Target cell or cell screening method and biological culture chip

Abstract

A method for screening target cells, a corresponding test kit and its use, a method for screening cells, and a biological culture chip and its preparation method and its use. A method for screening target cells includes culturing the candidate single cells in a culture chamber equipped with a signal screening layer, the signal screening layer containing 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. The cell screening method includes placing candidate cells in a culture chamber to form a target antibody-antigen signaling antibody compound; and determining whether the candidate cells are target cells based on the signal of the signaling molecule bound to the signaling antibody. The biological culture chip includes a matrix (100) and a biological culture space disposed on the surface of the matrix (100) and used for culturing biological cells.

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 uses thereof according to a first aspect of the present disclosure, a method for screening target cells, a kit, a method for producing antibodies, a method for screening monoclonal antibodies, monoclonal antibodies, antibody-antigen complexes, and methods for screening antibodies according to a second aspect of the present disclosure, and a biological culture chip, a method for producing the same, and uses thereof according to a third aspect of the present disclosure. [Background technology]

[0002] Limiting dilution is a commonly used cloning method, 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 hybrid animal cells. Monoclonal antibodies are highly homogeneous antibodies that target only a specific epitope and are produced by cloning a single B cell. Monoclonal antibodies are usually produced by hybridoma technology, which is based on cell fusion. Sensitized B cells capable of secreting specific antibodies are fused with myeloma cells, which have the ability to proliferate indefinitely, to form 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 simultaneously bind to one antigen molecule. Antibody pairs are typically used in double-antibody sandwich ELISA experiments. Antibody pair screening is generally performed using the double-antibody sandwich ELISA method. This screening is based on the following: After obtaining a monoclonal antibody, two types of monoclonal antibodies are separated from it: a capture antibody and a labeled antibody (HRP enzyme-labeled). 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 development 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 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 labeled antibody do not 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, resulting in a significant increase in screening costs.

[0006] In current biological and medical research, traditional two-dimensional (2D) adherent cell culture is often preferred for cytological drug detection. This is fundamentally different from the in vivo state of tissue cells (a 3D state in an ECM environment). While cells in vivo grow in a 3D microenvironment, cells grown in 2D culture are not in their natural state. The culture microenvironment is significantly different from the in vivo microenvironment, which affects cellular gene expression and signal transduction. Cultured cells gradually lose their in vivo biological characteristics and biological functions, making them unusable for research and applications. Furthermore, 2D cell culture has many serious drawbacks, such as cell heterogeneity, which makes it unconvenient for cell differentiation and related research. Furthermore, 2D cell culture has many significant drawbacks, such as cell heterogeneity, which makes it unconvenient for cell differentiation and related research. 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 fabrication method, and its use. The fabrication method for the 3D cell culture chip based on soft lithography includes: fabricating 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 of a defined thickness; solidifying the liquid layer and removing the mask to obtain a stamp having a stamp surface with a defined 3D structure; contacting the stamp surface of the stamp with a modifier to releasably attach the modifier to the stamp surface; and contacting the stamp surface with the surface of a selected substrate to remove the stamp from the selected substrate, separating at least a portion of the modifier from the stamp surface and attaching it to the surface of the selected substrate, thereby forming a pattern structure including an array of multiple patterns capable of adsorbing at least single cells, thereby obtaining a 3D cell culture chip.

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

[0009] [Patent Document 1] Chinese 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 includes culturing candidate single cells in a culture chamber under conditions suitable for secreting the target molecule, wherein the culture chamber includes a signal screening layer covering the candidate single cells, the signal screening layer including 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 in the signal screening layer. By this method, since only single cells are present in the culture chamber, the target molecule can be specifically captured by the signaling molecule under conditions that allow the single cells to 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 signaling molecule signal, it can be determined whether the single cell is capable of expressing the target molecule. Compared to the conventional limiting dilution method, this method is rapid, cost-effective, and efficient, and can achieve 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. The kit can be used to effectively implement the above-described method, thereby achieving the above-described effects. The features of the method described above are also suitable for the kit, and will not be described in detail herein.

[0013] According to another aspect of the present disclosure, there is further provided a method for producing an antibody, comprising screening target cells by the above method; and growing the target cells 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, there is further provided a method for screening monoclonal antibodies, which comprises distributing 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-mentioned method; isolating the positive hybridoma cells; and culturing the positive hybridoma cells to express antibodies to obtain monoclonal antibodies.As described above, the method of the present disclosure can efficiently obtain fusion cells that express monoclonal antibodies, and can further obtain monoclonal antibodies.

