CN110669672B

CN110669672B - Micro-fluidic chip for producing antibody

Info

Publication number: CN110669672B
Application number: CN201910975076.9A
Authority: CN
Inventors: 顾志鹏; 张意如; 陈跃东; 张瑜; 周侗
Original assignee: Dongguan Dongyangguang Diagnostic Products Co ltd
Current assignee: Dongguan Dongyangguang Diagnostic Products Co ltd
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2023-02-07
Anticipated expiration: 2039-10-14
Also published as: CN110669672A

Abstract

The invention relates to a micro-fluidic chip for producing antibodies, which comprises a chip body, wherein a liquid channel and a gas channel are arranged in the chip body; the liquid channel comprises a sample introduction channel, a mixing channel, a reaction channel and a collection channel which are sequentially communicated, wherein the sample introduction channel is provided with a sample introduction port, and the collection channel is provided with a collection port; the gas channel comprises a gas inlet, a gas path channel and a gas outlet which are communicated in sequence; and gas exchange can be carried out and liquid exchange is not carried out between the reaction channel and the gas channel. The microfluidic chip is used for producing the antibody, can realize continuous operation, avoids a plurality of manual operation steps of celline culture bottles, avoids the possibility of pollution, and has the advantages of low cost and high production efficiency.

Description

Microfluidic chip for producing antibody

Technical Field

The invention relates to the technical field of microfluidics, in particular to a microfluidic chip for producing antibodies.

Background

Antibodies are one of the most widely used tools in the diagnostic and therapeutic fields. Antibodies can now be prepared by both in vivo, i.e., by promoting ascites production in an animal, and in vitro, i.e., by producing a supernatant from tissue culture hybridoma cells.

The monoclonal antibody content in the supernatant of tissue culture hybridoma cells is usually less than 0.01mg/ml, while the monoclonal antibody content in ascites is 1-100.00mg/ml. Tissue culture of hybridoma cells usually takes 9 weeks, while ascites preparation does not take more than 4 weeks. The preparation of ascites is always subject to the disadvantages of animal protection because of the use of living animals. In addition, the cost of the method is very high whether the tissue culture method or the method for preparing ascites by animals, so that the problem of extremely high price of the antibody at present is caused.

At present, monoclonal antibodies are usually prepared by adopting celline culture bottles in an in-vitro means, the culture bottles are expensive, the operation process is extremely complex, continuous production cannot be realized, a large amount of manual operation is required in the culture process, and a strict flow is required to avoid pollution.

The microfluidic chip technology (microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip to automatically complete the whole analysis process, and can be used for many times. Because of the micron-scale structure, the fluid exhibits and develops specific properties therein that differ from the macro-scale, thus developing unique analytical-generated properties. The microfluidic chip has been widely used for separation, detection, reaction, cell culture and the like, however, at present, there is no microfluidic chip capable of continuously and efficiently producing antibodies.

Disclosure of Invention

The invention aims to provide the microfluidic chip for producing the antibody, which can be operated continuously, has low cost and no cross contamination risk, and is used for replacing the existing product for preparing the antibody by in vitro tissue culture.

To this end, in a first aspect of the present invention, there is provided a microfluidic chip for producing an antibody, comprising a chip body,

a liquid channel and a gas channel are arranged in the chip body;

the liquid channel comprises a sample introduction channel, a mixing channel, a reaction channel and a collection channel which are sequentially communicated, wherein the sample introduction channel is provided with a sample introduction port, and the collection channel is provided with a collection port;

the gas channel comprises a gas inlet, a gas path channel and a gas outlet which are communicated in sequence;

and gas exchange can be carried out and liquid exchange is not carried out between the reaction channel and the gas channel.

Further, gas exchange can be performed and liquid exchange is not performed between the mixing channel, the collecting channel and the gas channel.

Further, the number of the sample inlets is 1-5; when the number of the sample inlets is 2-5, each sample inlet is respectively connected with the mixing channel through a sample inlet channel.

The number of the sample inlets can be selected according to the type of the solution used in the antibody production process, and in a specific embodiment, the number of the sample inlets is 3, and the sample inlets respectively correspond to cell suspension (hybridoma cells), IMDM complete medium and Fetal Bovine Serum (FBS) required by antibody production.

Further, the chip body comprises a first core plate and a second core plate;

the liquid passages are disposed in the first core plate and the gas passages are disposed in the second core plate.

Further, the gas inlet and the gas outlet are arranged in the first core plate.

Further, the mixing channel is a snake-shaped structure, a human bone structure or a micro-column array.

Further, the aperture of the collecting channel gradually increases along the reaction channel in the direction of the collecting port.

