KR101723082B1 - Cell Separator - Google Patents

Abstract

The present invention relates to a device for separating a cell, and more particularly to a device for separating a cell, including a plurality of conduits including a sample injection section, a cell separation section, a cell separation section, and a conduit through which magnetic components are transferred, A cartridge including a fixed member fixed at a height, and a base plate coupled to one end of the cartridge and in contact with the conduit, the method comprising the steps of injecting a sample containing blood, Separating the cells, separating the cells including magnetic cells and non-magnetic cells at the same flow rate, sensing the presence of bubbles in the transferring step of the cells, and the like.

Description

[0001] Cell Separator [

The present invention relates to an apparatus for separating cells, and more particularly, to an apparatus and a method for separating cells from each other. More particularly, the present invention relates to an apparatus for separating cells, A cell separation device capable of maintaining the accuracy of separation by minimizing changes in pressure and flow rate, and a cell separation method based thereon.

Of the deaths in Korea in 2015, 27.9% of them died of cancer, and the number is 76,855. The incidence of cancer is highest in the lung cancer group with 17,399 people followed by liver cancer, stomach cancer, colon cancer, pancreatic cancer, etc. Most of these cancer patients die from the primary tumor of the patient. But died from metastasis of multiple, extensive tumor colonies formed by malignant cells that separate from the initial tumor and travel to the distal site through the blood. When the primary tumor is detected at an early stage, it may be removed by surgery, radiation therapy or chemotherapy. However, since cancer cells circulating in the blood as described above are at a low concentration and are difficult to detect themselves, The probability of success is low. The blood cancer cells described above are collectively referred to as cancer cells existing in the peripheral blood of a cancer patient and are defined as cancer cells which have been eliminated from the primary lesion or metastatic lesion. Recently, a method of using blood cancer cells as a potential biomarker for cancer diagnosis, treatment prognosis analysis, and micro-metastasis analysis has been developed. However, as described above, blood cancer cells have a blood distribution ratio of 1 per 1 billion cells or white blood cells It is present in trace amount of one level per 10,000,000 pieces, and it is difficult to analyze it accurately as an obstruction factor such as pollution and bubble in the analysis process.

Specifically, the process of separating cancer cells in the blood comprises three steps of preparing a blood sample, injecting the sample, and separating the cancer cells. The blood sample is specifically reacted with cancer cells in the blood for the subsequent separation of the cancer cells Antibody-magnetic material, and the like. The injection of the sample is performed by injecting the blood mixed with the magnetic material into the cancer cell separator as described above, and it is important to inject the precise amount at a constant flow rate while excluding the inflow of contaminants and disturbing factors such as bubbles. The separation process separates cancer cells using a magnetic material previously bound to blood, and is usually divided into a plurality of channels including cancer cells and non-cancer cells. The flow rate of the substances flowing through the respective channels is kept constant It is important to ensure the accuracy of separation.

In the prior arts studied so far, Patent Document 1 is directed to a method and a reagent for rapidly and efficiently separating circulating cancer cells. The blood and a ligand (a ligand that can specifically detect a reaction with cancer cells) However, the problem of contamination during the injection of the blood sample and securing accuracy in the separation process are not taken into consideration. In the case of Patent Document 2 and Patent Document 3, Although a technique for separating cancer cells using a ferromagnetic wire has been proposed, technical considerations for securing the same flow rate for each channel during the discharge process have not been taken into consideration. Particularly, when separated cancer cells are placed in a predetermined container , The flow rate of each flow channel depends on the length and height of the channel, There may be changes can cause poor accuracy of the separation problem.

WO 2003/065042 Korean Patent Publication No. 2015-0058955 (Patent unidentified 0003) Korean Patent No. 1667351

SUMMARY OF THE INVENTION The present invention has been devised to solve the problems described above, and it is an object of the present invention to prevent the change of internal pressure caused by a tube tube connected between a separation chip and a sample container of a separated sample, It is aimed at.

In addition, the cell separating apparatus according to the present invention is intended to prevent the air from being introduced into separated samples in the process of separating and transporting the cells, thereby improving the storage quality of the cells.

According to an aspect of the present invention, there is provided a cell separation apparatus comprising: a sample injection unit; Cell separator; A plurality of conduits connected to the cell separation unit and including a conduit through which magnetic components are transferred; A cartridge including a fixing member fixed at the same height as the plurality of channels, and a base plate coupled with one end of the cartridge and contacting the channel.

Here, the fixing member may include a first fixing member positioned between the contact portion of the cell separating portion and the base plate, and a second fixing member positioned at a rear end of the contact portion with the base plate, And has a shape of any one of circular, semicircular, and elliptical openings that are partly opened so that the channel can be inserted and removed.

A liquid sensor or a bubble sensor and a valve are disposed at a contact portion between the cartridge and the base plate of the cell separation device according to the present invention.

Further, the cell separating apparatus according to the present invention may include a step of checking whether or not bubbles are present on the pipeline by using a sensor; Closing the bubble-free channel with a valve to increase the pressure of the bubble-containing channel to remove bubbles when the bubble is present; An injection step of a sample containing blood; Separating the magnetic cells in the blood sample; The cells including the magnetic cells and the non-magnetic cells are transported at a flow rate and a flow rate equal to preset values and discharged to the outside.

