CN112795478A - Cell separation device, cell separation method and application - Google Patents

Cell separation device, cell separation method and application Download PDF

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Publication number
CN112795478A
CN112795478A CN202110109729.2A CN202110109729A CN112795478A CN 112795478 A CN112795478 A CN 112795478A CN 202110109729 A CN202110109729 A CN 202110109729A CN 112795478 A CN112795478 A CN 112795478A
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chip
channel
chambers
pressure
cell separation
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石剑
刘瑞
周威
韩超
陈勇
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Guangzhou Anfang Biotechnology Co ltd
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Guangzhou Anfang Biotechnology Co ltd
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    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C12M23/16—Microfluidic devices; Capillary tubes
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    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
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    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a cell separation device, a cell separation method and application. The cell separation device includes: the microfluidic chip comprises an upper chip, a filter membrane and a lower chip; an aspirator in communication with the microfluidic chip; the pressure control assembly comprises a pressure monitor and an air escape valve, and is communicated with the microfluidic chip; the air release pipeline is communicated with the microfluidic chip, and the pressure control assembly is arranged on the air release pipeline; the cell separation device provided by the invention can monitor the pressure difference in the micro-fluidic chip in real time, avoid cell selection omission or damage caused by overlarge pressure difference, can quickly and efficiently realize the separation of target cells, improves the efficiency of physical filtration, increases the purity of the separation of the target cells, can also be used for realizing the counting and detection of the cells, and has the advantages of simple method and low cost.

Description

Cell separation device, cell separation method and application
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a cell separation device, a cell separation method and application.
Background
Rare cells are present in very small amounts in blood and tissues but contain important genetic and expression information, such as stem cells, circulating endothelial cells, circulating tumor cells, fetal nucleated red blood cells in the mother, and residual diseased cells. The detection and analysis of these rare cells is critical to understanding the disease process and developmental mechanisms, and is more conducive to accurate medical practice. The detection and analysis of rare cells need to eliminate the interference of a large number of non-target cells, otherwise, the detection or misjudgment is very easy to cause. Therefore, the sorting and enriching technology of rare cells becomes especially critical and even is a decisive factor for the accuracy of the detection result. In recent years, a plurality of sorting and enriching means appear in the field, but the sorting and enriching means have advantages and disadvantages. Currently, there are two mainstream sorting methods: affinity enrichment and physical property enrichment. Affinity enrichment methods are mainly based on the isolation of target cells by protein biomarkers specifically expressed on the cell surface, and include positive enrichment methods for capturing target cells positively and negative enrichment methods for removing non-target cells negatively. The physical characteristic enrichment method mainly comprises the step of sorting according to physical characteristics such as size, density, mechanics, dielectric property and the like of target cells. The physical characteristic enrichment method comprises a centrifugation technology, a microfluidic technology, a membrane filtration separation technology, a fluid inertial focusing technology, an optical pressure difference technology and the like, and the enrichment method has the advantages of simple operation process, high capture efficiency, capability of realizing high-flux enrichment, low cost and no dependence on the expression of cell surface antigens. The microfluidic technology and the membrane filtration separation technology have the advantages of high integration level, high flux, visualization, good repeatability and the like due to the fact that the characteristic size can be accurately controlled, and become a preferred selection scheme for a plurality of researchers.
Although the advantages of the microfluidic technology and the filter membrane sorting technology are numerous, most of the application processes are that the sample is pushed by the external driving pressure to be sorted. At present, most researchers consider the importance of external driving pressure, and adopt accurate flow rate or pressure control equipment similar to a constant pressure pump, an injection pump, a plunger pump, a peristaltic pump and the like as sample driving external equipment. In most sorting unit, the sorting of cell is the dynamic process, the promotion of the transmembrane pressure difference of filter membrane must be caused in the entrapment of purpose cell, so lead to inside fluid hydraulic pressure and the extrusion force that the purpose cell received also can promote thereupon, external control equipment still operates according to the parameter that originally set for this moment, along with the lapse of time, the entrapment of a large amount of purpose cells will lead to inside cell external force to rise sharply, when reaching certain limit, the purpose cell of intercepting can be damaged or separated out because of can't bear huge external force, thereby lead to the omission of sorting and other testing results unsatisfactory. This is also the main reason why most of the current methods based on microfiltration membrane sorting cannot guarantee the cell activity and have high detectable rate. This problem necessitates some means to dynamically monitor and real-time regulate the pressure inside the microfluidic chip or filter during cell sorting.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a cell separation device, a cell separation method and application. The cell separation device can control the real-time pressure difference in the filtering process, judge the time node of the flow and protect cells from being missed and damaged due to overlarge pressure difference; based on this separator can realize the quick separation and the detection of cell.
