KR20190049603A - Minimal residual disease cell separate and culture apparatus - Google Patents

Abstract

Disclosed according to one embodiment of the present invention is a separating and culturing apparatus of minimal residual disease cells, comprising: a sample injection unit for injecting a sample containing minimal residual disease cells; a filter unit including a plurality of filters, one end of which is connected to the sample injection unit and are sequentially disposed according to the size of the gap; a sample collection unit connected to the other end of the filter unit and collecting a sample that has passed through the filter unit; a pump connected to at least one of the sample injection unit and the sample collection unit to allow the sample to pass through the filter unit at a predetermined flow rate; and a cell culture unit for receiving the filter separated from the filter unit and culturing the minimal residual disease cells remaining in the filter.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a micro-

More particularly, the present invention relates to a method for separating fine residual cancer cells present in the blood of a patient, thereby obtaining a micro residual cancer cell capable of selecting an anticancer drug optimized for the cancer cell that causes recurrence Separating incubator.

Even if chemotherapy has been successfully performed for cancer patients, there are frequent recurrences of cancer due to micro residual cancer cells in the presence of minimal residual die cell (CE) cells in the blood.

Since the residual cancer cells present in the patient's blood are resistant to anticancer drugs, they are considered to be resistant to the existing anti-cancer drugs, and follow-up is required.

In particular, genetic variations such as FLT3-ITD, KIT, RAS, WT1, NPM1, and BCOR have been reported in refractory hematologic malignancies with diverse genetic variants compared to other cancer types.

Therefore, in order to clarify the causative factors of cancer recurrence, it is necessary to clarify the relationship between the biological characteristics of the fine residual cancer cells and other anticancer drugs. Specifically, it is necessary to analyze the genetic variation of the fine residual cancer cells to determine whether a new gene mutation is formed by chemotherapy or whether the selection progrssion progresses in various tumors.

Accordingly, a need has arisen for a microdilution cancer cell differentiation incubator capable of not only measuring the biological action of the fine residual cancer cells but also monitoring the condition of the cells.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a micro-residual cancer cell isolation culture apparatus capable of separating minute residual cancer cells existing in the blood or bone marrow of a patient at a high recovery rate.

It is another object of the present invention to provide a micro-residual cancer cell culture incubator capable of culturing micro-residual cancer cells in a cell culture scaffold and tracking the growth of micro-residual cancer cells in real time.

The technical objects of the present invention are not limited to the above-mentioned technical problems, and other technical subjects not mentioned can be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, there is provided a microfluidic carcinoma cell culture incubator comprising: a sample injection unit for injecting a sample containing fine residual cancer cells; one end connected to the sample injection unit, A sample recovery unit connected to the other end of the filter unit to recover a sample having passed through the filter unit, and connected to at least one of the sample injection unit and the sample recovery unit, A pump for allowing the sample to pass through the filter unit at a predetermined flow rate, and a cell culture unit for receiving the filter separated from the filter unit and culturing the micro residual cancer cells remaining in the filter.

According to an embodiment of the present invention, a filter is disposed in the filter unit such that a size of a gap formed in the filter from the uppermost filter to the center filter is sequentially reduced with respect to the center filter, The filter may be arranged so that the size of the air gap sequentially increases up to the filter.

According to an embodiment of the present invention, the size of the cavity formed in the filter included in the filter unit may be 150 μm to 199 μm.

According to an embodiment of the present invention, the filter included in the filter unit may be a scaffold having a structure in which a plurality of tubular structures, which are fabric materials, are combined.

According to an embodiment of the present invention, the filter included in the filter portion may be arranged in parallel to a second direction perpendicular to the first direction on a plurality of tubular structure upper layers arranged in parallel to the first direction A plurality of tubular structures may be stacked.

According to an embodiment of the present invention, the filter included in the filter unit includes a first layer including a plurality of tubular structures, and a plurality of tubular structures included in the first layer, And a second layer including a tubular structure, wherein the tubular structure included in the first layer and the tubular structure included in the second layer are disposed to be shifted from each other.

According to one embodiment of the present invention, the pump is configured such that the sample is passed through the filter section at a rate of 4.5 ml / min. To 45 ml / min, or negative pressure may be applied to the sample.

According to the above-described micro-residual cancer cell separation incubator, not only the fine residual cancer cells present in the sample can be efficiently separated, but also the separated micro residual cancer cells can be cultured under the same conditions as the in vivo environment, And it is possible to select an optimal anticancer drug by analyzing the genome of the micro residual cancer cell.

