to claim 1, characterized in that: In the plane of the flow channel layer, and in the direction perpendicular to the cell culture screening cavity pointing to the cell export cavity, the width of the cell culture screening cavity is greater than the width of the cell export cavity.

5. The microfluidic chip according to claim 1, characterized in that: The single-cell inlet is used to receive single cells ejected from the single-cell printing chip.

6. The microfluidic chip according to claim 5, characterized in that: The single-cell printing chip includes a thermal bubble printing chip.

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

8. The microfluidic chip according to claim 1, characterized in that: The number of the functional units ranges from 10 to 10,000.

9. A method for single-cell culture, cell population screening and export, characterized in that, Includes the following steps: A microfluidic chip as described in any one of claims 1 to 8 is provided, wherein a single cell is injected into the common flow channel through the single cell inlet; After the cells settle naturally, the driving element is used to drive the liquid flow, introducing the single cells injected into the common flow channel into the cell culture screening chamber. After the cells have been cultured in the cell culture screening chamber for a preset time, screening reagents are introduced into the cell culture screening chamber through the liquid inlet channel to identify and screen out the target cell population. The target cell population is transferred into a designated container via the cell outlet.

10. The method for single-cell culture, cell population screening and export according to claim 9, characterized in that: Before injecting a single cell into the common channel through the single cell inlet, the cell culture medium is first perfused into the microfluidic chip to remove air bubbles.

11. The method for single-cell culture, cell population screening and export according to claim 10, characterized in that: After the single cells injected into the common channel are introduced into the cell culture screening chamber, cell culture medium is perfused again to culture the cells.

12. The method for single-cell culture, cell population screening and export according to claim 11, characterized in that: The cell culture medium was perfused using a syringe pump.

13. The method for single-cell culture, cell population screening and export according to claim 9, characterized in that: After the screening reagent is introduced into the cell culture screening chamber, the target cell population is screened out by performing fluorescence image characterization on the cell population in the cell culture screening chamber.

14. The method for single-cell culture, cell population screening and export according to claim 9, characterized in that: The single-cell culture, cell population screening and export method is used for screening monoclonal antibody cell populations.

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