CN112326964A

CN112326964A - Method for screening target cells, kit and application thereof

Info

Publication number: CN112326964A
Application number: CN202011297134.6A
Authority: CN
Inventors: 高晓航; 魏雨
Original assignee: Nanjing Likang Lide Biotechnology Co ltd
Current assignee: Nanjing Lingxin Biotechnology Co.,Ltd.
Priority date: 2020-08-04
Filing date: 2020-11-18
Publication date: 2021-02-05
Also published as: CN111999493A

Abstract

The present invention provides a method of screening target cells suitable for secreting a molecule of interest, comprising: culturing the candidate single cells in a culture chamber under conditions suitable for secreting a target molecule, wherein a signal screening layer is arranged in the culture chamber, covers the single cells, and contains a signal molecule specifically recognizing the target molecule; and selecting a target cell suitable for secreting the molecule of interest based on the signal of the signal molecule in the signal screening layer. The method can effectively screen target cells secreting target molecules.

Description

Method for screening target cells, kit and application thereof

Technical Field

The present invention relates to the field of biotechnology, specifically, the present invention relates to a method for screening target cells, a kit and use thereof, and more specifically, the present invention relates to a method for screening target cells, a kit, a method for preparing antibodies, a method for screening monoclonal antibodies, a monoclonal antibody, an antibody-antigen complex, and a method for screening antibodies.

Background

The limiting dilution method is a commonly used cloning method, and means that a cell line to be re-cloned is aspirated from a culture well and counted to count the number of cells of 1 mL. Commonly used to screen fused animal cells. Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology.

However, current methods for screening cells expressing a molecule of interest, particularly for screening fusion cells capable of monoclonal antibodies, remain to be improved.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the present invention provides a gene capable of efficient expression.

In one aspect of the present invention, the present invention provides a method of screening a target cell adapted to secrete a molecule of interest, comprising: culturing the candidate single cells in a culture chamber under conditions suitable for secreting a target molecule, wherein a signal screening layer is arranged in the culture chamber, covers the single cells, and contains a signal molecule specifically recognizing the target molecule; and selecting a target cell suitable for secreting the molecule of interest based on the signal of the signal molecule in the signal screening layer. With this method, since only a single cell exists in the culture chamber, during the culture, if the single cell can secrete the target molecule, the target molecule can be specifically captured by the signal molecule, whereby the signal molecule can be aggregated in a specific range, whereby the signal of the signal molecule can be amplified, whereby by detecting the signal difference of the signal molecule, it can be determined whether the single cell can express the target molecule. Compared with the existing limiting dilution method, the method is rapid, saves cost, improves efficiency and can realize high-throughput screening.

In a second aspect of the invention, the invention provides a kit comprising, according to an embodiment of the invention: a hydrogel; a signal molecule; and a biological culture chip. The kit can be used to effectively carry out the above-described method, thereby achieving the above-described effects, and the features described above for the method are also applicable to the kit, and are not described herein again.

The present invention also features a method of making an antibody, which, according to an embodiment of the invention, includes: screening the target cells using the methods described above; subjecting the target cells to amplification and expression of antibodies. Thus, a fusion cell capable of expressing a monoclonal antibody can be efficiently obtained.

The present invention also provides a method of screening for monoclonal antibodies, which, according to an embodiment of the present invention, includes: distributing a plurality of hybridoma cells on said biological culture chip, wherein each of said culture chambers contains at most one of said hybridoma cells per said chamber; selecting positive hybridoma cells according to the method described above; isolating the positive hybridoma cells; culturing and expressing the antibody of the positive hybridoma cell so as to obtain the monoclonal antibody. As described above, by the method of the present invention, a fusion cell expressing a monoclonal antibody can be efficiently obtained, and a monoclonal antibody can be further obtained.

The invention also provides a monoclonal antibody obtained by the method.

The invention also provides an antibody-antigen complex, wherein the antibody is the monoclonal antibody, and the monoclonal antibody is connected with a signal molecule. The antibody-antigen complex can be used to efficiently obtain a partner antibody, i.e., an antibody having an epitope different from that of a known monoclonal antibody.

The invention also provides a method for screening antibodies, which comprises the following steps: (1) placing the candidate cell in a culture chamber for culturing for a predetermined time under conditions suitable for the candidate cell to express the antibody; (2) adding a signal antibody-antigen complex to the culture chamber, the signal antibody being linked to a signal molecule, the signal antibody being the monoclonal antibody of claim 15; and (3) determining whether the candidate cell is a target cell that secretes an antibody having a different epitope from the monoclonal antibody based on the signal of the signal molecule.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic view showing the structure of a biological cell culture chip according to an embodiment of the present invention;

FIG. 2 is a schematic view showing the structure of a biological cell culture chip according to another embodiment of the present invention;

FIG. 3 is a schematic view showing the structure of a biological cell culture chip according to another embodiment of the present invention;

FIG. 4 is a schematic diagram showing the structure of a template for preparing a biological cell culture chip according to an embodiment of the present invention;

FIG. 5 shows a method of screening for monoclonal antibodies according to one embodiment of the invention;

FIG. 6 shows a method of screening for partner antibodies according to one embodiment of the present invention;

FIG. 7 shows a comparison of the dryness of cultured neuronal cells under suspension culture and chip culture conditions according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

Method for screening target cells

According to an embodiment of the invention, the signal screening layer is arranged in a matrix material of the culture chamber; the signal screening layer is arranged in the culture medium; the signal screening layer contains at least one of an antibody and a probe; the signal screening layer is formed of hydrogel; or the mechanical strength of the base material is higher than that of the signal screening layer. Therefore, molecules secreted by the single cells can conveniently enter the signal screening layer, and the screening efficiency is improved.

According to an embodiment of the invention, the culture chamber is arranged on a biological culture chip comprising: a base formed from a base material; and a micro-well formed at a surface of the substrate, and opened at the surface of the substrate, and defining the culture chamber. In the course of intensive research on three-dimensional cell culture chips, the present inventors have generally found that it is difficult for cells to exhibit biophysical behavior in an in vivo environment during cell culture or culture of biomaterials using conventional three-dimensional culture chips. For this reason, the inventors have conducted intensive studies and unexpectedly found that the matrix material of the existing three-dimensional culture chip has a significant negative effect on the effect of cell culture, particularly the achievement of biological functions of single-cell secretion factors (including antibodies). Furthermore, it has been found that the different positions of the cells in contact with the matrix in the existing three-dimensional culture chip can have obvious difference from the surrounding culture environment, and the obtained microenvironment for the different positions of the cells has increased difference and heterogeneity, for example, the cells can not contact with the support of sufficient extracellular matrix at the positions of the cells in contact with the matrix. In addition, the conventional three-dimensional culture chip mostly adopts a hard substrate, so that the hard substrate can form certain stress on cells in the culture process in the cell culture process, thereby influencing the biological behavior of the cells. These factors severely affect the ability and functional detection of single-cell factor or antibody secretion.

According to an embodiment of the invention, the micro-holes form a predetermined array pattern in the substrate. Therefore, the positive micropores can be conveniently positioned, and the subsequent operation of taking out single cells for amplification culture and the like is facilitated.