FIG. 1 is a functional block diagram for explaining a micro-residual cancer cell culture incubator according to an embodiment of the present invention.
2 is a cross-sectional view of a filter unit according to an embodiment of the present invention.
3 is a perspective view of a filter included in a filter unit according to an embodiment of the present invention.
4 is a cross-sectional view of a filter included in a filter unit according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

Unless defined otherwise, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by one of ordinary skill in the art to which this invention belongs. Also, commonly used predefined terms are not ideally or excessively interpreted unless explicitly defined otherwise.

Also, the singular forms herein may include plural forms unless specifically stated in the text. It is noted that the terms "comprises" and / or "comprising" used in the specification are intended to be inclusive in a manner similar to the components, steps, operations, and / Or additions.

FIG. 1 is a functional block diagram for explaining a micro-residual cancer cell culture incubator according to an embodiment of the present invention.

1 includes a sample injection unit 110, a filter unit 130, a sample collection unit 150, a pump 170, and a cell culture unit 190.

FIG. 1 illustrates only components according to an embodiment of the present invention. It will be apparent to those skilled in the art that additional general configurations other than those shown in FIG. 1 can be added have.

The sample injecting section 110 injects a sample containing the fine residual cancer cells. The sample injection unit 110 according to an embodiment of the present invention may be implemented in the form of a syringe.

The sample may be the blood or marrow of the patient suspected of containing the fine residual cancer cells, and may be injected with 5 ml to 10 ml of the sample.

The filter unit 130 includes a plurality of filters, one end of which is connected to the sample injecting unit 110 and the other end of which is connected to the sample collecting unit 150 and are sequentially disposed according to the size of the pores.

Generally, it is known that cancer cells existing in the blood are larger than the size of the differentiated blood cells. Therefore, a sample injection unit 110 injected with a sample is connected to one end of the filter unit 130, and a sample is passed through the filter unit 130 , It is possible to separate the fine residual cancer cells into an activated state.

The filter (not shown) included in the filter unit 130 is formed with voids so that the fine residual cancer cells larger in size than the voids formed in the filter are allowed to remain on the filter.

The fine residual cancer cells remaining in the filter are transferred to the cell culture unit 190 together with the filter and cultured in the same environment as the in vivo environment to be used as data for characterizing biological characteristics such as an anticancer reaction and metastasis formation do.

A specific method of arranging the filter according to the pore size of the filter disposed inside the filter unit 130 will be described in detail below.

The sample recovery unit 150 is connected to the other end of the filter unit 130 and collects the sample that has passed through the filter unit 130. The sample collecting unit 150 may be implemented in the form of a syringe as in the sample injecting unit 110, and may be detachably connected to the filter unit 130.

The pump 170 is connected to at least one of the sample injecting unit 110 and the sample collecting unit 150 to allow the sample to pass through the filter unit 130 at a predetermined flow rate.

When the flow rate of the sample flowing through the filter unit 130 is set to be faster than the reference value, the fine residual cancer cells larger than the pore size formed in the scaffold are pushed out due to the rapid flow rate, or the fine residual cancer cells are killed If the flow rate of the sample is insufficient, the sample may not pass through the scaffold.

The cell culture unit 190 receives the separated filter from the filter unit 130 and cultivates the fine residual cancer cells remaining in the filter in an in vivo environment. That is, the fine residual cancer cells remaining in the scaffold are cultured in an in-vivo environment.

For analysis of anti-cancer drugs in micro-residual cancer cells, micro-residual cancer cells isolated from the sample (blood or bone marrow) should be cultured in a structure similar to the bone marrow environment. Accordingly, the cell culture unit 190 according to an embodiment of the present invention receives the filter separated from the filter unit 130.

Since the fine residual cancer cells which have not passed through the gap formed in the filter remain in the filter separated by the filter unit 130, when the filter unit 130 in this state is placed in the cell culture unit 190, It is possible to cultivate the fine residual cancer cells.

Thereafter, a variety of anticancer drugs are added to the cultured tumor cells to measure the degree of apoptosis of the tumor cells, thereby allowing the patient to select an optimal anticancer drug.

According to the above-described fine residual cancer cell differentiation cell 100, it is possible not only to efficiently separate the fine residual cancer cells present in the sample but also to separate the fine residual cancer cells under the same conditions as the in vivo environment Thus, it is possible to analyze the growth process of tumor cells and to select an optimal anticancer agent through analysis of microorganism residual cancer cells.

2 is a cross-sectional view of a filter unit according to an embodiment of the present invention.

