CN113637637B - Method for efficiently separating, capturing and recovering rare cells in whole blood - Google Patents
Method for efficiently separating, capturing and recovering rare cells in whole blood Download PDFInfo
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- CN113637637B CN113637637B CN202110788130.6A CN202110788130A CN113637637B CN 113637637 B CN113637637 B CN 113637637B CN 202110788130 A CN202110788130 A CN 202110788130A CN 113637637 B CN113637637 B CN 113637637B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/20—Recycling
Abstract
The invention discloses a method for efficiently separating, capturing and recovering rare cells in whole blood, which comprises the following steps: the channel pipe is provided with a first pipe orifice and a second pipe orifice; a first inlet in communication with the first nozzle; a second inlet in communication with the first nozzle; a third inlet in communication with the second nozzle; an outlet in communication with the second nozzle; the target liquid channel is arranged in the channel pipe and is communicated with the first pipe orifice, a side liquid channel is formed between the channel pipe wall of the target liquid channel and the inner wall of the channel pipe in a surrounding mode, the side liquid channel is communicated with the second pipe orifice, a plurality of micropores communicated with the side liquid channel are formed in the channel wall of the target liquid channel, and the aperture of each micropore is smaller than the volume of a target cell. Step S1, separating and enriching target cells; step S2, flushing target cells; and S3, recovering target cells. And the separation, the capturing and the recovery of the target cells are completed rapidly.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to a method for efficiently separating, capturing and recycling rare cells in whole blood.
Background
The separation and enrichment of specific rare cells in blood has important medical value, such as the great potential of stem cells in the fields of treating different types of diseases, medical science and the like; the counting and characteristic analysis of the Circulating Tumor Cells (CTCs) can be used as important basis for breast cancer metastasis detection, recurrence monitoring, curative effect and personalized treatment prognosis judgment, etc. But the stem cells and Circulating Tumor Cells (CTCs) in blood are extremely small, the CTCs content is 1/100 ten thousand of that of blood cells, and the stem cells content only accounts for 0.01% of that of blood cells. In order to realize large-scale application of rare cells such as stem cells and CTCs, the rare cells need to be rapidly and efficiently isolated from whole blood.
At present, the separation of rare cells in blood is realized by adopting a microfluidic technology, and the separation is mainly divided into active separation and passive separation. Compared with the active separation mode which utilizes an additional dynamic field (electric field, magnetic field, sound wave and light) to realize cell separation, the passive separation mode which utilizes a design structure and fluid dynamics to realize separation has the advantages of no additional force field and additional control system, easy manufacture and assembly, simple and easy operation of the system and the like, and is widely applied. The filter microfluidic chip based on the size separation of target cells is the simplest to design and relatively flexible to use, and is a commonly used separation mode at present.
At present, a microfluidic chip based on filtration mainly comprises four modes of a weir, a column, cross flow and a membrane, but the microfluidic chip is easy to be blocked, the subsequent separation efficiency can be influenced after the blockage, rare cells are difficult to capture and analyze, and meanwhile, the efficient recovery of the filtration target cells in the chip is difficult to realize.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a method for efficiently separating, capturing and recovering rare cells in whole blood, which can efficiently separate, capture and recover target cells in whole blood.
