CN114686372A - Hybridoma cell screening chip and screening and collecting method thereof - Google Patents

Hybridoma cell screening chip and screening and collecting method thereof Download PDF

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CN114686372A
CN114686372A CN202210356239.7A CN202210356239A CN114686372A CN 114686372 A CN114686372 A CN 114686372A CN 202210356239 A CN202210356239 A CN 202210356239A CN 114686372 A CN114686372 A CN 114686372A
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hybridoma
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金庆辉
袁浩钧
蒋新华
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Shandong Fenghua Biotechnology Co ltd
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Abstract

The invention provides a hybridoma cell screening chip and a screening and collecting method thereof. The chip comprises a glass substrate and a PDMS sheet which are attached together, wherein the attaching surface of the PDMS sheet is provided with a first screening area, an incubation area, a second screening area and a collecting area which are sequentially connected; wherein the first screening zone is used for separating unfused single cells from fused cells; the incubation zone is pre-confluent with capture cells for separating tumor-tumor fused cells from B-B fused cells and hybridoma cells; a second screening zone for separating captured cells with target cells bound thereto from captured cells without target cells bound thereto; the collecting region is used for collecting hybridoma cells; also included are first, second, and third valves associated with the incubation region, and a fourth valve associated with the collection region. The invention realizes the screening of the single cell level of the hybridoma cells through reasonable chip structure design, and leads the screening efficiency and the screening effect of the hybridoma cells to be improved doubly.

Description

Hybridoma cell screening chip and screening and collecting method thereof
Technical Field
The invention relates to the technical field of microfluidic biochips, in particular to a hybridoma cell screening chip and a screening and collecting method thereof.
Background
In recent years, with the continuous improvement and development of microfluidic and micromachining technologies, the advantages of Micro total analysis systems (μ -TAS) in the fields of biological research and biochemical detection at the cellular level are becoming more and more significant. The micro-fluidic chip has the advantages of equivalent size of functional units and cells, high precision, rapid and convenient detection and the like, and has obvious advantages in the separation and detection of rare cells. All the existing cell analysis steps and processes (such as cell manipulation, cell capture/screening, cell recovery, online real-time dynamic monitoring analysis and the like) are integrated on one microchip, so that the integration of analysis operation is realized, and the damage and pollution to target cells in the sorting process can be reduced. Therefore, the micro-fluidic chip technology is used for carrying out specific capture and recovery of hybridoma cells, and the method has wide application prospect in the field of immunological detection and analysis.
The patent application with publication number CN 112920951 a discloses a cell screening chip and its manufacturing and cell screening collection method, the patent technology can screen cells with certain characteristics by using microfluidic chip, which is suitable for relatively simple cell population samples. However, the sample of hybridoma cells is relatively complex, and the chip cannot realize the screening of the hybridoma cells due to simple structure and single function.
Hybridoma cell screening is the most critical link for obtaining monoclonal antibodies. The whole process of the traditional hybridoma cell screening technology is basically carried out on a 384-well plate of the conventional cell culture, and because the influencing factors of the fusion process of the B lymphocyte and the bone marrow tumor cell are complex, most of the generated fusion cells have no antibody preparation function or low titer, and the hybridoma cells with relatively excellent performance can be obtained only by carrying out repeated circulation such as culture-identification on the fused cells, so the traditional technology consumes long time and has low screening efficiency. If the fused cells can be respectively subjected to single cell horizontal culture and identification, hybridoma cells can be selected in a targeted manner, and the screening efficiency can be obviously improved. Therefore, a simpler method for obtaining high purity and high titer hybridoma cells is desired.
Disclosure of Invention
The invention aims to provide a hybridoma cell screening chip and a screening and collecting method thereof, so as to solve the problems of long time consumption and low screening efficiency of the existing hybridoma cell screening technology and the problems of elimination or selection omission of high-yield hybridoma cells caused by the survival competition relationship among different cells.