Furthermore, a filtering membrane is arranged between the collecting channel and the collecting port, and the aperture of the filtering membrane is smaller than the diameter of the cell used for producing the antibody and is larger than 10kDa. In a specific embodiment, the cell used to produce the antibody is a hybridoma cell.

Furthermore, an elastic film is arranged between the reaction channel and the gas channel, and the film is permeable to gas and liquid and can deform when the gas pressure in the gas channel changes.

Furthermore, an elastic film is arranged among the mixing channel, the collecting channel and the air channel, the film is air-permeable and liquid-impermeable, and can deform when the air pressure in the air channel changes.

Further, the film is a silica gel film or 3M ^TM 3394 or PDMS film.

Further, the thickness of the film is 0.1-0.3mm.

Furthermore, the width of the air passage channel is 1-3mm, and the depth is 0.5-1.5mm.

Furthermore, the micro-fluidic chip except the filter membrane and the thin film is made of PDMS, PS, PMMA, COC, ABS, PC or glass.

In a second aspect of the invention, there is provided a device for producing antibodies, the device comprising a microfluidic chip according to the invention.

Further, the introduction port is connected with a liquid power source, and the liquid power source can continuously provide liquid inflow and flow control. In particular embodiments, the liquid power source is an injection pump, a peristaltic pump, or a constant pressure pump.

Further, the gas inlet and the gas outlet are connected to a gas power source capable of providing O ₂ And removing CO ₂ And can adjust O ₂ The pressure of (a).

In a third aspect of the invention, there is provided the use of a microfluidic chip or device according to the invention for the production of antibodies.

Compared with the prior art, the invention has the advantages that,

1. by applying the microfluidic chip disclosed by the invention, the continuous operation can be realized on the production of the antibody, so that a plurality of manual operation steps of celline culture bottles are avoided, and the possibility of pollution is avoided;

2. the micro-fluidic chip is provided with a gas exchange and temperature control system, so that the use of a large cell culture box is avoided in the production process of the antibody;

3. compared with a culture bottle, the micro-fluidic chip has small pipeline volume, hybridoma cells are more fully contacted with nutrient substances in a culture medium, and the antibody generation efficiency is higher.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1 is a schematic structural diagram of a microfluidic chip which is a two-layer core plate;

FIG. 2 is a cross-sectional view of a microfluidic chip;

FIG. 3 is a schematic view of a partial structure of a microfluidic chip;

FIG. 4 is a schematic diagram of the connection of the microfluidic chip to a power source;

FIG. 5 is a diagram showing the arrangement of observation points of cell density;

FIG. 6 is a schematic diagram of a structure in which the microfluidic chip is a three-layer core plate;

reference numerals:

100-a microfluidic chip;

110-a first core board; 111-sample inlet; 1111-a first sample inlet; 1112-a second sample inlet; 1113-third inlet; 112-a sample introduction channel; 1121-first sample introduction channel; 1122-a second sample injection channel; 1123-a third sample feed channel; 113-a mixing channel; 114-a reaction channel; 115-a collection channel; 116-a collection port; 117 — gas inlet; 118-a gas outlet; 119-a filtration membrane;

120-a second core board; 121-gas path channel; 122-a thin film;

130-a first power source; 140-a second power source; 150-a third power source; 160-a fourth power source; 170-a fifth power source; 180-first cell density observation point; 190-a second cell density observation point;

200-a microfluidic chip;

2101-first core board upper plate; 211-sample inlet; 2111-first sample inlet; 2112-a second sample inlet; 2113-third sample inlet; 216-a collection port; 217-gas inlet; 218-a gas outlet;

2102-a first core lower plate; 212-a sample introduction channel; 2121-a first sample injection channel; 2122-a second sample injection channel; 2123-a third sample entry channel; 213-a mixing channel; 214-a reaction channel; 215-a collection channel;

220-a second core board; 221-gas path channel.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the present invention, orientation words are defined, and in the case where no description is made to the contrary, the use of the orientation words such as "left, right, upper and lower" means that the microfluidic chip provided by the present invention is defined in the normal use condition, and these orientation words are used for the convenience of understanding, and thus do not limit the scope of the present invention.

Hereinafter, for convenience of description, the production of antibodies by hybridoma cells is described as an example, but it should be understood that the microfluidic chip of the present application is not limited thereto.