According to the cell separating apparatus of the present invention, the height of the channel connected between the separating chip and the receiving container of the separated sample can be kept constant to minimize the change in the internal pressure that can be caused in the discharging process, And the effect of preventing the change of the flow velocity in advance.

In addition, the cell separating apparatus according to the present invention includes a liquid or bubble sensor and constitutes a valve capable of controlling the discharge line, so that it is possible to judge and confirm the inflow of bubbles that can flow in the discharge process in advance It is possible to improve the separation performance of the cell.

1 is a top cross-sectional view of a cell separator according to an embodiment of the present invention.
2 shows an internal flow path of the separation chip according to an embodiment of the present invention.
3 is a top cross-sectional view of a base plate according to an embodiment of the present invention.
4 shows a sensor and a valve of a cell separation device according to an embodiment of the present invention.

Hereinafter, a cell separating apparatus according to the present invention will be described with reference to the accompanying drawings.

The use of the terms "comprises", "having", or "having" in this application is intended to specify the presence of stated features, integers, steps, components, parts, or combinations thereof, But do not preclude the presence or addition of features, numbers, steps, operations, components, parts, or combinations thereof.

Also, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The important point in the cell separating apparatus according to the present invention is that the change of the flow rate and the flow rate is minimized and the inflow of contaminants is checked in the entire process of injecting and separating the sample for separating a specific cell.

1, which shows an upper cross-sectional view of a cell separation device according to an embodiment of the present invention, the cell separation device of the present invention basically comprises a cartridge 10 and a base 10, The cartridge 10 has a polygonal shape and includes an injection part 20 and a separation chip 30. The separated specific cell is discharged through a discharge part 40 to the outside And stored in a container. More specifically, the cell separation apparatus according to the present invention includes a cartridge 10 including an injection unit 20, a separation chip 30, a channel, and a fixing member, and a discharge pipe (not shown) And a base plate (100) in contact with the base plate (41).

A sample to which the functional additive for separating the blood and the rear end is combined is introduced into the injection unit 20 together with the buffer solution. Normally, as shown in FIG. 1 showing one embodiment of the present invention, , 22). The injected blood sample is separated by an antibody exhibiting a specific reaction so as to be able to bind with cancer cells through the magnetic chip 200 coupled with the separation chip 30 via the sample loop 23 of FIG. 1 if necessary . In the above separation, an antibody exhibiting a specific reaction with a cancer cell and a magnetic substance are preliminarily bound, and the cancer cell is separated from the magnetic substance combined with the antibody and the magnetic chip 200. In this process, the injection unit 20 and the separation chip 30 constituting the cell separation device according to the present invention are connected by the sample loop 23, the sample transfer channel 24, and the buffer solution transfer channel 25, The cancer cells and the sample solution separated by the separation chip 30 and the magnetic chip 200 are stored in the outer container by the discharge pipe 41 of the discharge portion 40.

If the flow rate and the flow rate are changed during the injection and transfer, the separation of the cancer cells inside the separate chip 30 is affected and the accuracy of separation is lowered. 2, the separation chip 30 is coupled to the lower magnetic chip 200, and the separation chip 30 includes a channel through which a blood sample flows and is transported, The chip 200 is positioned below the flow path and serves to hold only cancer cells. More specifically, the separating chip 30 includes three divided channels for specifically separating cancer cells from blood samples. The center channel of the three channels captures cancer cells to separate cancer cells from blood samples Channel 33 and the two channels on either side are channels 34 through which the remaining blood sample from which the cancer cells are separated flows. Accordingly, the magnetic chip 200 is positioned under the channel while the cancer cells are flowing, and the cancer cells are separated based on the magnetic substance combined with the cancer cells. In order for the cancer cells to be accurately separated from the blood sample through the separation chip 30 and the magnetic chip 200, the flow rate and the flow rate of the blood sample flowing through the three channels must be equal to a preset value, If the flow rate or flow rate of some channels is changed, the other two channels are also affected, and the flow rate and flow rate of the sample flowing through each channel are changed. 1, the first and second fixing members 42 and 43 are separately formed in order to keep the flow rate and the flow rate in each channel inside the separating chip 30 constant, The fixing members 42 and 43 separately fix the three channels 41 connected to the three channels formed in the separating chip 30 and the height of the channel fixed by the fixing members 42 and 43 Can be kept the same. In the present invention, maintaining the height of the channel through which the cancer cells separated by the separation chip 30 and the sample flow are maintained to be the same can be achieved by maintaining the flow rate and flow rate of each channel of the separation chip 30 equal to each other If the height of one channel is higher than that of the other two channels, the flow rate of the channel connected to the channel having the increased height in the separating chip 30 becomes relatively slow as compared with other channels, The flow rate of the sample flowing into the channel becomes small, so that the cancer cell can not be accurately separated by the magnetic chip 200. In addition to the above height, when the piping connected to the separating chip 30 moves during the separation, the flow rate and the flow rate inside the piping can be changed, and a fixing member capable of fixing the piping at the same height is required . The first and second fixing members 42 and 43 of the cell separating apparatus according to the present invention may include a first fixing member 42 between the separating chip 30 and the contact portion 44 of the base plate 100 And a second fixing member 43 is disposed at the rear end of the contact portion 44 with the base plate 100. The fixing member is all formed on the cartridge 10. [ The fixing member may be configured to have any one of circular, semicircular, and elliptical shapes that are partially opened so that the channel can be inserted into the fixing member and then removed again.

