Target cell or cell screening method, and biological culture chip

Abstract

To provide a biological culture chip suitable even for complex detection of cell biological functions such as antibody secretion and growth factor secretion.SOLUTION: Provided is a biological culture chip including a matrix formed of a matrix material, and culture chambers formed on a surface of the matrix to define the culture chambers, the matrix material of the matrix having an equilibrium swelling ratio of 1.25 to 1.75 and the culture chambers being formed in openings configured on the surface of the matrix to define the culture chambers.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present disclosure relates to the field of biotechnology, and more particularly to a method for screening target cells, a kit and use thereof according to a first aspect of the present disclosure, a method for screening target cells, a kit, a method for producing an antibody, a method for screening a monoclonal antibody, a monoclonal antibody, an antibody-antigen complex and a method for screening an antibody according to a second aspect of the present disclosure, and a biological culture chip, a method for producing the same and use thereof according to a third aspect of the present disclosure. [Background technology]

[0002] Limiting dilution is a commonly used cloning method, which is accomplished by pipetting the cell line to be recloned from a culture well and measuring 1 mL of cells. This method is commonly used for screening fusion animal cells. Monoclonal antibodies are highly homogenous antibodies that target only a specific epitope and are produced by cloning a single B cell. Monoclonal antibodies are usually produced by hybridoma technology. Hybridoma technology is based on cell fusion. Sensitized B cells capable of secreting specific antibodies are fused with myeloma cells capable of unlimited proliferation to become B cell hybridomas.

[0003] However, existing methods for screening cells capable of expressing target molecules, particularly monoclonal antibody fusion cells, still need improvement.

[0004] Antibody pairs are widely used in protein quantification. Sandwich ELISA, chemiluminescence, and immunochromatography cannot be performed without high-quality antibody pairs. An antibody pair is a set of two antibodies that can bind to one antigen molecule at the same time. Antibody pairs are usually used in double antibody sandwich ELISA experiments. Generally, screening for antibody pairs is performed by double antibody sandwich ELISA. This screening is based on the following description. After obtaining a monoclonal antibody, two types of monoclonal antibodies, a capture antibody and a labeled antibody (HRP enzyme labeled), are separated from it. The capture antibody is coated on an antigen capture plate. First, the antigen is added and incubated, and unbound antigen is washed away. Next, the labeled antibody is added and incubated, and unbound labeled antibody is washed away. Finally, a color-developing solution is added to develop color. Under conditions that allow color development, the labeled antibody specifically binds to the antigen, indicating that the capture antibody and the labeled antibody form an antibody pair. Under conditions that do not allow color development, the labeled antibody cannot specifically bind to the antigen and is washed away, indicating that the capture antibody and the labeled antibody cannot form an antibody pair. Under conditions where the two selected monoclonal antibodies cannot be paired, the two monoclonal antibodies must be reselected for retesting until an antibody pair is found.

[0005] However, the screening efficiency of current antibody pairs is very low, which leads to a significant increase in screening costs.

[0006] In current biological and medical research, in most cases, it is preferred to carry out cytological testing of drugs for detection by traditional two-dimensional (2D) adherent cell culture. This is essentially different from the existing state of actual tissue cells in vivo (3D state under ECM environment). Cells in vivo grow in a 3D microenvironment, but cells growing in 2D culture are not in the natural state of cell growth. The culture microenvironment is significantly different from the in vivo microenvironment, which affects the gene expression and signal transduction of cells, and cultured cells gradually lose their biological characteristics and biological functions in vivo, losing the value of research and application. In addition, 2D cell culture has many serious disadvantages, such as cell heterogeneity, which does not contribute to cell differentiation research and related research. In addition, 2D Although a large number of cells are obtained through culture, it is difficult to select useful cells from among these, resulting in waste of reagents.

[0007] CN108102913A discloses a 3D cell culture chip based on soft lithography, its preparation method and its use. The preparation method of the 3D cell culture chip based on soft lithography includes: preparing a mask with a defined pattern structure by soft lithography; uniformly coating the mask with a liquid polymer compound or polymer compound solution to form a liquid layer with a defined thickness; solidifying the liquid layer and removing the mask to obtain a stamp with a stamp surface with a defined 3D structure; contacting the stamp surface of the stamp with a modifier, so that the modifier is releasably attached to the stamp surface; and contacting the stamp surface with the surface of a selected substrate, removing the stamp from the selected substrate, and separating at least a portion of the modifier from the stamp surface and attaching it to the surface of the selected substrate, forming a pattern structure including an array of a plurality of patterns that can adsorb at least a single cell, to obtain a 3D cell culture chip.

[0008] However, existing chips for culturing biocompatible materials need to be suitable not only for 2D arrays and 3D cultures of single cells, but also for the complex detection of cell biological functions, such as antibody secretion, growth factor secretion, etc., and further improvements are still needed. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] China Patent Application Publication No. 108102913 Summary of the Invention [Problem to be solved by the invention]

[0010] The present disclosure aims to solve at least one of the technical problems existing in the prior art. [Means for solving the problem]

[0011] Therefore, according to one aspect of the present disclosure, a method for screening target cells is provided, the target cells being suitable for secreting a target molecule, the method comprising culturing a candidate single cell in a culture chamber under conditions suitable for secreting the target molecule, wherein the culture chamber comprises a signal screening layer for covering the candidate single cell, the signal screening layer comprising a signaling molecule for specifically recognizing the target molecule; and selecting a target cell suitable for secreting the target molecule based on the signal of the signaling molecule of the signal screening layer. With this method, since only a single cell exists in the culture chamber, the target molecule can be specifically captured by the signaling molecule under conditions in which the single cell can secrete the target molecule during culture. Based on this, the signaling molecule can be aggregated to a specific range to amplify the signal of the signaling molecule. Therefore, based on the difference in the signal of the signaling molecule, it can be determined whether the single cell can express the target molecule. Compared with the limiting dilution method of the prior art, this method is rapid, cost-effective, efficient, and can realize high-throughput screening.

[0012] According to a second aspect of the present disclosure, the present disclosure provides a kit, comprising a hydrogel; a signaling molecule; and a biological culture chip. By using the kit, the above method can be effectively carried out, thereby obtaining the above effects. Note that the above-mentioned features of the method are also suitable for the kit, and will not be described in detail in this specification.

[0013] According to another aspect of the present disclosure, there is further provided a method for producing an antibody, comprising screening a target cell by the above method; and growing the target cell to express the antibody. Therefore, fused cells capable of expressing monoclonal antibodies can be obtained efficiently.

[0014] According to another aspect of the present disclosure, a method for screening monoclonal antibody is further provided, which includes dispensing a plurality of hybridoma cells onto a biological culture chip, where each culture chamber contains at most one hybridoma cell; selecting positive hybridoma cells by the above method; isolating the positive hybridoma cells; and culturing the positive hybridoma cells to express antibody to obtain monoclonal antibody.As described above, the method of the present disclosure can efficiently obtain fusion cells expressing monoclonal antibody, and further obtain monoclonal antibody.

[0015] According to another aspect of the present disclosure, there is further provided a monoclonal antibody obtainable by the above method.

[0016] According to another aspect of the present disclosure, an antibody-antigen complex is further provided. The antibody is the above-mentioned monoclonal antibody, and the monoclonal antibody is bound to a signaling molecule. By using the antigen-antibody complex, a pair of antibodies can be efficiently obtained, one of which has an antigenic determinant different from that of the known monoclonal antibody.

[0017] According to another aspect of the present disclosure, there is further provided a method for screening an antibody, comprising: (1) placing a candidate cell in a culture chamber and culturing the candidate cell for a predetermined time under conditions suitable for the candidate cell to express an antibody; (2) adding a signaling antibody-antigen complex to the culture chamber, wherein the signaling antibody is bound to a signaling molecule, and the signaling antibody is the monoclonal antibody described above; and (3) determining whether the candidate cell is a target cell based on a signal of the signaling molecule, wherein the target cell secretes an antibody having an antigenic determinant different from that of the monoclonal antibody.

[0018] According to another aspect of the present disclosure, there is provided a method for screening cells, comprising: (1) placing candidate cells in a culture chamber for a predetermined time under conditions suitable for the candidate cells to express an antibody, and culturing the candidate cells in the culture chamber for a predetermined time; (2) forming a target antibody-antigen signaling antibody complex in the culture chamber, where the signaling antibody is bound to a signaling molecule; and (3) determining whether the candidate cells are target cells based on the signal of the signaling molecule. This method allows for efficient antibody pair screening.