According to an embodiment of the present invention, the matrix material has an equilibrium swelling ratio of about 1.25 to about 1.75.

According to an embodiment of the invention, the opening of the micro-pores has a diameter of 8-25 microns and a depth of 15-35 microns.

According to an embodiment of the invention, the matrix material comprises at least one selected from the group consisting of collagen hydrogel, methylated cellulose, agarose gel, polyacrylamide gel, or the like.

According to the embodiment of the invention, the distance between at least two adjacent micropores is 10-50 microns.

According to an embodiment of the invention, the signal molecule is a fluorescent molecule.

According to an embodiment of the invention, the fluorescent molecule comprises at least one of FITC, AF 488.

According to an embodiment of the present invention, positive cells are localized using fluorescence microscopy.

The invention also provides a monoclonal antibody obtained by the method.

According to the embodiment of the invention, compared with the traditional limiting dilution method, the method disclosed by the invention has absolute advantages in both screening efficiency and screening cost. For example, in the case of screening monoclonal antibodies, the overall cost is reduced by about 90% while the screening efficiency is improved by at least 10 times. Under the condition that the screening is not completely developed, people can be matched more reasonably and the time can be arranged according to the urgency degree of the project, and the screening efficiency can be effectively improved to be more than 30 times on the basis.

Thus, the present invention proposes a method of screening a target cell, said target cell being suitable for secreting a molecule of interest, comprising: culturing the candidate single cells in a culture chamber under conditions suitable for secreting a target molecule, wherein a signal screening layer is arranged in the culture chamber, covers the single cells, and contains a signal molecule specifically recognizing the target molecule; and selecting a target cell suitable for secreting the molecule of interest based on the signal of the signal molecule in the signal screening layer.

It will be appreciated by those skilled in the art that the culture chamber may be a culture space in a biological culture chip as described herein. Thus, the advantages and features of the previously described biological cell culture chip are equally applicable to this screening method and will not be described in further detail herein. It is emphasized that, according to embodiments of the present invention, the signal screening layer may be used as a liquid or gel-like medium to cover the single cells, or as a solid or semi-solid substrate. According to an embodiment of the present invention, in consideration of the need to isolate single cells, a gel-like material having a lower mechanical strength than the matrix, for example, a material having a lower concentration of gel material controlled, may be selected as the signal screening layer.

The signal screening layer contains at least one of labeled antibodies, proteins, polypeptides, and probes.

According to an embodiment of the invention, the signal screening layer is formed by a first hydrogel, the culture chamber is arranged on a biological culture chip, the biological culture chip comprises:

a matrix formed from a second hydrogel; and

a micro-well formed at a surface of the substrate and opened at the surface of the substrate, and defining the culture chamber,

wherein the concentration of the first hydrogel is less than the concentration of the second hydrogel.

Thus, in a further aspect of the present invention, the present invention provides a kit comprising:

a hydrogel;

a signal molecule; and

biological cell culture chip.

The above method can be effectively carried out using the kit, and the features and advantages of the method are also applicable to the kit, and are not described herein again.

Further, the present invention provides a method for screening an antibody, comprising:

screening the target cells using the methods described above;

subjecting the target cells to amplification and expression of antibodies.

Referring to fig. 5, the present invention also provides a method for screening monoclonal antibodies derived from single cells, comprising:

distributing a plurality of hybridoma cells on said biological culture chip to form an array, wherein each of said culture chambers contains at most one of said hybridoma cells per said chamber;

according to the method, the cell chip containing the hybridoma single cell array is placed in a culture medium containing the marked target antigen (protein or polypeptide), and when an antigen-antibody reaction occurs, the marked positive hybridoma secreting the target antibody is selected;

isolating the positive hybridoma cells;

culturing and antibody expressing the positive hybridoma cells to obtain monoclonal antibodies of single cell origin.

Thus, the present invention also provides a monoclonal antibody of single cell origin, which is obtained by the foregoing method.

Thus, the present invention also provides an antibody-antigen complex, wherein the antibody is the monoclonal antibody described above, and the monoclonal antibody is linked to a signal molecule.

Further, referring to fig. 6, the present invention also provides a method for high throughput screening of paired antibodies (abbreviated as "paired antibodies") against the same antigen but different epitopes, comprising:

(1) placing the candidate cell in a culture chamber for culturing for a predetermined time under conditions suitable for the candidate cell to express the antibody;

(2) adding a signal antibody-antigen complex into the culture chamber, wherein the signal antibody is connected with a signal molecule, and the signal antibody is the single-cell source monoclonal antibody or the derivative thereof, including the antibody fragment which is combined with the antigen specific determinant, such as Fab (fragment of antigen binding), (Fab)2 and Fv; and

adding an antigen without a label into the culture chamber, if the cell secretes an antibody, the secreted antibody forms an antibody-antigen complex with the antigen and is captured around the cell, and then adding a signal antibody which is connected with a signal molecule and is the single-cell-derived monoclonal antibody in the culture medium of the cell chipAntibodies or derivatives thereof, including antibody fragments thereof which bind to specific epitopes of antigens, including Fab (fragment of antigen binding), (Fab)₂And Fv and the like

(3) Determining whether the candidate cell is a target cell that secretes an antibody having a different epitope from the single cell-derived monoclonal antibody based on a signal of the signal molecule.

For convenience of understanding, a culture chip applicable to the present invention will be described in detail below.

Biological cell culture chip

A biological cell culture chip according to an embodiment of the present invention is described below with reference to the accompanying drawings.

As shown in fig. 1 to 3, according to an embodiment of the present invention, a biological cell culture chip is provided, which includes: a substrate 100 and micro-pores 200, wherein the substrate 100 is formed of a substrate material having an equilibrium swelling ratio of about 1.25-1.75, the micro-pores 200 are formed on a surface of the substrate 100, and the micro-pores 200 are opened at the surface of the substrate 100, wherein the micro-pores 200 define a biological culture space for culturing cell biomaterials.

In the prior art, the interface compatibility between the chip matrix and the cell culture medium is poor. The hard matrix interface and the cell culture medium are incompatible, which can cause the nonuniformity of the three-dimensional culture microenvironment of the cells of the array, and the liquid layer between the interfaces can easily cause the sliding of the two interfaces, thus causing the damage of the cell array, influencing the realization of the cell biological function, and influencing the complete capture of cell detection signals and the sensitivity of detection, such as the detection of secreted proteins.

According to the embodiment of the invention, because the matrix material has a certain equilibrium swelling ratio, when the biological culture chip is used for culture, all surfaces of biological materials such as single cells can be in a state of being surrounded by liquid or gelatinous extracellular matrix material, so that a uniform microenvironment can be provided for culture of the biological cell materials, and the biological activity of the cells can be further improved in the process of culturing the cells, and the cells can be promoted to realize biological functions.