Specifically, the technical solution adopted by the present invention is as follows.
An object of one aspect of the present invention is to provide a cell separation apparatus comprising:
the microfluidic chip comprises an upper chip, a filter membrane and a lower chip;
an aspirator in communication with the microfluidic chip;
the pressure control assembly comprises a pressure monitor and an air escape valve, and is communicated with the microfluidic chip;
the air release pipeline is communicated with the microfluidic chip, and the pressure control assembly is arranged on the air release pipeline;
wherein the content of the first and second substances,
the upper chip is provided with: the first through hole is connected with the air leakage pipeline, the second through hole is connected with the aspirator, and the third through hole is used for connecting a sample container;
the lower layer chip is provided with: a chamber corresponding to the third through-hole; one end of the first channel is communicated with the first through hole of the upper chip, and the other end of the first channel is communicated with the cavity; one end of the second channel is communicated with the second through hole of the upper chip, and the other end of the second channel is communicated with the cavity;
the filter membrane is arranged between the third through hole of the upper layer chip and the cavity of the lower layer chip.
When the cell separation device is used, a sample containing cells to be separated is placed into a sample container, a pressure monitor is opened, an air escape valve is closed, a suction device is started, a cavity, a first channel and a second channel in a lower chip are in a negative pressure state, pressure difference exists on two sides of a filter membrane at the moment, the sample is rapidly filtered under the action of the pressure difference, the rare cells are left in the sample container due to the large volume, filtrate is pumped out by the suction device through the second channel, and rapid separation of the rare cells is realized.
The pressure monitor is used for monitoring the real-time pressure difference of the sample separation process and can be used for judging the time node of the process. The air release valve is used for protecting target cells from being missed and damaged due to overlarge pressure difference, and when the pressure difference exceeds a certain threshold value, the air release valve is automatically opened, so that the target cell capturing efficiency and the activity rate are improved. For example, when the air pressure is steep, the sample in the sample container is almost completely filtered, and the filtrate in the chamber still cannot be drawn under the action of surface tension. If the aspirator continues to provide negative pressure, the filtrate in the chamber will cause the pressure difference in the chamber to rise sharply until the break-through tension is reached because the filtrate cannot break through the surface tension of the liquid. The large pressure differential at this point results in a very severe loss rate and activity of the captured cells of interest. Therefore, the air release valve needs to be opened in time to enable the interior of the microfluidic chip to be directly communicated with air, redundant filtrate in the cavity can flow into the aspirator from the first channel, the cavity and the second channel along with air, and the cavity does not have any pressure difference exceeding the cell bearing range in the period.
According to some embodiments of the invention, the number of the third through holes is one or more, and may be 1, 2, 3, 4, or 5. The number of the third through holes can be increased to connect a plurality of sample containers, so that the amount of samples processed each time is increased, and the efficiency is improved.
According to some embodiments of the invention, the third through hole is threadedly connected to the sample container.
According to some embodiments of the invention, the number of chambers corresponds to the number of third through holes. The chamber is a temporary storage area for filtrate to be pumped out through the suction device, and the depth of the chamber can be determined according to actual conditions.
According to some embodiments of the present invention, the number of the first through holes is one or more, and may be 1, 2, 3, 4, or 5, and the number thereof may or may not be the same as the number of the third through holes. The first through hole connects the interior of the microfluidic chip with the pressure control assembly, so that the monitoring and the regulation of the pressure in the microfluidic chip are realized.
According to some embodiments of the invention, the vent line is connected to the first through hole by a luer lock.
According to some embodiments of the present invention, the number of the second through holes is one or more, and may be 1, 2, 3, 4, or 5, and the like, and the number may or may not be the same as the number of the third through holes. The second through hole connects the interior of the microfluidic chip with the aspirator, and the filtrate is pumped out of the microfluidic chip through the second through hole.
According to some embodiments of the invention, the second through hole is connected to the aspirator through a suction line.
According to some embodiments of the invention, the second through hole is connected to the suction line by a luer lock.