[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, there is further provided an antibody-antigen complex. The antibody is the monoclonal antibody described above, and the monoclonal antibody is bound to a signaling molecule. By using the antigen-antibody complex, it is possible to efficiently obtain a pair of antibodies, 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 candidate cells in a culture chamber and culturing the candidate cells for a predetermined period of time under conditions suitable for the candidate cells 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; and (3) determining whether the candidate cells are target cells based on a signal from the signaling molecule, wherein the target cells secrete 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 the predetermined time; (2) forming a target antibody-antigen signaling antibody complex in the culture chamber, wherein 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 screening to obtain antibody pairs.

[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 and obtain antibodies with antigenic determinants different from those of the signaling antibody. 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, there is provided a biological culture chip including a matrix formed from a matrix material, wherein the equilibrium swelling ratio of the matrix material is about 1.25 to 1.75; and a culture chamber formed on a surface of the matrix to define the culture chamber.

[0024] Because 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, which can further improve the biological activity of the cells during cell culture, promote the realization of cell biological functions (such as antibodies and factors), and facilitate accurate detection.

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

[0026] The above and / or further aspects and advantages of the present disclosure will become apparent from 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 one embodiment of the present disclosure. [Figure 2] FIG. 2 is a schematic structural diagram of a biological culture chip according to another embodiment of the present disclosure. [Figure 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 one embodiment of the present disclosure. [Figure 5] FIG. 5 is a diagram illustrating 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 between suspension culture and chip culture according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0027]

[0023] 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 designated by identical 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 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 is adapted to contain the candidate single cells. The method includes a signal screening layer for covering the target molecule, the signal screening layer containing 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. Since only a single cell is present 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 signaling molecule signal, it can be determined whether the single cell is able to express the target molecule. Compared to the limiting dilution method of the prior art, this method is rapid, cost-effective, and efficient, and can achieve high-throughput screening.

[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 in 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, thereby making it easier for molecules secreted from single cells to enter the signal screening layer, thereby improving screening efficiency.

[0030] According to one embodiment of the present disclosure, the culture chambers are disposed on a biological culture chip, which includes 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 will be briefly described here and then described in detail below. During extensive research into 3D cell culture chips, it was discovered that it is generally difficult for cells to exhibit biological behavior and physiological effects in an in vivo environment during cell culture on biocompatible materials using existing 3D culture chips. To this end, creative research has revealed that the matrix material of existing 3D culture chips affects the effectiveness of cell culture, particularly the realization of the biological functions of cell-secreted factors (including antibodies). Furthermore, it has been 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 cellular microenvironments obtained at different positions also exhibit significant differences and heterogeneity. For example, at the contact positions between cells and the matrix, cells cannot receive sufficient support from the extracellular matrix. Furthermore, many existing 3D culture chips employ hard matrices. Therefore, during cell culture, the hard matrix imposes a certain degree of stress on the cultured cells, affecting their biological behavior, and these conditions have a serious impact on the factor or antibody secretion ability and functional 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. The kit can be used to effectively implement the above-described method, thereby achieving the above-described effects. The features of the method described above are also suitable for the kit, and will not be described in detail herein.

[0035] According to another aspect of the present disclosure, there is further provided a method for producing an antibody, comprising screening target cells by the above method; and growing the target cells 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, there is further provided a method for screening monoclonal antibodies, which comprises distributing 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-mentioned method; isolating the positive hybridoma cells; and culturing the positive hybridoma cells to express antibodies to obtain monoclonal antibodies.As described above, the method of the present disclosure can efficiently obtain fusion cells that express monoclonal antibodies, and can further obtain monoclonal antibodies.

[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, there is further provided an antibody-antigen complex. The antibody is the monoclonal antibody described above, and the monoclonal antibody is bound to a signaling molecule. By using the antigen-antibody complex, it is possible to efficiently obtain a pair of antibodies, 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 candidate cells in a culture chamber and culturing the candidate cells for a predetermined period of time under conditions suitable for the candidate cells 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; and (3) determining whether the candidate cells are target cells based on a signal from the signaling molecule, wherein the target cells secrete 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 costs. Taking monoclonal antibody screening as an example, the overall cost is reduced by approximately 90%, while the screening efficiency is increased by at least 10 times. This result was obtained without comprehensive screening, and personnel and time can be more appropriately allocated according to the urgency of the project. Based on this, it is believed that screening efficiency can be effectively increased by more than 30 times.

[0041] Thus, a method for screening target cells is provided, wherein the target cells are 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-mentioned biological culture chip are also suitable for this 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 or gel medium for covering single cells, 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 single cells. For example, a material adjusted to have 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, 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 kit can be used to effectively carry out the above method, and the features and advantages of the method are also suitable for the kit and will not be described in detail herein.