[0019] According to one embodiment of the present disclosure, signaling antibodies; Antigens; and Biological Culture Chip A kit is provided comprising:

[0020] According to one embodiment of the present disclosure, Screening the target cells by the method described above; and Proliferating the target cells to express antibodies to obtain antibodies having antigenic determinants different from those of the signaling antibodies. A method for producing an antibody is provided, comprising:

[0021] According to one embodiment of the present disclosure, dispensing a plurality of hybridoma cells onto a biological culture chip, wherein each culture chamber contains at most one hybridoma cell; selecting positive hybridoma cells by the above method; isolating the positive hybridoma cells; and The positive hybridoma cells are cultured to express the antibody, thereby obtaining a monoclonal antibody having an antigenic determinant different from that of the signaling antibody. A method for screening a monoclonal antibody is provided, comprising:

[0022] The present disclosure further provides a monoclonal antibody obtained by the above method. The monoclonal antibody and the signaling antibody correspond to different antigenic determinants.

[0023] According to another aspect of the present disclosure, a biological culture chip is provided that includes a matrix formed from a matrix material, where the equilibrium swelling ratio of the matrix material of the matrix is ​​about 1.25 to 1.75; and a culture chamber formed on a surface of the matrix to define the culture chamber.

[0024] Since the matrix material has a certain equilibrium swelling ratio, the entire surface of the biocompatible material cultured using the biological culture chip, for example, the entire surface of a single cell, can be surrounded by a relatively uniform liquid or gel medium. Therefore, during cell culture, the biological activity of the cell can be further improved, the realization of cell biological functions (such as antibodies and factors) can be promoted, and accurate detection can be facilitated.

[0025] Additional aspects and advantages of the disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure. [Brief description of the drawings]

[0026] The above and / or further aspects and advantages of the present disclosure will become apparent and can be understood from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic structural diagram of a biological culture chip according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 is a schematic structural diagram of a biological culture chip according to another embodiment of the present disclosure. [Diagram 3] FIG. 3 is a schematic structural diagram of a biological culture chip according to another embodiment of the present disclosure. [Figure 4] FIG. 4 is a schematic structural diagram of a template for fabricating a biological culture chip according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 is a diagram showing a method for screening monoclonal antibodies according to one embodiment of the present disclosure. [Figure 6] FIG. 6 is a diagram illustrating a method for screening antibody pairs according to one embodiment of the present disclosure. [Figure 7] FIG. 7 is a diagram showing a comparison of stemness of neural stem cells in suspension culture and chip culture according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings, in which identical or similar elements or elements having identical or similar functions are indicated by the same or similar reference numerals throughout the description. The embodiments described below with reference to the accompanying drawings are exemplary and are used only to explain the present disclosure, and should not be construed as limiting the present disclosure.

[0028] Method for screening target cells According to one aspect of the present disclosure, there is provided a method for screening a target cell, the target cell being suitable for secreting a target molecule, the method comprising culturing a candidate single cell in a culture chamber under conditions suitable for secreting the target molecule, the culture chamber being adapted to culture the candidate single cell. and selecting a target cell suitable for secreting the target molecule based on the signal of ...

[0029] According to one embodiment of the present disclosure, the signal screening layer is disposed on the matrix material of the culture chamber; the signal screening layer is disposed on the culture medium; the signal screening layer comprises at least one antibody or probe; the signal screening layer is formed from a hydrogel; or the mechanical strength of the matrix material is higher than that of the signal screening layer, so that the molecules secreted from a single cell can easily enter the signal screening layer, thereby improving the screening efficiency.

[0030] According to one embodiment of the present disclosure, the culture chamber is disposed on a biological culture chip comprising a matrix formed from a matrix material; and a culture chamber formed on the surface of the matrix to define the culture chamber. The biological culture chip is briefly described here and will be described in detail below. In the course of detailed research on 3D cell culture chips, it was found that during the culture of cells or biocompatible materials using existing 3D culture chips, it is usually difficult for cells to exhibit biological behavior and physiological action under an in vivo environment. To this end, it was found through creative research that the matrix material of existing 3D culture chips affects the effect of cell culture, especially has a significant negative effect on the realization of the biological function of single cell secreted factors (including antibodies). Furthermore, it was found that in existing 3D culture chips, different contact positions between cells and the matrix are exposed to significantly different surrounding culture environments, and the microenvironments of cells obtained at different positions also have large differences and heterogeneities. For example, at the contact positions between cells and the matrix, cells cannot receive sufficient support from the extracellular matrix. Furthermore, many of the existing 3D culture chips adopt hard matrices. Therefore, during the cell culture process, the hard matrix exerts a certain degree of stress on the cultured cells, affecting their biological behavior, which in turn seriously affects the factor or antibody secretion ability and function detection of single cells.

[0031] According to one embodiment of the present disclosure, the signaling molecule is a fluorescent molecule.

[0032] According to one embodiment of the present disclosure, the fluorescent molecules include at least one of FITC or AF488.

[0033] According to one embodiment of the present disclosure, the method further comprises locating positive cells by fluorescence microscopy.

[0034] According to a second aspect of the present disclosure, the present disclosure provides a kit, comprising a hydrogel; a signaling molecule; and a biological culture chip. By using the kit, the above method can be effectively carried out, thereby obtaining the above effects. Note that the above-mentioned features of the method are also suitable for the kit, and will not be described in detail in this specification.

[0035] According to another aspect of the present disclosure, there is further provided a method for producing an antibody, comprising screening a target cell by the above method; and growing the target cell to express the antibody. Therefore, fused cells capable of expressing monoclonal antibodies can be obtained efficiently.

[0036] According to another aspect of the present disclosure, a method for screening monoclonal antibody is further provided, which includes dispensing a plurality of hybridoma cells onto a biological culture chip, where each culture chamber contains at most one hybridoma cell; selecting positive hybridoma cells by the above method; isolating the positive hybridoma cells; and culturing the positive hybridoma cells to express antibody to obtain monoclonal antibody.As described above, the method of the present disclosure can efficiently obtain fusion cells expressing monoclonal antibody, and further obtain monoclonal antibody.

[0037] According to another aspect of the present disclosure, there is further provided a monoclonal antibody obtainable by the above method.

[0038] According to another aspect of the present disclosure, an antibody-antigen complex is further provided. The antibody is the above-mentioned monoclonal antibody, and the monoclonal antibody is bound to a signaling molecule. By using the antigen-antibody complex, a pair of antibodies can be efficiently obtained, one of which has an antigenic determinant different from that of the known monoclonal antibody.

[0039] According to another aspect of the present disclosure, there is further provided a method for screening an antibody, comprising: (1) placing a candidate cell in a culture chamber and culturing the candidate cell for a predetermined time under conditions suitable for the candidate cell to express an antibody; (2) adding a signaling antibody-antigen complex to the culture chamber, wherein the signaling antibody is bound to a signaling molecule, and the signaling antibody is the monoclonal antibody described above; and (3) determining whether the candidate cell is a target cell based on a signal of the signaling molecule, wherein the target cell secretes an antibody having an antigenic determinant different from that of the monoclonal antibody.

[0040] According to one embodiment of the present disclosure, compared with the conventional limiting dilution method, the method of the present disclosure has absolute advantages in terms of screening efficiency and screening cost. Taking the screening of monoclonal antibodies as an example, the overall cost is reduced by about 90%, while the screening efficiency is increased by at least 10 times. This is merely a result obtained without comprehensive screening, and manpower and time can be more appropriately allocated according to the urgency of the project. Based on this, it is believed that the screening efficiency can be effectively increased by more than 30 times.

[0041] Thus, there is provided a method for screening target cells, the target cells being suitable for secreting a target molecule, the method comprising culturing candidate single cells in a culture chamber under conditions suitable for secreting the target molecule, wherein the culture chamber comprises a signal screening layer for covering the candidate single cells, the signal screening layer comprising a signaling molecule for specifically recognizing the target molecule; and selecting target cells suitable for secreting the target molecule based on the signal of the signaling molecule of the signal screening layer.

[0042] Those skilled in the art can understand that the culture chamber may be the culture space in the biological culture chip described herein. Therefore, the advantages and features of the above biological culture chip are also suitable for the present screening method, and will not be described in detail herein. It is emphasized that according to one embodiment of the present disclosure, the signal screening layer may be used as a liquid medium or gel medium to cover the single cell, or may be used as a solid or semi-solid matrix. According to one embodiment of the present disclosure, a gel material with lower mechanical strength than the matrix may be selected, taking into account the need to isolate the single cell. For example, a material adjusted to a lower concentration than the gel material is used as the signal screening layer.

