Microfluidic chip and method for culturing single cells and sorting and extracting cell populations - Patents.com

Abstract

A microfluidic chip and a method for culturing a single cell and selecting and extracting a cell group are provided. The chip (1) includes a substrate layer, a flow path layer, a liquid supply flow path (2), a liquid discharge flow path (3), a plurality of common flow paths (4), and a plurality of functional units (5). The flow path layer is located below the substrate layer. The material of the flow path layer includes at least one of silicon dioxide, spin-on glass, non-photosensitive epoxy resin, and non-photosensitive polyimide. Both ends of the common flow path (4) are connected to the liquid supply flow path (2) and the liquid discharge flow path (3), respectively. The functional unit (5) includes a single cell inlet (501), a cell culture selection chamber (502), a cell discharge chamber (503), a cell discharge port (504), and a drive member (505). The driving member (505) is used to supply power to the liquid to draw the single cell (6) introduced into the common flow channel (4) into the cell culture and selection chamber (502) and to discharge the cultured and selected target cell group from the cell outlet (504). The microfluidic chip can complete the culture of a single cell, the identification and selection of the cell group, and the discharge of the target cell group all on the chip, making experimental operations easier, reducing the risk of cross-contamination, and avoiding fluorescent interference from the flow channel layer itself.

Description

[Technical field]

[0001] The present invention is in the field of microfluidics, cell line development, monoclonal antibody selection, etc. and relates to microfluidic chips and methods for single cell culture and the selection and derivation of cell populations. [Background technology]

[0002] A cell is the basic unit of life activity. Therefore, research at the single cell level can reveal the laws of the development of life activity from a deeper level, and is widely applied in the fields of monoclonal antibody selection, cell line culture, etc. Single cell isolation is the basis and key point of single cell research. Currently, single cell isolation is mainly performed using micropipette aspiration, limiting dilution, microwell arrays, and microfluidics-based selection methods. However, current isolation methods face problems such as high operational difficulty, low efficiency, and the acquisition of multiple cells, which are inconvenient for subsequent culture and analysis. In addition, when applied to monoclonal antibody selection and cell line culture, the isolated single cells are often placed on a microplate and cultured, and the cell groups are subjected to potency and phenotype analysis, and then the cell groups with excellent performance are selected and cultured on a large scale. Therefore, the entire process requires a lot of effort, is complicated, takes a long time, and is low in efficiency. Therefore, particularly in single-cell research such as the selection of monoclonal antibodies and the development of cell lines, there is an urgent need for efficient research methods that are easy to manipulate and integrate the isolation and culture of single cells with the selection and derivation of cell populations. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of the above-mentioned shortcomings of the conventional techniques, an object of the present invention is to provide a microfluidic chip and a method for culturing single cells and selecting and extracting cell groups, in order to solve the problems that the conventional techniques for processes such as cell isolation and culture and selection of cell groups require a great deal of labor, complicated operations, long times, and low efficiency. [Means for solving the problem]

[0004] In order to achieve the above and other related objects, the present invention provides an integrated microfluidic chip, the integrated microfluidic chip comprising: a substrate layer; a flow path layer located below the substrate layer and made of at least one of silicon dioxide, spin-on glass, non-photosensitive epoxy resin, and non-photosensitive polyimide; a liquid supply flow path and a liquid discharge flow path located in the flow path layer and spaced apart; a plurality of common flow paths located in the flow path layer and spaced apart, the common flow path having both ends connected to the liquid supply flow path and the liquid discharge flow path, respectively; and a plurality of functional units including a single cell inlet, a cell culture selection chamber, a cell outlet chamber, a cell outlet, and a drive member, the single cell inlet being located in the substrate layer and connected to the common flow path, the cell culture sorting chamber, the cell outlet chamber and the cell outlet are all located in the flow path layer, and both ends of the cell culture sorting chamber are connected to the common flow path and the cell outlet chamber, respectively, the cell outlet is located below the cell outlet chamber and connected to the cell outlet chamber, the driving member is located in the cell outlet chamber and faces the cell outlet, and the driving member supplies power to a liquid to draw a single cell introduced into the common flow path through the single cell inlet into the cell culture sorting chamber and to discharge a group of target cells cultured and selected in the cell culture sorting chamber from the cell outlet.