[0047] Furthermore, 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 hybridoma cell array in a medium containing a labeled target antigen (protein or polypeptide), causing an antigen-antibody reaction, and selecting labeled-positive hybridoma cells that secrete the target antibody; isolating the positive hybridoma cells; and The positive hybridoma cells are cultured 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] Furthermore, 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 antigen; The monoclonal antibody or a 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. 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 derived from a single cell or a derivative thereof, for example, an antibody fragment containing an antigen-binding fragment (Fab), (Fab)2, etc., that binds to a specific antigen determinant; and (3) determining whether the candidate cell is a target cell based on the signal of the signaling molecule, wherein 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 Another aspect of the present disclosure provides a method for screening cells, including: (1) placing candidate cells in a culture chamber for a predetermined period of time under conditions suitable for the candidate cells to express an antibody; (2) forming a target antibody-antigen signaling antibody complex in the culture chamber, wherein the signaling antibody binds to a signaling molecule; and (3) determining whether the candidate cells are target cells based on the signal of the signaling molecule. This method can efficiently obtain antibody pairs through screening. In this method, candidate cells are placed in a culture chamber for single-cell culture, and antibodies secreted from the single cells form target antibody-antigen signaling antibody complexes, concentrating the antigen signaling antibody to a certain range, thereby effectively achieving screening for antibody pairs. Compared to the conventional limiting dilution method, this method is rapid, cost-effective, and efficient, and can achieve high-throughput screening.

[0053] According to one embodiment of the present disclosure, the predetermined time is 1 to 10 hours, preferably 2 to 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 a free antigen and a 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 antibodies, which have antigenic determinants distinct from the signaling antibody. Obtaining antibodies 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 obtainable by the above method, wherein the monoclonal antibody and the signaling antibody correspond to different antigenic determinants.

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

[0062] Those skilled in the art will 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-mentioned biological culture chip are also suitable for this 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 or gel medium for covering single cells, or 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 single cells. For example, a material with a lower concentration than the gel material may be 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 microwells 200. The matrix 100 is formed of a matrix material. The equilibrium swelling ratio of the matrix material is approximately 1.25 to 1.75. The microwells 200 are formed on the surface of the matrix 100, and the microwells 200 have openings on the surface of the matrix 100. The microwells 200 define a biological culture space for culturing a biocompatible material.

[0066] In conventional chips, the interface compatibility between the chip matrix and cell culture medium is poor. The incompatibility between the hard matrix interface and the cell culture medium leads to uneven 3D culture microenvironments for aligned cells. The interface between the matrix and the culture medium tends to slide due to the liquid layer between the two interfaces, which damages cell alignment and affects the realization of cellular 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, the matrix material has a certain equilibrium swelling ratio, so that 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 liquid or gel-like extracellular matrix material, providing a uniform microenvironment for the culture of the biocompatible material, thereby further improving the biological activity of the cells during cell culture and promoting the realization of cell biological functions.

[0068] According to this embodiment of the present disclosure, the matrix used in the present disclosure can deform as cells grow during culture on a biocompatible material. In other words, compared to conventional hard matrices such as glass, silicon wafers, and plastics, the matrix material used in the present disclosure is a soft matrix, which reduces the forces between the matrix and cells and between the matrix and the extracellular matrix material, thereby further improving the interfacial compatibility between cells and the matrix and between the matrix and the extracellular matrix material. Furthermore, according to this embodiment of the present disclosure, depressions are formed at the contact area between the matrix and cells during cell growth during the culture process. Those skilled in the art will understand that depressions are formed as cells grow toward the surface of the matrix, causing deformation of the matrix. Those skilled in the art can determine whether depressions are present by observing the inner surface of the matrix with a microscope. According to one embodiment of the present disclosure, the depth of the depressions is 5% or more, for example, 10% or 20% of the diameter of the cells. The term "depth of depressions" as used herein may be determined by comparing interface micrographs of a substrate without depressions with those of a substrate with depressions. 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 can easily adapt to and integrate with various extracellular matrix environments, providing a uniform and integrated 3D culture space for cells, creating a microenvironment that promotes survival, proliferation, migration, protein expression and secretion, and stem cell differentiation, as well as the detection of cell biological functions of 2D-arranged cells. Furthermore, considering that the matrix is ​​soft, it is easy to isolate single cells from the culture space without weakening the adhesive force between 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 to 45°C. According to this embodiment of the present disclosure, the matrix material can become liquid at 37°C. Note that the above temperature values ​​are measured under 1 atmosphere. Therefore, the matrix material can easily become liquid at temperatures moderately higher than room temperature and can easily solidify into a predetermined shape by further cooling. Because the matrix material has the above properties, cytokines can be easily added without inactivating them when the matrix material is in a liquid state. During the addition of liquid medium for cell culture, the biological culture chip absorbs the medium and further releases these cytokines into the culture space. Based on this, according to this embodiment of the present disclosure, the matrix further contains 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 of these in different ratios.