[0043] The signal screening layer comprises at least one labeled antibody, labeled protein, labeled polypeptide, or labeled probe.

[0044] According to one embodiment of the present disclosure, the signal screening layer is formed from a first hydrogel, and the culture chamber is provided on a biological culture chip, and the concentration of the first hydrogel is lower than the concentration of the second hydrogel.

[0045] Thus, according to yet another aspect of the present disclosure, Hydrogels; signaling molecules; and Biological Culture Chip A kit is provided comprising:

[0046] The above method can be effectively carried out by using the present kit, and the features and advantages of the present method are also suitable for the kit and will not be described in detail herein.

[0047] Further, according to another aspect of the present disclosure, Screening the target cells by the method; and Proliferating target cells to express antibodies Further provided is a method for screening an antibody, comprising:

[0048] Referring to FIG. 5, the present disclosure dispensing a plurality of hybridoma cells onto a biological culture chip to form an array, wherein each culture chamber of the biological culture chip contains at most one hybridoma cell; placing the biological culture chip containing the sequence of the hybridoma cells into a medium containing a labeled target antigen (protein or polypeptide) to select labeled positive hybridoma cells that undergo an antigen-antibody reaction and secrete the target antibody; isolating the positive hybridoma cells; and Cultivating the positive hybridoma cells to express the antibody and obtain a monoclonal antibody. The present invention further provides a method for screening a monoclonal antibody derived from a single cell, comprising:

[0049] Based on this, another aspect of the present disclosure further provides a monoclonal antibody from a single cell, obtained by the above method.

[0050] Thus, according to another aspect of the present disclosure, there is further provided an antibody-antigen complex, wherein the antibody is a monoclonal antibody as described above, and wherein the monoclonal antibody is bound to a signaling molecule.

[0051] Further, referring to FIG. 6, another embodiment of the present disclosure further provides a method for high throughput screening of paired antibodies (abbreviated as paired antibodies) against the same antigen but different antigenic determinants, comprising the steps of: (1) placing candidate cells in a culture chamber and culturing them for a predetermined period of time under conditions suitable for the candidate cells to express antibodies; (2) adding a signaling antibody-antigen complex to the culture chamber, wherein the signaling antibody is bound to a signaling molecule, and the signaling antibody is a single cell-derived supracellular antigen; The monoclonal antibody or derivative thereof, for example, an antibody fragment containing an antigen-binding fragment (Fab), (Fab)2, etc., that binds to a specific antigenic determinant; and An unlabeled antigen is added to the culture chamber, and under the condition that the cells secrete the antibody, the secreted antibody and the antigen form an antibody-antigen complex, and the antibody-antigen complex is captured around the cells, and then a signaling antibody is added to the medium of the biological culture chip, where the signaling antibody is bound to a signaling molecule, and the signaling antibody is the above-mentioned monoclonal antibody or its derivative derived from a single cell, for example, an antibody fragment containing an antigen-binding fragment (Fab), (Fab)2, etc., is bound to a specific antigen determinant; and (3) determining whether the candidate cell is a target cell based on the signal of the signaling molecule, where the target cell secretes an antibody having an antigenic determinant different from the monoclonal antibody derived from the single cell.

[0052] Antibody pair screening methods According to another aspect of the present disclosure, a method for screening cells is provided, comprising: (1) placing candidate cells in a culture chamber for a predetermined time under suitable conditions for the candidate cells to express an antibody; (2) forming a target antibody-antigen signaling antibody complex in the culture chamber, where the signaling antibody is bound to a signaling molecule; and (3) determining whether the candidate cell is a target cell based on the signal of the signaling molecule. This method can efficiently obtain antibody pairs by screening. This method places candidate cells in a culture chamber for single cell culture, and the antibody secreted from the single cell forms a target antibody-antigen signaling antibody complex, concentrating the antigen signaling antibody to a certain range, thereby effectively achieving screening of antibody pairs. Compared with the limiting dilution method of the prior art, this method is rapid, cost-effective, efficient, and can realize high-throughput screening.

[0053] According to one embodiment of the present disclosure, the predetermined time is between 1 and 10 hours, preferably between 2 and 5 hours, and more preferably 3 hours.

[0054] According to one embodiment of the present disclosure, a culture chamber is configured on a biological culture chip (described in detail below), and a target antibody-antigen signaling antibody complex is formed by adding an antigen-signaling antibody complex or sequentially adding free antigen and free signaling antibody to the culture chamber.

[0055] According to one embodiment of the present disclosure, the signaling molecule is a fluorescent molecule, preferably the fluorescent molecule comprises at least one of FITC or AF488.

[0056] According to one embodiment of the present disclosure, in step (3), the method further comprises locating positive cells based on the signal of the signaling molecule by fluorescence microscopy.

[0057] According to one embodiment of the present disclosure, signaling antibodies; Antigens; and Biological Culture Chip A kit is provided comprising:

[0058] According to one embodiment of the present disclosure, Screening the target cells by the method; and The target cells are grown to express an antibody having an antigenic determinant distinct from the signaling antibody. Obtaining antibodies that A method for producing an antibody is provided, comprising:

[0059] According to one embodiment of the present disclosure, dispensing a plurality of hybridoma cells onto a biological culture chip, wherein each culture chamber of the biological culture chip contains at most one hybridoma cell; selecting positive hybridoma cells by the above method; isolating the positive hybridoma cells; and The positive hybridoma cells are cultured to express the antibody, thereby obtaining a monoclonal antibody having an antigenic determinant different from that of the signaling antibody. A method for screening a monoclonal antibody is provided, comprising:

[0060] According to another aspect of the present disclosure, there is further provided a monoclonal antibody obtained by the above method. The monoclonal antibody and the signaling antibody correspond to different antigenic determinants.

[0061] The signaling antibodies used to screen for antibody pairs may be obtained or generated by the following methods.

[0062] Those skilled in the art can understand that the culture chamber may be the culture space in the biological culture chip described herein. Therefore, the advantages and features of the above biological culture chip are also suitable for the present screening method, and will not be described in detail herein. It is emphasized that according to one embodiment of the present disclosure, the signal screening layer may be used as a liquid medium or gel medium for covering the single cell, or may be used as a solid or semi-solid matrix. According to one embodiment of the present disclosure, a gel material with a lower mechanical strength than the matrix may be selected in consideration of the need to isolate the single cell. For example, a material adjusted to a lower concentration than the gel material is used as the signal screening layer.

[0063] The signal screening layer comprises at least one labeled antibody, labeled protein, labeled polypeptide, or labeled probe.For ease of understanding, the biological culture chip suitable for the present disclosure is described in detail below.

[0064] Biological Culture Chip A biological culture chip according to an embodiment of the present disclosure will be described with reference to the accompanying drawings.

[0065] As shown in Figures 1 to 3, according to one embodiment of the present disclosure, a biological culture chip is provided. The biological culture chip includes a matrix 100 and a microwell 200. The matrix 100 is formed by a matrix material. The equilibrium swelling ratio of the matrix material of the matrix is ​​about 1.25 to 1.75. The microwell 200 is formed on the surface of the matrix 100, and the microwell 200 has an opening on the surface of the matrix 100. The microwell 200 defines a biological culture space for culturing a biocompatible material.

[0066] In the prior art, the interface compatibility between the chip matrix and cell culture medium is low. The incompatibility between the hard matrix interface and the cell culture medium causes the non-uniformity of the 3D culture microenvironment of the aligned cells, and the interface between the matrix and the culture medium tends to slide due to the liquid layer between the two interfaces, which damages the cell alignment and affects the realization of cell biological functions, such as the sufficient capture of cell detection signals and the detection sensitivity of secreted proteins.

[0067] According to this embodiment of the present disclosure, since the matrix material has a certain equilibrium swelling ratio, the entire surface of the biocompatible material cultured using the biological culture chip, for example, the entire surface of a single cell, can be surrounded by the liquid or gel-like extracellular matrix material to provide a uniform microenvironment for the culture of the biocompatible material, which can further improve the biological activity of the cells during cell culture and promote the realization of the cell biological function.