A method for efficient separation, capture and recovery of rare cells in whole blood according to an embodiment of the first aspect of the present invention comprises:
the channel pipe is provided with a first pipe orifice and a second pipe orifice;
a first inlet in communication with the first nozzle;
a second inlet in communication with the first nozzle;
a third inlet in communication with the second nozzle;
an outlet in communication with the second nozzle;
the target liquid channel is arranged in the channel pipe and is communicated with the first pipe orifice, a lateral liquid channel is formed between the channel pipe wall of the target liquid channel and the inner wall of the channel pipe in a surrounding mode, the lateral liquid channel is communicated with the second pipe orifice, a plurality of micropores communicated with the lateral liquid channel are formed in the channel wall of the target liquid channel, and the aperture of each micropore is smaller than the volume of a target cell;
the method comprises the following steps:
step S1, separating and enriching target cells, wherein the second inlet and the third inlet are closed, the first inlet and the outlet are opened, a sample liquid is injected from the first inlet, filtrate is discharged from the outlet, and the target cells are captured in the target liquid channel to obtain primary target cells;
s2, flushing the target cells, wherein the first inlet and the third inlet are closed, the second inlet and the outlet are opened, flushing fluid is injected from the second inlet, and the flushing fluid is discharged from the outlet to finish flushing the target cells;
and S3, recovering the target cells, wherein the second inlet and the outlet are closed, the first inlet and the third inlet are opened, reverse flushing liquid is injected from the third inlet, and the reverse flushing liquid is mixed with the target cells of the target liquid channel and flows out of the first inlet, so that the target cells are obtained.
The chip for separating, capturing and recovering rare cells in whole blood according to the embodiment of the invention has at least the following beneficial effects:
when the target cells are separated and enriched, the second inlet and the third inlet are closed, the sample liquid flows into the target liquid channel through the first inlet and the first pipe orifice, waste liquid (liquid, background cells with smaller size and the like) in the sample liquid flows into the side liquid channel through the micropores, and then flows out through the second pipe orifice and the outlet, so that the background cells in the sample are removed efficiently, the target cells are captured in the target liquid channel, and primary target cells are obtained.
And then the first inlet is closed, the second inlet is opened, forward flushing liquid flows into the target liquid channel through the first inlet and the first pipe orifice, primary target cells in the target liquid channel are flushed, flushing waste liquid flows into the side liquid channel through the micropores, and then flows out through the second pipe orifice and the outlet, so that target cells are obtained.
When the target cells are recovered, the second inlet and the outlet are closed, the reverse flushing liquid flows into the side liquid channel and the target liquid channel after passing through the third inlet and the second pipe orifice, the reverse flushing liquid enters the target liquid channel through the micropores, and the target cells flow out through the first pipe orifice and the first inlet, so that the target cells are obtained.
According to some embodiments of the invention, the target liquid channel comprises a plurality of layers of channel walls, secondary target liquid channels are arranged between the channel walls, the micropores are arranged on each layer of channel walls, and the size of the micropores on the secondary target liquid channel is equal to or smaller than that of the micropores on the previous stage of the secondary target liquid channel, or is smaller than or equal to that of the micropores on the previous stage of the target liquid channel.
According to some embodiments of the invention, the channel wall is surrounded by a first micro-column array, the first micro-column array is formed by arranging a plurality of micro-columns at intervals, and the micro-holes are formed between the micro-columns at intervals.
According to some embodiments of the invention, the microcolumn is a rounded square microcolumn, an axis of the rounded square microcolumn is perpendicular to a line connecting the first pipe orifice and the second pipe orifice, and an included angle between a bottom edge of the rounded square microcolumn and the line connecting the first pipe orifice and the second pipe orifice is 45 degrees.
According to some embodiments of the invention, a second micro-column array is arranged in the target liquid channel, and the second micro-column array is formed by arranging a plurality of micro-columns at intervals.
According to some embodiments of the invention, the tube diameter of the secondary target liquid channel of the target liquid channel gradually decreases from the first tube orifice to the second tube orifice.
According to some embodiments of the invention, the tube diameter of the lateral liquid channel gradually decreases from the first tube orifice to the second tube orifice.
According to some embodiments of the invention, the first nozzle is connected with the inlet of the target liquid channel and the front end of the side liquid channel in a tangent curve;
the second pipe orifice is connected with the tail end of the target liquid channel and the outlet of the side liquid channel in a tangent curve.
According to some embodiments of the invention, the number of the channel pipes is a plurality, the channel pipes are arranged side by side, and each channel pipe is internally provided with the target liquid channel and the lateral liquid channel.