[0071] It should be noted that, according to this embodiment of the present disclosure, 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, methyl The soft matrix may be selected from the group consisting of polycellulose, agarose gel, and polyacrylamide gel, and mixtures of at least two of these in different proportions. 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 they can provide a space for the culture or growth and proliferation of single cells. According to this embodiment of the present disclosure, the microwell may have a structure such as 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. This allows single cells to be easily captured for culture without leakage 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. As used herein, the term "diameter" is defined as the diameter of a circle with the largest area that can fit into the opening or bottom. As used herein, the term "depth" of a microwell refers to the shortest distance between the cross-section of the opening 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 diameters 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] Furthermore, according to this embodiment of the present disclosure, the biological culture chip may include multiple microwells, where the microwells form a predetermined pattern and the distance between adjacent microwells is 10 to 100 μm. Therefore, multiple single cells can be cultured in batches. According to this embodiment of the present disclosure, when the distance between the microwells is 10 to 100 μm, the cells cultured in the microwells do not interfere with each other. This improves the uniformity of batch culture, enhances the accuracy and efficiency of detecting cell biological functions, and facilitates high-throughput collection and analysis of cell signaling data. 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 may be 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. Therefore, multiple microwells can be positioned via 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 allow 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 located and recorded, which further facilitates the subsequent isolation of positive cells or other components in the microwells.

[0075] Furthermore, 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 hybridoma cells and culture factors for the hybridoma cells are disposed on the inner surface of the microwell; the biocompatible material is MCF10A cells and insulin is disposed on the inner surface of the microwell; or the biocompatible material is B cells and at least one of CD40L, IL2, and IL10, 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 a cell array chip) with a soft matrix such as a solidified hydrogel is provided, which can solve a series of problems in 2D and 3D cell arraying in vitro, including cell capture, formation of a flux-controlled cell array, cytocompatibility of the chip material, compatibility of the chip material with cell culture media, and realization and accurate detection of the biological functions of the arrayed cells. Arrays with microwells of various diameters and depths can be provided on the chip with the 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 can be adjusted to achieve optimal mechanical strength for various cell types and various detection purposes of biological functions. Furthermore, the diameter and depth of the microwells can be adjusted to facilitate single-cell arraying of various cells or controlled amounts of multicellular aggregation in the microwells. The distance between the microwells can be adjusted to determine the cell array density on the chip and the cell array flux per unit area on the chip. The gel soft matrix can provide properties similar to those of the extracellular matrix. The gel-soft matrix has excellent cytocompatibility, providing cells with water, nutrients, and flexible space, thereby avoiding the inadequate mechanical strength that cannot be adjusted to accommodate the various cell types cultured in chips with hard matrices and avoiding the residual chemical molecules that are typically required for chips with hard matrices. The gel-soft matrix also has excellent compatibility with cell culture media or 3D culture hydrogel biocompatible materials, which ensures a uniform microenvironment around the arrayed cells and ensures the completeness and accuracy of cell function detection.

[0077] Fabrication of biological culture chips According to another aspect of the present disclosure, a template for fabricating the above-mentioned biological culture chip is provided. Referring to FIG. 4, according to one embodiment of the present disclosure, the template includes a substrate 300 and microcolumns 400 formed on the surface of the substrate 300, the microcolumns 400 being adapted to fit into the microwells 200 in a manner that fits into a mold. According to this embodiment of the present disclosure, the matrix material is solidified on the template, thereby enabling efficient fabrication of the above-mentioned biological culture chip.

[0078] According to this embodiment of the present disclosure, the microcolumns have a hydrophobic surface. Therefore, the formed biological culture chip can be effectively detached from the template. Furthermore, according to this embodiment of the present disclosure, the surface of the microcolumns is suitable for binding biological factors due to the 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 surfaces of the microwells. As described above, various biological factors can be provided according to various culture targets and purposes.

[0079] According to another aspect of the present disclosure, there is provided a method for producing the above-mentioned biological culture chip, comprising: (1) applying a liquid biocompatible material as a matrix material to the above-mentioned template and solidifying the matrix material; and (2) separating the solidified biocompatible material from the template to obtain a biological culture chip. This method allows the above-mentioned biological culture chip to be efficiently produced. According to this embodiment of the present disclosure, biological factors or reagents are pre-adsorbed onto 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) at a concentration of 1 μg / mL. The template is immersed in the denaturing solution and reacted at room temperature for 4 hours. After removal, the template is Rinse three times successively with fresh PBS and deionized water, 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 column-arranged template to cover the template. After the matrix material, such as the gel matrix, is cooled and solidified, the template is removed to form a soft matrix-based chip with a microwell array and a consistent thickness and hardness. Specifically, a 0.6% to 1.2% agarose gel liquid is poured into the template and left at room temperature for 5 minutes; after the liquid solidifies, 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] Culturing method using biological culture chip According to another aspect of the present disclosure, there is provided a method for culturing cells using the above-mentioned biological culture chip, comprising disposing cells in the microwells and exposing the biological culture chip to an environment under predetermined conditions. By using this method, a microenvironment such as a uniform extracellular matrix can be provided to the cultured cells, thereby effectively maintaining cell viability and improving cell culture efficiency. 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 are well compatible, which helps to provide a uniform and suitable microenvironment for high-throughput 3D culture of arrayed cells, helps to realize the biological functions of the arrayed cells, and helps to sufficiently capture and accurately analyze the biochemical reaction signals of the cells (e.g., secreted protein signals, etc.).