[0068] According to this embodiment of the present disclosure, further, the matrix used in the present disclosure can be deformed according to the growth of cells during the culture of the biocompatible material. In other words, compared with conventional hard matrices such as glass, silicon wafer, and plastic, the matrix material used in the present disclosure is a soft matrix, so that the force between the matrix and the cells and the force between the matrix and the extracellular matrix material can be reduced, thereby further improving the interface compatibility between the cells and the matrix and the interface compatibility between the matrix and the extracellular matrix material. Furthermore, according to this embodiment of the present disclosure, in the culture process, during cell growth, a depression is formed in the contact area between the matrix and the cells. Those skilled in the art can understand that the depression is formed by cells growing toward the surface of the matrix, causing deformation of the matrix. Those skilled in the art can observe the inner surface of the matrix with a microscope and determine whether there is a depression. According to one embodiment of the present disclosure, the depth of the depression is 5% or more, for example, 10% or 20% of the diameter of the cell. The term "depth of the depression" as used herein may be determined by comparing an interface micrograph of a substrate without a depression and an interface micrograph of a substrate with a depression. Depth values ​​herein are defined as the diameter of a sphere of maximum volume that can fit into the formed recess.

[0069] Therefore, according to this embodiment of the present disclosure, the soft matrix in the microwells, which has better biocompatibility, cell culture medium compatibility, and mechanical tension, can improve the efficiency of cell capture and culture, including single cells and a controlled amount of multiple aggregated cells. According to this embodiment of the present disclosure, the soft matrix is ​​easy to match and blend with various extracellular matrix environments, providing a uniform and integrated 3D culture space for cells, creating a microenvironment conducive to survival, proliferation, migration, protein expression and secretion, and differentiation of stem cells, as well as a microenvironment conducive to detection of cell biological functions of 2D-arranged cells. Furthermore, considering that the matrix is ​​a soft matrix, it is also easy to isolate single cells from the culture space without weakening the adhesive force between the cells and conventional matrix materials.

[0070] According to this embodiment of the present disclosure, the matrix material has a solid-liquid phase transition temperature of 40-45°C. According to this embodiment of the present disclosure, the matrix material can become liquid at 37°C. The above temperature values ​​are measured under 1 atmosphere. Therefore, the matrix material can easily become liquid at a temperature moderately higher than room temperature, and can be easily solidified into a predetermined shape by further cooling. Since the matrix material has the above properties, when the matrix material is in a liquid state, cytokines can be added easily without inactivating them. During addition to the liquid medium for cell culture, the biological culture chip can absorb the medium and further release these cytokines into the culture space. On this basis, according to this embodiment of the present disclosure, the matrix further comprises cytokines corresponding to the biocompatible material. According to this embodiment of the present disclosure, the matrix is ​​further selected from the group consisting of fetal bovine serum (FBS), protein A / G, collagen, gelatin, or bovine serum albumin (BSA), and mixtures of at least two different ratios thereof.

[0071] According to this embodiment of the present disclosure, it should be noted that the specific type of matrix material that can be used is not particularly limited as long as it can meet the above performance requirements. In consideration of better biocompatibility and performance adjustment, the matrix material can be collagen hydrogel, methylcellulose, etc. The soft matrix may be selected from the group consisting of polycellulose, agarose gel, and polyacrylamide gel, and mixtures of at least two of them in different ratios. Those skilled in the art will appreciate that the concentrations of these materials can be adjusted to obtain the desired properties of the soft matrix, such as the solid-liquid phase transition temperature.

[0072] Furthermore, according to this embodiment of the present disclosure, the structure and size of the microwell are not particularly limited as long as it can provide a space for the culture or growth and proliferation of a single cell. According to this embodiment of the present disclosure, the microwell may have a structure such as, for example, a rectangular parallelepiped, a cube, or a cylinder. Furthermore, referring to FIG. 2, according to this embodiment of the present disclosure, the diameter of the opening of the microwell 200 is smaller than the diameter of the bottom of the microwell 200. Therefore, the single cell can be easily captured for culture without the cells leaking out during culture. More specifically, according to this embodiment of the present disclosure, the diameter of the opening of the microwell is 80% or less, preferably 50%, of the diameter of the bottom of the microwell. The term "diameter" as used herein is defined as the diameter of a circle with the largest area that can fit into the opening or bottom. The term "depth" of the 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 of the microwell. According to this embodiment of the present disclosure, the diameter of the opening of the microwell 200 is 8 to 25 μm, and the depth is 15 to 35 μm. It is also understood that different opening sizes may be set for different cells. For example, according to this embodiment of the present disclosure, the biocompatible material is B cells and the opening diameter is 8-12 μm; or the biocompatible material is hybridoma cells or tumor cells and the opening diameter is 15-25 μm.

[0073] Further, according to this embodiment of the present disclosure, the biological culture chip may include a plurality of microwells, the microwells forming a predetermined pattern, and the distance between two adjacent microwells is 10-100 μm. Therefore, a plurality of single cells can be cultured in a batch. According to this embodiment of the present disclosure, when the distance between the microwells is 10-100 μm, the cells cultured in the microwells do not interfere with each other. Therefore, the uniformity of the batch culture can be improved, the accuracy and efficiency of the detection of cell biological functions can be increased, and high-throughput collection and analysis of cell signal data can be facilitated. According to this embodiment of the present disclosure, referring to FIG. 3, the microwells on the biological culture chip may form an array. For example, there is an array of tens of thousands or hundreds of thousands of microwells on the chip.

[0074] According to this embodiment of the present disclosure, the biological culture chip may further include positioning markers formed on the matrix. Thus, the multiple microwells can be positioned through these positioning markers. The shape of the positioning markers is not particularly limited. According to this embodiment of the present disclosure, fluorescent positioning markers may be provided to perform automatic and accurate positioning under a microscope, or coordinate lines may be provided to position each microwell. When positive cells are observed, each positive microwell can be quickly positioned and recorded, which further facilitates the subsequent isolation of the positive cells or other components of the microwells.

[0075] Further, according to this embodiment of the present disclosure, a reagent or biological factor useful for culturing the biocompatible material may be further disposed on the inner surface of the microwell. According to this embodiment of the present disclosure, the biocompatible material is a hybridoma cell and a culture factor for the hybridoma cell is disposed on the inner surface of the microwell; the biocompatible material is a MCF10A cell and insulin is disposed on the inner surface of the microwell; or the biocompatible material is a B cell and at least one of CD40L, IL2, or IL10 is disposed on the inner surface of the microwell, or a mixture of at least two of them in different ratios is disposed on the inner surface of the microwell.

[0076] According to this embodiment of the present disclosure, a biological culture chip (also called cell array chip) with a soft matrix such as solidified hydrogel is provided, which can solve a series of problems in 2D arraying and 3D culture of cells in vitro, including cell capture, formation of flux-controlled cell array, cytocompatibility of chip material, compatibility of chip material with cell culture medium, realization and accurate detection of biological functions of arrayed cells, etc. Arrays with microwells of various diameters and depths can be provided on the chip with gel soft matrix to meet various needs. The mechanical strength of the microwells can be determined by the concentration of the gel soft matrix. The mechanical strength is adjusted according to various detection purposes of different cell types and biological functions to obtain the optimal mechanical strength of the microwells. Furthermore, the diameter and depth of the microwells can be adjusted to facilitate single cell arraying of various cells or aggregation of multi-cells in a controlled amount in the microwells. The distance between the microwells can be adjusted to determine the density of the cell array on the chip and the flux of the cell array on the chip per unit area. The gel soft matrix can provide properties similar to those of the extracellular matrix. The gel soft matrix has good cytocompatibility and provides water, nutrients, and flexible space for cells, thereby avoiding inadequate mechanical strength that cannot be adjusted according to various cells cultured in chips with hard matrices, and also avoiding residual chemical molecules due to chemical modification that is usually required for chips with hard matrices. The gel soft matrix has excellent compatibility with cell culture medium or 3D culture hydrogel biocompatible materials, which ensures a uniform microenvironment around the arrayed cells and guarantees the completeness and accuracy of detection of cell functions.

[0077] Fabrication of biological culture chips According to another aspect of the present disclosure, a template for fabricating the above biological culture chip is provided. Referring to FIG. 4, according to one embodiment of the present disclosure, the template includes a substrate 300; and a micro-column 400 configured on the surface of the substrate 300, the micro-column 400 being fitted to the micro-well 200 in a manner that fits the mold. According to this embodiment of the present disclosure, the matrix material is solidified on the template, so that the above biological culture chip can be fabricated efficiently.

[0078] According to this embodiment of the present disclosure, the micro-columns have a hydrophobic surface. Therefore, the formed biological culture chip can be effectively peeled off from the template. Furthermore, according to this embodiment of the present disclosure, the surface of the micro-columns is suitable for binding biological factors by hydrophobic effect. Then, by applying a matrix material, the biological factors can be transferred to the matrix material as biological factors that bind to the inner surface of the microwell. As mentioned above, various biological factors can be provided according to various culture subjects and purposes.

