KR101873315B1 - Method for recovering target cell - Google Patents
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- KR101873315B1 KR101873315B1 KR1020150079329A KR20150079329A KR101873315B1 KR 101873315 B1 KR101873315 B1 KR 101873315B1 KR 1020150079329 A KR1020150079329 A KR 1020150079329A KR 20150079329 A KR20150079329 A KR 20150079329A KR 101873315 B1 KR101873315 B1 KR 101873315B1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0693—Tumour cells; Cancer cells
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Abstract
A target cell recovery method for separating target cells from fluid samples such as blood, physiological fluids, and then efficiently recovering while preventing damage to the separated target cells is disclosed. The method for recovering target cells according to the present invention is a method for recovering target cells isolated from a cell mixture, comprising the steps of: moving a biochip in which the separated target cells are located into a container; Injecting a buffer into the biochip; And repeating the movement in a vertical direction along a straight line in a predetermined area inside the biochip, and sucking the buffer and collecting the buffer in the collection space.
Description
The present invention relates to a target cell recovery method. More particularly, the present invention relates to a method for recovering a target cell from a fluid sample such as blood, physiological fluid, or the like, and efficiently recovering the target cell while preventing damage to the separated target cell.
Recently, regulations for animal tests and clinical trials to treat human diseases have been strengthened. In order to replace these animal tests and clinical trials, studies and techniques for collecting live cells from human blood have been actively developed. The collection of cells is performed by various cell collecting devices such as a microfluidic device, a CTC chip, a circulating tumor cell chip, and a filter.
As an example of a cell collection device, United States Patent Application Publication No. 2007 / 0025883A1 discloses a parylene membrane filter for filtering cells from a fluid. The membrane filter is mounted in a chamber and has a plurality of pores that are formed so that a cell, for example, a cancer cell, is prevented from passing therethrough.
As another example of a cell collection device, US Patent Application Publication No. 2009 / 0188864A1 discloses a method and apparatus for microfiltration to perform cell separation. A plurality of filter patches are installed in the central square hole of the microfilter. Filter patches consist of membranes with multiple pores for filtration of cells. In order to collect the cancer cells filtered on the filter from the chamber or the central square hole and collect the solution, for example, water is supplied to the chamber in the opposite direction and the solution is forcibly discharged out of the chamber Therefore, there is a problem that it is very difficult to collect and collect cancer cells from the filter. In addition, there is a problem that the cancer cells are easily damaged in the process of discharging from the chamber.
Therefore, there is a need for a method for recovering a target cell from a fluid sample such as a blood or a physiological fluid, which can efficiently recover the target cell while preventing damage to the target cell.
The object of the present invention is to solve the above-mentioned problems, and it is an object of the present invention to provide a method for separating target cells from a fluid sample such as blood, physiological fluid, etc., will be.
It is to be understood that the technical objectives of the present invention are not limited to the above-mentioned technical objects and other technical objects which are not mentioned in the following description are to be understood from the following description, It will be understood clearly by those with knowledge.
According to another aspect of the present invention, there is provided a method for recovering target cells isolated from a cell mixture, comprising the steps of: Moving; Injecting a buffer into the biochip; And repeating the movement in a vertical direction along a straight line in a predetermined area in the biochip, and sucking the buffer and collecting the buffer in a recovery space, wherein the step of injecting into the biochip comprises: The step of injecting a buffer contained in a second container other than the container into the biochip, wherein the step of aspirating the buffer and collecting the biochip in the recovery space comprises: introducing non-target cells through a filtration hole provided at a lower end of the biochip; And sucking the target cells remaining on the upper surface of the filtration hole by the sucking means together with the buffer while not sucking the buffer through the filtration hole and collecting the buffer into the collection space, The distance between the lower end of the suction device and the distal end of the suction device is maintained at a predetermined distance, And the suction means is repeatedly moved in the vertical direction along a straight line in a predetermined area inside the buffer, and the buffer is sucked.
In this case, the step of moving the bio chip into the inside of the container may include moving the bio chip into a container containing a buffer, and injecting the bio chip into the bio chip, And a buffer is injected into the biochip.
In this case, the step of moving the bio chip to the inside of the container may include moving the bio chip into a container not containing a buffer, and injecting the bio chip into the bio chip. And injecting a buffer contained in the container into the biochip through the mesh at the lower end of the biochip.
delete
At this time, in the step of injecting into the biochip, the buffer is injected into the inner wall of the biochip, which is the lateral direction of the inside of the biochip, so that the buffer falls into the inside of the biochip on the inner wall of the biochip can do.