In order to solve the technical problem, the invention adopts the following technical scheme:
according to a first aspect of the invention, a hybridoma cell screening chip is provided, which comprises a glass substrate and a PDMS sheet which are attached together, wherein the attaching surface of the PDMS sheet is provided with a first screening area, an incubation area, a second screening area and a collecting area which are sequentially connected to complete hybridoma cell screening; wherein the first screening zone comprises: the first screening area separates unfused single cells from fused cells after fusion based on cell size difference; the incubation area is pre-filled with capture carriers, wherein the capture carriers are capture cells or magnetic beads, and the tumor-tumor fusion cells are further separated from the B-B fusion cells and the hybridoma cells through the capture carriers; the second screening zone includes: a second main channel, a plurality of second screening columns respectively arranged at both sides of the second main channel, and a second waste liquid channel, wherein the second screening area separates the capture carrier to which the target cells are bound from the capture carrier to which the target cells are not bound based on the cell size difference; the collecting region is used for collecting hybridoma cells; the hybridoma cell screening chip further comprises: a first valve, a second valve, a third valve in communication with the incubation region, and a fourth valve in communication with the collection region.
The first valve is used for controlling the sample from the first screening area to enter the incubation area, the second valve and the third valve are used for controlling the capture carrier, the buffer and the trypsin to enter the incubation area, and the fourth valve is used for controlling the sample from the second screening area to enter the collecting area.
The first screening district links to each other with first introduction port for treat the entering of screening sample, treat that the screening sample includes: hybridoma cell, tumor-tumor fused cell, B-B fused cell, tumor cell single cell, and B cell single cell.
In the first screening area, the distance between any two adjacent first screening columns is larger than the diameter of the unfused single cell and smaller than the diameter of the fused cell after fusion.
The incubation area is connected with a second sample inlet, and the second sample inlet is controlled by a second valve and is used for the entrance of a capture carrier, a PBS buffer solution or trypsin.
In the second screening zone, the distance between any two adjacent second screening columns is larger than the diameter of the capture carrier without the target cells bound thereto and smaller than the diameter of the capture carrier with the target cells bound thereto.
According to a preferred embodiment of the present invention, the width of the first main channel is 100 to 500 micrometers, the size of the first screening pillars is 10 to 50 micrometers, and the distance between the first screening pillars is typically 8 to 30 micrometers depending on the size of the unfused single cells. Because the diameter of the unfused single cells is smaller, and the diameter of the fused cells is larger, the spacing of the screening columns of the first screening area is generally larger than the diameter of the unfused single cells and smaller than the diameter of the fused cells, so that the target cells can be ensured to enter the incubation area and cannot flow out through the waste liquid channel of the first screening area.
According to a preferred embodiment of the present invention, the width of the second main channel is 100 to 500 micrometers, the size of the second screening pillars is 10 to 50 micrometers, and the interval between the second screening pillars is determined according to the size of the capture carrier to which the target cells are not bound, and is generally 8 to 30 micrometers. Because the diameter and the mass of the capture carrier which is not combined with the target cells are smaller, and the diameter and the mass of the capture carrier which is combined with the target cells are larger, the spacing of the screening columns of the second screening area is generally larger than the diameter of the capture carrier and smaller than the diameter of the capture carrier which is captured with the target cells, so that the capture carrier which is combined with the target cells can be ensured to enter the collecting area and not flow out through the waste liquid channel of the second screening area.
According to a second aspect of the present invention, there is provided a method for screening and collecting hybridoma cells, comprising the steps of: s1: providing a hybridoma cell screening chip as described above; s2: closing the first valve and the fourth valve, opening the second valve and the third valve, and introducing a capture carrier into the incubation region, wherein the capture carrier is a capture cell or a magnetic bead; s3: opening the first valve, closing the second valve, and introducing a sample to be screened into the first screening area to separate unfused single cells from fused cells, wherein hybridoma cells, tumor-tumor fused cells and B-B fused cells enter the incubation area through a flow channel, and tumor cell single cells and B cell single cells flow out of the first waste liquid channel; s4: after the fused cells enter the incubation area, closing the first valve and the fourth valve, opening the second valve and the third valve, combining the hybridoma cells and the B-B fused cells with a capture carrier in the incubation area, not combining the tumor-tumor fused cells with the capture carrier, and introducing a PBS buffer solution into the incubation area through the second sample inlet so as to enable the tumor-tumor fused cells to flow out; s5: then, introducing trypsin or removing the magnetic force of a magnet into the incubation area through a second sample inlet, so that the sample enters a second screening area, the capture carrier of the unbound cells flows out of a second waste liquid channel according to the different sizes of the capture carrier bound with the cells and the capture carrier of the unbound cells, and the capture carrier bound with the hybridoma cells and the B-B fusion cells further enters a collection area; s6: the B-B fusion cells are naturally apoptotic in the collecting region, and then the collection of the hybridoma cells is realized.