[0079] According to another aspect of the present disclosure, there is provided a method for preparing the above biological culture chip, comprising: (1) applying a liquid biocompatible material as a matrix material to the above template and solidifying the matrix material; and (2) separating the solidified biocompatible material from the template to obtain a biological culture chip. This method can efficiently prepare the above biological culture chip. According to this embodiment of the present disclosure, a biological factor or reagent is pre-adsorbed on the surface of the template.

[0080] More specifically, according to this embodiment of the present disclosure, the method for fabricating the above biological culture chip includes the following steps. Step 1. Modify the template. Cytokines and other substances required in the biological culture microenvironment are bound to the template by hydrophobic effect. Specifically, the materials used for modification are prepared in a denaturing solution (containing PBS solvent) with a concentration of 1 μg / mL; the template is immersed in the denaturing solution and reacted at room temperature for 4 hours; after being taken out, the template is Rinse successively with fresh PBS and deionized water three times, blow dry with nitrogen gas, and store in a dry place until further use. Step 2. A molten matrix material, such as a gel matrix, is placed on the template with a column arrangement to cover the template; the matrix material, such as a gel matrix, is cooled and solidified, and then the template is removed to form a soft matrix-based chip with a microwell arrangement with a certain thickness and hardness. Specifically, an agarose gel liquid with a concentration of 0.6% to 1.2% is poured into the template and left at room temperature for 5 minutes; after the liquid is solidified, the template is removed to form a biological culture chip. Each microwell of the chip contains a factor or therapeutic agent required for the microenvironment.

[0081] Cultivation method using biological culture chip According to another aspect of the present disclosure, there is provided a method for culturing cells using the above biological culture chip, comprising: disposing cells in the microwells; and exposing the biological culture chip to an environment with a predetermined condition. By using this method, a uniform extracellular matrix-like microenvironment can be provided for the cultured cells, which can effectively maintain the viability of the cells and increase the efficiency of the cell culture. According to this embodiment of the present disclosure, at most one cell is disposed in each microwell.

[0082] According to this embodiment of the present disclosure, the soft matrix of the chip and the cell culture medium have good compatibility, which is helpful to provide a uniform and suitable microenvironment for high-throughput 3D culture of aligned cells, which is helpful to realize the biological functions of aligned cells, and which is helpful to sufficient capture and accurate analysis of cellular biochemical reaction signals (e.g., secreted protein signals, etc.).

[0083] Furthermore, according to this embodiment of the present disclosure, the chip having a soft matrix can better generate an extracellular matrix-like interface effect between the cells and the chip, allowing the 3D cultured cells to grow in an environment that is closer to their living environment in vivo, contributing to cell survival and biological function.

[0084] According to this embodiment of the present disclosure, cell arrays on chips with soft matrices rely on cell gravity and biocompatible materials, such as extracellular matrices, added to the microwells to attract and stabilize the cells, instead of chemically modifying the chip.

[0085] According to this embodiment of the present disclosure, the chip with soft matrix is ​​conducive to picking positive cells (single cells or multicellular spheroids) under a microscope using a glass needle. However, it is difficult to pick cells from the chip with hard matrix under a microscope using a glass needle, because the hard matrix interface is prone to damage the glass needle or the cells. EXAMPLES

[0086] Example 1 Preparation of a Chip 1.1 Preparation of chips with soft matrices Templates having the column arrangement shown in FIG. 4 were selected and classified into the following two types depending on the cell type. A. A template suitable for general cells (typically 15-20 μm in diameter) with microcolumns 35 μm in height and 20 μm in cross-sectional diameter, with a distance of 30 μm between adjacent microcolumns; and B. A template suitable for small cells (typically 8-10 μm in diameter) with microcolumns 20 μm in height and 10 μm in cross-sectional diameter, with a distance of 30 μm between adjacent microcolumns.

[0087] Denaturing solutions containing methylcellulose or low melting point agarose were prepared using PBS as the solvent.

[0088] After soaking the template in the denaturing solution for 4 hours at room temperature, it was removed and rinsed three times successively with fresh PBS and deionized water, blown dry with nitrogen gas, and stored in a dry place for use. Thus, the template used to fabricate the chip is modified so that cytokines and other substances required in the microenvironment bind to the template by hydrophobic effect.

[0089] The molten hydrogel matrix was placed on the modified template (with column array) to cover the template. After the hydrogel matrix was cooled and solidified, the template was removed to form a soft matrix-based chip with a microwell array and a thickness of about 300-500 μm. Specifically, an agarose gel liquid with a concentration of 0.6%-1.2% (swelling ratio of about 1.25-1.75) was poured into the template and left at room temperature for 5 minutes; after the liquid was solidified, the template was removed to form a biological culture chip. Each microwell of the chip contained a factor or therapeutic agent required for the microenvironment. The following table shows the composition of the chip with a soft matrix.

[0090] [Table 1]

[0091] 1.2 Preparation of chips with hard matrices According to the method described in CN108102913A "3D cell culture chip based on soft lithography, its preparation method and its application", a chip with hard matrix 1 and a chip with hard matrix 2 were prepared using hard matrix materials glass and silicon wafer, respectively, but the details are not described in this specification.

[0092] Example 2 Comparison of chips with soft and hard matrices in cell culture 2.1 Comparison of array formation efficiency According to the following procedure, mouse hybridoma cells, human tumor cells (MCF7), human umbilical cord mesenchymal stem cells, and mouse B cells were cultured using chips 1, 2, and 3 having soft matrices, and the above cells were also cultured using chips having a hard matrix (glass substrate). These cells were cultured in parallel using type 1. These cells were cultured for 15 minutes using conventional cell culture methods (see the Cellular and Molecular Biology Experiment Guide for details) and the sequence efficiency was determined. The sequence efficiency was determined as follows. The amount of single cells per unit area (1 mm × 1 mm) was observed and counted, and divided by the total volume of the chamber to obtain the efficiency of single-cell array acquisition. The chip specifications were adjusted to limit the space on the chip so that each chamber could accommodate only one cell, and very small amounts of doublet cells caused by uncontrollable factors were easily identified under a microscope and excluded from the statistical range. The results of the above decisions are as follows:

[0093] [Table 2]

[0094] *Single cell cultures were cultured on chips (20μm / 30μm, 10μm / 30μm). The first number represents the diameter of the chamber, and the second number represents the distance between adjacent chambers. In the above table, except for mouse B cells, which were cultured using the 10μm / 30μm chip, other types of cells were cultured using the 20μm / 30μm chip.

[0095] From the above data, it can be seen that the array efficiency of the chip with hard matrix is ​​about 32-50%, indicating that the chip with hard matrix does not contribute to the formation of cell arrays. The array efficiency of the chip with soft matrix is ​​very high, about 91-98%, indicating that the chip with soft matrix is ​​very effective in the formation of cell arrays.

[0096] The inventor believes that the above difference is mainly due to the hard matrix of the chip, such as glass or silicon wafer. The material of the hard matrix has no voids or water permeability, so the gas in the small chamber cannot be completely discharged. As a result, the cells seeded thereafter cannot take the appropriate position by gravity sedimentation. However, the soft matrix of the chip, such as hydrogel or other biocompatible materials, has a certain pore size and water permeability, so that when the cells are seeded, the medium can permeate from the periphery of the chip and completely discharge the gas from the small chamber, promoting cell sedimentation and forming an array. Based on the above, there is a large difference in the proportion of chambers used to efficiently form cell arrays between chips with hard matrix and chips with soft matrix, which have the same amount of chambers. Therefore, the efficiency of the chip with hard matrix in cell screening is much higher than that of the chip with soft matrix. It becomes slightly lower.

[0097] 2.2 Comparison of cell viability on chip According to the method of 2.1, cells were cultured using chips with hard matrix and chips with soft matrix, respectively, and cell viability was detected according to the following method. Cell viability on the chip was determined by live / dead assay (Thermo, L34951). Dead cells show red fluorescence, and live cells show green fluorescence. The results are as follows.