In this case, the step of sucking the buffer and collecting in the collection space may include the step of sucking the buffer in a state in which the distance between the lower end of the biochip and the distal end of the sucking means for sucking the buffer is maintained at 0.02 mm to 1.0 mm .
According to another aspect of the present invention, there is provided a method for recovering target cells isolated from a cell mixture, comprising the steps of: ; ≪ / RTI > Injecting a buffer into the biochip first; And dividing the area inside the biochip into a plurality of sub-areas having a matrix of 2.times.2 or 3.times.3, sequentially moving each of the plurality of sub-areas along a straight line in a vertical direction, Wherein the step of firstly injecting the biochip into the biochip is performed by injecting a buffer contained in a second container, which is a container other than the container or the container, into the biochip Wherein the step of primarily sucking the buffer and collecting the biochip in the recovery space comprises passing non-target cells through a filtration hole provided at a lower end of the biochip, and passing the non-target cells through the filtration hole, The remaining target cells are sucked by the suction means together with the buffer, and further, Wherein the step of recovering the biochip includes maintaining a distance between a lower end of the biochip and a distal end of the suction means at a predetermined distance so that an area inside the biochip is divided into a plurality of sub regions having 2
In this case, the step of secondly injecting the buffer into the biochip after the step of first sucking the buffer and collecting the buffer in the collection space; And secondly sucking the buffer in a predetermined area inside the biochip and collecting the buffer in a collection space.
The method further includes the step of sucking the buffer in a predetermined area in the biochip after the step of sucking the buffer and collecting the buffer in the recovery space and collecting the buffer in the recovery space in a tertiary manner. can do.
The method further includes the step of sucking the buffer in a predetermined area in the biochip after the step of sucking the buffer in a tertiary manner and collecting the same in a recovery space, can do.
At this time, the biochip includes a sleeve; And a chip coupled to a lower end of the sleeve.
At this time, the target cell may be characterized as being blood tumor cells (CTCs; Circulating Tumor Cells).
The present invention has the following effects.
According to the present invention, target cells can be separated from a fluid sample such as blood or physiological fluid, and then efficiently recovered while preventing damage to the separated target cells.
1 is a view for explaining a structure of a biochip.
FIG. 2 is a view for explaining how a target cell is separated from a cell mixture using a biochip. FIG.
Figures 3 and 4 are flow charts of a method for recovering target cells according to the present invention.
5 and 6 are views for explaining an embodiment of a target cell recovery method according to the present invention.
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail.
It should be understood, however, that the invention is not intended to be limited to the particular embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Like reference numerals are used for like elements in describing each drawing.
It is to be understood that when an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, . On the other hand, when an element is referred to as being "directly connected" or "directly connected" to another element, it should be understood that there are no other elements in between.
The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Hereinafter, the same reference numerals will be used for the same constituent elements in the drawings, and redundant explanations for the same constituent elements will be omitted.
1 is a view for explaining a structure of a biochip. FIG. 2 is a view for explaining how a target cell is separated from a cell mixture using a biochip. FIG.
The term "target cell" in the present invention means preferably CTCs (Circulating Tumor Cells) and is not necessarily limited to blood tumor cells. The blood tumor cells are a primary tumor cell, a small number of tumor cells that move away from the primary tumor and circulate in the blood, which is known as a key factor of metastatic cancer. Statistics show that about 90% of cancer patients die from metastatic cancer.
Blood tumor cells range from one to several thousand per ml of blood, depending on the type of cancer. Recently, various attempts have been made to effectively isolate blood tumor cells.
The collection of cells is carried out by various cell collecting devices such as a biochip and a microfluidic device. Biochips are classified into microarrays and microfluidic devices. The microarray can be formed by arranging DNA, Deoxyribonucleic acid (DNA), Protein and the like and extracting DNA, protein, enzyme from sample such as physiological fluid and blood of human saliva and sweat, (DNA chip), a protein chip (protein chip), and the like.
A microfluidic device is a device for analyzing a target reacting with a sensor or a biomolecule while flowing a sample, and is also called a microfluidic chip or a lab-on-a-chip.
