CN102703373A - Method and system for separating image recognition cell of computer - Google Patents

Abstract

The invention discloses a method and a system for separating an image recognition cell of a computer. The cell dyeing principle is applied; different dyes are selected and added to different target cells to be separated; the dyed cells display different colors due to the difference of the cell varieties; different colors of cells are recognized by using the image color recognition technology of the computer, and the electrophoresis separation technology is combined so as to achieve the purpose of separating various cells. The method and the system for separating the image recognition cell of the computer have the advantages of high automatic degree, high efficiency, wide application, high cleanliness, small cell pollution, simple device, low cost and the like.

Description

A computer image recognition cell separation method and system

technical field

The invention relates to biological cell separation technology, in particular to a technology for separating cells based on cell color.

Background technique

Biological cell separation has a wide range of applications and important values in biology and medicine, such as clinical blood analysis, disease diagnosis, bacterial analysis, single-cell gene diagnosis, drug injection, etc.

At present, there are many existing cell separation methods. The commonly used cell separation technology is mainly flow cytometry, but flow cytometry equipment is expensive, bulky, requires special personnel to operate, and consumes a large amount of cells, which is difficult to use in laboratories and hospitals. be widely used. There are also other separation methods based on the physical or chemical characteristics of cells, such as: centrifugation, using the density physical characteristics of different cells, using centrifugal technology to stratify the cells, so as to achieve the purpose of separation. Due to the variety of cells, the density characteristics are very It is difficult to separate them completely by centrifugal technology, and there are disadvantages such as poor resolution and incomplete separation. There is also the use of the principle of cell-to-laser scattering to separate cells. This separation method needs to collect weak scattered light to distinguish different cells. Therefore, the detection environment and equipment accuracy are very high, so that the equipment is huge, costly, and anti-interference. Difference.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention proposes a computer image recognition cell separation method and system in order to achieve a simplified separation system with high automation, high efficiency, wide application, high cleanliness, small cell pollution and simple equipment , low cost and other advantages.

In order to achieve the purpose of the present invention, the idea of the present invention is to apply the principle of cell staining. After adding different chromatin to different cells, due to the different types of cells, the dyed cells have different colors, such as color A, color B, and color C. , color D, etc., which can use computer image color recognition technology to separate different types of cells.

The present invention proposes the following technical solutions:

A computer image recognition method for cell separation, said method comprising the following steps:

(1) Stain the cells with different dyes for different types of cells contained in the cell suspension;

(2) Inject the stained cell suspension into the cell separation chip, and use the sheath flow method to make the cell flow flow as a single cell in the cell solution channel of the chip. There are multiple bifurcated channels on the cell solution channel, each A pair of microelectrodes are set at the bifurcation position, and during the flow process of the cell suspension, the computer image automatic recognition device is used to identify the color of the flowing cells in real time at each bifurcation channel position;

(3) At the bifurcation position of the channel, when the computer recognizes that the color of the cell is not color A, it will issue an instruction to apply a pulse voltage signal to the microelectrode pair at the position of the bifurcation channel through the signal generator, so that the cell that is not color A Under the action of electrophoretic force, it deflects and flows forward along the bifurcated channel; when the computer recognizes that the cell is color A, the computer does not issue an instruction, and the cell flows forward along the straight channel here, thereby separating the color A cell;

(4) According to the process of step (2), when the cell solution reaches the position of the next bifurcated channel, the computer recognizes it again, and when it recognizes that the color of the cell is not color B, it issues an instruction to apply The pulse voltage makes the cells that are not color B deflect under the action of electrophoretic force, and flow out along the bifurcated channel at this position; when the computer recognizes that the cell is color B, the computer does not issue instructions, and the cells follow the straight channel here Forward flow, thereby separating cells of color B;

(5) By analogy, separate the cells of the third, fourth and more colors, until the cells of different colors that need to be separated in the cell suspension to be separated are completely separated.