According to some embodiments of the invention, the number of the first channels is one or more, and may be 1, 2, 3, 4, or 5, etc. The number of the first channels can be consistent with the number of the chambers, or can be inconsistent;
when the number of the first channels is smaller than that of the chambers, the first channels are connected with one of the chambers, the chambers are connected through a third channel, and the second channels are connected with one of the chambers, so that the intercommunication inside the microfluidic chip is realized;
when the number of the first channels is the same as that of the chambers, one end of each first channel is connected with the chamber, and the other end of each first channel can be connected with the same first through hole in an intersecting manner or connected with different first through holes in an intersecting manner;
when the number of the first channels is larger than that of the chambers, one end of the first channel is connected with the chambers, and the other end of the first channel can be intersected and connected with the same first through hole or respectively connected with different first through holes.
According to some embodiments of the invention, the number of the second channels is one or more, and may be 1, 2, 3, 4, or 5, etc. The number of the second channels can be consistent with the number of the chambers or not;
when the number of the second channels is smaller than that of the chambers, the second channels are connected with one of the chambers, the chambers are connected through a third channel, and the first channel is connected with one of the chambers, so that the intercommunication of the interior of the microfluidic chip is realized;
when the number of the second channels is consistent with that of the chambers, one end of each second channel is connected with the chamber, and the other end of each second channel can be connected with the same second through hole in an intersecting manner or connected with different second through holes in an intersecting manner;
when the number of the second channels is larger than that of the chambers, one end of the second channel is connected with the chambers, and the other end of the second channel can be intersected and connected with the same second through hole or respectively connected with different second through holes.
According to some embodiments of the invention, the filter membrane is fixed by press-fitting the lower surface of the upper chip and the upper surface of the lower chip.
According to some embodiments of the invention, the lower surface of the upper chip and the upper surface of the lower chip are further provided with silicone films bonded to each other. Further, the bonding may be chemical bonding or thermal bonding. The tightness of the upper chip and the lower chip when the upper chip and the lower chip are tightly attached can be ensured to a greater extent through the bonding effect between the silica gel films.
According to some embodiments of the invention, the upper chip and the lower chip have a thickness of 1 to 10 mm.
According to some embodiments of the present invention, the material of the upper chip and the lower chip includes PDMS, PC, PMMA, COC, PS, and the like.
According to some embodiments of the present invention, the upper chip and the lower chip may be reversibly packaged.
According to some embodiments of the invention, the upper chip and the lower chip are injection molded.
According to some embodiments of the invention, the sample container is a biocompatible material such as PE.
According to some embodiments of the invention, the sample container is open at an upper end.
According to some embodiments of the invention, the diameter of the first channel and the second channel is between 100 μm and 2 mm; preferably, the first and second channels are symmetrically distributed on both sides of the chamber.
According to some embodiments of the invention, the filter membrane is a conical filter membrane.
According to some embodiments of the invention, the filter membrane has a pore size in the range of 1 μm to 100 μm.
According to some embodiments of the invention, the pressure control assembly has a pressure control range of-30 Kpa to 30 Kpa.
According to some embodiments of the present invention, a signal processor is further disposed in the pressure control assembly for receiving signals and issuing commands. When the micro-fluidic chip is filtered and sorted, the air release valve is closed, and the lower cavity of the micro-fluidic chip forms negative pressure under the action of the aspirator. The pressure monitor detects and filters negative pressure in real time, and the signal processor records and makes logic judgment to the acquisition signal, and sends out an instruction to control the working mode of the air escape valve after the judgment condition is satisfied.
According to some embodiments of the present invention, the pressure monitor detects the pressure in the chamber of the microfluidic chip in real time, transmits a pressure signal to the signal processor, and the signal processor records and makes a logic judgment, and sends an instruction to control the operation mode of the air release valve after the judgment condition is satisfied.
According to some embodiments of the invention, the signal logic is judged to be a human being as an edit function, including but not limited to linear, non-linear, fitting, etc. mathematical models. The judgment condition is an artificial editing function, and includes but is not limited to linear, nonlinear, fitting and other mathematical models. The operation modes of the air release valve include, but are not limited to, valve opening, valve closing, valve opening before closing, valve closing before opening, and the like.
According to some embodiments of the invention, the pressure monitor is connected to the bleed line.
According to some embodiments of the invention, the pressure monitor is connected to the first through hole and the release valve through a three-way joint; the air release valve is communicated with the atmosphere. The working mode of the air release valve is controlled, the gas exchange between the micro-fluidic chip cavity and the atmosphere is realized, and the pressure balance between the cavity and the atmosphere is realized.