Preferably, step S6 further includes: and carrying out subsequent treatment on the hybridoma cells in the collecting region according to requirements.
As described in the background section of the present invention, the existing cell screening chip cannot screen a sample containing a large number of cells with similar attributes, such as a hybridoma cell sample, and the key point of the present invention is to design a microfluidic chip capable of directly screening hybridoma cells from a hybridoma cell sample by using the sample characteristics of the hybridoma cells and the biochemical characteristics of the hybridoma cells, and to realize the in-slice screening of the hybridoma cells by the functions of different partitions and the control of different partitions and samples.
The invention utilizes the micro-fluidic chip to screen the hybridoma cells, realizes the screening of the hybridoma cells at the single cell level through reasonable chip structure design because the internal structure of the micro-fluidic chip is close to the size of the cells, overcomes the problem that the high-yield hybridoma cells are eliminated or selected in a missing way due to the existence competition relationship among different cells in the prior art, and ensures that the screening efficiency and the screening effect of the hybridoma cells are improved.
Compared with the prior art, the hybridoma screening chip and the screening and collecting method thereof have the advantages that the method for screening the hybridoma through multiple cell culture and detection has the following advantages:
1) in the prior art, time-consuming and labor-consuming operations such as plate laying, culture and the like are required, about 2-3 months are required, and the hybridoma cell screening chip can realize successful screening of the hybridoma cells in only 4 hours, so that the screening efficiency is greatly improved;
2) the prior art cultures the whole cells, but the screening of the invention is to the screening of the single cell level of the hybridoma cells, is more accurate, and the flux can reach 107Magnitude;
3) the prior art can not determine the recovery efficiency of the hybridoma cells by carrying out overall culture on a sample, but the recovery rate of the hybridoma cell screening chip reaches more than 90 percent;
4) in the prior art, because repeated screening is needed, the sample has higher pollution risk and low cleanliness, and the hybridoma cell screening chip provided by the invention only needs one-time in-chip screening, so that the pollution risk is greatly reduced and the cleanliness is high.
In conclusion, the invention provides the hybridoma cell screening chip and the screening and collecting method thereof, which have the advantages of short time consumption, high efficiency, good screening effect and low pollution risk.
Drawings
FIG. 1 is a plan view of a hybridoma cell screening chip according to a preferred embodiment of the present invention.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
It should be understood that the hybridoma cell sample requires a large amount of B cells and mouse tumor cells in the preparation process, and since the fusion probability is not high, most of the obtained cells are unfused B cells and mouse tumor cells, and a small amount of B-B fusion cells, tumor-tumor fusion cells and target cell hybridoma cells are obtained. The invention particularly designs a micro-fluidic chip capable of directly screening the hybridoma cells from the hybridoma cell sample by utilizing the sample characteristics of the hybridoma cells and the biochemical characteristics of the hybridoma cells.
As shown in fig. 1, a hybridoma cell screening chip according to a preferred embodiment of the present invention is formed by bonding a glass substrate and a PDMS sheet, and includes: the device comprises a first screening area 1, an incubation area 2, a second screening area 3, a collecting area 4, a first sample inlet 5 connected with the first screening area 1, a second sample inlet 6 connected with the incubation area 2, a first valve 7, a second valve 8 and a third valve 9 connected with the incubation area 2, a fourth valve 10 connected with the collecting area 4 and a sample outlet 11 connected with the collecting area 4, wherein the first screening area 1, the incubation area 2, the second screening area 3 and the collecting area 4 are sequentially connected. Wherein, the sample inlet, the sample outlet and the valve are formed by punching. It should be understood that the power source of the chip is provided by a positive pressure pump connected to the first and second sample inlets 5 and 6, or a negative pressure pump connected to the sample outlet 11.
Wherein the first screening zone 1 comprises: the first main channel 101, a plurality of first screening columns 102 respectively arranged at two sides of the first main channel 101, a first waste liquid channel 103 connected with the end region of the setting region of the first screening columns 102, and a first waste liquid port 104 positioned at the end of the first waste liquid channel 103, the first screening region 1 is used for separating unfused single cells from fused cells through cell size difference.
The incubation area 2 is pre-filled with capture cells or magnetic beads, and the tumor-tumor fusion cells are separated from the B-B fusion cells and the hybridoma cells through the capture cells or magnetic beads.