According to some embodiments of the invention, the channel tube is made of polydimethylsiloxane or plexiglass.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of a chip structure for efficient separation, capture and recovery of rare cells in whole blood according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing a connection structure between a channel pipe and a target liquid channel and a side liquid channel according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing a connection structure of a channel pipe and a multi-stage target liquid channel and a side liquid channel according to an embodiment of the present invention;
FIG. 4 is an enlarged schematic view of the portion A in FIG. 2;
fig. 5 is an enlarged schematic view of the portion B in fig. 2.
Reference numerals:
100. a channel tube; 110. a first nozzle; 120. a second nozzle; 200. a first inlet; 300. a second inlet; 400. a third inlet; 500. an outlet; 600. a target liquid channel; 610. a lateral liquid channel; 620. micropores; 630. a microcolumn; 640. a secondary target fluid channel; 700. and a second array of micropillars.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, it should be understood that references to orientation descriptions such as upper, lower, front, rear, left, right, etc. are based on the orientation or positional relationship shown in the drawings, are merely for convenience of description of the present invention and to simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the present invention.
In the description of the present invention, a number means one or more, a number means two or more, and greater than, less than, exceeding, etc. are understood to not include the present number, and above, below, within, etc. are understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed 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 explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present invention can be reasonably determined by a person skilled in the art in combination with the specific contents of the technical scheme.
In addition, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the technical solutions, and when the technical solutions are contradictory or cannot be implemented, the combination of the technical solutions should be considered as not existing, and not falling within the scope of protection claimed by the present invention.
As shown in fig. 1 and 2, a method for efficient separation, capture and recovery of rare cells in whole blood according to an embodiment of the present invention includes:
a channel tube 100, the channel tube 100 being provided with a first nozzle 110 and a second nozzle 120;
a first inlet 200 in communication with first nozzle 110;
a second inlet 300 in communication with first nozzle 110;
a third inlet 400 in communication with the second nozzle 120;
an outlet 500 in communication with the second nozzle 120;
the target liquid channel 600, the target liquid channel 600 is arranged in the channel tube 100, the target liquid channel 600 is communicated with the first tube orifice 110, a side liquid channel 610 is enclosed between the channel tube wall of the target liquid channel 600 and the inner wall of the channel tube 100, the side liquid channel 610 is communicated with the second tube orifice 120, a plurality of micropores 620 communicated with the side liquid channel 610 are arranged on the channel wall of the target liquid channel 600, and the aperture of the micropores 620 is smaller than the volume of the target cells;
the method comprises the following steps:
step S1, separating and enriching target cells, wherein a second inlet 300 and a third inlet 400 are closed, a first inlet 200 and an outlet 500 are opened, a sample liquid is injected from the first inlet 200, filtrate is discharged from the outlet 500, and the target cells are captured in a target liquid channel 600 to obtain primary target cells;
step S2, flushing the target cells, wherein the first inlet 200 and the third inlet 400 are closed, the second inlet 300 and the outlet 500 are opened, flushing fluid is injected from the second inlet 300, and the flushing fluid is discharged from the outlet 500, so that the flushing of the target cells is completed;
in step S3, the target cells are recovered, the second inlet 300 and the outlet 500 are closed, the first inlet 200 and the third inlet 400 are opened, the reverse flushing fluid is injected from the third inlet 400, and the reverse flushing fluid is mixed with the target cells in the target fluid channel 600 and flows out from the first inlet 200, thereby obtaining the target cells.
Specifically, when the target cells are separated and enriched, the second inlet 300 and the third inlet 400 are closed, the sample liquid flows into the target liquid channel 600 through the first inlet 200 and the first nozzle 110, the waste liquid in the sample liquid, the background cells with smaller size and the like flow into the lateral liquid channel 610 through the micropores 620, and then flow out through the second nozzle 120 and the outlet 500, so that the background cells in the sample are removed efficiently, and the target cells are captured in the target liquid channel 600, and the primary target cells are obtained.