According to some embodiments of the present invention, the pressure monitor can dynamically detect the pressure of the chamber in the microfluidic chip in real time, so as to achieve the purpose of detecting the filtration pressure of the filter membrane in real time. The dynamic detection can artificially set the detection time interval, and the detection time interval can comprise equidistant time setting and also can comprise non-equidistant time setting. The detection data can be stored in a data end to establish a curve, and can also be used as a judgment basis of a signal processor.
According to some embodiments of the invention, the number of pressure monitors is one or more; preferably, the number of the pressure monitors corresponds to the number of the first through holes.
According to some embodiments of the invention, the number of the relief valves is one or more; preferably, the number of the air escape valves corresponds to the number of the pressure monitors. The air release valve can be connected with the inner spaces of two or more relatively independent microfluidic chips, and the air pressure of the two independent spaces can be balanced by the work of the air release valve.
According to some embodiments of the invention, the aspirator is a syringe pump.
Another aspect of the present invention is to provide a method for separating cells using the above cell separation apparatus, comprising the steps of:
adding a sample into a sample container, opening a pressure monitor, closing a gas escape valve, and starting an aspirator;
the filtrate is pumped out by the aspirator through the chamber and the second channel;
and in the filtering process, the pressure monitor detects the pressure of the cavity in real time, and when the pressure is higher than a preset threshold value, the air escape valve is started to work.
According to some embodiments of the invention, after the filtration is completed, the method further comprises the step of adding a buffer to the sample container to wash the cells of interest.
According to some embodiments of the invention, after the filtration is completed, the air release valve is opened, so that the residual filtrate in the chamber is pumped out.
According to some embodiments of the invention, the release valve is opened when the air pressure rises sharply or exceeds-1 kPa.
According to some embodiments of the invention, the release valve is opened when the air pressure exceeds-1 kPa during the flushing.
It is an object of another aspect of the present invention to provide a use of the cell separation apparatus as described above or the cell separation method as described above for cell separation.
It is an object of another aspect of the present invention to provide a cell separation device as described above or a method of cell separation as described above for use in cell assays.
It is an object of another aspect of the present invention to provide a cell separation device as described above or a method for rare cell separation as described above for use in cell counting.
It is an object of another aspect of the present invention to provide a cell separation device as described above or a method for rare cell separation as described above for use in microorganism separation and microorganism detection.
The invention has at least the following beneficial effects:
the cell separation device provided by the invention can monitor the pressure difference in the micro-fluidic chip in real time, avoid cell selection omission or damage caused by overlarge pressure difference, can quickly and efficiently realize the separation of target cells, improves the efficiency of physical filtration, increases the purity of the separation of the target cells, can also be used for realizing the counting and detection of the cells, and has the advantages of simple method and low cost.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic structural diagram of one embodiment of the present invention;
FIG. 2 is a schematic structural diagram of an upper chip and a lower chip according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of an upper chip and a lower chip according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of an upper chip and a lower chip according to an embodiment of the present invention.
Wherein, the corresponding relation between the reference numbers and the part names in the figure is as follows:
a sample container 1; an aspirator 2; a suction line 21; a pressure control assembly 3; a gas bleed line 31; a pressure monitor 32; a release valve 33; a microfluidic chip 4; an upper chip 41; a filter membrane 42; a lower chip 43; a third through hole 411; a first via 412; a second through hole 413; a chamber 431; a first channel 432; a second channel 433; a third channel 434.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it should be understood that the orientation or positional relationship referred to in the description of the orientation, such as the upper, lower, front, rear, left, right, etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the description of the present invention, the meaning of a plurality of means is one or more, the meaning of a plurality of means is two or more, and larger, smaller, larger, etc. are understood as excluding the number, and larger, smaller, inner, etc. are understood as including the number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, unless otherwise explicitly limited, terms such as arrangement, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.
EXAMPLE 1 cell separation apparatus
As shown in fig. 1 to 4, some embodiments of the present invention provide a cell separation device including: the microfluidic chip 4 comprises an upper chip 41, a filter membrane 42 and a lower chip 43; the aspirator 2 is communicated with the microfluidic chip 4; the pressure control assembly 3 comprises a pressure monitor 32 and an air escape valve 33, and the pressure control assembly 3 is communicated with the microfluidic chip 4; the air release pipeline 31, the air release pipeline 31 communicates with microfluid chip 4, and the pressure control assembly 3 is set up on the air release pipeline 31; wherein, the upper chip 41 is provided with: a first through hole 412 connected to the air escape pipe 31, a second through hole 413 connected to the aspirator 2, a third through hole 411 for connecting to the sample container 1; the lower chip 43 is provided with: a chamber 431 corresponding to the third through hole 411; a first passage 432, one end of the first passage 432 communicating with the first through hole 412 of the upper chip 41, and the other end communicating with the chamber 431; a second channel 432, one end of the second channel 432 is communicated with the second through hole 413 of the upper chip 41, and the other end is communicated with the chamber 431; the filter 42 is disposed between the third through hole 411 of the upper chip 41 and the chamber 431 of the lower chip 43.