Referring to fig. 1, an embodiment of the present invention provides a microfluidic chip 100, including a chip body, wherein a liquid channel and a gas channel are disposed in the chip body; the liquid channel comprises a sample injection channel 112, a mixing channel 113, a reaction channel 114 and a collection channel 115 which are sequentially communicated, wherein the sample injection channel 112 is provided with a sample inlet 111, and the collection channel 115 is provided with a collection port 116; the gas channel comprises a gas inlet 117, a gas channel 121 and a gas outlet 118 which are communicated in sequence; gas exchange and no liquid exchange can be performed between the reaction channel 114 and the gas channel 121.

In further embodiments, gas exchange may be performed and liquid exchange may not be performed between the mixing channel 113, the collection channel 115, and the gas channel 121.

In a further embodiment, the number of the sample inlets 111 can be selected according to the kind of the solution used in the antibody production process, as shown in fig. 1, the number of the sample inlets is 3, and the first sample inlet 1111, the second sample inlet 1112 and the third sample inlet 1113 correspond to the IMDM complete medium, the cell suspension and the Fetal Bovine Serum (FBS) required for antibody production, respectively; the first sample inlet 1111 passes through the first sample channel 1121, the second sample inlet 1112 passes through the second sample channel 1122, and the third sample inlet 1113 is communicated with the mixing channel 113 through the third sample channel 1123.

The mixing channel 113 can be a common mixing structure in the field of microfluidic chips, and only needs to ensure that the added liquid is fully and uniformly mixed, and specifically, the mixing channel 113 is a common mixing structure such as a snake-shaped structure, a human bone structure and a micro-column array.

In a further embodiment, as shown in FIG. 2, the aperture of the collection channel 115 increases gradually along the reaction channel 114 towards the collection port 116. A filtering membrane 119 is arranged between the collecting channel 115 and the collecting port 116, the aperture of the filtering membrane 119 is smaller than the diameter of the hybridoma cell, and the protein molecules with the aperture larger than 10kDa, namely below 10kDa, can pass through the filtering membrane 119.

In the reaction channel 114, the hybridoma cells continue to produce antibodies during growth, and when collection is performed, the antibodies in the solution and the hybridoma cells simultaneously flow toward the collection port 116, and since the pore size of the collection channel 115 gradually increases toward the collection port 116 along the reaction channel 114, as shown in fig. 2, the upper wall of the collection channel 115 is inclined upward, and the flow rate of the liquid gradually decreases in this direction. Due to the high density of hybridoma cells, which tend to settle to the bottom of the collection channel 115, it is primarily the antibodies and other solutions produced that collect at the filter membrane 119. The pore size of the filter membrane 119 is smaller than the diameter of the hybridoma cells and larger than 10kDa, ensuring that all hybridoma cells remain in the collection channel 115, while the antibody is able to pass through the filter membrane 119 and the target antibody is finally collected through the collection port 116. The filter membrane 119 needs to cover the entire area of the collection port 116.

In a further embodiment, an elastic membrane 122 is disposed between the reaction channel 114 and the air channel 121, and the membrane 122 is air-permeable and liquid-impermeable and can deform when the pressure of the air in the air channel 121 changes. The membrane 122 is required to cover the gas channel 121 and the reaction channel 114.

In a further embodiment, an elastic membrane 122 is disposed between the mixing channel 113, the collecting channel 115 and the air channel 121, and the membrane 122 is air-permeable and liquid-impermeable and can deform when the pressure of the gas in the air channel 121 changes. The membrane 122 needs to cover the gas channel 121 and the mixing channel 113, the collecting channel 115.

As shown in FIG. 3, gas inlet 117 provides O at a pressure ₂ The pressure of which can be regulated by a power source at the inlet. At normal pressure, the membrane 122 is in a normal state; when O is present ₂ When the pressure is low, the film 122 deforms toward the gas channel 121; when O is present ₂ When the high pressure state is present, the membrane 122 is deformed toward the reaction channel 114 side.

The thin film 122 is a silica gel film, 3M ^TM 3394 or PDMS membrane with thickness of 0.1-0.3mm, good gas permeability and liquid impermeability ₂ On the one hand, O is promoted when the pressure is changed ₂ Enters the reaction channel 114 through the membrane 122, promotes growth of hybridoma cells, and forces CO ₂ Out of the reaction channel 114 through the membrane 122 to avoid excessive CO ₂ The resulting pH change affects the growth environment of the hybridoma cells, and in particular, because the membrane 122 is gas permeable, it may be used by increasing O during use ₂ Partial pressure, first forcing O ₂ Through the membrane 122 into the reaction channel 114; then reducing O ₂ Partial pressure of O ₂ The partial pressure is less than that of the mixed gas in the reaction channel 114, the pressure of the mixed gas in the reaction channel 114 is higher than that of the gas channel 121, the mixed gas passes through the film 122, and part of CO ₂ Discharging, by repeating the above process, to obtain O ₂ Into the reaction channel 114, CO ₂ Is discharged from the reaction channel 114; on the other hand, the membrane 122 can be deformed up and down by air pressure adjustment, so that the flow of cells in the reaction channel 114 is promoted, a large number of cells are prevented from being gathered in the collection channel 115, and the hybridoma cells can be promoted by regular deformation of the membrane 122Are uniformly distributed in the whole reaction channel 114, and are beneficial to fully contacting the hybridoma cells with nutrient substances such as culture media.