[0005] Optionally, the liquid supply flow path, the liquid discharge flow path, the common flow path, the cell culture selection chamber and the cell discharge chamber all penetrate a surface of the flow path layer facing the substrate layer to expose the substrate layer. The driving member is bonded to a surface of the substrate layer.

[0006] Optionally, the material of the substrate layer includes silicon.

[0007] Optionally, the extending direction of the liquid supply flow path and the extending direction of the liquid discharge flow path are parallel to each other.

[0008] Optionally, the extension direction of the common flow channel and the extension direction of the liquid supply flow channel are perpendicular to each other.

[0009] Optionally, the thickness of the cell culture sorting chamber is such that it can accommodate only a monolayer of cells.

[0010] Optionally, in a plane in which the flow path layer is located and in a direction perpendicular to a direction in which the cell culture selection chamber faces the cell discharge chamber, the width of the cell culture selection chamber is larger than the width of the cell discharge chamber.

[0011] Optionally, the single cell inlet is used to receive a single cell ejected from a single cell printing tip.

[0012] Optionally, the single cell printing chip comprises a thermal bubble printing chip.

[0013] Optionally, the actuation member includes any of a heating film, a piezoelectric nozzle, a PDMS microvalve, a solenoid valve, and a peristaltic pump.

[0014] Optionally, the number of said functional units is in the range of 10 to 10,000.

[0015] The present invention further provides a method for culturing a single cell and selecting and extracting a cell population, comprising the steps of providing any one of the microfluidic chips described above, injecting a single cell from the single cell inlet into the common flow channel, drawing the single cell injected into the common flow channel into the cell culture and selection chamber by causing a liquid to flow with the driving member after the cells have naturally settled, culturing the cells in the cell culture and selection chamber for a predetermined time, introducing a selection reagent into the cell culture and selection chamber through the liquid supply flow channel to identify and select a target cell population, and transferring the target cell population into a predetermined container through the cell outlet.

[0016] Optionally, a cell culture medium is flowed into the integrated microfluidic chip to remove air bubbles before injecting a single cell into the common channel through the single cell inlet.

[0017] Optionally, the single cells injected into the common flow channel are drawn into the cell culture selection chamber, and then a cell culture medium is again poured in to culture the cells.

[0018] Optionally, a syringe pump is used to pump the cell culture medium.

[0019] Optionally, a target cell population is selected by introducing a sorting reagent into the cell culture sorting chamber and then performing a fluorescent image characterization of the cell population within the cell culture sorting chamber.

[0020] Optionally, the method for culturing single cells and selecting and deriving cell populations is used to select cell populations for monoclonal antibodies. Effect of the Invention

[0021] As described above, the present invention provides a microfluidic chip for use in the isolation and culture of single cells and the selection and extraction of target cell groups. The chip allows single cell culture, identification and selection of cell groups, and extraction of target cell groups to all be completed on the chip, improving cell throughput, simplifying experimental operations, and reducing the amount of reagent used and the risk of potential cross-contamination. In addition, the combination of thermal bubble printing technology makes the entire process easier to control, simple, and highly efficient. In addition, the microfluidic chip of the present invention uses a silicon dioxide layer, a spin-on glass layer, a non-photosensitive epoxy resin material layer, or a non-photosensitive polyimide material layer for the flow channel layer instead of the conventional dry film, making it possible to avoid fluorescence interference from the flow channel layer itself. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows a planar layout diagram of channels in a microfluidic chip of the present invention. [Diagram 2] FIG. 2 shows a vertical layout diagram of channels of functional units in a microfluidic chip of the present invention. [Diagram 3] FIG. 3 shows a flow chart of the method for single cell culture and cell population selection and derivation in the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Hereinafter, the embodiments of the present invention will be described with reference to specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Furthermore, the present invention may be implemented or applied in other different specific embodiments. In addition, various supplements or modifications may be made to each of the details in this specification based on different perspectives and applications without departing from the spirit of the present invention.