According to the embodiment of the present invention, further, the substrate used in the present invention can generate corresponding deformation according to the growth of cells during the cultivation process of the biological cell material. In other words, compared with the traditionally used hard matrix such as glass, silicon chip, plastic and the like, the matrix material adopted by the invention belongs to a soft matrix, so that the acting force between the matrix and the cells and the extracellular matrix biological material for cell culture can be reduced, and the interface compatibility between the cells and the matrix and the extracellular matrix can be further improved. In addition, according to an embodiment of the present invention, the substrate may be recessed in a region contacting the cell during the growth of the cell during the culture. As will be appreciated by those skilled in the art, the depressions are formed by the growth of cells which abut the surface of the matrix and thereby deform the matrix. One skilled in the art can determine whether a depression is formed by microscopic observation of the interior surface of the substrate. According to an embodiment of the invention, the depth of the depression can be up to 5%, e.g. 10%, 20% of the cell diameter. The term "depth of the depression" as used herein may be determined by comparing the substrate interface map where no depression is formed with the substrate interface map where the depression is formed. The depth value is defined herein as the diameter of the largest volume sphere that can be accommodated in the formed depression.

Therefore, according to the embodiment of the invention, the microporous soft matrix with better biocompatibility, cell culture medium compatibility and mechanical tension can capture and culture cells (including single cells and a plurality of cells with controllable quantity aggregation) more efficiently, and according to the embodiment of the invention, the soft matrix is easy to be compatible and fused with different extracellular matrix environments, and a uniform and fused three-dimensional culture space is provided for the cells, so that a microenvironment beneficial to survival, proliferation, migration, protein expression and secretion, stem cell differentiation and the like and beneficial to biological function detection of array cells are created for the cells in a two-dimensional array. In addition, considering that the substrate is a soft substrate, it can be easily realized also when separating single cells from the culture space without the need to overcome the adsorption force between the conventional substrate material and the cells.

According to the embodiment of the invention, the matrix material has a solid-liquid phase change transformation temperature of 40-45 ℃. According to an embodiment of the invention, the matrix material is capable of being in a liquid state at 37 degrees celsius. The above temperatures were measured under 1 atmosphere. Thus, the matrix material can be easily made to be in a liquid state at a temperature appropriately higher than room temperature, and further cured into a predetermined shape by lowering the temperature. Since the matrix material has the above-described properties, when the matrix material is in a liquid state, cytokines can be easily added without inactivating the cytokines. During the cell culture process by adding the culture medium, the cell culture chip can absorb the liquid culture medium, thereby further releasing the cytokines into the culture space. Thus, according to an embodiment of the invention, the matrix further comprises a cytokine corresponding to the biological material. According to an embodiment of the invention, the matrix further comprises at least one selected from the group consisting of FBS, protein a/G, collagen, gelatin and BSA, or a mixture of at least two thereof in different ratios.

It should be noted that, according to the embodiment of the present invention, the specific type of the matrix material that can be used is not particularly limited as long as the above performance requirements can be satisfied. The matrix material includes at least one selected from collagen hydrogel, methylated cellulose, agarose gel, polyacrylamide gel, etc., or a mixture of at least two thereof in different ratios, in consideration of better biocompatibility and adjustment ability of properties. It will be appreciated by those skilled in the art that the desired soft matrix properties, such as solid-to-liquid phase transition temperature, etc., can be achieved by adjusting the concentration of these materials.

In addition, according to an embodiment of the present invention, the structure and size of the microwell are not particularly limited as long as a culture or growth amplification space can be provided for a single cell. According to embodiments of the present invention, the microwells may have a rectangular parallelepiped, cubic, cylindrical, or the like structure. In addition, referring to fig. 2, according to an embodiment of the present invention, the opening diameter of micro-well 200 is smaller than the bottom diameter of micro-well 200. Therefore, the single cells can be conveniently captured for culture, and the escape behavior of the cells can not occur in the culture process. In particular, according to an embodiment of the invention, the opening diameter of the microwell is at most 80%, preferably 50%, of the diameter of the bottom of the microwell. The term "diameter" as used herein is defined as the diameter of the largest area circle that the opening or bottom can accommodate. The term "depth" of a microwell as used herein refers to the shortest distance between the cross section of the opening of the microwell and the cross section of the bottom. According to an embodiment of the present invention, the openings of microwells 100 have a diameter of 8-25 microns and a depth of 15-35 microns. In addition, the inventors have found that different opening diameters can be set for different cells, for example, according to an embodiment of the present invention, the biomaterial is B cells, the opening diameter is 8-12 microns; or the biological material is hybridoma cells or tumor cells, and the diameter of the opening is 15-25 micrometers.

In addition, according to an embodiment of the present invention, the biological culture chip may include a plurality of micro wells, the micro wells constituting a predetermined pattern, and a distance between two adjacent micro wells being 10 to 100 μm. Thus, the culture of a plurality of single cells can be processed in a batch. According to the embodiment of the invention, when the distance between the micropores is 10-100 microns, the cells cultured by the micropores cannot interfere with each other between the micropores, so that the batch processing uniformity can be improved, the accuracy and the efficiency of cell biological function detection can be improved, and the high-throughput collection and analysis of cell signal data are facilitated. According to an embodiment of the present invention, referring to FIG. 3, the wells on a biological growth chip may be organized in an array, for example, an array of tens of thousands or even hundreds of thousands and more wells on a single chip.

According to the embodiment of the invention, the biological cell culture chip can be further provided with a positioning mark, and the positioning mark is formed on the substrate. Thus, the plurality of micropores can be located by these locating marks. The form of the positioning mark is not particularly limited, and according to the embodiment of the present invention, the positioning mark of the fluorescent material may be set so as to perform automatic and precise positioning under a microscope, and the coordinate line may also be set so as to position each of the micropores, so that each of the micropores can be quickly positioned and recorded after positive cells are observed. Thereby further facilitating subsequent separation of positive cells or other components in the microwells.

In addition, according to an embodiment of the present invention, a reagent or a biological factor for facilitating culture of a biomaterial, which is a hybridoma cell according to an embodiment of the present invention, may be further disposed on the inner wall surface of the micro-well; the biological material is MCF10A cells, and insulin is arranged on the inner wall surface of the micropores; or the biological material is B cells, and the inner surface of the micropore is provided with at least one of CD40L, IL2 and IL10, or a mixture of at least two of the above in different proportions.

According to the embodiment of the invention, the invention provides a biological cell culture chip (also called as a cell array chip) based on a soft matrix such as solidified hydrogel, which can well solve a series of problems in two-dimensional array and three-dimensional culture of cells in vitro, and comprises the following steps: cell capture, formation of a flux-controllable cell array, cell compatibility of a chip material, compatibility of the chip material and a cell culture medium, biological function realization and accurate detection of array cells, and the like. The inventors could meet different needs by implementing microwell arrays of different diameters and depths on gel-like soft matrix chips. The mechanical strength of the micropores can be determined by the concentration of the gel soft matrix, and the optimal mechanical strength of the micropores is adjusted according to different cell types and different biological function detection purposes; in addition, the diameter and depth of the microwells can be controlled to target single cell arrays of different cells or the aggregation of multiple cells of controllable cell numbers in the microwells; the micropore spacing is controllable, and the density of the cell array on the chip and the cell array flux on the chip per unit area are determined by the size of the spacing; the gel soft matrix can simulate the characteristics of the extracellular matrix, has good cell compatibility, can provide water, nutrition and soft space for cells, avoids the stiff mechanical strength of the hard matrix cell chip which can not be adjusted according to different cells, and avoids chemical molecule residues which are usually required by chemical modification on the hard matrix chip; the gel soft substrate has excellent compatibility with cell culture solution or three-dimensional culture hydrogel biological materials, ensures a uniform microenvironment around the array cells, and ensures the integrity and accuracy of cell function detection.