As described above, the channel connected to the separation chip 30 is fixed at the same height to keep the flow rate and the flow rate of each channel in the separation chip 30 constant and check whether the contaminant is introduced or not. It is important. In order to prevent contaminants from entering the cell separator according to the present invention, all of the connecting parts are sealed, so that when there is no abnormal state such as malfunction, there is no case where contaminants are introduced. However, A considerable error may occur in the analysis to be performed later. In many cases, the contaminants are already introduced into the pipeline before the separation process, not during the separation process. In the present invention, it is checked whether contaminants have flowed into the pipeline before starting the separation process . Contaminants that can enter the separation process of the blood sample are introduced into the gaseous phase unless otherwise specified, and thus have the shape of a bubble in a liquid blood sample. In the present invention, a liquid sensor is used to confirm whether bubbles have been introduced before or during the separation step. 3 and 4, the sensor 300 is positioned above the base plate 100, and the discharge path 41 included in the cartridge 10 is moved from the contact portion 44 to the sensor 300 . More specifically, as shown in FIGS. 1, 3 and 4, in the process of coupling the cartridge 10 including the discharge pipe line 41 to the base plate 100 having the liquid sensor 300 The discharge pipe passage 41 of the cartridge 10 is engaged at the contact portion 44 so as to be in contact with the liquid sensor 300 located at the upper portion of the base plate 100. [ Accordingly, in the process of coupling the cartridge 10 and the base plate 100, the discharge pipe passage 41 is configured to be in contact with the liquid sensor 300, thereby detecting whether only the liquid flows in the discharge pipe passage 41 . In the present invention, a liquid sensor is used to detect whether bubbles are included in the buffer solution. The above sensing is usually performed in the process of confirming whether bubbles are present on the pipeline before separation. If it is determined that bubbles are present on the pipeline, The pressure of the bubbling line is increased by closing the discharge line without the valve. In this case, the pressure of the duct with the bubbles increases, and the bubbles are discharged to the outside. In the present invention, the liquid sensor is an OPB 350 product of OPTEK Technology Co., and all of the liquid sensors are known to be commercially available, and a detailed description thereof will be omitted in the present invention. In the present invention, when bubbles are contained in the discharge pipe, or when it is necessary to control the discharge pipe, the flow in the pipe can be controlled by using the valves shown in FIGS. 3 and 4, , It is preferable to use a pinch valve capable of simply opening and closing the pipe.

A method of separating a cell in a cell separating apparatus according to the present invention comprises the steps of: checking whether or not bubbles are present on a channel by using a sensor; blocking a bubble-free channel with a valve to remove a bubble- Removing the bubbles by raising the temperature of the blood sample, injecting the sample containing blood, separating the magnetic cells in the blood sample, transferring the magnetic cells and the non-magnetic cells to the same flow rate and discharging the same .

Having thus described a particular portion of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby, It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the invention, and that such modifications and variations are intended to fall within the scope of the appended claims.

10: Cartridge 100: Base plate
20: injection part 21: injection part sample injection port
22: Injection Buffer Inlet 23: Injection Sample Loop
24: sample transfer conduit 25: buffer solution transfer conduit
30: Separation chip
31: separation chip buffer injection port 32: separation chip sample injection section
33: cancer cell isolation channel 34: blood sample flow channel
200: Magnetic chip
40: discharge part 41: discharge part pipe
42: first fixing member 43: second fixing member
300: sensor 400: valve

Claims (6)

A sample injector including a sample loop;
A cell separator disposed at a rear end of the sample injecting unit and separating a magnetic component;
A plurality of conduits connected to the cell separation unit and including a conduit through which magnetic components are transferred;
Wherein the plurality of channels are fixed at the same height so that all of the plurality of channels including the magnetic component can be transported at the same flow rate;
And a base plate coupled to one end of the cartridge and contacting the channel, wherein a liquid sensor and a valve are disposed at a contact portion between the cartridge and the base plate.
delete The cell separation device according to claim 1, wherein the fixing member has a shape of a circular, semicircular, or elliptical shape that is partially open to allow the channel to be inserted and removed.
delete The cell separation device according to claim 1, wherein a bubble sensor is used instead of the liquid sensor.
Checking whether there is air bubbles on the pipeline using the sensor;
Closing the bubble-free channel with a valve to increase the pressure of the bubble-containing channel to remove bubbles when the bubble is present;
An injection step of a sample containing blood;
Separating the magnetic cells in the blood sample;
Wherein the magnetic and non-magnetic cells are transported at the same flow rate and discharged to the outside.

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