The present invention further proposes a computer image recognition cell separation system for realizing the above method, the system includes a computer image recognition system and a cell separation chip, a plurality of bifurcated channels are set on the cell solution channel of the cell separation chip, and each bifurcated channel The bifurcation position is provided with microelectrodes; the image acquisition device of the computer image recognition system is aligned with the cell separation chip for collecting cell images, and the analysis software in the computer image recognition system is used to identify the color of the cells in the designated area. The image recognition system controls the signal generator of the cell separation chip, and the signal generator is electrically connected with the microelectrodes on the cell separation chip, and applies electric signals to the microelectrodes according to the instructions of the computer.

The above method and system proposed by the present invention have the following obvious advantages:

(1) High degree of automation . Because it is automatically recognized by the computer, the cell separation is automatically controlled without manual intervention, and the process from cell injection to automatic separation can be fully automated;

(2) High efficiency . Computer high-speed identification and control can quickly identify and separate different types of cells, which is conducive to maintaining cell viability; One-time separation efficiency of multiple cells;

(3) Wide range of applications . All cells can be stained and electrophoresis, so all cells of different types can be separated by the technology of the invention;

(4) High cleanliness and little cell pollution. Due to the use of the microchip system, the cells are controlled by the electric field around the microelectrode in a small closed space, the chip is easily sterilized, the working environment is clean, and the cells are less polluted;

(5) The equipment is simple and the cost is low . The system is simple in composition, the cost of separation chips, signal generators and other equipment is low, and the requirements for manufacturing technology are not high.

Description of drawings

Fig. 1 is the system principle diagram realizing the present invention;

Fig. 2 is a partial schematic diagram of a cell separation chip.

Detailed ways

Further illustrate the present invention below in conjunction with accompanying drawing:

This method is based on the principle of cell staining in biology. After adding different dyes to different cells, the dyed cells have different colors due to the different types of cells. Using computer image color recognition technology, the purpose of separating different types of cells is achieved.

Referring to FIG. 1 , the computer image recognition cell separation system includes a computer image recognition system and a cell separation chip 2 .

As shown in Figure 2, the cell separation chip uses the sheath flow method to make the cells flow as single cells in the cell solution channel of the chip, and adopts the form of sequentially setting multiple bifurcated channels on the cell solution channel. Microelectrodes A1-K1 and A2-K2 are arranged on both sides of the channel at the bifurcation position of the channel. The image acquisition device 1 (including a microscope and a CCD camera) of the computer image recognition system is aligned with the cell separation chip 2 for collecting cell images, and the image recognition and analysis software in the computer 4 is used to analyze the color of the cells according to the image, computer image recognition The computer 4 of the system is connected to the signal generator 3 of the cell separation chip, and the signal generator 3 is electrically connected to the dielectrophoretic electrodes A1-K1 and A2-K2 on the cell separation chip 2, and the electrodes are applied according to the instructions of the computer 4. electric signal.

The method of cell separation is as follows:

1. Cell staining:

Take the isolation of human peripheral blood lymphocytes as an example. We know that in human peripheral blood, there are mainly various lymphocytes such as T cells, B cells, and natural killer (NK) cells. The classification of these cells is mainly determined by their surface antigens. For example, mature T cells express CD3 antigen, B cells are CD19 positive cells, and most of the rest are NK cells (CD3-/CD56+ cells in peripheral blood are generally considered to be NK cells, So if you need to identify and isolate true NK cells, you also need to identify CD56 antigen).

(1) Primary sorting.

Prepare FITC (excitation light 488nm, emission light 525nm, green light) labeled CD3 antibody (CD3-FITC, green) and PE (excitation light 488nm, emission light 575nm, orange light) labeled CD19 (CD19-PE, Orange) After the antibody is mixed with peripheral blood, according to the specificity of the self-antigens on the cell surface, these antigens will recognize and bind to the corresponding antibody, thereby indirectly marking and staining the cells, such as B cells binding to CD19-PE, which will appear orange. Flow the cell suspension through the cell separation chip at a constant speed. When mature T cells (already carrying green light) pass the signal generator, they enter the green channel directly. When the B cells carrying the red light marker pass by, they will be deflected and enter another channel. In the same way, when B cells enter the next signal detector of another channel, they will be correctly identified. Finally, unlabeled cells are the NK cell-dominated population.