The second screening zone 3 comprises: a second main channel 301, a plurality of second screening pillars 302 respectively disposed at both sides of the second main channel 301, a second waste liquid channel 303 connected to an end region of a region where the second screening pillars 302 are disposed, and a second waste liquid port 304 at an end of the second waste liquid channel 303, for further separating the capture carrier to which the target cells are bound from the capture carrier to which the target cells are not bound by a cell size difference.
The collection region 4 is used for collecting hybridoma cells and performing post-treatment on the hybridoma cells.
The first sample inlet 5 is connected with the first screening area 1, and is used for the entry of the sample to be screened, and the sample to be screened comprises: hybridoma cell, tumor-tumor fused cell, B-B fused cell, tumor cell single cell, and B cell single cell. The second sample inlet 6 is connected with the incubation area 2 and is used for the entrance of capture cells or magnetic beads, PBS buffer solution and trypsin.
In the first screening area 1, the distance between any two adjacent first screening columns 102 is larger than the diameter of the unfused single cell and smaller than the diameter of the fused cell after fusion, so that the target cell can be ensured to enter the incubation area and not flow out through the waste liquid channel of the first screening area.
According to the preferred embodiment, the width of the first main channel 101 is 100-500 microns, the size of the first screening pillars 102 is 10-50 microns, and the spacing between the first screening pillars 102 is determined according to the size of the unfused single cell.
In the second screening region 3, the distance between any two adjacent second screening columns 302 is larger than the diameter of the capture carrier without the target cells bound thereto and smaller than the diameter of the capture carrier with the target cells bound thereto, so that it is ensured that the capture carrier with the target cells bound thereto enters the collection region and does not enter the waste liquid channel of the second screening region to flow out.
According to the preferred embodiment, the width of the second main channel 301 is 100 to 500 micrometers, the size of the second screening pillars 302 is 10 to 50 micrometers, and the interval between the second screening pillars 302 is determined according to the size of the capture carrier to which the target cells are not bound.
Wherein the first valve 7 is used to control the sample from the first screening zone 1 to enter the incubation zone 2, the second valve 8 and the third valve 9 are used to control the capture cells or magnetic beads, the buffer and the trypsin to enter the incubation zone 2, and the fourth valve 10 is used to control the sample from the second screening zone 3 to enter the collection zone 4.
According to the preferred embodiment, the hybridoma cell screening chip is prepared by the following steps:
the preparation of PDMS sheet and the preparation of glass base and the bonding of the two are carried out respectively without sequence;
the preparation of the PDMS sheet comprises the preparation of a silicon substrate as a mould thereof and the preparation of the PDMS sheet;
the silicon substrate manufacturing method specifically comprises the following steps: adopting four-inch monocrystalline silicon as a substrate, spin-coating 30-micron thick photoresist on a silicon wafer by using a rotary glue coating machine, and pre-drying: baking at 65 ℃ for 30 minutes; then, photoetching is carried out according to the designed layout; and (3) after photoetching is finished, post-drying: postbaking for 30 minutes at 95 ℃; developing by using a developing solution, removing the photoresist and cleaning to obtain a microstructure with a designed height; then hard-baking for 30 minutes at 95 ℃; obtaining silicon chip molds with microstructures of different heights;
the PDMS sheets were made as follows: putting the prepared silicon chip mold into a fluorosilane atmosphere for incubation for 4 hours so as to facilitate the subsequent stripping between the PDMS chip and the mold; according to the weight ratio of 15: 1, weighing PDMS prepolymer and curing agent, placing the PDMS prepolymer and the curing agent in a container, uniformly stirring the PDMS prepolymer and the curing agent by using a glass rod, placing the container in a vacuum drier, vacuumizing until the vacuum degree is 13psi, and standing for 30min to remove bubbles; taking out the PDMS mixture, placing the silicon wafer mold on a horizontal table, pouring PDMS, and standing for 30min to fill the PDMS in the mold; putting the mould into an oven, heating for 1h at the temperature of 80 ℃, and carefully stripping the PDMS from the silicon wafer after the PDMS is completely cured; punching holes at the positions of a first sample inlet 5 and a second sample inlet 6, a first waste liquid inlet 104, a second waste liquid inlet 304 and a sample outlet 11 on the PDMS chip by using a needle with a specific aperture, and punching holes at the air inlets corresponding to the valves; placing the PDMS chip into a plasma cleaning machine, keeping the PDMS chip in vacuum for 1h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a PEG 6-9-siloxane and acetone mixed solution (v: v is 1:1) at the sample inlet, filling the whole chip inner cavity and pipeline with the mixed solution by using the negative pressure in the PDMS chip as a driving force, and incubating for 1h at room temperature; and then, washing with ultrapure water to ensure that the surfaces of all the inner cavities and pipelines in the PDMS chip are hydrophilic so as to reduce the adhesion of all the inner cavities and pipelines to cells.