Then the first inlet 200 is closed, the second inlet 300 is opened, forward flushing liquid flows into the target liquid channel 600 through the second inlet 300 and the first pipe orifice 110, primary target cells in the target liquid channel 600 are flushed, flushing waste liquid flows into the side liquid channel 610 through the micropores 620, and then flows out through the second pipe orifice 120 and the outlet 500, so that flushing of the target cells is completed.
When the target cells are recovered, the second inlet 300 and the outlet 500 are closed, and the reverse flushing liquid flows into the lateral liquid channel 610 through the third inlet 400 and the second nozzle 120, and the reverse flushing liquid enters the target liquid channel 600 through the micro-holes 620, so that the target cells in the target liquid channel 600 flow out through the first nozzle 110 and the first inlet 200, thereby obtaining the target cells.
Specifically, the chip of this embodiment includes a first chip board and a second chip board, where the surface of the first chip board is a groove with a consistent pattern of the channel tube 100 and the target liquid channel 600, and one surface of the second chip board is a smooth surface, and the second chip board is covered on the groove of the first chip board to jointly enclose the closed channel tube 100 and the target liquid channel 600.
In an exemplary embodiment, a concentration of mouse embryonic fibroblast cell line 3T3 is introduced from the first inlet 200 at a rate of 0.2mL/hr, the second inlet 300 is closed, the sample fluid fills the target fluid channel 600, and the fluid is then exuded through the microwells 620. In this process, individual 3T3 cells are gradually trapped at microwells 620. Subsequently, when all the microwells 620 capture single cells, 3T3 cells gradually accumulate at the microwells 620, gradually capture target cells, and the capture effect is greater than 80%.
In the case of a two-stage target liquid channel, the separation effect is greater than 90%. Subsequently, the reverse flushing fluid was introduced at a rate of 0.2mL/hr from the third inlet, allowing rapid recovery of 3T3 cells, up to 90% recovery.
The whole blood of the mouse is introduced from the first inlet 200 at a rate of 0.2mL/hr, the second inlet 300 is closed, the sample fluid fills the target fluid channel 600, and then the fluid is oozed out through the micro-holes 620. And in this process, more than 99% of the blood cells ooze out of the micropillar array, with only very individual larger cells captured to microwells 620.
Compared with the existing microfluidic separation and filtration platform for rare cells in blood, the separation and capture rate of the invention is up to 90%, and the recovery rate is 90%. The single cell can be captured for subsequent single cell analysis, the separation efficiency is 72% compared with the existing microfluidic filter platform, and the single cell capture and the rear end target cell online processing analysis cannot be realized. The recovery efficiency is obviously improved, and single cells can be captured for subsequent online analysis.
As shown in fig. 3, in some embodiments of the present invention, the target liquid channel 600 includes a plurality of channel walls, between which secondary target liquid channels 640 are provided, and each of the channel walls has micropores 620, and the size of the micropores in the secondary target liquid channel 640 is equal to or smaller than the size of the micropores in the secondary target liquid channel 640 of the previous stage, or is smaller than or equal to the size of the micropores in the target liquid channel 600 of the previous stage.
Specifically, when the chip has only one secondary target fluid channel 640, the secondary target fluid channel 640 of the next stage is smaller than or equal to the size of the micro-hole in the target fluid channel 600 of the previous stage. When a plurality of secondary target fluid passages 640 are included, the first secondary target fluid passage 640 adjacent to the target fluid passage 600 is less than or equal to the size of the micropores in the target fluid passage 600. And the subsequent secondary target fluid passage 640 has a pore size smaller than or equal to the secondary target fluid passage 640 of the previous stage.