The material of the microfluidic chip, except for the filtering membrane 119 and the thin film 122, may be PDMS, PS, PMMA, COC, ABS, PC, or glass, and the like, in which case PDMS or PS is preferably selected and manufactured by injection molding or imprint process. It should be noted that the microfluidic chip of the present invention may be formed by one, two, three or more layers, and for convenience of processing, in some embodiments, it is preferably a two-layer or three-layer core plate structure, and the structure such as the flow channel and the hole is processed on each layer of the core plate by a conventional processing method according to the requirement, and the core plates are sealed by a conventional means such as hot pressing, gluing or plasma bonding.

In one embodiment, referring to fig. 1, the microfluidic chip 100 has a two-layer core plate structure, and is fabricated on the first core plate 110 according to the widths and depths of the sample injection channel 112, the mixing channel 113, the reaction channel 114, and the collection channel 115, and the sample injection port 111, the collection port 116, the gas inlet 117, and the gas outlet 118 of the perforated structure are provided to ensure the entry and circulation of liquid and gas.

In another embodiment, referring to fig. 6, the microfluidic chip 200 is a three-layer core plate structure, and the first core plate 210 is divided into two independent structures, namely a first core plate upper plate 2101 and a first core plate lower plate 2102, wherein the first core plate upper plate 2101 is provided with a sample inlet 211, a collection port 216, a gas inlet 217 and a gas outlet 218, which are all of a perforated structure; the depths of the structures of the sample inlet channel 212, the mixing channel 213, the reaction channel 214 and the collection channel 215 arranged on the first core lower plate 2102 are consistent with the thickness of the first core lower plate 2102, so that the processing surface of the structures can be reduced, the roughness of the pipeline is reduced, and the production of antibodies is facilitated; the second core plate 220 is provided with air passage channels 221.

The operation of the microfluidic chip of the present invention is described below with the CHO cell line as an example and with reference to FIGS. 1 to 5:

the chip is sterilized before use, and the sterilization mode can adopt alcohol or ultraviolet lamp for sterilization.

As shown in fig. 4, the first sample inlet 1121, the second sample inlet 1122, and the third sample inlet 1123 are respectively connected to a first power source 130, a second power source 140, and a third power source 150, wherein the first power source 130 provides an IMDM complete medium, the second power source 140 provides a hybridoma cell suspension, and the third power source 150 provides Fetal Bovine Serum (FBS). The power source can continuously provide the three solutions, and the flow rates of the three solutions can be adjusted.

The collection port 116 of the chip is connected to a fourth power source 160, and the first power source 130 is connected to the fourth power source 160.

The gas inlet 117 and the gas outlet 118 are connected to a fifth power source 170, and the fifth power source 170 is capable of providing O at a certain temperature and pressure ₂ And removing CO ₂ (ii) a And fifth power source 170 is capable of controlling and regulating O ₂ The pressure of (a).

As shown in fig. 5, a first cell density observation point 180 is provided above the mixing channel 113, a second cell density observation point 190 is provided at the junction of the reaction channel 114 and the collection channel 115, the cell density per unit time at the observation point can be observed by a microscope, and whether the number of hybridoma cells entering the reaction channel 114 reaches an optimum condition can be estimated by observing the first cell density observation point 180; the growth of the hybridoma cells can be monitored by observing the second cell density observation point 190. And nutrient supplement or serum collection is carried out in time according to the cell number.

The fifth power source 170 is first turned on to provide gas and temperature control for the entire chip, maintaining the chip temperature at 37 ℃.

Then, the first power source 130, the second power source 140 and the third power source 150 are started, the IMDM complete medium, the hybridoma cell suspension and the fetal bovine serum are continuously pumped in a certain proportion, and the three solutions respectively enter the mixing channel 113 through the first sample feeding channel 1121, the second sample feeding channel 1122 and the third sample feeding channel 1123 and are fully mixed in the mixing channel 113.

The first cell density observation point 180 is observed by a microscope to estimate the number of hybridoma cells entering the reaction channel 114, thereby ensuring that an optimal number of hybridoma cells enter the reaction channel 114.