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

[0084] According to this embodiment of the present disclosure, cell arrays on chips with soft matrices are formed by relying on cell gravity and biocompatible materials, such as extracellular matrix, 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 a soft matrix is ​​useful for collecting positive cells (single cells or multicellular spheroids) under a microscope using a glass needle. However, it is difficult to collect cells from the chip with a hard matrix under a microscope using a glass needle because the hard matrix interface is prone to damage the glass needle or the cells. [Example]

[0086] Example 1: Chip Preparation 1.1 Fabrication of chips with soft matrices Templates having the column arrangement shown in Figure 4 were selected and classified into the following two types depending on the cell type. A. A template suitable for general cells (generally 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 immersing the template in the denaturing solution for 4 hours at room temperature, the template was removed and rinsed three times with fresh PBS and deionized water, blown dry with nitrogen gas, and stored in a dry place for further use. Thus, the template used to fabricate the chip was modified so that cytokines and other substances required in the microenvironment could bind to the template via hydrophobic effects.

[0089] A molten hydrogel matrix was placed on the modified template (with a columnar arrangement) to cover the template. After the hydrogel matrix cooled and solidified, the template was removed to form a soft-matrix-based chip with a microwell array and a thickness of approximately 300-500 μm. Specifically, an agarose gel liquid with a concentration of 0.6% to 1.2% (swelling ratio of approximately 1.25-1.75) was poured into the template and left at room temperature for 5 minutes; after the liquid 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 table below shows the composition of the chip with soft matrix.

[0090] [Table 1]

[0091] 1.2 Fabrication of chips with hard matrices According to the method described in CN108102913A "3D cell culture chip based on soft lithography, its fabrication method and its application", chip 1 with hard matrix and chip 2 with hard matrix were fabricated 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 a soft matrix, 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 sequencing efficiency was determined. The sequencing efficiency was determined as follows. The amount of single cells per unit area (1 mm × 1 mm) was observed and counted, and the result was divided by the total volume of the chamber to obtain the single-cell array acquisition efficiency. The chip specifications were adjusted to limit the chip space so that each chamber could accommodate only one cell, and the very small number of doublets caused by uncontrollable factors were easily identified using 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 indicates the diameter of the chamber, and the second number indicates the distance between adjacent chambers. In the table above, except for mouse B cells, which were cultured using a 10μm / 30μm chip, all other cell types were cultured using a 20μm / 30μm chip.

[0095] The above data show that the arraying efficiency of the chip with hard matrix is ​​about 32-50%, indicating that the chip with hard matrix is ​​not conducive to cell array formation. The arraying efficiency of the chip with soft matrix is ​​very high, about 91-98%, indicating that the chip with soft matrix is ​​very effective in cell array formation.

[0096] The inventors believe that the above differences are primarily due to the hard matrix of the chip, such as glass or silicon wafer. Because the hard matrix material lacks porosity and water permeability, it is unable to completely vent the gas in the small chambers. As a result, cells subsequently seeded cannot properly position themselves by gravitational sedimentation. However, the soft matrix of the chip, such as hydrogel or other biocompatible materials, has a certain pore size and water permeability. Therefore, once cells are seeded, the medium permeates from the periphery of the chip, allowing the gas to completely vent from the small chambers, promoting cell sedimentation and forming an array. Based on the above, chips with hard and soft matrices, even though they have the same number of chambers, have a large difference in the proportion of chambers used to efficiently form cell arrays. Therefore, the efficiency of chips with hard matrices in cell screening is significantly higher than that of chips with soft matrices. It becomes slightly lower.

[0097] 2.2 Comparison of cell viability on chip Cells were cultured on chips with hard and soft matrices according to the method described in 2.1, and cell viability was detected as follows. Cell viability on the chip was determined using a live / dead assay (Thermo, L34951). Dead cells exhibited red fluorescence, while live cells exhibited green fluorescence. The results are as follows:

[0098] [Table 3]

[0099] The above data demonstrate that chips with hard matrices are not conducive to cell culture and survival, readily inducing cell apoptosis and necrosis, and are therefore not useful for cell proliferation. The main reason for this is that hard matrix materials lack physical elasticity and are unable to provide a uniform, extracellular matrix-like microenvironment (with properties similar to the living space in vivo) required for 3D cell culture. This alters the pathways of cell survival, proliferation, senescence, and apoptosis, leading to cell senescence, apoptosis, or necrosis. However, soft matrices have better plasticity, sufficient water and support space, and can adjust to changing cell survival space requirements. They can also be compatible with and integrate with ECM media for 3D cell culture, providing cells with a uniform and appropriate microenvironment, thereby supporting cell survival and biological functions (e.g., factor and antibody secretion). Furthermore, during the fabrication of chips with soft matrices, various factors or materials necessary for the culture of various cells can be appropriately added to create a microenvironment for cell culture and survival, significantly improving cell survival and proliferation. However, this is difficult to achieve with hard matrix materials. 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 distribute the factors or materials around the cells, or residual chemicals can be toxic to the cells.