The channel walls of the target liquid channel may be replaced with 2 or more layers, the channel walls enclosing the target liquid channel, the second channel wall and the first channel wall enclosing the secondary target liquid channel 640, and the nth channel wall and the n-1 th channel wall enclosing the n-1 th channel wall.
When the target cells are separated and enriched, the second inlet 300 and the third inlet 400 are closed, the sample liquid flows into the target liquid channel 600 through the first inlet 200 and the first pipe orifice 110, the waste liquid in the sample liquid, the background cells with smaller size and the like flow into the side liquid channel 610 through the micropores 620 and then flow out through the second pipe orifice 120 and the outlet 500, and the efficient removal of the background cells in the sample is completed, so that the target cells are captured in the target liquid channel 600, and the primary target cells are obtained.
The waste liquid flows into the next secondary target liquid channel 640 through the micropores 620 on the channel wall, further capturing the overflow target cells until the waste liquid flows into the lateral liquid channel 610 through the last channel wall, and obtaining the primary target cells. And then repeating the flushing and back flushing to finally obtain the target cells.
The method adopts a multi-stage channel filtration mode to further capture the waste liquid containing a small amount of exuded target cells. Filtering the waste liquid, capturing target cells exuded by the superior target liquid channel due to overlarge pressure, improving capturing efficiency, and further, between the adjacent channel walls, the pore diameter of the micropores 620 of the next layer of channel wall is smaller than that of the micropores 620 of the previous layer, so that capturing efficiency can be further improved.
As shown in fig. 3 in combination with fig. 4 and 5, in some embodiments of the present invention, the channel wall is surrounded by a first micro-pillar array, and the first micro-pillar array is formed by arranging a plurality of micro-pillars 630 at intervals, and micro-holes 620 are formed between the micro-pillars 630 at intervals.
Further, the microcolumn 630 is a rounded square microcolumn, the axis of the rounded square microcolumn is perpendicular to the line connecting the first nozzle 110 and the second nozzle 120, and the included angle between the bottom edge of the rounded square microcolumn and the line connecting the first nozzle 110 and the second nozzle 120 is 45 degrees.
The smaller the aspect ratio between the two microcolumns 630 and the gap, the easier the low aspect ratio liquid will enter the side liquid channel through the target liquid channel (depending on the wetting and hysteresis of the fluid on the rough solid surface), so the design can avoid the problem that the liquid is difficult to exude due to the larger aspect ratio (relative to the fluid direction). The square spacing of the round corners can be adjusted according to the size of target cells, and the sizes of the square corners and the round corners can be adjusted according to the machining precision.
In some embodiments of the present invention, a second micro-column array 700 is disposed in the target fluid channel 600, and the second micro-column array 700 is formed by arranging a plurality of micro-columns 630 at intervals.
The second micro-column arrays 700 having the same pitch and the same size are disposed at the middle position of the target liquid channel 600 to prevent the supporting effect of the target liquid channel 600. And changing the direction of fluid in the target fluid channel 600, to facilitate the exudation of waste fluid from the interstices of the microcolumn 630.
In some embodiments of the present invention, the tube diameters of the target fluid channel 600 and the secondary target fluid channel 640 gradually decrease from the first nozzle 110 to the second nozzle 120.
The pipe diameter of the target liquid channel 600 is continuously reduced, and the pipe pressure is continuously increased, so that the seepage speed of the micropores 620 and the reverse flushing liquid entering the target liquid channel 600 through the gap are increased. Specifically, the target liquid channel 600 is surrounded by a micro-column array to form a pipeline with a smaller pipe diameter.
In some embodiments of the present invention, the tube diameter of lateral fluid channel 610 decreases gradually from first tube orifice 110 to second tube orifice 120. Likewise, the decreasing tube diameter of the lateral fluid channel 610 helps to increase the rate of permeate out of the micro-holes 620 and reverse rinse fluid flow through the gap into the target fluid channel 600.