The glass substrate is manufactured by selecting and cutting the glass substrate material.
The bonding process of the PDMS sheet and the glass substrate comprises the following steps: placing the PDMS sheet and the glass substrate with the bonding surfaces facing upwards into a plasma cleaning machine for cleaning, irradiating for 2min, taking out, rapidly bonding together, and heating on a hot plate at 90 ℃ for 20min to 30min to enhance the bonding degree of the PDMS sheet and the glass substrate.
And finishing the chip manufacturing.
There is also provided in accordance with a preferred embodiment of the present invention a method for screening and collecting hybridoma cells, including the steps of:
s1: a hybridoma cell screening chip as shown in FIG. 1 is provided.
S2: before the hybridoma cell sample to be screened enters the chip, the first valve 7 and the fourth valve 10 are closed, the second valve 8 and the third valve 9 are opened, and the capture cells are introduced into the incubation area 2 through the second injection port 6, so that the capture cells are paved and attached to the wall in the incubation area 2, wherein the capture cells are formed by combining the mouse fibroblasts and the antigen combined with the virus peptide, and after the antigen and the virus peptide are combined, the virus peptide can be coupled to the HLA2 site on the mouse fibroblast surface, so that the cells have the capacity of capturing the cells producing the antigen-corresponding antibody, and are called as capture cells.
S3: after the captured cells are attached to the wall, the first valve 7 is opened, the second valve 8 is closed, the sample to be screened is introduced into the first screening area 1 through the first sample inlet 5, the unfused single cells are separated from the fused cells, the hybridoma cells, the tumor-tumor fused cells and the B-B fused cells enter the incubation area 2 through a self-flow channel, and the tumor cell single cells and the B cell single cells flow out from the first waste liquid port 104.
S4: after the fused cells enter the incubation area 2, the first valve 7 and the fourth valve 10 are closed, the second valve 8 and the third valve 9 are opened, the hybridoma cells and the B-B fused cells are combined with the capture cells in the incubation area, while the tumor-tumor fused cells with the competitive survival advantage are not combined with the capture cells, because the tumor cells do not generate antibodies and do not react with the capture cells; after the sample is completely injected, PBS buffer solution is introduced into the incubation area 2 through the second injection port 6, and the unbound tumor-tumor fused cells are flushed out through the second waste liquid port 304 of the second screening area 3.
S5: then, trypsin is introduced into the incubation area 2 through the second sample inlet 6, so that adherent captured cells are suspended and enter the second screening area 3, screening can be performed through the second screening area 3 according to the difference of the sizes of the captured cells combined with the cells and the captured cells not combined with the cells, the captured cells not combined with the cells flow out of the second waste liquid port 304, and the captured cells combined with hybridoma cells and B-B fusion cells further enter the collection area 4 through a flow channel.
S6: the hybridoma cells are collected in the collection region 4 and subjected to various subsequent treatments as required.
Example 1: screening of hybridoma cells for production of anti-aflatoxin antibody
The mice were immunized with aflatoxin to give mouse spleens, from which B cells were obtained. And carrying out a cell fusion experiment on the mouse tumor cell and the mouse B cell to obtain a sample of the hybridoma cell, wherein the sample contains the B-B fusion cell, the tumor-tumor fusion cell, the hybridoma cell, the B cell and the tumor cell. Wherein the size of the B cell is 12-13 microns, the size of the tumor cell is 11-12 microns, the size of the B-B fusion cell is 17-18 microns, the size of the hybridoma cell is 16-17 microns, and the size of the tumor-tumor fusion cell is 15-16 microns.
A hybridoma cell screening chip was prepared according to the above-described embodiment, in which the size of the first screening pillars 102 was set to 13 micrometers and the size of the second screening pillars 302 was set to 16 micrometers.
The mouse fibroblast marked aflatoxin is used as a capture cell, and the size of the capture cell is 13-14 microns.