Preparation of biological culture chip

In a second aspect of the invention, the invention provides a template for preparing a biological cell culture chip as described above. Referring to fig. 4, according to an embodiment of the present invention, the template includes: a substrate 300; and a micro-column 400, the micro-column 400 being disposed on the surface of the substrate 300, the micro-column 300 having a size matching the size of the micro-well 200. According to the embodiments of the present invention, the aforementioned biological culture chip can be efficiently prepared by solidifying a matrix material on a template.

According to an embodiment of the present invention, the microcolumn has a hydrophobic surface. Thus, the template and the formed biological cell culture chip can be efficiently detached. In addition, according to an embodiment of the present invention, the surface of the microcolumn is adapted to bind the biological factor by a hydrophobic force. Thus, subsequent application of the matrix material may transfer the biological agent to the matrix material as the biological agent bound to the interior surfaces of the pores. As described above, different biological agents can be set depending on different culture objects and purposes.

In a third aspect of the present invention, the present invention provides a method for preparing the above-mentioned biological cell culture chip, which, according to an embodiment of the present invention, comprises: (1) applying the biomaterial in liquid form to the template as described above and allowing the matrix material to solidify; and (2) separating the solidified biological material from the template so as to obtain the biological cell culture chip. By this method, the above-described biological cell culture chip can be efficiently produced. According to the embodiment of the invention, the biological factors or reagents are adsorbed on the surface of the template in advance.

Specifically, according to an embodiment of the present invention, the method for preparing the biological cell culture chip comprises:

the first step is as follows: and modifying the template. So that the substances such as cytokines required in the biological culture microenvironment are bound on the template through hydrophobic force. Specifically, the material to be modified is prepared into a solution (the solvent is PBS) according to the concentration of 1 ug/ml; soaking the template in the modifying solution, and reacting for 4 hours at room temperature; after being taken out, the mixture is rinsed for 3 times by fresh PBS and deionized water in sequence, is dried by nitrogen, and is stored in a dry place for later use.

And secondly, covering the melted matrix material such as the gel matrix on the template with the columnar array, and removing the template after the matrix material such as the gel matrix is cooled and solidified to form the soft matrix micropore array chip with certain thickness and hardness. Specifically, 0.6% -1.2% agarose gel liquid can be poured on the template and placed for 5 minutes at room temperature; and removing the template after solidification to form the cell chip. And each micro-pore in the chip contains factors required by micro-environment or treatment drugs.

Culture method for biological culture cell culture chip application

In a fourth aspect of the present invention, there is provided a method for culturing cells using the biological cell culture chip of any one of the above, comprising: placing cells in the microwells; and subjecting the biochip to predetermined environmental conditions. By using the method, the cells can have an extracellular matrix microenvironment which is uniform all around in the culture process, so that the activity of the cells can be effectively maintained, and the cell culture efficiency can be improved, wherein according to the embodiment of the invention, at most 1 cell is arranged in each micropore.

According to the embodiment of the invention, the chip soft matrix has better compatibility with a cell culture medium, is favorable for providing a uniform and proper microenvironment for three-dimensional culture of high-throughput array cells, is favorable for realizing biological functions of the array cells, and is favorable for completely capturing biochemical reaction signals (such as secretory protein signals) of the cells and accurately analyzing the biochemical reaction signals.

In addition, according to the embodiment of the invention, the chip with the soft matrix can better form the extracellular matrix-like interface effect of the cell-chip, so that the three-dimensional culture of the cells is closer to the living environment of the cells in vivo, and the survival and biological function realization of the cells are facilitated.

According to the embodiment of the present invention, the cell array of the soft matrix chip is formed independent of chemical modification of the chip, and attracts and stabilizes cells by cell gravity and biomaterials such as extracellular matrix added to the microwells.

According to the embodiment of the invention, the soft substrate chip is beneficial to the glass suction needle micro-picking of positive cells (single cells or multi-cell spheres), while the hard substrate chip is difficult to micro-pick by using the glass suction needle, and the hard substrate interface of the hard substrate chip is easy to cause the breakage of the glass needle or the rupture and damage of the cells.

Example 1 chip preparation

1.1 preparation of Soft matrix chips

The templates with the columnar arrays as shown in FIG. 4 were selected and classified into the following two types according to the cell types to be used:

A. the micro-column is suitable for conventional cells (the diameter is usually 15-20 microns), the height of the micro-column is 35 microns, the diameter of the cross section is 20 microns, and the distance between the micro-columns is 30 microns.

B. The micro-column is suitable for small cells (the diameter is usually 8-10 microns), the height of the micro-column is 20 microns, the diameter of the cross section is 10 microns, and the distance between the micro-columns is 30 microns.

Preparing a modification solution by using PBS as a solvent, wherein the modification solution comprises: methylated cellulose or low melting agarose.

And (3) soaking the template in the modification solution for 4 hours at room temperature, taking out the template, sequentially rinsing the template for 3 times by using fresh PBS and deionized water, drying the template by blowing with nitrogen, and storing the dried template for later use. Therefore, the modification of the chip template is realized, and substances such as cytokines and the like required in a microenvironment are combined on the template through hydrophobic acting force.

Covering the melted hydrogel matrix on the modified template (with the columnar array), and removing the template after the gel matrix is cooled and solidified to form the soft matrix micropore array chip with the thickness of about 300-. Specifically, the inventors poured agarose gel liquid (swelling ratio about 1.25-1.75) with a concentration of 0.6% -1.2% onto the template, and left it at room temperature for 5 minutes; and removing the template after solidification to form the cell chip. And each micro-pore in the chip contains factors required by micro-environment or treatment drugs.

The composition of the soft matrix chip is summarized in the following table:

1.2 preparation of hard matrix chips

Hard matrix materials of glass and silicon wafers are adopted to prepare a hard matrix chip 1 and a hard matrix chip 2 respectively, and the preparation method and the application of the three-dimensional cell culture chip based on the soft lithography technology are referred to CN108102913A for preparation, and are not described herein again.

Example 2 comparison of Soft matrix chip and hard matrix chip in cell culture

2.1 array formation efficiency comparison

Mouse hybridoma cells, human tumor cells (MCF7), human umbilical cord mesenchymal stem cells and mouse B cells were cultured using soft matrix chips 1, 2 and 3 according to the following procedure, while the above cells were subjected to parallel experiments using hard matrix chip 1 (glass substrate). The culture conditions are conventional cell culture methods (the specific method refers to cell and molecular biology experimental guidelines), and after 15 minutes of culture, the array efficiency is determined. The detection method of the array efficiency is as follows:

the number of single cells per unit area (1 mm. times.1 mm) was observed and counted, and divided by the total number of cells, thereby obtaining the yield of the single cell array.