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

[0101] [Table 4]

[0102] The inventors have investigated the reason for the low success rate of single cell collection in chips with hard matrices. This is mainly because all cells are cultured in suspension without any constraints, so when using a needle to collect target cells, non-target cells nearby the target cells are likely to be collected; furthermore, when the needle approaches the target cells, even a slight force (force generated by the movement of the needle through the liquid) can cause the target cells to separate from the chip or be pushed away, leading to collection failure. However, the soft matrix material of the chip is a hydrogel, which easily creates a moderate adhesive effect with the cells and does not completely culture the cells in suspension, thereby solving the problems caused by hard matrices and increasing the success rate of collection.

[0103] Furthermore, to improve efficiency, consumable needles can be replaced at any time during cell inoculation if they become contaminated, deformed, or have other problems. However, needle breakage or other problems due to improper operation significantly affect collection efficiency. This is because it takes time to replace the needle and accurately adjust its position. Hard matrices lack elasticity. During operation under a microscope, the needle must be placed very close to the tip or even touch the surface of the tip to release as much weak suction force as possible to the target cells for successful collection. However, because manual or semi-automated operations are difficult to complete accurately every time, it is common for the needle to deform or break when touching the tip. Due to its good physical elasticity, soft matrices are highly resistant to excessive needle contact, allowing the operator to 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 housed in an animal care room for one week to acclimate to the environment. Six-week-old mice were then prepared for immunization. Novel coronavirus S1 protein purchased from Novoprotein Technology Co., Ltd. was dissolved in PBS and adjusted to a concentration of 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 mice via the orbit and the antibody titers in the serum were measured. The antigen dose for the third or additional immunization was adjusted depending on the antibody titer until the immune response of the mice to the antigen reached an optimal level.

[0107] 3.2 Cell fusion Based on the serum titer measured 3 days after the booster immunization, mice with the highest titers were selected for subsequent fusion. Prior to fusion, all mice were bled to obtain serum.

[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 before splenectomy.

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

[0110] The removed spleen was placed on a 40 μm sieve and then placed in a 50 mL centrifuge tube filled with blank 1640 medium. Care was taken not to overfill the spleen during trituration to prevent overflow of the medium. The spleen needed to be fully exposed to the medium during trituration. To facilitate isolation of splenocytes by trituration, a small hole was punctured in the spleen using a syringe needle or several small incisions were made in the spleen using scissors. The spleen was triturated to completely pass the contents through the sieve, and any remaining connective tissue was discarded. The triturated spleen was centrifuged twice at 1300 rpm at room temperature for 8 minutes to collect the spleen cells. After removing the supernatant, 10 mL of PBS was added to resuspend the splenocytes. 30 mL of red blood cell lysis buffer was added and lysed at 4°C for 15 minutes, mixing evenly twice during lysis. The resulting mixture was centrifuged at 1300 rpm at room temperature for 8 minutes to collect the splenocytes. The splenocytes were resuspended in PBS and centrifuged twice until the lysis buffer was completely removed.

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

[0112] The pipette tip was placed as close as possible to the cells, and 1 mL of PEG was dispensed dropwise along the tube wall within 1 minute using a pipette. During the PEG dispensing, the centrifuge tube was rotated and slightly shaken 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 minute. During the addition of the medium, the centrifuge tube was rotated and slightly shaken to ensure that the cells were completely and evenly exposed to the medium. 2 mL of pre-warmed blank 1640 medium was added dropwise within 1 minute. During the addition of the medium, the centrifuge tube was rotated and slightly shaken to ensure that the cells were completely and evenly exposed to the medium. 5 mL of pre-warmed blank 1640 medium was added dropwise within 1 minute. During the addition of the medium, the centrifuge tube was rotated and slightly shaken to ensure that the cells were completely and evenly exposed to the medium. Approximately 21 mL of preheated blank 1640 medium was added dropwise. While adding the medium dropwise, the centrifuge tube was rotated and slightly shaken to ensure that the cells were completely and evenly exposed to the medium. After fusion was complete, 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, with the medium changed once during that time.