When the cell separation device is used, a sample containing cells to be separated is placed into the sample container 1, the pressure monitor 32 is opened, the air release valve 33 is closed, the aspirator 2 is started, the cavity 431, the first channel 432 and the second channel 433 in the lower chip 43 are in a negative pressure state, at the moment, pressure difference exists on two sides of the filter membrane 42, the sample is rapidly filtered under the action of the pressure difference, the rare cells are left in the sample container 1 due to the large volume, and the filtrate is drawn out by the aspirator 2 through the second channel 433, so that rapid separation of the rare cells is realized.
Pressure monitor 32 is used to monitor the real-time differential pressure during the sample separation and detection process and may be used to determine the time node of the process. The air release valve 33 is used for protecting target cells from being leaked and damaged due to overlarge pressure difference, and when the pressure difference exceeds a certain threshold value, the air release valve 33 is automatically opened, so that the target cell capturing efficiency and the activity rate are improved. For example, when the air pressure is steep, the sample in the sample container 1 is almost completely filtered, and the filtrate in the chamber 431 still cannot be drawn under the action of the surface tension. If the aspirator 2 continues to provide negative pressure, the filtrate in the chamber 431 will cause the pressure differential in the chamber 431 to rise sharply until the break-through tension, because the liquid surface tension cannot be broken through. The large pressure differential at this point results in a very severe loss rate and activity of the captured cells of interest. Therefore, it is necessary to open the release valve 33 in time to directly communicate the interior of the microfluidic chip 4 with air, and the excess filtrate in the chamber 431 will flow with the gas from the first channel 432, the chamber 431, the second channel 433 to the aspirator 2, during which the chamber 431 does not have any pressure difference beyond the cell bearing range.
Further, the number of the third through holes 411 is one or more, and may be 1, 2, 3, 4, or 5. Increasing the number of third through holes 411 allows to connect a plurality of sample containers 1, thereby increasing the amount of samples processed each time and improving the efficiency.
Further, the third through hole 411 may be hermetically coupled to the sample container 1, and preferably, the third through hole 411 is threadedly coupled to the sample container 1.
Further, the number of the chambers 431 corresponds to the number of the third through holes 411. The chamber 431 is a buffer zone where the filtrate is drawn off via the aspirator 2, the depth of which can be determined according to the actual situation.
Further, the number of the first through holes 412 is one or more, and may be 1, 2, 3, 4, or 5, and the number thereof may be the same as or different from the number of the third through holes 411. The first through hole 412 connects the inside of the microfluidic chip 4 and the pressure control assembly 3 together, so that the monitoring and the regulation of the pressure inside the microfluidic chip 4 are realized.
Further, the air release pipe 31 is connected to the first through hole 412 by a luer lock.
Further, the number of the second through holes 413 is one or more, and may be 1, 2, 3, 4, or 5, and the number thereof may be the same as or different from the number of the third through holes 411. The second through hole 413 connects the inside of the microfluidic chip 4 with the aspirator 2, and the filtrate is extracted from the microfluidic chip 4 through the second through hole 413.
Further, the second through hole 413 is connected to the aspirator 2 through the suction line 21.
Further, the second through hole 413 is connected to the suction line 21 by a luer lock.
Further, the number of the first passages 432 is one or more, and may be 1, 2, 3, 4, or 5, etc. The number of first channels 432 may or may not be the same as the number of chambers 431; when the number of the first channels 432 is smaller than that of the chambers 431, the chambers 431 can be connected through the third channel 434, for example, when the number of the first channels 432 is 1 and the number of the chambers 431 is 2, the first channel 432 can be connected with one of the chambers 431, the two chambers 431 are connected through the third channel 434, and the other chamber 431 is connected with the second channel 433, so that the intercommunication inside the microfluidic chip 4 is realized. When the number of the first passages 432 is equal to the number of the chambers 431, for example, when the number of the first passages 432 is 2 and the number of the chambers 431 is 2, one end of each of the first passages 432 is connected to two of the chambers 431, and the other end may intersect and connect the same first through hole 412, or may intersect and connect two first through holes 412. When the number of the first passages 432 is greater than the number of the chambers 431, for example, when the number of the first passages 432 is 2 and the number of the chambers 431 is 1, one end of the first passage 432 is connected to the chambers 431, and the other end may intersect and connect with the same first through hole 412, or may intersect and connect with two first through holes 412.