To achieve that only one cell can be contained in each chamber, we define the chamber space by controlling the chip specification. In addition, the few double cells caused by uncontrollable factors can be easily judged and eliminated in a statistical range through a microscope.

The above test results are summarized as follows:

single cell culture was performed on chips (20 μm/30 μm, 10 μm/30 μm). The first number represents the diameter of the cell and the second number represents the distance between adjacent cells. In the above table, 20 μm/30 μm size chips were used for the remaining cell types, except for the mouse B cells using 10 μm/30 μm size chips.

As can be seen from the above data, the hard matrix chip is not conducive to the formation of cell arrays, and the array efficiency is about 32-50%. The array efficiency of the soft matrix chip is very high, about 91-98%, and is very effective for forming a cell array.

The inventors believe that the main reason for the above differences is that the hard substrate chip, such as a glass or silicon wafer, due to its material not having voids and water permeability, cannot and hardly completely remove the gas in its small chamber, and consequently the subsequently seeded cells cannot occupy the right place by gravity sedimentation. However, for soft matrix chips, such as hydrogels, biological materials have certain pore sizes and water permeability, so that when cells are seeded, culture solution can permeate from the periphery of the chip, so that gas in the cells is completely removed, thereby facilitating the sedimentation of the cells to form an array. Thus, the ratio of the chambers effective for forming the cell array is greatly different between the hard substrate chip and the soft substrate chip having the same number of chambers, and thus the efficiency of the hard substrate chip is much lower than that of the soft substrate chip when the cell screening test is performed.

2.2 cell viability comparison on chip

According to the method of 2.1, cell culture is carried out by using a hard matrix chip and a soft matrix chip respectively, and cell viability is detected according to the following method,

we used the dead-alive assay (Thermo, L34951) to identify the viability of the cells on the chip. Where red is considered dead cells and green is considered live cells.

The results are summarized below:

the above data show that the hard matrix chip does not utilize the culture and survival of cells, easily causes the occurrence of apoptosis and necrosis of cells, and is disadvantageous for the proliferation of cells. The main reason is that the hard matrix material has no physical elasticity, and cannot provide a uniform extracellular matrix microenvironment (similar to the living space characteristics in vivo) required by 3D culture for cells in all directions during the cell culture process, so that the survival, proliferation, aging and apoptosis pathways of the cells are changed to cause aging, apoptosis or necrosis. However, the soft matrix material has good plasticity and sufficient water and scaffold space, can be adjusted along with the change of space requirement in the cell survival process, can be compatible and fused with the ECM culture medium for 3D culture of cells, provides a uniform and appropriate microenvironment for the cells, and is beneficial to the realization of cell survival and biological functions (such as factors, antibody secretion and the like). In addition, in the process of manufacturing the soft matrix chip, the inventor can properly add various factors or materials required by adapting to different cell cultures, thereby constructing a microenvironment suitable for the survival of the cell cultures and greatly improving the survival and proliferation capacity of the cells. However, hard matrix materials are difficult to do, typically by adding to culture media or chemically modifying the surface of the material, which makes it difficult to achieve either strict dispersion around cells or toxic effects on cells from residual chemicals.

2.2 comparison of Capacity to pick Single cells on chip

The purpose of a single-cell chip is generally to obtain and analyze information on a single cell of particular interest. Therefore, the objective single cell needs to be isolated for subsequent operations. For this reason, the inventors tried to perform single cell picking operation on each of the soft substrate chip and the hard substrate chip, and performed single cell picking using a capillary picking needle.

The comparative results are shown below:

chip type	Picking success rate (Single cell)	Reuse efficiency of the pick needle
			Hard matrix chip	23.5％	Is low in
Soft matrix chip	95.6％	Height of

The inventor believes that the low success rate of picking single cells on a hard substrate chip is mainly caused by that the cells are completely cultured in suspension without any restriction, so that when picking the needle to pick the cells, the nearby non-target cells are also easily picked; in addition, when the picking needle approaches the cell, the target cell is easily detached from the chip or pushed away by a slight force (force generated by movement of the picking needle in the liquid), resulting in failure in picking. However, the soft matrix chip is made of hydrogel, so that proper adhesion is easily generated between the soft matrix chip and cells, and the cells are not completely in a suspension culture environment, so that the problem of a hard matrix is solved, and the success rate of picking is improved.

In addition, the picking needle is used as a consumable, and if pollution, deformation or other problems are found, the picking needle can be replaced at any time in the cell picking process, so that the efficiency is improved. However, if the picking needle is broken due to improper operation, the picking efficiency will be greatly affected. Because the replacement of the pick-up needle and the precise adjustment of the position require time to operate. The hard matrix has no elasticity, and in the micromanipulation process, the picking needle and the chip need to be kept at a very tight distance and even contact the surface of the chip, so that weak suction force can be released to target cells as much as possible, and the target cells can be successfully picked. However, manual or semi-automatic operations are difficult to be performed precisely each time, and thus, it is often the case that the picking needle contacts the chip to cause deformation or breakage. And the soft substrate has good material elasticity, so that the soft substrate has high tolerance on the excessive contact of the picking needle, and an operator can timely find and make adjustment.

Example 3: screening of monoclonal antibodies

In this example, a single-cell-derived monoclonal antibody screening against the S1 protein of the novel coronavirus was prepared on a soft matrix chip by the method of example 1

3.1 immunization of mice

Mice were purchased from Shanghai Si Laike laboratory animals, Inc., 5 week old BALB/C female mice. After obtaining the mice, the mice were kept in an animal house for one week for acclimatization. Mice of 6 weeks of age were prepared for immunization. The novel coronavirus S1 protein was purchased from near-shore protein science and technology Co., Ltd, dissolved in PBS, and adjusted to a concentration of 1mg/ml before immunization. Emulsification was performed by syringe mixing emulsification method before antigen injection. The specific immunization scheme and conditions are as follows:

number of immunizations	Time of day	Amount of antigen used	Adjuvant selection and dosage	Immunization regimen
					One need not	Day 0	100ug	Complete adjuvant, 100ul	Abdominal cavity
Two exempt from	Day 14	50ug	Incomplete adjuvant, 100ul	Abdominal cavity
					Sanwu	Day 28	50ug	Incomplete adjuvant, 100ul	Abdominal cavity
Reinforcement	Day 35	100ug	Without adjuvant	Abdominal cavity

After the mice are subjected to the second-immunization and the third-immunization, orbital blood collection is respectively carried out to determine the titer of the antibody in serum. According to the titer, the dosage of the antigen for the triple immunity or the boosting immunity is adjusted. Thereby the immune response of the mice to the antigen reaches the optimal state.

3.2 cell fusion

According to the results of the serum titers 3 days after the boost immunization, the mice with the highest titer were selected for subsequent fusion. Before fusion, whole blood of the mice is collected and serum is obtained for standby.

SP2/0 cells were prepared in advance (3-5 days, note early resuscitation). 50ml of blank 1640 medium and 500ml of sterile water were placed in the incubator in advance, pre-warmed overnight, and prepared for the next day of fusion. The PEG used in the fusion process was preheated in an incubator before the spleen was removed.