[0024] Please refer to Figures 1 to 3. It should be noted that the drawings provided in this embodiment are merely for a schematic explanation of the basic idea of ​​the present invention. The drawings only show assemblies related to the present invention, and are not based on the number, shape and size of the assemblies in actual implementation. The form, number and ratio of each assembly in actual implementation may be changed arbitrarily, and the layout and shape of the assemblies may be more complicated. EXAMPLES

[0025] In this embodiment, a microfluidic chip is provided, which includes a substrate layer, a flow channel layer, a liquid supply channel, a liquid discharge channel, a plurality of common channels, and a plurality of functional units. The flow channel layer is located below the substrate layer.

[0026] Specifically, the material of the flow path layer includes at least one of silicon dioxide, spin-on glass (SOG, a polyoxysilane-based spin-coating material), non-photosensitive epoxy resin, and non-photosensitive polyimide. Instead of a conventional dry film, a non-photosensitive material layer such as a silicon dioxide layer, a spin-on glass layer, a non-photosensitive epoxy resin material layer, or a non-photosensitive polyimide material layer is used for the flow path layer of the microfluidic chip in the present invention, so that the problem of fluorescence interference of the flow path layer itself can be avoided.

[0027] As an example, the material of the substrate layer includes silicon, and desired circuit elements and circuits can be fabricated on the layer. In addition, since a silicon dioxide layer, a spin-on glass layer, a non-photosensitive epoxy resin material layer, or a non-photosensitive polyimide material layer is used for the flow path layer, not only can the problem of fluorescence interference of the flow path layer itself be avoided, but also good bonding with the silicon substrate can be achieved. In addition, it is also easy to process the flow path layer on the silicon substrate using micro-nano technology to form the desired flow path. For example, first, a first flow path layer is formed on the substrate layer, and the first flow path layer is patterned to obtain the desired flow path. Next, a second flow path layer is formed on the second flow path layer to block the flow path in the first flow path layer.

[0028] As an example, refer to Figures 1 and 2. Figure 1 shows a planar layout diagram of channels in the microfluidic chip 1, and Figure 2 shows a vertical layout diagram of channels of functional units in the microfluidic chip.

[0029] Specifically, the liquid supply flow path 2 and the liquid discharge flow path 3 are both located in the flow path layer and are provided with a gap therebetween. In addition, the plurality of common flow paths 4 are located in the flow path layer and are provided with a gap therebetween. Both ends of the common flow path 4 are respectively connected to the liquid supply flow path 2 and the liquid discharge flow path 3. The plurality of functional units 5 are arranged in an array format. The functional units 5 include a single cell inlet 501, a cell culture selection chamber 502, a cell discharge chamber 503, a cell discharge outlet 504, and a drive member 505. The single cell inlet 501 is located in the substrate layer and is connected to the common flow path 4. The cell culture selection chamber 502, the cell discharge chamber 503, and the cell discharge outlet 504 are all located in the flow path layer. In addition, both ends of the cell culture selection chamber 502 are respectively connected to the common flow path 4 and the cell discharge chamber 503. The cell outlet port 504 is located below the cell outlet chamber 503 and communicates with the cell outlet chamber 503. The driving member 505 is located at the bottom of the cell outlet chamber 503 and faces the cell outlet port 504. The driving member 505 is used to draw the single cell 6 introduced into the common flow channel 4 through the single cell inlet 501 into the cell culture and sorting chamber 502 by supplying power to a liquid, and to discharge the target cell group cultured and sorted in the cell culture and sorting chamber 502 from the cell outlet port 504.

[0030] As an example, the liquid supply flow path 2, the liquid discharge flow path 3, the common flow path 4, the cell culture selection chamber 502, and the cell discharge chamber 503 all penetrate the surface of the flow path layer facing the substrate layer to expose the substrate layer. Also, the driving member 505 is bonded to the surface of the substrate layer.

[0031] Specifically, the functional unit 5 has a function of isolating and culturing a single cell, and selecting and extracting a cell group. If necessary, a large number of the functional units 5 can be integrated into the integrated microfluidic chip, thereby ensuring high throughput in culturing and selecting a single cell. As an example, the number of the functional units 5 ranges from 10 to 10,000, e.g., 1,000, 2,000, 5,000, etc.

[0032] As an example, the flow path of the liquid is indicated by dashed arrows in Fig. 1. The inlet of the liquid supply flow path 2 and the outlet of the liquid discharge flow path 3 can be adjusted as necessary, and may be, for example, at one end of the flow path or at a predetermined position in the center of the flow path, but this is not shown in the figure. In addition, the culture solution and reagent may be operated by an external power source (e.g., a syringe pump) to ensure cell culture and selection.