Further, the number of the second channels 433 is one or more, and may be 1, 2, 3, 4, or 5, etc. The number of the second channels 433 may or may not be the same as the number of the chambers 431; when the number of the second channels 433 is less than that of the chambers 431, the chambers 431 may be connected through a third channel 434, for example, when the number of the second channels 433 is 1 and the number of the chambers 431 is 2, the second channel 433 may be connected to one of the chambers 431, the two chambers 431 are connected through the third channel 434, and the other chamber 431 is connected to the first channel 432, so as to achieve the intercommunication inside the microfluidic chip 4. When the number of the second channels 433 is equal to the number of the chambers 431, for example, when the number of the second channels 433 is 2 and the number of the chambers 431 is 2, one end of each of the second channels 433 is connected to two of the chambers 431, and the other end may intersect and connect to the same second through hole 413, or may intersect and connect to two second through holes 413. When the number of the second channels 433 is greater than the number of the chambers 431, for example, when the number of the second channels 433 is 2 and the number of the chambers 431 is 1, one end of the second channel 433 is connected to the chambers 431, and the other end may intersect and connect with the same second through hole 413, or may intersect and connect with two second through holes 413.
Further, the filter 42 is fixed by press-fitting the lower surface of the upper chip 41 and the upper surface of the lower chip 43.
Further, the lower surface of the upper chip 41 and the upper surface of the lower chip 43 are also provided with silicone films bonded to each other. Further, the bonding may be chemical bonding or thermal bonding. The sealing performance of the upper chip 41 and the lower chip 43 when they are closely adhered can be ensured to a greater extent by the bonding action between the silicone films.
Further, the thickness of the upper chip 41 and the lower chip 43 is 1 to 10 mm.
Further, the material of the upper chip 41 and the lower chip 43 includes PDMS, PC, PMMA, COC, PS, and the like.
Further, the upper chip 41 and the lower chip 43 may be reversibly packaged.
Further, the upper chip 41 and the lower chip 43 are injection molded.
Further, the sample container 1 is of a biocompatible material, such as PE. Further, the sample container 1 is open at the upper end.
Further, the diameter of the first channel 432 and the second channel 433 is 100 μm to 2 mm; preferably, the first channel 432 and the second channel 433 are symmetrically distributed on both sides of the chamber 431.
Further, the filter membrane 42 is a conical filter membrane; the pore size of the filter membrane 42 ranges from 1 μm to 100. mu.m.
Further, the pressure control range of the pressure control assembly 3 is-30 Kpa to 30 Kpa.
Further, a signal processor for receiving signals and issuing commands is also arranged in the pressure control assembly 3. When the microfluidic chip 4 is filtered and sorted, the air escape valve 33 is closed, and the chamber 431 of the microfluidic chip 4 forms negative pressure under the action of the aspirator. The pressure monitor 32 detects the filtering negative pressure in real time, the signal processor records the acquired signal and makes logic judgment, and the signal processor sends an instruction to control the working mode of the air escape valve 33 after the judgment condition is satisfied.
Further, the pressure monitor 32 detects the pressure in the chamber 431 of the microfluidic chip 4 in real time, transmits a pressure signal to the signal processor, the signal processor records and makes a logic judgment, and sends an instruction to control the working mode of the air release valve 33 after the judgment condition is satisfied. The operation of the bleed valve 33 includes, but is not limited to, opening, closing, opening before closing, closing before opening, and the like.
Further, the signal logic is judged to be a human being as an editing function, including but not limited to linear, non-linear, fitting, etc. mathematical models. The judgment condition is an artificial editing function, including but not limited to linear, nonlinear, fitting and other mathematical models.
Further, a pressure monitor 32 is connected to the bleed air line 31. The pressure monitor 32 is respectively connected with the first through hole 412 and the air release valve 33 through a three-way joint; the air release valve 33 is open to the atmosphere. And controlling the working mode of the air release valve 33 to realize the gas exchange between the chamber 431 in the microfluidic chip 4 and the atmosphere, so that the pressure of the chamber 431 and the atmosphere is balanced.