The immunized mice were killed by decapitation and sterilized by soaking in 75% ethanol for 3-5 minutes. The spleen harvesting process was operated in a biosafety cabinet. The sterilized mice were removed, drained of ethanol, and placed on paper towels. The abdomen was exposed face up. A small opening (1cm) was cut in the left upper abdomen with scissors, and the spleen (posterior lower left lobe of liver) was removed with forceps. During the harvesting process, care was taken not to puncture the spleen and remove excess connective tissue.

The removed spleen was placed in a 40um sieve placed in a 50ml centrifuge tube filled with 1640 blank medium. Note that the liquid should not be added too full to prevent media spillage during spleen trituration. During the milling process, the spleen was allowed to contact the medium well. To facilitate grinding and isolation of splenocytes, a small hole is punctured with a syringe needle or several small holes are cut with scissors before grinding. Grinding, passing spleen contents completely through a screen, and discarding the remaining connective tissue and the like. Spleen cells were collected by centrifugation at 1300rmp for 8 min at room temperature. And repeating the steps once. After removal of the supernatant, 10ml of PBS was added to resuspend the splenocytes. 30ml of erythrocyte lysate are added, lysed at 4 ℃ for 15 minutes, during which they are mixed 2 times. Spleen cells were collected by centrifugation at 1300rmp for 8 min at room temperature. PBS resuspension and centrifugation, repeated 2 times, will lysate will be removed.

Spleen cells and tumor cells were counted separately. The final splenocytes: the ratio of tumor cells was adjusted to about 5: 1. The two cells were mixed in a 50ml centrifuge tube at room temperature at 1300rmp, centrifuged for 8 minutes and the cells were collected. The supernatant was completely removed (residues affected the fusion efficiency). The centrifugal tube is afraid of being hit by hands at the bottom, or the centrifugal tube is repeatedly moved on a safety cabinet ventilation screen plate, so that the settled cells are scattered, and the final state is uniform slurry or cell slurry. The fusion was performed using PEG preheated at 37 degrees celsius in a 37 degree water bath and terminated with blank 1640 medium. The specific fusion steps are as follows:

dripping PEG, wherein the gun tip is as close to the cells as possible along the tube wall, dripping 1ml within 1 minute, rotating the centrifugal tube while dripping, and slightly shaking to ensure that cell homogenate can fully and uniformly contact the PEG;

standing for 1 minute at room temperature;

dropping preheated blank 1640 culture medium, dropping 1ml within 1 minute, rotating the centrifuge tube while dropping, and slightly shaking to ensure that cells can fully and uniformly contact the culture medium;

preheated blank 1640 medium was added dropwise, and 2ml was added dropwise within 1 minute. The centrifugal tube is rotated while dropwise adding, and meanwhile, the centrifugal tube is slightly shaken, so that the cells can be ensured to be fully and uniformly contacted with the culture medium;

dropping preheated blank 1640 culture medium, dropping 5ml within 1 minute, rotating the centrifuge tube while dropping, and slightly shaking to ensure that cells can fully and uniformly contact the culture medium;

add preheated blank 1640 medium, about 21ml, rotate the centrifuge tube while dropping, while shaking slightly, to ensure that the cells are in full and uniform contact with the medium. After fusion, 1300rmp at room temperature, centrifugation for 8 min, and cell collection;

resuspend the fused cells in complete medium (20% FBS, 1% P/S, 1 XHAT, 1 × Hybridoma Growth Factor (HGF)) (gentle pipetting was noted), add medium at 1 × 10 in terms of splenocytes⁶Spleen cells/ml. The resuspended cells were seeded into a culture dish. The fused cells were cultured for 3-5 days, during which time the cells were changed.

3.3 chip preparation

Preparing and modifying a layer of HGF (a PBS (phosphate buffer solution) containing HGF is used as a modifying solution, which is beneficial to the survival and proliferation of hybridoma cells), sucking about 500 microliters of chip gel (sterile operation), dripping the chip gel into the center of the stamp, standing for 10 minutes at room temperature (which can effectively prevent excessive drying), slightly separating the template (sometimes also called the stamp) and the chip after solidification, and avoiding displacement in the whole separation process, otherwise, damaging the integrity of the structure. The chip is manufactured and placed in a culture dish with the diameter of 10cm for storage and standby.

4.4 Forming cell arrays

The fused cells obtained in 4.3 were collected and washed 1-2 times with PBS or medium to remove excess culture medium (antibodies in the culture supernatant would cause a high background for detection). It is recommended that the cell concentration be adjusted to 8X 10⁵---1×10⁶And/ml. The cell suspension was added dropwise to the chip surface at approximately 500. mu.l/chip. The incubator was allowed to stand for 10 minutes. The excess cell suspension is recovered by a pipette, then 500. mu.l of the culture medium is sucked up and down by tilting the chip about 30-45 degrees, the excess cells are slowly rinsed from above by dropping (without allowing the liquid to flow out of the chip), and the excess liquid is removed by a vacuum pump. And observing the condition of the cell array under a microscope, and judging whether to perform subsequent operation.

4.5 Primary labeling and Positive hybridoma screening

Currently used is the Thermo protein labeling kit (a30006), the basic operating procedures being performed according to the instructions.

Briefly, within 10 minutes of waiting for the cell array to form, the ice box was removed, while the labeled antigen and home-made 3D culture hydrogel were removed and placed in the ice box for use. A1.5 ml sterile centrifuge tube was prepared, 50. mu.l complete medium was added and placed in an ice-box until use. After the cell array was formed, 50. mu.l of hydrogel was added to each centrifuge tube and mixed well. And (3) taking 70-80 microliters of uniformly mixed hydrogel, dropwise adding the hydrogel from one corner of the chip, and slightly shaking the chip to enable the liquid to completely cover the structural area. The incubator was allowed to stand for 5 minutes, and the whole process was carefully kept horizontal. The chip was transferred to a 3.5cm diameter petri dish using a syringe needle and forceps, and placed in an incubator and then a selection medium (2 ml in volume, fluorescently labeled antigen: complete medium: 1: 200-1000) was added. Standing for at least 3 hours.

4.6 Single cell picking

After screening for 3-5 hours, the signal condition was first observed under a fluorescent microscope. And (5) determining that positive signals appear and the proportion meets the requirement, and preparing for cell picking.

4.7 monoclonal culture and identification

Cells were picked and released in 96-well plates and microscopic examination was performed within 1-2 hours to determine the presence of single cells in each well. After continuous culture for 7-14 days, ELISA test is carried out on the cell hole supernatant which is stably proliferated, and whether the cell hole supernatant is secreting antigen specific antibody or not is judged, namely positive clone is obtained. And continuously expanding and culturing the positive clone, freezing and storing, and identifying the secretion condition of the antibody again before freezing and storing.

4.8 screening results

Through screening, 15 cell strains capable of secreting specific antibodies, namely 14 monoclonal cell strains, are temporarily obtained in the project. All the obtained clones are derived from different mice or different single hybridoma cells of the same mouse, so that the diversity of the clones can be fully ensured.