(2) Secondary sorting.

Take sorting of T cell subsets as an example. Because mature T cells can only express CD4 or CD8 molecules, CD3+ mature T cells can be divided into two subtypes: helper T cells (Th) and killer T cells (Ts) according to the differences in CD4 and CD8 antigens. CD4 antibody (denoted as CD4-PE-Cy5, red) labeled with PE-Cy5 (excitation light 488nm, emission light 670nm, red light). Similarly, the PE-labeled CD8 antibody is recorded as CD8-PE (orange). [Note, because the re-sorted CD3+ cells have FITC-labeled green fluorescence at this time, they cannot be labeled with FITC-labeled antibodies. 】According to the specificity of the self-antigens on the cell surface, these antigens will recognize and bind to the corresponding antibodies, thereby indirectly marking and staining the cells. After these different fluorescently labeled antibodies were added to the above separated CD3+ cell suspension, they were mixed evenly and flowed through the cell separation chip. Among CD3+ cells, Th cells (CD3+/CD4+, the cells are red-green) and Ts cells (CD3+/CD8+, yellow-green) are sorted according to the difference in the expression of CD4 and CD8; when the cells pass through the signal detector, they are It will recognize red and orange for screening.

Similarly, CD3- cells can be further sorted according to the difference between CD16 and CD56. CD16 is expressed on most NK cells, but also on neutrophils. This antigen is weakly expressed on NK cells and lost upon NK cell activation. CD56 is expressed on most (but not all) NK cells and also on some T lymphocytes. Used in combination with CD3, it can differentiate between CD3+/CD56+ T lymphocytes and CD3-/CD56+ NK cells. Combination of the three mAbs can most completely identify all NK cells. NK cells express either CD16 or CD56, but they do not express CD3. The combination of CD16 and CD56 distinguishes NK cells from double-negative cells based on fluorescence intensity. Using this set of reagents in this way, NK cells can form an independent population and be distinguished from other cells.

2. Inject the cell suspension into the cell separation chip. The cell suspension flows in from the first area. When the cell suspension is about to enter the second area (that is, the image recognition area shown in the dotted line in the figure), it is about to enter When the position of the first bifurcated channel is reached, the image acquisition device 1 of the computer collects the cell image, and the image recognition and analysis software analyzes the color of the cell according to the image. When the cell is recognized as non-green, the computer sends an instruction to make the signal generator A pulse voltage signal is applied to the pair of electrodes A1-K1, the cells are deflected under the action of electrophoretic force, and flow forward along the first bifurcated channel CH2; when the computer recognizes that the cells entering here are green, the computer does not send Instructions, the signal generator does not apply a voltage signal to A1-K1, and the green cells flow forward in a straight line through the first straight channel CH1, thereby separating the green cells.

3. Same as the above principle, when the cell solution enters the third area (that is, the second image recognition area shown in the dotted line in the figure), when the computer recognizes that the color of the cell is not red, it sends an instruction to the signal generator, and the A2 - K2 applies a pulse voltage to deflect the non-red cells under the action of electrophoretic force and flow out along the next bifurcated channel CH3. Otherwise, the computer does not issue an instruction, and the red blood cells flow forward in a straight line through the second straight channel CH4, that is, the red blood cells are separated.

By analogy, the separation of cells of different colors can be achieved.

The structure of the cell separation chip of the present invention includes a PDMS channel layer and a bottom electrode layer. The PDMS channel layer can be made by micromachining technology. The PDMS channel layer and the bottom electrode layer are registered and bonded by micro-registration. The PDMS channel layer can be treated with plasma surface before registration to improve the PDMS channel layer and The bond strength of the bottom electrode layer.