Further, the pressure monitor 32 can dynamically detect the pressure in the chamber 431 of the microfluidic chip 4 in real time, so as to achieve the purpose of detecting the pressure filtered by the filter membrane in real time. The dynamic detection can artificially set the detection time interval, and the detection time interval can comprise equidistant time setting and also can comprise non-equidistant time setting. The detection data can be stored in a data end to establish a curve, and can also be used as a judgment basis of a signal processor.
Further, the number of pressure monitors 32 is one or more; preferably, the number of pressure monitors 32 corresponds to the number of first through holes 412.
Further, the number of the air escape valves 33 is one or more; preferably, the number of relief valves 33 corresponds to the number of pressure monitors 32. The air release valve 33 can be connected with the internal spaces of two or more relatively independent microfluidic chips 4, and the air pressure of the two independent spaces can be balanced by the work of the air release valve 33.
Further, the aspirator 2 is a syringe pump.
Example 2A method for cell separation Using the cell separation apparatus of example 1
Opening the pressure monitor 32, closing the air escape valve 33, adding the sample into the sample container 1, and starting the aspirator 2;
filtrate is drawn out by the aspirator 2 via the chamber 431, the second channel 433;
and in the filtering process, the pressure monitor detects the pressure of the cavity in real time, and when the pressure is higher than a preset threshold value, the air escape valve is started to work.
Taking the isolation of circulating tumor cells as an example:
adding a sample into a sample container, opening a pressure monitor, closing a gas escape valve, and starting an aspirator;
the filtrate is pumped out by the aspirator through the chamber and the second channel, and when the air pressure value rises sharply or exceeds-1 kPa, the air escape valve is opened;
then, adding PBS buffer solution into the sample container to wash the separated circulating tumor cells, and opening the air escape valve when the air pressure value is steeply increased or exceeds-1 kPa;
example 3 some working procedures of the cell separation apparatus of example 1
1. And (3) infiltration process:
opening the pressure monitor 32, closing the air release valve 33, adding 2mL PBS into the sample container 1, starting the aspirator 2 to extract liquid (speed 1mL/min), forcibly stopping the aspirator 2 after 1min of liquid extraction, opening the air release valve 33, waiting for 30s, and closing the air release valve 33.
2. And (3) a capturing process:
adding a sample (5mL of whole blood +5mLISET) into the sample container 1, pumping liquid (500 mu L/min) by the aspirator 2, recording the highest pressure value Pmax of the end of the air escape valve 33 within 18min, opening the air escape valve 33 when judging that the pressure value of the end of the air escape valve 33 is less than-1 Kpa, waiting for 30s, forcibly stopping the aspirator 2, and closing the air escape valve 33.
3. A capture cleaning process:
adding 2ml PBS into the sample container 1, sucking liquid (500 mu L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value of the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, closing the air release valve 33, and repeatedly cleaning for 2 times.
4. The pretreatment process comprises the following steps:
adding 150 mu L of the pretreatment solution into the sample container 1, incubating the pretreatment solution for 10min, pumping the solution (200 mu L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
5. The pretreatment cleaning process comprises the following steps:
adding 300 μ L PBS into the sample container 1, sucking the liquid (200 μ L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
6. And (3) a sealing process:
adding 150 mu L of sealing liquid into the sample container 1, carrying out sealed incubation for 30min, pumping the liquid (200 mu L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
7. Antibody process:
the aspirator 2 firstly pumps 500 μ L of air (ensuring that the antibody is not diluted), then 170 μ L of the antibody is added into the sample container 1, the antibody is incubated for 60min, the aspirator 2 pumps liquid (200 μ L/min) through the second through hole 413, when the pressure value at the end of the air escape valve 33 is judged to be less than-1 Kpa, the air escape valve 33 is opened, the time is waited for 5s, the aspirator 2 is forcibly stopped, and the air escape valve 33 is closed.