1, the
2, the blood (cell mixture solution) 2 includes a plurality of
The
The diameters of the filtration holes are smaller than the diameters of the cancer cells (4). The diameter of the filtration holes is 5 to 2 탆. Preferably, the diameter of the filtration holes is 7 to 2 탆. The
The blood (cell mixture solution) 2 is supplied to the
The
On the other hand, the
Other examples of non-target cells (6) are leukocytes, which vary in shape and size, the shape and number of nuclei, the presence or absence of granules, and are about 10 to 21 μm in diameter. Leukocytes easily pass through the filtration holes of a diameter smaller than their diameter by the deformation of the cytoplasm. The largest macrophage of leukocytes is 21 ㎛ in diameter. When the diameter of the filtration holes is 20 탆, most of the leukocytes having 7,000 ~ 8,000 cells per blood pass through the filtration holes 16.
Thus, when the
Here, a method for recovering target cells (cancer cells) remaining in the
Hereinafter, a first embodiment of the target cell recovery method according to the present invention will be described with reference to FIG. 3 and FIG.
3 is a flow chart of a method for recovering target cells according to the present invention. 5 is a view for explaining an embodiment of a target cell recovery method according to the present invention.
As shown in FIG. 3, the first embodiment of the method for recovering target cells according to the present invention is a method for recovering target cells isolated from a cell mixture, comprising the steps of: (S100) to the inside of the container; Injecting a buffer into the biochip (S110); And a step (S120) of repeatedly moving in a vertical direction along a straight line in a predetermined area inside the biochip, and sucking the buffer and collecting the buffer in a collection space.
More specifically, in connection with the embodiment of step S100, the biochip may be moved into a container containing a buffer or may be moved to a container containing no buffer.
Also, with respect to the embodiment of step S110, as a method of injecting a buffer into a biochip, a buffer contained in the container may be injected into the biochip, and in a second container other than the container The buffer containing the biochip may be injected into the biochip. The method of injecting the buffer into the biochip may include a method of injecting the buffer directly from the container or the second container, or by inserting the biochip into the container containing the buffer, The buffer contained in the container may be injected into the biochip through a mesh formed in the biochip.
In addition, as a method of injecting the buffer into the biochip, a plurality of injections can be performed by suitably selecting or combining the above methods. That is, it should be understood that the buffer may be additionally injected through different methods or the same method.
The method of injecting a buffer contained in the container or the second container directly into the biochip may include injecting a buffer into the inner wall of the biochip in a lateral direction of the inside of the biochip, The chip can be dropped on the inner wall of the bio chip. As described above, since the buffer is injected along the side wall of the biochip without injecting the buffer material vertically into the lower end of the biochip, damage to the target cell can be prevented. In addition, the shape of the lower end of the biochip may be circular or may be a square or a triangle depending on the embodiment.
5, in a step S120 of sucking the buffer and collecting it in a collection space, the biochip is repeatedly moved in the vertical direction along a straight line in a predetermined region A of the biochip, The buffer is sucked and collected in the recovery space. Depending on the embodiment, the direction of repeated movement in the up and down direction along the straight line may be changed. That is, it may move from the upper side to the lower side along the straight line shape, or may move from the lower side to the upper side along the straight line shape. It is also possible to move the upper side from the lower side and the lower side from the upper side repeatedly a plurality of times.
It is also preferable that the buffer is sucked while maintaining a distance between the lower end of the biochip and the distal end of the suction means for sucking the buffer between 0.02 mm and 1.0 mm. When the buffer is sucked at a distance of less than 0.02 mm, the target cell may be damaged because the distance between the distal end of the suction means (such as a pipette) and the lower end of the biochip is too close. , The effective suction is not performed.
Hereinafter, a second embodiment of the target cell recovery method according to the present invention will be described with reference to FIG. 4 and FIG.
4 is a flow chart of a method for recovering target cells according to the present invention. 6 is a view for explaining an embodiment of a target cell recovery method according to the present invention.
As shown in FIG. 4, the second embodiment of the method for recovering target cells according to the present invention is a method for recovering target cells isolated from a cell mixture, wherein the separated target cells are placed in a biochip, (S200) the inside of the container; Injecting a buffer into the biochip (S210); Regions in the biochip are divided into a plurality of sub-regions having a matrix of 2.times.2 or 3.times.3, and each of the sub-regions is sequentially moved in a vertical direction along a straight line, (S220) of sucking and collecting in a collection space; Injecting a buffer into the biochip (S230); (S240) of sucking the buffer in a predetermined area inside the biochip and collecting the buffer in a recovery space; A step (S250) of sucking the buffer in a predetermined area inside the biochip and collecting the buffer in a recovery space; And a step (S260) of sucking the buffer quaternary in a predetermined area inside the biochip and collecting the buffer in a collection space.