[0098] [Table 3]

[0099] From the above data, it can be seen that chips with hard matrices are not conducive to cell culture and survival, and easily cause cell apoptosis and necrosis, making them unhelpful for cell proliferation. The main reason for this is that hard matrix materials lack physical elasticity and cannot provide a uniform extracellular matrix-like microenvironment (having similar characteristics to the living space in the body) required for 3D cell culture in all directions, which changes the pathways of cell survival, proliferation, aging, and apoptosis, leading to cell aging, apoptosis, or necrosis. However, soft matrices have better plasticity, have sufficient water and support space, can adjust to the changes in the requirements of cell survival space, and can also be compatible and fused with ECM medium for 3D cell culture to provide a uniform and appropriate microenvironment for cells, thereby helping to realize cell survival and biological functions (such as factors and antibody secretion). In addition, during the fabrication of chips with soft matrices, various factors or materials required for the culture of various cells can be appropriately added to construct a microenvironment for cell culture and survival, which greatly improves cell survival and proliferation. However, it is difficult for hard matrix materials to achieve this. Typically, various factors or materials are added to the culture medium or the surface of the hard matrix material is chemically modified, which can make it difficult to sufficiently disperse the factors or materials around the cells or can result in residual chemicals that are toxic to the cells.

[0100] 2.2 Comparison of single cell collection ability on chip Single-cell chips are generally used to obtain and analyze single-cell information of special significance. Therefore, it is necessary to isolate target cells for subsequent manipulation. Based on this, the present inventors have attempted to use a capillary needle to collect single cells from chips with soft and hard matrices, respectively. The comparison results are as follows:

[0101] [Table 4]

[0102] The inventors have investigated the reason why the success rate of collecting single cells from chips with hard matrices is low. believes that mainly because all cells are cultured in suspension without any constraints, when using a needle to collect target cells, non-target cells near the target cells are easily collected; furthermore, when the needle approaches the target cells, the target cells may be affected by a slight force (the force generated by the movement of the needle in the liquid) and may be pushed away or separated from the chip, leading to collection failure. However, the material of the soft matrix of the chip is a hydrogel, which is easy to produce a moderate adhesive effect with the cells and does not completely culture the cells in suspension, thus solving the problems caused by the hard matrix and increasing the success rate of collection.

[0103] In addition, the consumable needle can be replaced at any time during cell inoculation if there is contamination, deformation, or other problems to increase efficiency. However, needle breakage or other problems due to improper operation will greatly affect the collection efficiency. This is because it takes time to replace the needle or adjust its position accurately. Hard matrices have no elasticity. During operation under a microscope, the distance between the needle and the tip needs to be very close or even touch the surface of the tip so that weak suction force can be released as much as possible to the target cells for successful collection. However, since it is difficult to complete manual or semi-automatic operation accurately every time, it is common for the needle to be deformed or broken when touching the tip. Due to good physical elasticity, soft matrices have high resistance to excessive contact of the needle, which the operator can detect and adjust in time.

[0104] Example 3 Screening of monoclonal antibodies In this example, a chip having a soft matrix was prepared by the method of Example 1 to screen single cell-derived monoclonal antibodies against the novel coronavirus S1 protein. 3.1 Immunization of mice Five-week-old BALB / c female mice purchased from Shanghai Silaike Experiment Animal Co., Ltd. were used. The mice were kept in an animal breeding room for one week to acclimate to the environment. Six-week-old mice were then prepared for immunization. The novel coronavirus S1 protein purchased from Novoprotein Technology Co., Ltd. was dissolved in PBS and the concentration was adjusted to 1 mg / mL before immunization. The antigen was emulsified by mixing with a syringe before injection. The specific immunization schedule and conditions are as follows:

[0105] [Table 5]

[0106] After the second and third immunizations, blood was collected from the mouse via the orbit and the antibody titers in the serum were measured. The antigen dose for the third or additional immunization was adjusted according to the antibody titers until the immune response of the mouse to the antigen reached an optimal state.

[0107] 3.2 Cell fusion According to the serum titer results 3 days after the booster immunization, mice with the highest titers were selected for subsequent fusions. Prior to fusion, all blood from the mice was collected to obtain serum for use.

[0108] SP2 / 0 cells were prepared in advance (3-5 days previously thawed). 50 mL of blank 1640 medium and 500 mL of sterile water were pre-warmed in an incubator overnight for fusion the next day. PEG used for fusion was pre-warmed in an incubator prior to splenectomy.

[0109] Immunized mice were cervically dislocated and immersed in 75% ethanol for 3-5 minutes for sterilization. Splenectomy was performed in a biosafety cabinet. Sterilized mice were removed and left to drain the ethanol, then placed abdominally up on a paper towel. A small incision (1 cm) was made in the left upper abdomen using scissors, and the spleen (inferior to the left liver lobe) was removed using forceps. Care was taken not to puncture the spleen or remove excess connective tissue during removal.

[0110] The removed spleen was placed on a 40 μm sieve, and then the sieve was placed in a 50 mL centrifuge tube filled with blank 1640 medium. During the grinding of the spleen, care should be taken not to add too much liquid to prevent the medium from overflowing. During grinding, the spleen needs to be completely exposed to the medium. To facilitate the isolation of splenocytes by grinding, a small hole was made in the spleen using a syringe needle or several small cuts were made in the spleen using scissors before grinding. The spleen was ground to completely pass the spleen contents through the sieve, and the remaining connective tissue, etc. were discarded. The ground spleen was centrifuged twice at room temperature at 1300 rpm for 8 minutes to collect the splenic alveoli. After removing the supernatant, 10 mL of PBS was added to resuspend the splenocytes. 30 mL of red blood cell lysis buffer was added to dissolve at 4 °C for 15 minutes, and mixed evenly twice during lysis. The resulting mixture was centrifuged at room temperature at 1300 rpm for 8 minutes to collect the splenocytes. The splenocytes were resuspended in PBS and centrifuged twice until the lysis buffer was completely removed.

[0111] The splenocytes and tumor cells were counted separately. The ratio of splenocytes to tumor cells was adjusted to 5:1. The splenocytes and tumor cells were mixed in a 50 mL centrifuge tube at the above ratio and centrifuged at room temperature at 1300 rpm for 8 minutes to collect the mixed cells. The supernatant was completely removed (because the residual supernatant would affect the fusion efficiency). The bottom of the centrifuge tube was gently tapped by hand or moved back and forth on a mesh ventilated mesh plate in a biosafety cabinet to disperse the precipitated cells, and finally a cell homogenate was obtained. Cell fusion was performed using PEG preheated to 37°C in a 37°C water bath and terminated with blank 1640 medium. The specific procedure of fusion is as follows:

[0112] With the outlet tip of the pipette as close as possible to the cells, the pipette was used to dispense 1 mL of PEG dropwise along the tube wall within 1 min. The centrifuge tube was rotated and shaken slightly during the PEG dispense to ensure complete and even exposure of the cell homogenate to the PEG. The cell homogenate was allowed to stand at room temperature for 1 minute. 1 mL of pre-warmed blank 1640 medium was added dropwise within 1 min. The centrifuge tube was rotated and slightly shaken during the medium addition to ensure that the cells were fully and evenly exposed to the medium. 2 mL of pre-warmed blank 1640 medium was added dropwise within 1 min. The centrifuge tube was rotated and slightly shaken during the medium addition to ensure that the cells were fully and evenly exposed to the medium. 5 mL of pre-warmed blank 1640 medium was added dropwise within 1 min. The centrifuge tube was rotated and slightly shaken during the medium addition to ensure that the cells were fully and evenly exposed to the medium. Approximately 21 mL of pre-warmed blank 1640 medium was added dropwise. While adding the medium, the centrifuge tube was rotated and slightly shaken to ensure that the cells were completely and evenly exposed to the medium. After the fusion was completed, the resulting mixture was centrifuged at room temperature at 1300 rpm for 8 minutes to collect the fused cells.

[0113] Fusion cells, 1 x 10 6 Complete medium (20% FBS, 1% The cells were resuspended (with gentle purging) in 1×P / S, 1×HAT, and 1× hybridoma growth factor (HGF). The resuspended cells were seeded onto culture dishes and cultured for 3 to 5 days, during which the medium was changed once.

[0114] 3.3 Chip fabrication A template was prepared and modified with an HGF layer (using HGF-containing PBS solution as a denaturing solution beneficial to the survival and proliferation of hybridoma cells). To fabricate the chip, approximately 500 μL of gel was taken (by aseptic technique), dropped onto the center of the template (sometimes called a stamp), and left at room temperature for 10 minutes (to effectively prevent excessive drying). After the gel solidified, the template and the chip were gently separated from each other. In this case, displacement must be avoided, otherwise the integrity of the structure will be damaged. The fabricated chip was stored in a 10 cm diameter culture dish for use.