In some embodiments of the present invention, first nozzle 110 is connected to the inlet of target fluid channel 600 and the front end of side fluid channel 610 in a tangential curve;
the second nozzle 120 is connected to the tail end of the target liquid passage 600 and the outlet 500 of the side liquid passage 610 in a tangential curve. The resistance to inflow of liquid can be reduced, facilitating the outflow of waste liquid out of the microcolumn gap and the entry of backflushing liquid through the gap into the target liquid channel 600.
As shown in fig. 1, the number of the channel pipes 100 is plural, the channel pipes 100 are arranged side by side, and each channel pipe 100 is provided with a target liquid channel 600 and a side liquid channel 610.
In this embodiment, a plurality of channel pipes 100 are provided, the channel pipes 100 are arranged side by side, each channel pipe 100 is provided with a target liquid channel 600 and a side liquid channel 610, and the first inlet 200, the second inlet 300, the third inlet 400, the outlet 500 and each channel pipe 100 are all communicated, so that the separation efficiency is geometrically multiplied.
In some embodiments of the invention, the channel tube 100 is made of polydimethylsiloxane or plexiglass. Polydimethylsiloxane (PDMS) is a polymeric organosilicon compound, commonly referred to as a silicone. Has the characteristics of optical transparency, inertness, innocuity and nonflammability. The organic glass has the advantages of better transparency, chemical stability, mechanical property, weather resistance, easy dyeing, easy processing, beautiful appearance and the like.
The device for separating, capturing and recovering the rare cells in the whole blood can efficiently and rapidly finish the separation, capturing and recovering of the target cells, and has high recovery rate.
In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (5)
1. A method for efficient separation, capture and recovery of rare cells in whole blood, comprising:
the channel pipe is provided with a first pipe orifice and a second pipe orifice;
a first inlet in communication with the first nozzle;
a second inlet in communication with the first nozzle;
a third inlet in communication with the second nozzle;
an outlet in communication with the second nozzle;
the target liquid channel is arranged in the channel pipe and is communicated with the first pipe orifice, a lateral liquid channel is formed between the channel pipe wall of the target liquid channel and the inner wall of the channel pipe in a surrounding mode, the lateral liquid channel is communicated with the second pipe orifice, a plurality of micropores communicated with the lateral liquid channel are formed in the channel wall of the target liquid channel, and the aperture of each micropore is smaller than the volume of a target cell;
the method comprises the following steps:
step S1, separating and enriching target cells, wherein the second inlet and the third inlet are closed, the first inlet and the outlet are opened, a sample liquid is injected from the first inlet, filtrate is discharged from the outlet, and the target cells are captured in the target liquid channel to obtain primary target cells;
s2, flushing the target cells, wherein the first inlet and the third inlet are closed, the second inlet and the outlet are opened, flushing fluid is injected from the second inlet, and the flushing fluid is discharged from the outlet to finish flushing the target cells;
s3, recovering target cells, wherein the second inlet and the outlet are closed, the first inlet and the third inlet are opened, reverse flushing liquid is injected from the third inlet, and the reverse flushing liquid is mixed with the target cells of the target liquid channel and flows out from the first inlet, so that the target cells are obtained;
the channel wall is surrounded by a first micro-column array, the first micro-column array is formed by arranging a plurality of micro-columns at intervals, and micropores are formed among the micro-columns at intervals; the micro-column is a round-corner square micro-column, the axis of the round-corner square micro-column is perpendicular to the connecting line of the first pipe orifice and the second pipe orifice, and the included angle between the bottom edge of the round-corner square micro-column and the connecting line of the first pipe orifice and the second pipe orifice is 45 degrees; the pipe diameter of the target liquid channel is gradually reduced from the first pipe orifice to the second pipe orifice; the pipe diameter of the side liquid channel is gradually reduced from the first pipe orifice to the second pipe orifice; the first pipe orifice is connected with the inlet of the target liquid channel and the front end of the side liquid channel in a tangent curve; the second pipe orifice is connected with the tail end of the target liquid channel and the outlet of the side liquid channel in a tangent curve.