More specifically, in connection with the embodiment of step S200, the biochip may be moved into a container containing a buffer, or may be moved to a container containing no buffer.
Also, with respect to the embodiment of step S210, as a method of firstly injecting the buffer into the biochip, the buffer contained in the container may be injected into the biochip, The buffer contained in the container may be injected into the biochip. The method of injecting the buffer into the biochip may include a method of injecting the buffer directly from the container or the second container, or by inserting the biochip into the container containing the buffer, The buffer contained in the container may be injected into the biochip through a mesh formed in the biochip.
In addition, as a method of injecting the buffer into the biochip, a plurality of injections can be performed by suitably selecting or combining the above methods. That is, it should be understood that the buffer may be additionally injected through different methods or the same method.
The method of injecting a buffer contained in the container or the second container directly into the biochip may include injecting a buffer into the inner wall of the biochip in a lateral direction of the inside of the biochip, The chip can be dropped on the inner wall of the bio chip. As described above, since the buffer is injected along the side wall of the biochip without injecting the buffer material vertically into the lower end of the biochip, damage to the target cell can be prevented. In addition, the shape of the lower end of the biochip may be circular or may be a square or a triangle depending on the embodiment.
In addition, the region inside the biochip is divided into a plurality of sub-regions having a matrix of 2.times.2 or 3.times.3, and each of the plurality of sub-regions is sequentially repeatedly moved up and down along a straight line, (S220), the area inside the biochip is divided into a matrix of 2 x 2, as shown in Fig. Here, the division is not limited to division through a specific structure or configuration, but may be divided into a specific scale at the bottom of the biochip or a means for displaying the display so that the user can visually recognize it. Also, it should be understood that it can be divided depending on the user's eyes only.
6, it can be confirmed that the region inside the biochip is divided into the a region, the b region, the c region, and the d region. In order to primarily suck up the buffer and collect it in the recovery space, it may be performed in the order of the area a, the area b, the area c and the area d, or the order of the area d, area c, area b, and area a.
That is, the order in which each area is performed can be changed variously. In addition, it can be implemented by an optional combination of directions in which the regions are repeatedly moved in a vertical direction along a straight line.
FIG. 6 shows an embodiment in which a and c regions are linearly moved in an upward and downward repeated movement direction, and b regions are linearly moved in different spaces in an upward and downward repeated movement. it can be seen that the operation is performed in a direction moving from the lower side to the upper side in a linear shape in the d region and in the direction moving from the upper side to the lower side in different spaces.
In the same manner as the above-described method, the region inside the biochip may be divided into a plurality of sub-regions having a 3 x 3 matrix. In this case, it will be divided into nine sub-regions, and the buffer is repeatedly moved in the vertical direction along a straight line for each region, and is collected in the recovery space.
After the step S220, the buffer is injected into the biochip (S230). With respect to the embodiment of step S230, as a method of secondly injecting the buffer into the biochip, the buffer contained in the container may be injected into the biochip, and the buffer may be contained in a second container other than the container The buffer may be injected into the biochip. The method of injecting the buffer into the biochip may include a method of injecting the buffer directly from the container or the second container, or by inserting the biochip into the container containing the buffer, The buffer contained in the container may be injected into the biochip through a mesh formed in the biochip.
In addition, as a method of injecting the buffer into the biochip, a plurality of injections can be performed by suitably selecting or combining the above methods. That is, it should be understood that the buffer may be additionally injected through different methods or the same method.
The method of injecting a buffer contained in the container or the second container directly into the biochip may include injecting a buffer into the inner wall of the biochip in a lateral direction of the inside of the biochip, The chip can be dropped on the inner wall of the bio chip. As described above, since the buffer is injected along the side wall of the biochip without injecting the buffer material vertically into the lower end of the biochip, damage to the target cell can be prevented.
After the step S230, a step S240 is performed in which the buffer is secondarily sucked in a predetermined area in the biochip and collected in a collection space. In step S240, a predetermined area in the biochip (S250) is performed in which the buffer is sucked in a tertiary manner and is collected in a recovery space, and after the step S250, the buffer is quadratically sucked in a predetermined region in the biochip and collected in a recovery space Step S260 is proceeded.
The steps of S240, S250 and S260 are the same as those of S220.