The specific processing process of the PDMS channel layer of the chip can be in the following form:

A. Select silicon wafer or glass wafer as the substrate;

B. Suspension coating of SU-8 photoresist on the surface of the substrate;

C. After prebaking, photolithography, postbaking, developing, and hard-baking to form the male mold required for the channel;

D. Fix the positive mold obtained on a petri dish;

E. Pour in the mixed PDMS glue, and vacuum it after standing still;

F. Place it on a heating table at 75°C for curing;

G. Remove the cured PDMS, cut it according to the shape of the underlying structure, and remove the glue on the inlet and outlet.

The bottom electrode layer of the cell separation chip is made by corrosion and other methods. The specific processing process is as follows:

A. Select the surface silicon oxide sheet or quartz glass sheet as the substrate;

B. Sputter gold or platinum to form a conductive layer on the surface of the substrate. In order to improve the adhesion of gold and platinum to the substrate, chromium or titanium can be sputtered on the surface of the substrate first;

C. Hang coating AZ4620 and other photoresists on the surface of the conductive layer;

D. Photolithography development to obtain the same pattern as the microelectrode;

E. Use wet etching to remove the conductive layer outside the photoresist image;

F. Use acetone to remove the photoresist to obtain the microelectrode structure.

Claims (3)

1. computer picture recognition cell isolation method; It is the application cell dyeing theory, treats to difference that the separate targets cell is chosen and adds different dyestuffs, because cell category is different; Make that dyeing the cell that comes presents distinct colors; Utilize computer picture color recognition technology, distinguish the cell of different colours, and combine electrophoretic separation technique to reach the purpose of separating the different sorts cell; Said method comprising the steps of:

(1) to the different sorts cell that comprises in the cell suspension, dyes with different dyestuff pair cells;

(2) painted cell suspension is injected the cellular segregation chip together,, make stream of cells in the cell solution passage of chip, form unicellular flowing through sheath stream method; The cell solution passage is provided with a plurality of bifurcated passages, and each bifurcation site is provided with a pair of microelectrode, in the cell suspension flow process, with the flow through color of cell of computer picture automatic identification equipment Real time identification before each bifurcated passage position;

(3) in the passage bifurcation site; When computer Recognition when the cell of this position of flowing through is not color A; Just send instruction; Under the electrophoretic force effect, deflect for the microelectrode that is positioned at this bifurcated passage position to applying pulse voltage signal, make the cell that is not color A through signal generator, flow forward along bifurcated passage; And when computer Recognition went out cell and is color A, computingmachine did not send instruction, and cell flows forward along the craspedodrome passage here, thereby isolated the cell of color A;

(4) set by step the process of (2); Before cell solution arrived next bifurcated passage position, computingmachine was discerned again, when identifying the cell color and be not color B; Then send instruction; Microelectrode to this position deflects under the electrophoretic force effect to applying pulsed voltage, make the cell that is not color B, along the bifurcated passage outflow of this position; And when computer Recognition went out cell and is color B, computingmachine did not send instruction, and cell flows forward along the craspedodrome passage here, thereby isolated the cell of color B;

(5) by that analogy, isolate respectively the third, the cell of the 4th kind and more colors, the cellular segregation that in will treating the isolated cell suspension, needs isolating different colours fully till.

2. computer picture recognition cellular segregation system that realizes the said method of claim 1; It comprises computer picture recognition system and cellular segregation chip; It is characterized in that: in the said cellular segregation chip cell solution passage is arranged; Many bifurcated passages are set on the cell solution passage, microelectrode are set in the bifurcation site correspondence of each bifurcated passage; The image collecting device of said computer picture recognition system is aimed at the cellular segregation chip; Be used for the collection of cells image; The intrasystem analysis software of computer picture recognition is used for going out according to image analysis the color of cell, and said computer picture recognition system connects and the signal generator of control cellular segregation chip, and signal generator carries out electrical signal with microelectrode on the cellular segregation chip and is connected; Instruction according to computingmachine applies electrical signal to electrode, realizes treating the deflection operation of isolated cell.

3. computer picture recognition cellular segregation according to claim 2 system, it is characterized in that: said microelectrode is arranged on the passage both sides of bifurcation site.

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