[0033] 1, the extension direction of the liquid supply flow path 2 and the extension direction of the liquid discharge flow path 3 are parallel, and the extension direction of the common flow path 4 and the extension direction of the liquid supply flow path 2 are perpendicular. This allows a plurality of functional units 5 to be arranged in a regular rectangular array, which is convenient for cooperation with a printing nozzle of a single cell printing chip and for cooperation with a cell receiving device. In other embodiments, the communication method of the liquid supply flow path 2, the liquid discharge flow path 3, and the common flow path 4 may be adjusted as necessary, and is not limited to this embodiment.

[0034] As an example, as shown in Fig. 2, the thickness of the cell culture sorting chamber 502 is set so as to accommodate only a single layer of cells. For example, the thickness of the cell culture sorting chamber 502 may be equal to the size of the cells that need to be sorted. This ensures that the cells are arranged in a single layer during the cell culture process, which is convenient for subsequent cell counting and fluorescence analysis.

[0035] As an example, as shown in FIG. 1, in the plane in which the flow path layer is located and in a direction perpendicular to the direction in which the cell culture sorting chamber 502 faces the cell discharge chamber 503, the width of the cell culture sorting chamber 502 is greater than the width of the cell discharge chamber 503, so that the sorted cells can easily enter the cell discharge chamber 503 in sequence and be discharged.

[0036] As an example, the driving member 505 may include any one of a heating film, a piezoelectric nozzle, a PDMS (polydimethylsiloxane) microvalve, a solenoid valve, and a peristaltic pump. In this embodiment, it is preferable to use a heating film for the driving member 505 to configure a thermal bubble nozzle together with the cell outlet chamber 503 and the cell outlet 504. The thermal bubble nozzle is manufactured by micro-nano processing technology and integrated into the bottom of the microchannel. The thermal bubble nozzle uses the instantaneous high temperature of the heating film to vaporize the liquid above, generating bubbles to cause the liquid to flow and to be ejected from the nozzle. Then, the subsequent liquid is replenished by the action of capillary force, providing power for continuously flowing the liquid. The thermal bubble nozzle is controlled by a circuit in the lower layer.

[0037] As an example, as shown in FIG. 2, the single cell inlet 501 is used to receive a single cell 6 discharged from a single cell printing chip 7. The position of the single cell inlet 501 is set so that the sedimentation position of the single cell is the inlet region of the cell culture selection chamber 502, so that the single cell enters the common flow channel from the single cell inlet 501 and then accurately enters the cell culture selection chamber 502 by the driving action of the driving member 505. The single cell printing chip 7 may be a single cell printing chip disclosed in Chinese Patent Publication CN108330065A or other suitable single cell printing chip. This realizes the separation of single cells and their introduction into the single cell inlet 501. In this embodiment, it is preferable to use a single cell printing chip including a thermal bubble printing chip that causes little damage to cells and can introduce single cells efficiently and gently into the single cell inlet 501.

[0038] The integrated microfluidic chip of the present invention can be used for the isolation and culture of single cells, as well as the selection and extraction of target cell populations. This allows the culture of single cells, the identification and selection of cell populations, and the extraction of target cell populations to all be completed on the chip, improving cell throughput, simplifying experimental operations, reducing the amount of reagent used, and reducing the risk of potential cross-contamination. In addition, the combination of thermal bubble printing technology makes the entire process easier to control, simple, and efficient. EXAMPLES

[0039] In this example, a method for single cell culture and cell population selection and derivation is provided. Reference is now made to Figure 3, which shows a flow chart of the method. The method comprises the following steps:

[0040] Step S1 includes providing the integrated microfluidic chip described in Example 1, and injecting a single cell into the common channel through the single cell inlet.

[0041] After the cells have naturally settled, the driving member is used to move the liquid to draw the single cells injected into the common flow channel into the cell culture and selection chamber, in step S2.

[0042] After culturing the cells in the cell culture selection chamber for a predetermined period of time, a selection reagent is introduced into the cell culture selection chamber through the liquid supply flow path, thereby identifying and selecting a target cell group, in step S3.

[0043] Step S4: transferring the target cell group into a predetermined container through the cell outlet.