[0115] 4.4 Formation of cell arrays The fused cells obtained in 4.3 were collected and washed once or twice with PBS or medium to remove excess culture components (antibodies in the culture supernatant cause high background detection). The cell concentration was adjusted to 8 × 10 5 〜1×10 6 It is recommended to adjust the cell suspension to 1 mL / mL. The cell suspension was dropped onto the surface of the chip at about 500 μL / chip. The chip was placed in an incubator and left to stand for 10 minutes. Excess cell suspension was collected using a pipette, and then about 300 to 500 μL of medium was taken out and dropped onto the chip tilted at about 30 to 45° from above to wash away excess cells (so that the medium does not flow out of the chip), and then the excess medium was removed using a vacuum pump. The formation of cell arrays was observed under a microscope to determine whether to proceed with the subsequent operations.

[0116] 4.5 Antigen labeling and screening of positive hybridoma cells A protein labeling kit (A30006) from Thermo Scientific was used, and the basic operating procedures were carried out according to the manufacturer's instructions. That is, within 10 minutes of waiting for the formation of the cell array, the freezer was taken out and the labeled antigen and the self-made 3D culture hydrogel were arranged in the freezer for use. 50 μL of complete medium was added to a 1.5 mL sterile centrifuge tube and placed in the freezer for use. After the cell array was formed, 50 μL of hydrogel was added to each centrifuge tube and mixed evenly. 70-80 μL of well-mixed hydrogel was taken out and added dropwise from one corner of the chip. Meanwhile, the chip was gently shaken to ensure that the hydrogel completely covered the structure. The chip was then placed in an incubator and left to stand for 5 minutes, during which the chip was held horizontally. The chip was transferred to a 3.5 cm diameter culture dish using a syringe needle and tweezers, placed in an incubator, and then the screening solution (2 mL volume with a ratio of fluorescently labeled antigen: complete medium = 1: (200-1000)) was added. The chip was left to stand for at least 3 hours.

[0117] 4.6 Single-cell selection After screening for 3 to 5 hours, the signal status was observed under a fluorescent microscope. After observing until a signal appeared and reached the required ratio, the cells were ready to be selected.

[0118] 4.7 Monoclonal culture and identification Cells with positive signals were selected and released into 96-well plates, then examined microscopically within 1-2 hours to determine whether a single cell was present in each well. After 7-14 days of culture, the supernatants from the stably growing cells in each well were tested by ELISA to determine whether the cells secreted antigen-specific antibodies, i.e., whether the cells were positive clones. The positive clones were expanded in continuous culture and the secretion of antibodies was identified again before cryopreservation.

[0119] 4.8 Screening Results Through screening, 15 cell lines capable of secreting specific antibodies were obtained, including 14 monoclonal cell lines. All the obtained clones were derived from different mice or different hybridoma cells of the same mouse, which ensured sufficient clonal diversity. Compared with the conventional method (limiting dilution method), the method of the present disclosure has significant advantages, specifically as follows.

[0120] [Table 6]

[0121] Therefore, compared with the conventional limiting dilution method, the method disclosed herein has absolute advantages in terms of screening efficiency and screening cost, and the overall cost is reduced by about 90%, while the screening efficiency is increased by at least 10 times. This is merely a result obtained without comprehensive screening, and manpower and time can be better allocated according to the urgency of the project. Based on this, it is believed that the screening efficiency can be effectively increased by more than 30 times.

[0122] Example 5: Screening of paired antibodies 5.1 Obtaining labeled antibodies and fluorescent labeling A monoclonal cell line with a high antibody titer was selected according to the screening results in Example 4. Ascites was expressed as follows. (i) Two female B / C mice aged 8 to 10 weeks were prepared for each group. For sensitization, Freund's incomplete adjuvant was intraperitoneally injected into the mice at 500 μL / mouse. 5 The cells were inoculated intraperitoneally. NOTE: Fewer cells injected will result in slower ascites production but higher titers; hybridoma cells should be injected to detect antibody titers. Prior to inoculation, cells should be washed 2-3 times with approximately 0.2-1 mL serum-free medium per mouse.

[0123] (ii) Two days after inoculation, the mice were closely observed for ascites and survival. After about seven days, the mice were viable. If the mouse is active and has obvious abdominal dullness fluctuations, the ascites may be drained twice; once the mouse is found to be lethargic, the mouse should be sacrificed and the ascites drained.

[0124] Draining ascites from the body: Wipe the area where the needle is to be inserted (near the right side of the midline of the abdomen) with a cotton ball soaked in alcohol, insert the syringe with air in it, then slowly withdraw the plunger, which will slowly drain the ascites due to the pressure created in the abdomen. While inserting the needle, you may lift the syringe slightly until no blockage is felt.

[0125] Drainage of ascites after sacrifice: Using scissors and tweezers, gently make a small incision in the abdominal skin, gently pull apart both sides of the skin using two hemostats to expose the abdominal cavity, cut a small area of ​​the dermis using another pair of scissors and another pair of tweezers, and drain the ascites using a pipette or straight glass tube. Usually, 2-3 mL of ascites fluid can be collected from each mouse at one time. If the mouse is in good condition, the ascites fluid can be collected every other day. The antibody concentration was 0.5-5 mg / mL.

[0126] (iii) Ascites was collected in a 15 mL centrifuge tube and centrifuged at 800 rpm for 30 min (or 2000 rpm for 5 min) to remove cells and fat. Freshly collected ascites contains a large amount of impurities, such as fat, macrophages, hybridoma cells, epithelial cells in the peritoneal cavity, and cellular components of blood, especially cellular components. If these are not removed, the cellular components present in the ascites for a long time may slowly burst, releasing proteins into the ascites, increasing the impurities in the ascites and making purification difficult. Therefore, the collected ascites, whether for long-term storage or immediate purification, must first be centrifuged to remove cellular components and fat (fat has low density and generally floats on the surface of the ascites, so it can be sucked out using a pipette). Purification of the antibodies and preparation of fluorescently labeled antibodies were performed according to the specific operating procedures in accordance with the instructions of the Thermo kit (89953 A30006).

[0127] 5.2: Screening of paired antibodies The specific procedure was the same as that described in Example 4, except that the fluorescently labeled antibody was replaced with the antigen-fluorescently labeled primary antibody complex in a 2:1 ratio. In this procedure, the following other method, which has the same specific procedure as described in Example 4, can also achieve the same screening purpose and effect: 3D medium containing antigen is added to the chip containing 2D arrayed hybridoma cells, and cultured for 2 hours, antigen is completely bound to the antibody secreted by hybridoma cells, and then 3D liquid medium is removed, and 3D medium containing fluorescently labeled primary antibody is added, and cultured for 2 to 3 hours.

[0128] 5.4 Screening Results Two high titer antibody clones against the novel coronavirus S1 protein obtained in Example 4, namely S1-6S and RBD-10S, were selected as primary antibodies. After the above screening, the results are as follows:

[0129] [Table 7]

[0130] There were four antibody pairs against the S1 protein and three antibody pairs against the RBD protein. The paired antibodies were derived from different mice or different hybridoma cells of the same mouse, which can ensure the diversity of the antibody pairs.

[0131] Example 6: Screening of single cells secreting specific factors using chips with soft matrices With reference to Examples 4 and 5, CHO cells stably expressing VEGFA were obtained by screening. Specifically, the chip preparation, 2D array of single cells, antigen-antibody reaction of single cells, identification and collection of positive single cells, etc. were the same as those described in Examples 4 or 5. The difference is that in this example, the fluorescent-labeled antigen for immunofluorescence was replaced with a fluorescent-labeled specific antibody for VEGFA secretion. The results of screening are as follows:

[0132] [Table 8]

[0133] Two VEGFA antibodies from different sources were used in the test. After drug selection, 58 and 49 positive cells (capable of secreting the factor) were collected separately. Subsequent culture and validation yielded 52 and 42 CHO cell lines capable of stably expressing and secreting VEGFA with a positive rate of 89.6% and 85.7%, respectively. Traditional screening and isolation of stably transfected CHO cell lines is still performed by limiting dilution as described above. Our technique shows great advantages in terms of time, material and labor costs.

[0134] Furthermore, single cells capable of secreting other factors were also obtained by screening using Examples 4 and 5. Various cells capable of expressing secretory proteins include HEK293, CHO, Hela, MCF10A, HFF, etc. Factors include various growth factors and cytokines secreted outside the cells, such as VEGF, SDF-1a, PEDF, and FGF. Labeled proteins that specifically bind to proteins secreted from cells include antibodies specific to secretory proteins and receptor binding domains. Details will not be described in this specification.