Compared with the traditional method (limited dilution method), the method of the invention has very significant advantages, which are as follows:

screening method	Time of day	Number of clones	Number of 96 well plates	Personnel situation
					Limiting dilution	3-4 months	2 to 5	Hundreds of	More than 3 people
Method of the present patent application	1.5-2 months	At least 15-50	1-2	Single person

Therefore, compared with the traditional limiting dilution method, the method of the invention has absolute advantages in both screening efficiency and screening cost. The screening efficiency is improved by at least 10 times while the overall cost is reduced by about 90%. Under the condition that the screening is not completely developed, people can be matched more reasonably and the time can be arranged according to the urgency degree of the project, and the screening efficiency can be effectively improved to be more than 30 times on the basis.

Example 5: screening for paired antibodies

5.1 obtaining of labeled antibodies and fluorescent labeling

According to the screening results in example 4, a monoclonal cell line with high antibody titer was selected. Ascites is prepared by the following steps:

(ii) 2 mice were prepared per group of cells, 8-10 weeks old, B/C, female. Incomplete Freund's adjuvant is injected intraperitoneally to reach allergy, 500 ul/mouse.

Abdominal inoculation of cells 5X 10⁵. Note that: few cells are injected, ascites formation is slow but titer is higher; before the hybridoma cells are injected, the titer of the antibody needs to be detected; before inoculation, the cells need to be washed repeatedly 2-3 times with serum-free medium. About 0.2-1ml per mouse.

② ascites and survival condition of the mice are closely observed from the 2 nd day after inoculation. After about 7 days, if the activity is good, the ascites with obvious mobility can be released for about 2 times; once mice were found to be restricted in activity, ascites were sacrificed immediately.

Ascites is extracted from a living body: the position of the needle to be inserted (probably at the right side of the midline of the abdomen) is wiped by an alcohol cotton ball, then a little air is left in the syringe, then the syringe is inserted, then the syringe is pushed backwards slowly, the ascites can flow outwards slowly under pressure in the abdominal cavity, and the needle needs to be lifted upwards gently during insertion, so that the user feels good without hindrance.

Sacrifice and ascites collection: firstly, a small opening is cut on the outer skin of the abdominal cavity of the mouse by scissors and tweezers, then two hemostatic forceps are pulled open gently at two sides to expose the abdominal part, then the inner skin is cut by the other scissors and tweezers, the ascites can be obtained by a gun or a glass straight tube without cutting all the inner skin.

Normally, 2-3ml of ascites can be collected per mouse per time, and if the mouse is in good condition, the ascites can be collected every other day. The antibody concentration is 0.5-5 mg/ml.

③ ascites is collected into a 15ml centrifuge tube, centrifuged at 800rpm for 30min (2000r/min, 5 min) to remove cells and grease. The ascites fluid which is just collected contains a large amount of impurities such as fats and oils, macrophages, hybridoma cells, epithelial cells in the abdominal cavity, and cell components in blood, and particularly cell components, which may be gradually broken down during long-term storage of the ascites fluid without removal, protein components in the cells are released to the abdominal edema, and impurities in the ascites fluid are increased, making purification difficult. Therefore, after the ascites is collected, whether the ascites is stored for a long time or purified immediately, the cell components are removed by centrifugation, and meanwhile, the grease can be removed (the density of the grease is relatively low, and the grease generally floats on the surface of the ascites and is sucked out by a gun).

Antibody purification and fluorescent labeling preparation of labeled antibodies were performed using Thermo's kit (89953A30006), the detailed procedures are as follows.

5.2 paired antibody screening

The specific procedure was as described in example 4. Wherein the fluorescent labeled antigen is replaced by a complex of an antigen and a labeled antibody. The adding proportion is antigen: fluorescent primary antibody 2: 1.

In this step, the same screening purpose and effect can be achieved by another alternative method, that is, the specific steps are the same as those described in example 4. Adding a 3D culture medium containing an antigen on a hybridoma single cell 2D array chip, incubating for 2 hours to ensure that the antigen is fully combined with an antibody secreted by the single cell, removing the liquid 3D culture medium, adding the 3D culture medium containing a fluorescence labeling first antibody, and incubating for 2-3 hours.

5.3 screening results

Two clones which secreted high titer antibodies against the novel coronavirus S1 protein, selected in example 4, were selected as primary antibodies, S1-6S and RBD-10S, respectively. Through the screening, the final results are as follows:

at present, the inventors screened 4 groups of antibody pairs against the S1 protein and 3 groups of antibody pairs against the RBD protein. The paired antibodies are respectively derived from different mice or different hybridoma cells of the same mouse, so that the diversity of the antibody pairs is ensured.

Example 6: screening single cell secreting specific factor by using soft matrix chip

Referring to examples 4 and 5, CHO cells stably expressing VEGFA factor were screened. The specific steps, such as chip preparation, single cell 2D array, antigen-antibody reaction of single cell, identification and picking of positive single cell are the same as those described in example 4 or 5. In this example, the difference is the secretion of VEGF factor protein, and the fluorescent-labeled antigen used for immunofluorescence reaction is replaced by a specific antibody labeled with fluorescence. The screening results were as follows:

the inventors have performed experiments with VEGFA antibodies from two different sources simultaneously. After killing, 58 positive cells (secretion factors) and 49 positive cells (secretion factors) are respectively picked out, and after post-culture and verification, 52 CHO cell strains and 42 CHO cell strains which can stably express and secrete VEGFA are finally obtained, wherein the positive rates are respectively up to 89.6% and 85.7%. The traditional screening and separation of stable CHO cell strain still adopts a limited dilution method, as mentioned above, so that the technology also embodies great advantages in time, material consumption and personnel consumption.

In addition, the inventors also referred to examples 4 and 5, and screened single cells capable of secreting other factors, including, for example, all of the various cells expressing secreted proteins including: HEK293, CHO, Hela, MCF10A, HFF and other cells, factors including various growth factors and cytokines which can be secreted out of the cells including VEGF, SDF-1a, PEDF, FGF and the like, and marker proteins which specifically bind to proteins secreted by the cells including specific antibodies and receptor binding domains corresponding to the secreted proteins, which are not described in detail herein.

Example 7: mouse neural stem cell balling culture

Neural stem cells (neural stem cells) refer to a cell population that exists in the nervous system, has the potential to differentiate into neurons, astrocytes and oligodendrocytes, and thus is capable of generating a large amount of brain cell tissue, and is capable of self-renewal, and sufficiently providing a large amount of brain tissue cells. Therefore, how to better culture the neural stem cells in vitro is the basis and foundation for scientific research.