As described above, the target cell recovery method according to the present invention is advantageous in that target cells can be separated from a fluid sample such as blood or physiological fluid, and recovered efficiently while preventing damage to the separated target cells .
The description sets forth the best mode of the invention, and is provided to illustrate the invention and to enable those skilled in the art to make and use the invention. The written description is not intended to limit the invention to the specific terminology presented.
Thus, while the present invention has been described in detail with reference to the above examples, those skilled in the art will be able to make adaptations, modifications, and variations on these examples without departing from the scope of the present invention. In other words, in order to attain the intended effect of the present invention, all the functions shown in the drawings are separately included or all the steps shown in the drawings are not necessarily followed in the order shown, and the technical scope of the present invention In the following description.
100: Biochip
110: Sleeve
120: Chip
121: Lower end
4: Target cells (cancer cells)
6: Non-target cells
2: Cell mixture (blood)
12: Surface
14:
A: a predetermined area inside the biochip
a, b, c, d: a plurality of sub areas
Claims (12)
Moving the biochip where the separated target cell is located into the inside of the container;
Injecting a buffer into the biochip; And
And repeatedly moving upward and downward along a straight line in a predetermined area within the biochip, and sucking the buffer and collecting the buffer in a collection space,
The step of injecting into the biochip is to inject a buffer contained in the container or a container other than the container into the biochip,
The step of aspirating the buffer and collecting the buffer in the collection space may include passing non-target cells through a filtration hole provided at a lower end of the biochip, allowing the target cells remaining on the upper surface of the filtration hole to pass through the filtration hole, And is sucked by the suction means,
The step of sucking the buffer and collecting the biochip in the recovery space may include the steps of maintaining the distance between the lower end of the biochip and the distal end of the suction means for sucking the buffer between 0.02 mm and 1.0 mm, The suction means is repeatedly moved in a vertical direction along a straight line to suck the buffer,
Wherein the target cell is a CTCs (Circulating Tumor Cells).
The method of claim 1,
The biochip is moved to the inside of the container containing the buffer,
The method of claim 1,
Wherein a buffer contained in the container is injected into the biochip through a mesh at the lower end of the biochip.
The method of claim 1,
The biochip is moved into a container not containing a buffer,
The method of claim 1,
Injecting a buffer into the vessel; And
And injecting a buffer contained in the container into the biochip through a mesh at the lower end of the biochip.
The method of claim 1,
Wherein the buffer is injected into the inner wall of the biochip in a lateral direction inside the biochip so that the buffer falls down into the biochip on the inner wall of the biochip.
Moving the biochip where the separated target cell is located into the inside of the container;
Injecting a buffer into the biochip first; And
Regions in the biochip are divided into a plurality of sub-regions having a matrix of 2.times.2 or 3.times.3, and each of the sub-regions is sequentially moved in a vertical direction along a straight line, And sucking the recovered liquid into the recovery space,
The step of firstly injecting the biochip into the biochip may include injecting a buffer contained in the container or a container other than the container into the biochip,
The step of primarily sucking the buffer and collecting in the collection space may include passing non-target cells through a filtration hole provided at a lower end of the biochip, passing through the filtration hole, The cells are inhaled by the suction means together with the buffer,
The step of primarily sucking the buffer and collecting the biochip in the recovery space may include the steps of maintaining the distance between the lower end of the biochip and the distal end of the suction means for sucking the buffer between 0.02 mm and 1.0 mm, Regions in the biochip are divided into a plurality of sub-regions having a matrix of 2.times.2 or 3.times.3, and the suction means are repeatedly moved in the up-and-down direction sequentially along the linear shape in each of the plurality of sub- Inhaling the buffer,
Wherein the target cell is a CTCs (Circulating Tumor Cells).
After the step of sucking the buffer primarily and recovering it to the collection space,
Injecting a buffer into the biochip; And
Further comprising the step of secondarily sucking the buffer in a predetermined region inside the biochip and collecting the buffer in a collection space.
After the step of sucking the buffer secondarily and returning it to the collection space,
Further comprising the step of sucking the buffer in a predetermined region of the biochip in a tertiary manner and collecting the buffer in a collection space.
After the step of sucking the buffer tertiaryly and collecting it in the collection space,
Further comprising the step of fourtharily sucking the buffer in a predetermined region inside the biochip and collecting the buffer in a collection space.
In the biochip,
Sleeve; And
And a chip coupled to a lower end of the sleeve.
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