After the desired number of hybridoma cells has entered the reaction channel 114, the second power source 140 is turned off. With continued supply of O at fifth power source 170 ₂ Under the conditions of (1), hybridoma cells in the reaction channel 114 continuously produce antibodies in a liquid environment of IMDM complete medium and FBS, and proliferate to produce CO ₂ Enters the air channel 121 through the film 122 to prevent the growth environment of the hybridoma cells from being affected by CO ₂ Influence.

In the antibody production process, two solutions, i.e., IMDM complete medium and FBS, are continuously/intermittently introduced into the chip at a low flow rate, and each time after IMDM complete medium and FBS are introduced into the chip, since the chip is a closed cavity, hybridoma cells and the produced antibody solution enter the collection channel 115 through the reaction channel 114 due to the introduction of new liquid, and since the aperture of the collection channel 115 gradually increases from the reaction channel 114 to the collection port 116, the liquid flow rate gradually decreases in the direction. Since the density of hybridoma cells is high and they tend to settle at the bottom of the collection channel 115, the produced antibodies and other solutions mainly permeate the filter membrane 119 and then enter the fourth power source 160 through the collection port 116. Wherein the produced antibodies are collected in the fourth power source 160 and the filtered medium may be transported to the first power source 130 for reuse as desired. In the above process, the flow rate can be adjusted by observing the density of the hybridoma cells at the second cell density observation point 190, so as to determine the cell density of the entire reaction channel, and thus determine whether to adjust the flow rate of the IMDM complete medium and the FBS.

Generally, IMDM complete medium and FBS solution favor the proliferation and antibody production of hybridoma cells, and hybridoma cells consume a large amount of medium and FBS when proliferating and producing antibodies in large quantities. Therefore, whether to continuously/intermittently supply the culture medium and FBS into the chip can be determined according to the density of the hybridoma cells.

As shown in FIG. 3, if a large amount of cells are accumulated at the collecting channel 115 or the filtering membrane 119 during the collecting process, the supply of O from the fifth power source 170 may be adjusted ₂ Such that it is moved with a high/low pressure in a certain cycle to periodically deform the membrane 122, thereby causing a fine lineThe cells are oscillated and uniformly distributed again throughout the reaction channel 114.

The parts not referred to in the present invention are the same as or can be implemented using the prior art.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A micro-fluidic chip for producing antibodies comprises a chip body and is characterized in that,

a liquid channel and a gas channel are arranged in the chip body from top to bottom;

the liquid channel comprises a sample introduction channel, a mixing channel, a reaction channel and a collection channel which are sequentially communicated, wherein the sample introduction channel is provided with a sample introduction port, and the collection channel is provided with a collection port; a filtering membrane is arranged between the collecting channel and the collecting port; the aperture of the collecting channel is gradually increased towards the collecting port along the reaction channel;

the reaction channel and the gas channel can exchange gas and do not exchange liquid; an elastic film is arranged between the reaction channel and the gas channel, and the film is permeable to gas and liquid and can deform when the gas pressure in the gas channel changes;

the gas exchange can be carried out and the liquid exchange is not carried out among the mixing channel, the collecting channel and the gas channel; an elastic film is arranged among the mixing channel, the collecting channel and the air channel, the film is air-permeable and liquid-tight and can deform when the air pressure in the air channel changes.

2. The microfluidic chip of claim 1, wherein the chip body comprises a first core plate and a second core plate;

the liquid channel is arranged in the first core plate, and the air channel is arranged in the second core plate.

3. The microfluidic chip of claim 2, wherein the gas inlet and the gas outlet are disposed in the first core plate.

4. The microfluidic chip according to claim 1, wherein the pore size of the filter membrane is smaller than the diameter of the cells used for antibody production and larger than 10kDa.

5. The microfluidic chip according to claim 1, wherein the thin film is a silicone film, 3M ^TM 3394 or PDMS film.

6. The microfluidic chip according to claim 1, wherein said thin film has a thickness of 0.1 to 0.3mm.

7. An apparatus for producing an antibody, comprising the microfluidic chip according to any one of claims 1 to 6.

8. The device of claim 7, wherein the sample inlet is connected to a liquid power source capable of continuously providing liquid inflow and flow control.

9. The apparatus of claim 7, wherein the gas inlet and the gas outlet are connected to a gas power source capable of providing O ₂ And removing CO ₂ And can adjust O ₂ Pressure and/or temperature of (a).

10. Use of a microfluidic chip according to any of claims 1 to 6 or a device according to any of claims 7 to 9 for the production of antibodies.