(1) Extracting fetal rat neural stem cells. C57BL/6 mice with 14 days gestational age are killed by dislocation of cervical vertebrae, soaked in 75% alcohol, then the abdomen is cut off, a string of fetal mice (umbilical cord is cut off) is taken out and placed in a culture dish containing precooled PBS. The placenta is stripped by forceps, and the fetal rat is taken out and placed in a new culture dish containing precooled PBS (generally, one pregnant rat has 8-11 fetal rats, less 6 rats and more 13 rats). The fetal rat heads were unscrewed and placed in new dishes containing PBS (2 fetal rats were removed for manipulation each time). Taking out cerebral cortex under stereomicroscope, and removing brain tissue connected with cerebral cortex such as olfactory bulb and meninges. The cerebral cortex was placed in a 15mL centrifuge tube, repeatedly pipetted with a Pasteur pipette to a suspension without macroscopic tissue mass, and centrifuged at 800rpm for 2 min. The supernatant was removed for further use, and the pellet was added with fresh PBS buffer and blown into a suspension, and centrifuged again at 800rpm for 2 min. The supernatant was aspirated and mixed with the supernatant aspirated by the first centrifugation and centrifuged at 1200rpm for 2 min. The supernatant was discarded, and the precipitate was blown off with an enrichment medium to obtain an NPC suspension, which was then filtered through a filter having a pore size of 40 μm to remove undispersed clumps. The NPC suspension is aspirated and placed in a culture flask of T25 for suspension culture. The flasks were not shaken two days before the start of the culture, and passage was started on day 6 or 7, depending on the size of the cell pellet. The cells were collected, centrifuged at 800rpm for 2min and the supernatant removed. The pellet was blown up by adding PBS buffer and centrifuged at 800rpm for 2min to remove the remaining medium. Removing supernatant, adding 1-2ml of Accutase or tryplE cell digestive juice, and digesting in 37 deg.C incubator for 5-10min, wherein the middle part can be flicked by hand. Digestion was stopped by adding 5 volumes of Ca/Mg2+ -free PBS (or 4-5mL resuspended in pre-warmed medium), vortexed repeatedly, and centrifuged at 1200rpm for 2 min. The supernatant was discarded, 1mL of multiplication medium (more was added to avoid further loss of filtration) was added, and the pellet was pipetted down to suspension. Filtering with filter membrane with pore size of 40 μm to remove undigested cell balls to obtain NPC suspension. The cell concentration of the NPC suspension was counted using a hemocytometer, and 10X 10^6 cells were seeded and cultured in a new T25 flask.

(2) And (5) inoculating the chip. The specification of the chip related to the embodiment is 12 μm/30 μm, the types of the factors added into the soft matrix are EGF and bFGF, and the final concentration is 20ng/mL and 10ng/mL respectively as described in embodiment 4 or 5.

(3) And (6) analyzing results. By comparing the conventional culture method with the method of the present invention, specific test data are shown below, and it can be seen that the soft matrix chip culture contributes to the improvement of the survival rate of cells while maintaining the cell dryness well.

Example 8: tumor cell balling culture (MCF7)

Studies of tumor growth and drug sensitivity have relied largely on in vitro monolayer tumor models (2D cell cultures) that lack many of the disease characteristics, such as hypoxia, changes in cell-cell contacts, and changes in metabolism. Through years of research, scientists believe that the phenotype of a 3D tumor model is closer to real cancer tissues, and more accurate models and data can be provided for drug screening.

(1) MCF7 cell culture and chip inoculation. MCF7 cells were cultured in DMEM medium containing 10% FBS and 1% P/S. When the culture area reaches 80% of the surface area of the culture dish, the chip is digested and inoculated. The specific steps of chip inoculation are described in example 4 or 5.

(2) And (4) continuously culturing. The chip containing the cells is placed in an incubator for continuous culture for 3-5 days, and the specific proliferation condition of the cells is continuously observed in the period. After culture, we realized an array of single MCF7 cell-derived cell spheres on a chip.

This three-dimensional culture (or "cell-mass" culture) of tumor cells closely replicates many of the key attributes of the original tumor. Meanwhile, the array culture is suitable for large-scale drug screening to detect drug sensitivity related to gene change, establish experimental basis for personalized treatment methods, and can optimize clinical results of cancer patients.

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention. Furthermore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified. In the description of the present invention, the first feature being "on" or "under" the second feature may include the first and second features being in direct contact, and may also include the first and second features being in contact with each other not directly but through another feature therebetween.

In the description of the invention, "above", "over" and "above" a first feature in a second feature includes the first feature being directly above and obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method of screening target cells for the secretion of a molecule of interest, comprising:

culturing the candidate single cells in a culture chamber under conditions suitable for secreting a target molecule, wherein a signal screening layer is arranged in the culture chamber, covers the single cells, and contains a signal molecule specifically recognizing the target molecule; and

selecting a target cell suitable for secreting the molecule of interest based on the signal of the signal molecule in the signal screening layer.

2. The method of claim 1, wherein the signal screening layer is disposed in a matrix material of a culture chamber;

the signal screening layer is arranged in the culture medium;

the signal screening layer contains at least one of an antibody and a probe;

the signal screening layer is formed of hydrogel; or

The mechanical strength of the base material is higher than that of the signal screening layer.

3. The method of claim 1, wherein the culture chamber is disposed on a biological culture chip comprising:

a base formed from a base material; and

a micro-well formed at a surface of the substrate and opened at the surface of the substrate, and defining the culture chamber.

4. The method of claim 3, wherein the microwells form a predetermined array pattern on the substrate.

5. The method of claim 3, wherein said matrix material has an equilibrium swelling ratio of about 1.25 to about 1.75.

6. The method of claim 3, wherein the opening of the microwell has a diameter of 8-25 microns and a depth of 15-35 microns.

7. The method according to claim 3, wherein the matrix material comprises at least one selected from the group consisting of collagen hydrogel, methylated cellulose, agarose gel, polyacrylamide gel, and the like.

8. The method of claim 3, wherein the pitch of at least two adjacent micropores is 10 to 50 μm.

9. The method of claim 1, wherein the signal molecule is a fluorescent molecule.

10. The method of claim 9, wherein the fluorescent molecule comprises at least one of FITC, AF 488.

11. The method of claim 1, wherein the positive cells are localized using a fluorescence microscope.

12. A kit, comprising:

a hydrogel;

a signal molecule; and

a biological culture chip.

13. A method of producing an antibody, comprising:

screening a target cell by the method according to any one of claims 1 to 11;

subjecting the target cells to amplification and expression of antibodies.

14. A method of screening for monoclonal antibodies comprising:

distributing a plurality of hybridoma cells on said biological culture chip, wherein each of said culture chambers contains at most one of said hybridoma cells per said chamber;

the method according to claims 1 to 11, selecting positive hybridoma cells;

isolating the positive hybridoma cells;

culturing and expressing the antibody of the positive hybridoma cell so as to obtain the monoclonal antibody.

15. A monoclonal antibody obtained by the method of claim 14.

16. An antibody-antigen complex, wherein the antibody is the monoclonal antibody of claim 15, and wherein the monoclonal antibody has a signal molecule attached thereto.

17. A method of screening for an antibody comprising:

(2) adding a signal antibody-antigen complex to the culture chamber, the signal antibody being linked to a signal molecule, the signal antibody being the monoclonal antibody of claim 15; and

(3) determining whether the candidate cell is a target cell that secretes an antibody having a different epitope from the monoclonal antibody based on the signal of the signal molecule.