[0044] As an example, in step S1, before a single cell is injected from the single cell inlet into the common flow channel, a cell culture solution is poured into the integrated microfluidic chip to remove air bubbles.

[0045] As an example, in step S2, the single cells injected into the common flow channel are drawn into the cell culture selection chamber, and then a cell culture medium is poured in again to cause nutrients to diffuse and enter the cell culture selection chamber, thereby culturing the cells. In this embodiment, after all the single cells have entered the corresponding cell culture selection chamber, the cell culture medium is poured in using a syringe pump.

[0046] For example, in step S3, a selection reagent is introduced into the cell culture selection chamber, and then a fluorescent image characteristic evaluation is performed on the cell population in the cell culture selection chamber to select a target cell population. The selection reagent may be a phenotyping antibody or other target, and is specifically determined according to the cells to be selected. In this embodiment, after the cells are cultured in the cell culture selection chamber for several days, a phenotyping antibody or other target is injected into the chip, and a target cell population with good performance is selected based on the fluorescent characteristics of the cell population.

[0047] As an example, in step S4, the target cell group is preferably transferred into a certain container by thermal bubble printing for subsequent culture or analysis. The thermal bubble printing method has the advantages of small cell damage, fast response, strong driving force, easy control, easy integration and miniaturization, which ensures the easy and efficient implementation of the whole process.

[0048] As an example, the methods for culturing single cells and selecting and deriving cell populations in this example can be used to select cell populations for monoclonal antibodies or other types of cell populations. EXAMPLES

[0049] In this embodiment, the microfluidic chip in Example 1 is used to select monoclonal cell lines. First, cell culture fluid is poured into the chip to remove air bubbles. Next, the pouring of culture fluid is stopped, and the transfected cells are placed in the single cell inlet of the chip in the form of single cells by passing them through the single cell printing chip. Then, the operation of the thermal bubble nozzle is controlled by the circuit in the lower layer to allow the single cells to enter the cell culture selection chamber. In order to obtain a cell group in which the cells are arranged in a single layer, the height of the cell culture selection chamber may be adjusted according to the size of the cells used. Next, the culture fluid is poured again, and the cells are cultured until they form a certain colony, and then the potency of the antibody secreted by each cell group is characterized by injecting the relevant fluorescent antibody into the reagent inlet. Then, the target cell group is selected as necessary. In this case, the thermal bubble nozzle at the cell outlet is used again to transfer the target cell group into a specified container, completing the selection of the cell group for the monoclonal antibody.

[0050] In summary, the present invention provides an integrated microfluidic chip for the isolation and culture of single cells and the selection and extraction of target cell groups. The chip allows single cell culture, cell group identification and selection, and target cell group extraction to all be completed on the chip, improving cell throughput, simplifying experimental operations, and reducing the amount of reagent used and the risk of potential cross-contamination. In addition, the combination of thermal bubble printing technology makes the entire process more controllable, simple, and highly efficient. In addition, the microfluidic chip of the present invention uses a silicon dioxide layer, a spin-on glass layer, a non-photosensitive epoxy resin material layer, or a non-photosensitive polyimide material layer for the flow path layer instead of the conventional dry film, so that the fluorescence interference of the flow path layer itself can be avoided. Therefore, the present invention effectively solves various shortcomings in the prior art and has high industrial applicability.

[0051] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Those familiar with the art can supplement or modify the above embodiments without departing from the spirit and scope of the present invention. Therefore, any equivalent supplement or modification completed by a person skilled in the art without departing from the spirit and technical ideas disclosed in the present invention is still included in the scope of the claims of the present invention. [Explanation of symbols]

[0052] 1. Microfluidic chip 2 Liquid supply channel 3. Discharge flow path 4 Common flow path 5 Functional Units 501 Single cell inlet 502 Cell Culture Selection Room 503 Cell extraction room 504 Cell outlet 505 Driving member 6. Single Cells 7. Single-cell printing chip S1~S4 steps