The specific flow is that firstly, all valves are opened, the sample is injected from the second injection port 6, the whole chip is filled with fibroblast culture medium, the first valve 7 is closed, the captured cells enter the incubation area 2 through the second injection port 6 and adhere to the wall, after 2 hours and the adherence is finished, the first valve 7 is opened, the second valve 8 is closed, the sample enters the first injection port 5, the unfused single cells pass through the first screening column 102 in the first screening area 1 and enter the two side auxiliary channels, then the single cells are discharged through the first waste liquid port 104, the three fused cells enter the incubation area 2, the incubation is carried out for 10 minutes at normal temperature in the incubation area, the captured cells can be combined with the hybridoma cells and the B-B fused cells, in the process, the first valve 7 and the fourth valve 10 are closed, the PBS solution enters the second injection port 6 for flushing, the tumor-tumor fused cells which are not combined with the captured cells are flushed out, then the fourth valve 10 is opened, trypsin solution is fed into the second sample inlet 6, digestion is carried out for 3 minutes, the size of the captured cells is 13-14 microns, the size of the bound cell mass of the captured cells and the fused cells is 18-20 microns, the size of the second screening column 302 is 16 microns, therefore, the captured cells which are not captured to the target cells flow out from the collecting area 304, the captured cells which are captured to the target cells cannot pass through the second screening column 302 due to the large volume of the captured cells and enter the collecting area 4, and the B-B fused cells cannot be cultured continuously, so that the cells naturally die in the collecting area, and only hybridoma cells are left, thereby completing cell screening.
Example 2: screening of hybridoma cells for production of anti-aflatoxin antibody
The mice were immunized with aflatoxin to give mouse spleens, from which B cells were obtained. And carrying out a cell fusion experiment on the mouse tumor cell and the mouse B cell to obtain a sample of the hybridoma cell, wherein the sample contains the B-B fusion cell, the tumor-tumor fusion cell, the hybridoma cell, the B cell and the tumor cell. Wherein the size of the B cell is 12-13 microns, the size of the tumor cell is 11-12 microns, the size of the B-B fusion cell is 17-18 microns, the size of the hybridoma cell is 16-17 microns, and the size of the tumor-tumor fusion cell is 15-16 microns.
A hybridoma cell screening chip was prepared according to the above embodiment, in which the size of the first screening pillars 102 was set to 13 micrometers and the size of the second screening pillars 302 was set to 18 micrometers.
Magnetic beads are used for marking aflatoxin as a capture carrier, and the size of the capture carrier is 15 microns.
The specific flow is that firstly, all valves are opened, the sample is injected from the second injection port 6, the whole chip is filled with fibroblast culture medium, the first valve 7 is closed, the magnetic beads are fed from the second injection port 6 to be fully paved in the incubation region 2, then the first valve 7 is opened, the second valve 8 is closed, the sample is fed in the first injection port 5, the unfused single cells pass through the first screening column 102 in the first screening region 1 to enter the two side auxiliary channels, and then are discharged through the first waste liquid port 104, the three fused cells enter the incubation region 2, the incubation is carried out for 10 minutes at normal temperature in the incubation region 2, the magnetic beads can be combined with the hybridoma cells and the B-B fused cells, in the process, the first valve 7 and the fourth valve 10 are closed, the PBS solution is fed in the second injection port 6 to be washed, the tumor-tumor fused cells which are not combined with the magnetic beads are washed out, then the fourth valve 10 is opened to remove the magnetic force of the magnet, the PBS solution is flushed, the magnetic beads leave the incubation area, the size of the magnetic beads is 15 microns, the size of the cell mass of the magnetic beads combined with the fused cells is 22-24 microns, and the size of the second screening column 302 is 18 microns, so the magnetic beads which do not capture the target cells flow out from the second waste liquid port 304, the magnetic beads which capture the target cells cannot pass through the second screening column 302 due to the large volume of the magnetic beads, enter the collection area 4, and the B-B fused cells cannot be cultured continuously, so the cells naturally die in the collection area, and only the hybridoma cells are left, thereby completing the screening of the cells.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.