[0114] 3.3 Chip fabrication A template was prepared and modified with an HGF layer (using a denaturing solution containing HGF in PBS, beneficial for the survival and proliferation of hybridoma cells). To fabricate the chip, approximately 500 μL of gel was dispensed (using aseptic technique) onto the center of the template (also called a stamp) and allowed to stand at room temperature for 10 minutes (to effectively prevent excessive drying). After the gel solidified, the template and chip were gently separated from each other. Care was taken to avoid displacement, otherwise the structural integrity would be compromised. The fabricated chip was stored in a 10 cm diameter culture dish for further use.

[0115] 4.4 Cellular arrangement formation 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 may 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 approximately 500 μL per 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 approximately 300 to 500 μL of medium was taken from above and dropped onto the chip tilted at approximately 30 to 45 degrees to wash away excess cells (so as not to let the medium flow out of the chip). The excess medium was then removed using a vacuum pump. The formation of cell arrays was observed under a microscope to determine whether to proceed with subsequent procedures.

[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. Within 10 minutes of waiting for the cell array to form, the cryocontainer was removed and the labeled antigen and homemade 3D culture hydrogel were placed in the cryocontainer for use. 50 μL of complete medium was added to 1.5 mL sterile centrifuge tubes and placed in the cryocontainer for use. After the cell array was formed, 50 μL of hydrogel was added to each centrifuge tube and mixed uniformly. 70-80 μL of well-mixed hydrogel was removed and added dropwise from one corner of the chip. During this time, the chip was gently shaken to ensure the hydrogel completely covered the structure. The chip was then placed in an incubator and left to stand for 5 minutes, while the chip was held horizontally. The chip was then transferred to a 3.5 cm diameter culture dish using a syringe needle and tweezers, placed in the incubator, and then a screening solution (2 mL volume with a fluorescently labeled antigen: complete medium ratio of 1:(200-1000)) was added. The chip was left to stand for at least 3 hours.

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

[0118] 4.7 Monoclonal Culture and Identification Cells with positive signals were selected and placed in 96-well plates. Microscopic examination was then performed within 1 to 2 hours to determine whether single cells were present in each well. After 7 to 14 days of culture, the supernatant from stably growing cells in each well was tested by ELISA to determine whether the cells secreted antigen-specific antibodies, i.e., whether the cells were positive clones. Positive clones were expanded by continued culture and re-identified for antibody secretion 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 of the clones obtained were derived from different mice or different hybridoma cells of the same mouse, ensuring sufficient clonal diversity. Compared to 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 of the present disclosure has absolute advantages in terms of screening efficiency and screening costs, reducing the overall cost by about 90% while increasing the screening efficiency by at least 10 times. This is merely a result obtained without comprehensive screening, and personnel 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.

[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 × 10 5 The cells were inoculated intraperitoneally. NOTE: Injecting fewer cells 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 of 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 there is obvious abdominal fluctuation of the dullness, the ascites may be drained twice; if the mouse is found to be lethargic, the mouse should be sacrificed and the ascites drained.

[0124] Draining ascites from the body: Wipe the injection site (near the right side of the abdominal midline) with a cotton ball soaked in alcohol, insert the syringe containing the remaining air, and then slowly withdraw the plunger, which will cause the ascites to slowly flow out due to the pressure generated in the abdomen. During the insertion of the needle, you may lift the syringe slightly until you no longer feel any blockage.

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

[0126] (iii) The ascites fluid was collected in a 15 mL centrifuge tube and centrifuged at 800 rpm for 30 minutes (or 2000 rpm for 5 minutes) to remove cells and fat. Freshly collected ascites fluid contains a large amount of impurities, especially cellular components, such as fat, macrophages, hybridoma cells, peritoneal epithelial cells, and blood cellular components. If these components are not removed, cellular components present in the ascites fluid for a long time may slowly rupture, releasing proteins into the fluid and increasing the impurities in the fluid, making purification difficult. Therefore, whether collected ascites fluid is intended for long-term storage or immediate purification, it must first be centrifuged to remove cellular components and fat (fat has low density and generally floats on the surface of the ascites fluid, so it can be aspirated using a pipette). Antibody purification and preparation of fluorescently labeled antibodies were performed according to the specific operating procedures in accordance with the instructions for 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 an antigen-fluorescently labeled primary antibody complex at a ratio of 2:1. In this procedure, the following other method, which has the same specific procedure as that 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 to allow the antigen to completely bind to the antibody secreted by the hybridoma cells, and then the 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 from the same mouse, ensuring diversity of the antibody pairs.

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

[0132] [Table 8]

[0133] Two VEGFA antibodies from different sources were used in the study. 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 positive rates of 89.6% and 85.7%, respectively. Conventional screening and isolation of stably transfected CHO cell lines is still performed by limiting dilution, as described above. Our technique offers significant advantages in terms of time, materials, 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 secreted proteins include HEK293, CHO, HeLa, MCF10A, and HFF. Factors include various growth factors and cytokines secreted extracellularly, such as VEGF, SDF-1α, PEDF, and FGF. Labeled proteins that specifically bind to proteins secreted from cells include antibodies specific to secreted proteins and receptor-binding domains. Details are not provided herein.