There are cells of various sizes in the sample (blood or bone marrow). For example, if the sample is blood, it is important to select a filter capable of removing minute residual cancer cells, since there are various sizes of cells including red blood cells.

Accordingly, the filter unit 130 according to an embodiment of the present invention has a structure in which a plurality of filters 135 having different cavities are sequentially arranged. At this time, the filter is arranged such that the size of the air gap is sequentially reduced from the uppermost filter to the center filter with respect to the center filter, and the filter is arranged so that the size of the air gap from the center filter to the lowest filter is sequentially increased.

Therefore, the fine residual cancer cells remain in the filter having the pores smaller than the size of the fine residual cancer cells. The remaining cells other than the fine residual cancer cells remain in the other filters having the pores smaller than the size, so that the fine residual cancer cells can be efficiently separated.

According to an embodiment of the present invention, the gap formed in the filter included in the filter unit 130 has a value between 150 mu m and 199 mu m. Table 1 below shows the recovery rates of the fine residual cancer cells according to the size of pores formed in the filter.

Cell line Number of cells () Filter size Number of cells recovered () Recovery rate (%) HL-60 10,000 300 μm to 450 μm 4,842 48.4 1,000 475 47.5 100 23 23.0 10,000 200 μm to 299 μm 5,638 56.4 1,000 564 56.4 100 34 34.0 10,000 150 μm to 199 μm 8,521 85.2 1,000 867 86.7 100 81 81.0 10,000 100 μm to 149 μm 6,375 63.8 1,000 671 67.1 100 19 19 KG1 10,000 300 μm to 450 μm 3,482 34.8 1,000 532 56.2 100 0 0 10,000 200 μm to 299 μm 5,632 56.3 1,000 463 46.3 100 53 56.0 10,000 150 μm to 199 μm 8,721 87.2 1,000 874 87.4 100 83 83.0 10,000 100 μm to 149 μm 5,635 56.3 1,000 613 61.3 100 43 43.0 NB4 10,000 300 μm to 450 μm 3728 37.3 1,000 362 36.2 100 12 12.0 10,000 200 μm to 299 μm 4832 48.3 1,000 575 57.5 100 52 52.0 10,000 150 μm to 199 μm 8759 87.6 1,000 872 87.2 100 82 82.0 10,000 100 μm to 149 μm 5942 59.4 1,000 422 42.2 100 3 3.0 K562 300 μm to 450 μm 4,326 43.2 1,000 342 34.2 100 34 34.0 10,000 200 μm to 299 μm 2,352 32.5 1,000 46 46.0 100 5 5.0 10,000 150 μm to 199 μm 7,435 74.3 1,000 765 76.5 100 75 75.5 10,000 100 μm to 149 μm 4,536 45.3 1,000 435 43.5 100 35 35.0

As shown in Table 1, it can be seen that when the pores formed in the filter have a value between 100 μm and 200 μm, the recovery rate of various kinds of fine residual cancer cells can be remarkably increased.

The filter unit 130 according to an embodiment of the present invention includes an upper layer filter having a gap of 199 m, an upper layer filter having a gap of 175 m, an intermediate filter having a gap of 150 m, a lower layer filter having a gap of 175 m, Filters may be arranged in a sequential order.

3 is a perspective view of a filter included in a filter unit according to an embodiment of the present invention.

According to an embodiment of the present invention, the filter included in the filter unit 130 may be a scaffold structure having a structure in which a plurality of tubular structures, which are fabric materials, are combined. A scaffold is a support structure in which a cell can grow.

The scaffold is a fabric material and has a structure in which tubular structures 131 are sequentially laminated. Specifically, a plurality of tubular structures 131 arranged in parallel so as to be parallel to the first direction are arranged in parallel to a second direction perpendicular to the first direction, and a plurality of tubular structures 131 are stacked I have. Thereafter, a plurality of tubular structures 131 arranged in the first direction and a plurality of tubular structures 131 arranged in the second direction are alternately stacked.

At this time, the voids formed in the scaffold can be determined by the diameter of the tubular structure and the separation distance between the tubular structures.

4 is a cross-sectional view of a filter included in a filter unit according to an embodiment of the present invention.

As described above, the filter has a structure in which a tubular structure made of a fabric material is woven and a gap is formed between the structures. In this case, when the diameter of the tubular structure or the spacing between the structures is adjusted, the size of the pores formed in the filter can be adjusted.

At this time, the tubular structures arranged in the same direction and belonging to different layers are arranged to be offset from each other.