Claims (10)

1. A hybridoma cell screening chip comprises a glass substrate and a PDMS sheet which are attached together, and is characterized in that the attachment surface of the PDMS sheet is provided with a first screening area, an incubation area, a second screening area and a collecting area which are sequentially connected to complete hybridoma cell screening; wherein the content of the first and second substances,
the first screening zone includes: the first screening area separates unfused single cells from fused cells after fusion based on cell size difference;
the incubation area is pre-filled with capture carriers, wherein the capture carriers are capture cells or magnetic beads, and the tumor-tumor fusion cells are further separated from the B-B fusion cells and the hybridoma cells through the capture carriers;
the second screening zone includes: a second main channel, a plurality of second screening columns respectively arranged at both sides of the second main channel, and a second waste liquid channel, wherein the second screening area separates the capture carrier to which the target cells are bound from the capture carrier to which the target cells are not bound based on the cell size difference;
the collecting region is used for collecting hybridoma cells;
the hybridoma cell screening chip further comprises: a first valve, a second valve, a third valve in communication with the incubation region, and a fourth valve in communication with the collection region.
2. The hybridoma cell screening chip of claim 1, wherein the first valve controls the sample from the first screening region to enter the incubation region, the second and third valves control the capture carrier, buffer, and trypsin to enter the incubation region, and the fourth valve controls the sample from the second screening region to enter the collection region.
3. The hybridoma cell screening chip of claim 1, wherein the first screening region is connected to a first sample inlet for entry of a sample to be screened, the sample to be screened comprising: hybridoma cell, tumor-tumor fused cell, B-B fused cell, tumor cell single cell, and B cell single cell.
4. The hybridoma cell screening chip according to claim 1, wherein in the first screening region, the distance between any two adjacent first screening columns is larger than the diameter of an unfused single cell and smaller than the diameter of a fused cell after fusion.
5. The hybridoma cell screening chip of claim 1, wherein the incubation region is connected to a second inlet port, and the second inlet port is controlled by the second valve for the entrance of the capture carrier, the PBS buffer, and the trypsin.
6. The hybridoma cell screening chip of claim 1, wherein in the second screening region, the distance between any two adjacent second screening pillars is larger than the diameter of the capture carrier to which target cells are not bound and smaller than the diameter of the capture carrier to which target cells are bound.
7. The hybridoma cell screening chip according to claim 4, wherein the width of the first main channel is 100 to 500 micrometers, the size of the first screening pillars is 10 to 50 micrometers, and the distance between any two adjacent first screening pillars is determined according to the size of the unfused single cell.
8. The hybridoma cell screening chip according to claim 6, wherein the width of the second main channel is 100 to 500 μm, the size of the second screening pillars is 10 to 50 μm, and the distance between any two adjacent second screening pillars is determined according to the size of the capture carrier to which the target cells are not bound.
9. A method for screening and collecting hybridoma cells, which is characterized by comprising the following steps:
s1: providing a hybridoma cell screening chip according to any one of claims 1 to 8;
s2: closing the first valve and the fourth valve, opening the second valve and the third valve, and introducing a capture carrier into the incubation region, wherein the capture carrier is a capture cell or a magnetic bead;
s3: opening the first valve, closing the second valve, and introducing a sample to be screened into the first screening area to separate unfused single cells from fused cells, wherein hybridoma cells, tumor-tumor fused cells and B-B fused cells enter the incubation area through a flow channel, and tumor cell single cells and B cell single cells flow out of the first waste liquid channel;
s4: after the fused cells enter the incubation area, closing the first valve and the fourth valve, opening the second valve and the third valve, combining the hybridoma cells and the B-B fused cells with the capture carrier in the incubation area, not combining the tumor-tumor fused cells with the capture carrier, and introducing PBS buffer solution into the incubation area through the second sample inlet so as to enable the tumor-tumor fused cells to flow out;
s5: then, introducing trypsin or removing the magnetic force of a magnet into the incubation area through a second sample inlet, so that the sample enters a second screening area, the capture carrier of the unbound cells flows out of a second waste liquid channel according to the different sizes of the capture carrier bound with the cells and the capture carrier of the unbound cells, and the capture carrier bound with the hybridoma cells and the B-B fusion cells further enters a collection area;
s6: the B-B fusion cells are naturally apoptotic in the collecting region, and then the collection of the hybridoma cells is realized.
10. The method for screening and collecting hybridoma cells according to claim 9, wherein step S6 further comprises: the hybridoma cells are subsequently processed as required in the collection region.
CN202210356239.7A 2022-04-06 2022-04-06 Hybridoma cell screening chip and screening and collecting method thereof Pending CN114686372A (en)

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