[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 they self-renew to provide a large number of cells to brain tissue. Therefore, better methods for culturing neural stem cells outside the body are a key focus of 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. The abdomen was opened, and a series of fetal mice (with the umbilical cord severed) were removed and placed in a culture dish containing pre-chilled PBS. The placenta was removed 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 to 11 fetal mice, with at least 6 and at most 13). The fetal heads were twisted and placed in a new culture dish containing PBS (two fetal mice per dish). The cerebral cortices were removed under a stereomicroscope, and brain tissue connected to the cerebral cortex, such as the olfactory bulbs and meninges, was removed. The cerebral cortices were placed in a 15 mL centrifuge tube, purged with a Pasteur pipette until no tissue fragments were visible, and centrifuged at 800 rpm for 2 minutes. The supernatant was removed for use. Fresh PBS buffer was added to the precipitate and purged into a suspension. The suspension was centrifuged at 800 rpm for 2 minutes. The supernatant was removed and mixed with the supernatant from the first centrifugation, followed by centrifugation 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 two days after the initiation of culture. Generally, depending on the size of the cell spheroids, cells begin passage on day 6 or 7. The cells were harvested and centrifuged at 800 rpm for 2 minutes. The supernatant was removed. PBS buffer was added to purge the precipitate, and the remaining medium was centrifuged at 800 rpm for 2 minutes to remove the remaining medium. The supernatant was removed. 1 to 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 to 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 PBS containing PEG-100 (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 may be added to avoid further loss in the subsequent filtration) to purge the precipitate into the suspension. The suspension was filtered through a 40 μm filter membrane to remove incompletely dissociated cell spheroids, yielding an NPC suspension. The cells in 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 Examples 4 and 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 are used to compare the method of the present disclosure with conventional culture methods, which shows that the chip with a soft matrix is ​​helpful in increasing cell survival rate and maintaining good stemness of cells.

[0139] Example 8: Sphere formation of tumor cells (MCF7) Studies of tumor growth and drug sensitivity have relied heavily on in vitro monolayer tumor models (2D cell cultures). These models lack many characteristics of disease, such as hypoxia, altered cell-cell contact, and metabolic changes. Through years of research, it has become clear that 3D tumor models offer a phenotype closer to that of actual cancer cells, providing 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 seeding procedure on the chip was performed with reference to Examples 4 and 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 time specific cell proliferation was constantly monitored. Cell spheroid arrays derived from single MCF7 cells were formed on the chip.

[0142] These 3D cultures (or "cell cluster" cultures) of tumor cells can faithfully recapitulate many important characteristics of the original tumor. Furthermore, 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 describing the present disclosure, it should be understood that orientations or positions indicated by terms such as "center," "longitudinal," "lateral," "length," "width," "thickness," "top," "bottom," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" are based on the orientations or positions shown in the accompanying drawings and are used solely for ease and brevity of illustration and description of the present disclosure, without indicating or implying that the referenced devices or components must have a particular orientation or be constructed and operated in a particular orientation. Thus, such terms should not be construed as limiting the present disclosure. Furthermore, features defined as "first" and "second" may explicitly or implicitly include one or more such features. In describing the present 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 direct contact between the first and second features, or may include contact between the first and second features that is not in direct contact but is achieved through the use of other features between them.

[0144] In the description of this disclosure, a first feature being "above," "on," 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] It should be noted that in the description of the present disclosure, unless otherwise expressly stated or defined, 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 via an intermediate, or an internal communication between two components. Those skilled in the art can understand the specific meanings of the aforementioned terms in the present disclosure according to specific circumstances.

[0146] In describing 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 disclosure. As used herein, exemplary descriptions of the foregoing terms 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.

[0147] While embodiments of the present disclosure have been shown and described, it is understood that those skilled in the art will readily appreciate that certain modifications may 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

[Claim 1] A method for screening a target cell, comprising: the target cell is adapted to secrete a target molecule; The method comprises: culturing the 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; Including, the signaling molecule is a fluorescently labeled antigen; the target molecule is a monoclonal antibody capable of binding to the signaling molecule; the signal screening layer is disposed in a medium contained in a culture chamber defined by a matrix formed from a matrix material; the signal screening layer is formed from a hydrogel; and the signal screening layer has a mechanical strength lower than that of the matrix material; the matrix material has an equilibrium swelling ratio of 1.25 to 1.75; The method, wherein the matrix material is selected from the group consisting of collagen hydrogel, methylcellulose, agarose gel, and polyacrylamide gel.

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