4, the tubular structure 130 belonging to the first layer 410 and the tubular structure belonging to the second layer 430 are arranged to face the same direction.

At this time, the tubular structure belonging to the first layer 410 and the tubular structure belonging to the second layer 430 are arranged to be offset from each other. When the sample passes through the filter, Cells are trapped in the filter.

On the other hand, samples such as blood or bone marrow have a predetermined flow rate and pass through the filter. When the flow rate is too fast, the fine residual cancer cells larger than the size of the pores formed in the filter are pushed out or not filtered, It may happen that it is killed. Or, if the flow rate is insufficient, the sample may not pass through the filter.

Table 2 below shows the mortality of the fine residual cancer cells filtered by the filter according to the flow rate of the sample. In this embodiment, to observe the mortality of the fine residual cancer cells according to the flow rate, the microscopic residual cancer cells fixed on the filter of the filter unit 130 were stained and the cell morphology was observed in a manner of observing the cell morphology.

Flow rate # of loading cell # of Apoptotic cell % of Apoptotic cell HL-60 50ml / min 300 182 61 45ml / min 300 45 15 35ml / min 300 42 14 10ml / min 300 39 13 4.5 ml / min 300 54 18 2 ml / min 300 164 55 KG-1 50ml / min 400 224 56 45ml / min 400 58 15 35ml / min 400 46 12 10ml / min 400 59 15 4.5 ml / min 400 49 12 2 ml / min 400 187 47 NB4 50ml / min 700 489 70 45ml / min 700 92 13 35ml / min 700 104 15 10ml / min 700 97 14 4.5 ml / min 700 94 13 2 ml / min 700 562 80 K562 50ml / min 400 235 59 45ml / min 400 52 13 35ml / min 400 59 15 10ml / min 400 67 17 4.5 ml / min 400 53 13 2 ml / min 400 164 41

Accordingly, the pump 170 according to the embodiment of the present invention is capable of supplying a sample of 4.5 ml / min. The sample can be pressurized at a predetermined pressure or a negative pressure can be applied so as to pass through the filter unit 130 at a flow rate of 45 ml / min.

Here, the pressure of the sample means that the sample is pushed into the filter portion 130 by applying a predetermined positive pressure to the sample, and the meaning of applying the negative pressure means that the sample is discharged from the outlet of the filter portion 130 Inhaling the sample.

According to the above-described micro-residual cancer cell differentiation culture apparatus, since the micro cancer cells remaining in the sample can be effectively separated, various anticancer drugs can be added to the cultured tumor cells to measure the degree of apoptosis of the tumor cells. Can be provided.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed methods should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A sample injection unit for injecting a sample containing fine residual cancer cells;
A filter unit including a plurality of filters, one end of which is connected to the sample injecting unit and are sequentially arranged according to the size of the gap;
A sample recovery unit connected to the other end of the filter and recovering a sample having passed through the filter unit;
A pump connected to at least one of the sample injecting unit and the sample collecting unit to allow the sample to pass through the filter unit at a predetermined flow rate; And
And a cell culture unit for receiving the filter separated from the filter unit and culturing the fine residual cancer cells remaining in the filter. The method according to claim 1,
The filter unit includes:
The filter is arranged such that the size of the air gap formed in the filter from the uppermost filter to the center filter is sequentially reduced on the basis of the center filter located in the middle, and the filter is arranged so that the size of the air gap from the center filter to the lowermost filter is sequentially increased Which is a microorganism of the present invention. The method according to claim 1,
Wherein the size of the pores formed in the filter included in the filter unit is 150 mu m to 199 mu m. The method according to claim 1,
Wherein the filter included in the filter unit comprises:
Wherein the scaffold is a structure in which a plurality of tubular structures as a fabric material are combined. 5. The method of claim 4,
Wherein the filter included in the filter unit comprises:
Wherein a plurality of tubular structures arranged in parallel to a second direction perpendicular to the first direction are stacked on top of a plurality of tubular structures arranged in parallel so as to be parallel to the first direction. 6. The method of claim 5,
Wherein the filter included in the filter unit comprises:
A first layer comprising a plurality of tubular structures; And
And a second layer including a plurality of tubular structures arranged in the same direction as the plurality of tubular structures included in the first layer,
Wherein the tubular structure included in the first layer and the tubular structure included in the second layer are disposed to be shifted from each other. The method according to claim 1,
The pump includes:
The sample was passed through the filter section at 4.5 ml / min. To 45 ml / min, or the negative pressure is applied to the sample.

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