2. The method for efficient separation, capture and recovery of rare cells in whole blood of claim 1, wherein: the target liquid channel comprises a plurality of layers of channel walls, secondary target liquid channels are arranged between the channel walls, micropores are arranged on each layer of channel wall, and the size of each micropore on the secondary target liquid channel is equal to or smaller than that of the corresponding secondary target liquid channel or is smaller than or equal to that of the corresponding target liquid channel of the previous stage.
3. The method for efficient separation, capture and recovery of rare cells in whole blood of claim 1, wherein: and a second micro-column array is arranged in the target liquid channel and consists of a plurality of micro-columns which are arranged at intervals.
4. The method for efficient separation, capture and recovery of rare cells in whole blood of claim 1, wherein: the number of the channel pipes is multiple, the channel pipes are arranged side by side, and each channel pipe is internally provided with the target liquid channel and the lateral liquid channel.
5. The method for efficient separation, capture and recovery of rare cells in whole blood of claim 1, wherein: the channel tube is made of polydimethylsiloxane or plexiglass.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106190774A (en) * | 2016-07-22 | 2016-12-07 | 深圳睿思生命科技有限公司 | For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method |
| CN206244772U (en) * | 2016-11-10 | 2017-06-13 | 上海美吉逾华生物医药科技有限公司 | It is a kind of for cell capture, the micro-fluidic chip of fluorescent staining |
| CN106989970A (en) * | 2017-04-01 | 2017-07-28 | 苏州汶颢微流控技术股份有限公司 | Integrated device and circulating tumor cell catching method are dyed in cell separation film-making |
| CN107402295A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell is automatically separated purifying micro-fluidic chip and its isolation and purification method |
| CN107723207A (en) * | 2017-11-01 | 2018-02-23 | 深圳市瑞格生物科技有限公司 | A kind of chip of separating trap cell and its application in tumour cell sorting |
| CN108034586A (en) * | 2018-01-02 | 2018-05-15 | 清华大学深圳研究生院 | A kind of micro-fluidic chip for unicellular seizure and culture |
| CN110093247A (en) * | 2019-05-07 | 2019-08-06 | 西安交通大学 | A kind of micro-fluidic chip of enrichment capture different specification size target cell |
| CN111718836A (en) * | 2020-06-16 | 2020-09-29 | 东南大学 | Microfluidic chip for rare cell acquisition and single cell packaging |
Family Cites Families (1)
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Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107402295A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell is automatically separated purifying micro-fluidic chip and its isolation and purification method |
| CN106190774A (en) * | 2016-07-22 | 2016-12-07 | 深圳睿思生命科技有限公司 | For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method |
| CN206244772U (en) * | 2016-11-10 | 2017-06-13 | 上海美吉逾华生物医药科技有限公司 | It is a kind of for cell capture, the micro-fluidic chip of fluorescent staining |
| CN106989970A (en) * | 2017-04-01 | 2017-07-28 | 苏州汶颢微流控技术股份有限公司 | Integrated device and circulating tumor cell catching method are dyed in cell separation film-making |
| CN107723207A (en) * | 2017-11-01 | 2018-02-23 | 深圳市瑞格生物科技有限公司 | A kind of chip of separating trap cell and its application in tumour cell sorting |
| CN108034586A (en) * | 2018-01-02 | 2018-05-15 | 清华大学深圳研究生院 | A kind of micro-fluidic chip for unicellular seizure and culture |
| CN110093247A (en) * | 2019-05-07 | 2019-08-06 | 西安交通大学 | A kind of micro-fluidic chip of enrichment capture different specification size target cell |
| CN111718836A (en) * | 2020-06-16 | 2020-09-29 | 东南大学 | Microfluidic chip for rare cell acquisition and single cell packaging |
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