8. And (3) antibody cleaning process:
adding 300 μ L PBS into the sample container 1, sucking the liquid (200 μ L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
9. The nucleus dyeing process:
adding 150 mu L of the nuclear staining reagent into the sample container 1, incubating for 5min, sucking liquid (200 mu L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
10. Dyeing and cleaning the core:
adding 300 μ L PBS into the sample container 1, sucking the liquid (200 μ L/min) by the aspirator 2 through the second through hole 413, opening the air release valve 33 when the pressure value at the end of the air release valve 33 is judged to be less than-1 Kpa, waiting for 5s, forcibly stopping the aspirator 2, and closing the air release valve 33.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A cell separation device, comprising:
the microfluidic chip comprises an upper chip, a filter membrane and a lower chip;
an aspirator in communication with the microfluidic chip;
the pressure control assembly comprises a pressure monitor and an air escape valve, and is communicated with the microfluidic chip;
the air release pipeline is communicated with the microfluidic chip, and the pressure control assembly is arranged on the air release pipeline;
the upper chip of the microfluidic chip is provided with: the first through hole is connected with the air leakage pipeline, the second through hole is connected with the aspirator, and the third through hole is connected with a sample container;
the lower chip of the microfluidic chip is provided with: a chamber corresponding to the third through-hole; one end of the first channel is communicated with the first through hole of the upper chip, and the other end of the first channel is communicated with the cavity; one end of the second channel is communicated with the second through hole of the upper chip, and the other end of the second channel is communicated with the cavity;
the filter membrane is arranged between the third through hole of the upper layer chip and the cavity of the lower layer chip.
2. The cell separation device according to claim 1, wherein the number of the third through holes is one or more; the number of the chambers corresponds to the number of the third through holes.
3. The cell separation device according to claim 1, wherein the number of the first channels is one or more;
when the number of the first channels is smaller than that of the chambers, the first channels are connected with one of the chambers, the chambers are communicated through a third channel, and the second channels are connected with one of the chambers, so that the intercommunication inside the microfluidic chip is realized;
when the number of the first channels is the same as that of the chambers, one end of each first channel is connected with the chamber, and the other end of each first channel can be connected with the same first through hole in an intersecting manner or connected with different first through holes in an intersecting manner;
when the number of the first channels is larger than that of the chambers, one end of the first channel is connected with the chambers, and the other end of the first channel can be intersected and connected with the same first through hole or respectively connected with different first through holes.
4. The cell separation device according to claim 1, wherein the number of the second channels is one or more;
when the number of the second channels is smaller than that of the chambers, the second channels are connected with one of the chambers, the chambers are communicated through a third channel, and the first channels are connected with one of the chambers, so that the intercommunication inside the microfluidic chip is realized;
when the number of the second channels is consistent with that of the chambers, one end of each second channel is connected with the chamber, and the other end of each second channel can be connected with the same second through hole in an intersecting manner or connected with different second through holes in an intersecting manner;
when the number of the second channels is larger than that of the chambers, one end of the second channel is connected with the chambers, and the other end of the second channel can be intersected and connected with the same second through hole or respectively connected with different second through holes.
5. The cell separation device according to any one of claims 1 to 4, wherein the lower surface of the upper chip and the upper surface of the lower chip are further provided with silicone films bonded to each other; preferably, the microfluidic upper chip and the microfluidic lower chip can be reversibly packaged.
6. The cell separation device according to any one of claims 1 to 4, wherein a signal processor for receiving signals and issuing commands is further provided in the pressure control assembly; preferably, the pressure monitor detects the pressure in the cavity of the microfluidic chip in real time, transmits a pressure signal to the signal processor, records and makes a logic judgment by the signal processor, and sends an instruction to control the working mode of the air escape valve after the judgment condition is satisfied.
7. The cell separation apparatus according to claims 1 to 4, wherein the pressure monitor is connected to the first through hole and the air escape valve through a three-way joint, respectively; the air release valve is communicated with the atmosphere.
8. The cell separation apparatus according to claims 1 to 4, wherein the number of the pressure monitors is one or more; preferably, the number of the pressure monitors is consistent with the number of the first through holes; more preferably, the number of the pressure monitors is consistent with the number of the air escape valves.
9. The method for cell separation by the cell separation device according to any one of claims 1 to 8, comprising the steps of:
adding a sample into a sample container, opening a pressure monitor, closing a gas escape valve, and starting an aspirator;
the filtrate is pumped out by the aspirator through the chamber and the second channel;
and in the filtering process, the pressure monitor detects the pressure of the cavity in real time, and when the pressure is higher than a preset threshold value, the air escape valve is started to work.
10. Use of the cell separation device according to any one of claims 1 to 8 for cell separation, cell detection, cell counting, microorganism separation, and microorganism detection.
CN202110109729.2A 2021-01-27 2021-01-27 Cell separation device, cell separation method and application Pending CN112795478A (en)

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Application publication date: 20210514