[0135] Example 7: Sphere formation of mouse neural stem cells Neural stem cells are a population of cells present in the nervous system that have the pluripotency to differentiate into neurons, astrocytes, and oligodendrocytes to generate a large number of brain cells, and can self-renew to provide a large number of cells to brain tissue. Therefore, methods to better cultivate neural stem cells outside the body are an important step in scientific research. It is the foundation and base.

[0136] (1) Extraction of neural stem cells from fetal mice. C57BL / 6 mice at 14 days of gestation were cervically dislocated and immersed in 75% ethanol, and the abdomen was incised, and a series of fetal mice (umbilical cord was cut) were removed and placed in a culture dish containing pre-chilled PBS. The placenta was peeled off from the fetal mice using tweezers, and the fetal mice were placed in a new culture dish containing pre-chilled PBS (typically, a pregnant mouse has 8-11 fetal mice, with at least 6 and at most 13). The heads of the fetal mice were twisted off and placed in a new culture dish (2 fetal mice per dish) containing PBS. The cerebral cortex was removed under a stereomicroscope, and brain tissues connected to the cerebral cortex, such as olfactory bulbs and meninges, were removed. The cerebral cortex was placed in a 15 mL centrifuge tube, purged with a Pasteur pipette until no tissue pieces were visible, and centrifuged at 800 rpm for 2 min. The supernatant was removed for use. Fresh PBS buffer was added to the sediment and purged into the suspension. The suspension was centrifuged at 800 rpm for 2 min. The supernatant was removed and mixed with the supernatant from the first centrifugation, then centrifuged at 1200 rpm for 2 minutes. The supernatant was removed. The precipitate was purged with growth medium to obtain an NPC suspension. The NPC suspension was filtered through a 40 μm filter membrane to remove undispersed clumps. The NPC suspension was aspirated into a T25 flask for suspension culture. The flask was not shaken for 2 days after the start of culture. Generally, cells start to be passaged on the 6th or 7th day, depending on the size of the cell spheroids. The cells were harvested and centrifuged at 800 rpm for 2 minutes. The supernatant was removed. PBS buffer was added to purge the precipitate, centrifuged at 800 rpm for 2 minutes, and the remaining medium was removed. The supernatant was removed. 1-2 mL of Accutase or TryplE cell dissociation solution was added to the tube containing the precipitate to dissociate the cells, and the tube was placed in a 37°C incubator for 5-10 minutes. The tube may be flicked with a finger during this time. Ca / Mg 2+Dissociation was stopped by adding 5 times the volume of the above solution (or adding 4-5 mL of pre-warmed medium for resuspension), purging multiple times, and centrifuging at 1200 rpm for 2 minutes. The supernatant was removed. 1 mL of growth medium was added (or more growth medium could be added to avoid more losses in the following filtration) to purge the precipitate into the suspension. The suspension was filtered through a 40 μm filter membrane to remove cell spheroids that were not completely dissociated, and an NPC suspension was obtained. The cells of the NPC suspension were counted using a hemocytometer, and the cell concentration was calculated. 10×10 6 The cells were seeded into a new T25 flask for culture.

[0137] (2) Inoculation onto the chip. The specific procedures were carried out with reference to Example 4 or 5. In this example, the chip specifications were 12 μm / 30 μm, and the factors added to the soft matrix were EGF and bFGF, with final concentrations of 20 ng / mL and 10 ng / mL, respectively.

[0138] (3) Results and Analysis. The following specific test data is used to compare the method of the present disclosure with the conventional culture method, which shows that the chip with soft matrix is ​​helpful to improve cell survival rate and maintain the stemness of cells well.

[0139] Example 8: Sphere formation of tumor cells (MCF7) Tumor growth and drug sensitivity studies have relied heavily on in vitro monolayer tumor models (2D cell cultures). This model lacks many characteristics of the disease, such as hypoxia, altered cell-cell contacts, and metabolic changes. Through years of research, it is believed that the phenotype of 3D tumor models is closer to real cancer cells and can provide more accurate models and data for drug screening.

[0140] (1) MCF7 cell culture and seeding on the chip. MCF7 cells were cultured in DMEM medium containing 10% FBS and 1% P / S. When the culture area reached 80% of the surface area of ​​the culture dish, the MCF7 cells were dissociated and seeded onto the chip. The specific procedure of seeding onto the chip was carried out with reference to Example 4 or 5.

[0141] (2) Continuous culture. The chip containing the cells was placed in an incubator and continuously cultured for 3 to 5 days, during which the specific proliferation of the cells was constantly observed. An array of cell spheroids derived from a single MCF7 cell was formed on the chip by the culture.

[0142] These 3D cultures (or "cell clump" cultures) of tumor cells can faithfully recapitulate many important properties of the original tumor. Moreover, arrayed cultures are suitable for large-scale drug screening to detect drug sensitivity associated with genetic alterations, establishing an experimental basis for personalized medicine and optimizing clinical outcomes for cancer patients.

[0143] In the description of this disclosure, it should be understood that the orientation or positional relationship indicated by terms such as "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" are based on the orientation or positional relationship shown in the accompanying drawings, and are used only for ease and brevity of illustration and description of the disclosure, without indicating or implying that the devices or components referred to need to have a particular orientation or be constructed and operated in a particular orientation. Thus, such terms should not be construed as limiting the disclosure. Furthermore, features defined as "first" and "second" may explicitly or implicitly include one or more such features. In the description of this disclosure, unless otherwise specified, "plurality" means two or more. In the context of this disclosure, a first feature being "above" or "below" a second feature may include the first feature and the second feature being in direct contact, or the first feature and the second feature being not in direct contact, but in contact through the use of another feature between them.

[0144] In the context of this disclosure, a first feature being "on," "above," or "above" a second feature includes the first feature being directly above and diagonally above the second feature, or simply indicates that the first feature is higher than the second feature.

[0145] In the description of the present disclosure, unless otherwise expressly specified or defined, it should be noted that terms such as "attach", "connect" and "connection" should be understood in a broad sense. For example, the connection may be a fixed connection, a detachable connection, or an integral connection; or the connection may be a mechanical connection or an electrical connection; or the connection may be a direct connection, an indirect connection through an intermediate, or an internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in the present disclosure according to the specific situation.

[0146] In the description of the present disclosure, a description using the terms "one embodiment," "some embodiments," "exemplary embodiments," "example," "particular examples," or "some examples" means that the particular feature, structure, material, or characteristic described with reference to the embodiment or example is included in at least one embodiment or example of the present disclosure. Exemplary descriptions of the foregoing terms herein do not necessarily refer to the same embodiment or example. Moreover, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0147] Although embodiments of the present disclosure have been shown and described, it is understood that those skilled in the art will understand that certain modifications and variations can be made without departing from the principles and spirit of the present disclosure. It should be understood that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the spirit and scope of the present disclosure, and that the scope of the present disclosure is defined by the appended claims and their equivalents.

Claims

1. A matrix formed from a matrix material; and A culture chamber formed on a surface of the matrix to define the culture chamber. Including, the matrix material of the matrix has an equilibrium swelling ratio of 1.25 to 1.75; The culture chamber is formed over an opening configured on a surface of the matrix to define the culture chamber. Biological culture chip.

2. 2. The biological culture chip according to claim 1, wherein the matrix material has a solid-liquid phase transition temperature of 40 to 45°C.

3. The biological culture chip of claim 1 , wherein during cell proliferation, depressions are formed in the contact areas between the matrix and the cells.

4. The biological culture chip according to claim 3 , wherein the depth of the recess is 5% or more of a diameter of the cell.

5. 2. The biological culture chip of claim 1, wherein the matrix material is selected from the group consisting of collagen hydrogel, methylcellulose, agarose gel, polyacrylamide gel, and a mixture of at least two of them in different proportions.

6. The biological culture chip of claim 1 , wherein the matrix further comprises a cytokine corresponding to the biocompatible material.

7. 2. The biological culture chip of claim 1, wherein the matrix is ​​further selected from the group consisting of fetal bovine serum, protein A / G, collagen, gelatin, bovine serum albumin, and a mixture of at least two thereof.

8. The biological culture chip of claim 1 , wherein the diameter of the opening of the culture chamber is smaller than the diameter of the bottom of the culture chamber.

9. 2. The biological culture chip of claim 1, wherein the opening has a diameter of 8 to 25 μm and a depth of 15 to 35 μm.

10. 2. The biological culture chip according to claim 1, wherein the culture chamber is composed of a plurality of microwells, the microwells form a predetermined pattern, and the distance between two adjacent microwells is 10 to 100 μm.

11. The biological culture chip of claim 10 , further comprising a positioning marker formed on the matrix.

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