Claims

1. A substrate layer; a flow path layer located below the substrate layer and made of at least one of silicon dioxide, spin-on glass, non-photosensitive epoxy resin, and non-photosensitive polyimide; A liquid supply flow path and a liquid discharge flow path are located in the flow path layer and are spaced apart from each other; a plurality of common flow paths located in the flow path layer and spaced apart from each other, the common flow paths each having both ends communicating with the liquid supply flow path and the liquid discharge flow path, a cell culture sorting chamber, a cell outlet chamber, a cell outlet, and a driving member, the cell culture sorting chamber, a cell outlet, and a driving member, the cell culture sorting chamber, a cell outlet, and a driving member are arranged on the flow path layer, the cell culture sorting chamber, the cell outlet chamber, and the cell outlet are all arranged on the flow path layer, and both ends of the cell culture sorting chamber are respectively arranged on the common flow path and the cell outlet chamber, the cell outlet is arranged below the cell outlet chamber, and is arranged on the cell outlet chamber, the driving member is arranged in the cell outlet chamber, and faces the cell outlet, the driving member supplies power to a liquid, the driving member being a functional unit used for drawing a single cell introduced into the common flow path through the single cell inlet into the cell culture sorting chamber, and for discharging a group of target cells cultured and selected in the cell culture sorting chamber from the cell outlet.

2. 2. The microfluidic chip according to claim 1, wherein the liquid supply flow path, the liquid discharge flow path, the common flow path, the cell culture selection chamber and the cell discharge chamber all penetrate a surface of the flow path layer facing the substrate layer to expose the substrate layer, and the driving member is bonded to a surface of the substrate layer.

3. The microfluidic chip according to claim 1 , wherein the material of the substrate layer includes silicon.

4. 2. The microfluidic chip according to claim 1, wherein the liquid supply channel and the liquid discharge channel extend in a direction parallel to each other.

5. The microfluidic chip according to claim 4 , wherein the extension direction of the common channel and the extension direction of the liquid supply channel are perpendicular to each other.

6. 2. The microfluidic chip according to claim 1, wherein the thickness of the cell culture selection chamber is set so as to be capable of accommodating only a monolayer of cells.

7. 2. The microfluidic chip according to claim 1, wherein the width of the cell culture selection chamber is larger than the width of the cell discharge chamber in a plane on which the flow path layer is located and in a direction perpendicular to the direction in which the cell culture selection chamber faces the cell discharge chamber.

8. The microfluidic chip according to claim 1 , wherein the single cell inlet is used to receive a single cell ejected from a single cell printing chip.

9. The microfluidic chip of claim 8 , wherein the single cell printing chip comprises a thermal bubble printing chip.

10. The microfluidic chip according to claim 1 , wherein the actuation member comprises any one of a heating film, a piezoelectric nozzle, a PDMS microvalve, an electromagnetic valve, and a peristaltic pump.

11. The microfluidic chip according to claim 1, wherein the number of said functional units is in the range of 10 to 10,000.

12. Providing the microfluidic chip according to any one of claims 1 to 11, comprising the steps of injecting a single cell from the single cell inlet into the common channel; After the cells have naturally settled, the driving member is used to move the liquid to draw the single cells injected into the common flow channel into the cell culture selection chamber; Cultivating cells in the cell culture and selection chamber for a predetermined period of time, and then introducing a selection reagent into the cell culture and selection chamber through the supply fluid flow path to identify and select a target cell group; A method for culturing a single cell and selecting and extracting a cell population, comprising the step of transferring a target cell population into a specified container through the cell outlet.

13. The method for culturing single cells and selecting and extracting cell populations as described in claim 12, characterized in that before injecting a single cell from the single cell inlet into the common flow path, a cell culture medium is poured into the microfluidic chip to remove air bubbles.

14. The method for culturing single cells and selecting and extracting cell groups as described in claim 13, characterized in that after the single cells injected into the common flow path are drawn into the cell culture selection chamber, cell culture medium is again poured in to culture the cells.

15. The method for culturing a single cell and selecting and extracting a cell population according to claim 14, characterized in that the cell culture medium is pumped in using a syringe pump.

16. The method for culturing single cells and sorting and extracting cell populations as described in claim 12, characterized in that after introducing a sorting reagent into the cell culture sorting chamber, a target cell population is sorted by performing fluorescent image characteristic evaluation of the cell population in the cell culture sorting chamber.

17. The method for culturing single cells and selecting and deriving cell populations according to claim 12, characterized in that the method for culturing single cells and selecting and deriving cell populations is used for selecting cell populations of monoclonal antibodies.

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