KR20210061565A - Separating filter for capturing cell, separating apparatus and method using it - Google Patents

Abstract

The present invention relates to a separation filter for capturing cells using a photosensitive resin cured film as a filter material while preventing deformation of and damage to blood cancer cells by controlling the shape of a penetration unit through which separation occurs, and a device and a method for separating cells using the same. The separation filter for capturing cells has a plurality of penetration units in the thickness direction of a sheet.

Description

Cell capture and separation filter, cell separation apparatus and method using the same TECHNICAL FIELD [Separating filter for capturing cell, separating apparatus and method using it}

The present invention relates to a cell capture separation filter, and a cell separation apparatus and method using the same.

Recently, cancer has emerged as a major cause of death worldwide, and socio-economic costs are increasing significantly. Accordingly, there is a need to develop technologies for effective diagnosis and prognosis related to cancer treatment. As a result, securing technology related to liquid biopsy that enables non-invasive sample collection is becoming important.

Liquid biopsy refers to a test that obtains cancer information from cancer-related biomarkers from the human body such as blood and urine, and is used for personalized prognosis prediction and diagnosis such as cancer diagnosis, treatment effect analysis, and metastasis recurrence prediction.

The biomarkers used for the liquid biopsy are typically circulating tumor cells (CTC), circulating tumor DNA (ctDNA), nano vesicles (exosome), and the like.

Circulating Tumor Cells (CTCs, hereinafter referred to as blood cancer cells) refer to cancer cells that circulate in the blood after being separated from the primary cancer. The blood cancer cells are present in a very small amount of 1 to 10 per billion blood cells in the blood, and their size also overlaps with the size of white blood cells in the order of several µm to 15 µm. Therefore, since it must have a high sensitivity for the separation of blood cancer cells, there is a great difficulty in the development of a physical separation method and apparatus for the separation.

The separation methods that are currently being widely studied include methods using antibodies that respond to specific cancer cells, separation methods using a physical filter or centrifugal force using a size difference, methods using optical specificity, and other microfluidic bases, using electric or magnetic fields, etc. Several methods have been suggested.

Korean Patent Publication No. 10-2014-0074321 discloses that it is possible to efficiently capture circulating cancer cells in the blood while presenting a metal filter made of copper having a plurality of through holes. A metal filter for capturing cancer cells in which the opening of the hole is corrugated and the surface of the metal sheet is gold plated is proposed.

The metal filters proposed in these patents have a problem in that the thickness of the metal filters is thin, so that cancer cells can pass through the thin film due to deformability, cells can be damaged, and the manufacturing process is complicated.

In Korean Patent Application Laid-Open No. 10-2017-0042685, materials such as polyethylene terephthalate, polycarbonate, polyimide, polytetrafluoroethylene, polyurethane, polylactic acid, and parylene were mentioned as filter materials. However, there is only mention of these polymer materials, and the manufacturing method or advantages using them are not considered at all.

Republic of Korea Patent Publication No. 10-2014-0074321 (2014.06.17), Method of manufacturing a metal filter Korean Patent Laid-Open No. 10-2015-0020290 (20015.02.25), cancer cell trapping metal filter, cancer cell trapping metal filter sheet, cancer cell trapping device, and manufacturing method thereof Korean Patent Laid-Open Publication No. 10-2017-0042685 (2019.04.19), cell capture method, specific cell capture complete device manufacturing method, and specific cell-containing solution manufacturing method

The present inventors designed a new type of filter by designing a process that dramatically simplified the complexity of the manufacturing process to prevent deformation or damage of blood cancer cells, which is a problem occurring in the existing metal filter, and use this to separate blood cancer cells. In this case, it was confirmed that the separation efficiency is high, and high reliability of the obtained result can be secured.

Accordingly, it is an object of the present invention to provide a cell capture separation filter of a new material.

In addition, another object of the present invention is to provide a method and apparatus for separating blood cancer cells using the cell capture separation filter.

In order to achieve the above object, the present invention provides a cell capture separation filter in which a plurality of penetrations are formed in a thickness direction of a sheet to separate cells to be separated from a fluid sample.

In this case, the sheet includes a material cured with a photosensitive resin.

The through part has a tapered structure in which the width of the lower opening gradually decreases compared to the width of the upper opening through which the fluid sample is introduced.

At this time, the width (W2) of the upper opening of the penetrating part is 8.5 μm to 18 μm, the length (L2) is 10 μm to 70 μm, the width (W1) of the lower opening is 3 μm to 8.4 μm, and the length (L1) Has a range of 20 μm to 60 μm.

In addition, the cell capture separation filter according to an embodiment of the present invention

Providing a substrate;

Laminating an uncured photosensitive resin film on a substrate;

An exposure step after placing a photomask on the uncured photosensitive resin film;

A developing step for removing the uncured region of the uncured photosensitive resin film; And

It manufactures including; separating the cured film formed with the through portion from the substrate.

At this time, it includes the step of further performing a process of attaching the release film to the substrate before the lamination.

In addition, it includes the step of further performing a hard baking process after the developing process.

In addition, the cell capture separation filter according to an embodiment of the present invention

Drying after coating the photosensitive resin composition on a substrate;

An exposure step after disposing a photomask on the obtained uncured photosensitive resin film;

A developing step for removing the uncured region in the cured film; And

It manufactures including; separating the cured film formed with the through portion from the substrate.

At this time, it further includes forming a sacrificial layer on the substrate before applying the photosensitive resin composition.

In addition, the present invention includes a housing having at least one fluid inlet and a fluid outlet, respectively; And a filter for separating cells installed in the housing, wherein the filter provides a cell separating device having the above-described cell capture and separating filter.

In addition, the present invention prepares a cell separation device equipped with the cell capture and separation filter, inserts a fluid sample into the fluid inlet of the cell separation device, passes through the cell capture separation filter, and selectively selects cells to be separated from the fluid sample. It provides a method of capturing, capturing cells.

The cell capture and separation filter according to the present invention is made of a polymer material, and by controlling the shape of the penetrating portion in which the separation occurs, it is possible to prevent deformation and damage of blood cancer cells, and at the same time, increase separation efficiency and reliability.

In particular, as the photosensitive resin is used as the filter material, there is an advantage in that it is possible to manufacture a filter through a simplified process compared to the patterning process of a conventional metal or polymer filter.

1 is a schematic diagram showing a cell capture separation filter according to an embodiment of the present invention.
2 is a cross-sectional view of FIG. 1.
3 is a schematic diagram showing an exemplary shape of a penetrating portion of the cell capture separation filter of the present invention.
4 is a view showing a manufacturing process of the cell capture separation filter according to the first embodiment of the present invention.
5 is a view showing a manufacturing process of the cell capture separation filter according to the second embodiment of the present invention.
6 is a view showing a manufacturing process of the cell capture separation filter according to the third embodiment of the present invention.
7 is a view showing a manufacturing process of the cell capture separation filter according to the fourth embodiment of the present invention.
8 is a schematic diagram showing a separation device equipped with a cell capture separation filter of the present invention.

With reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

In the drawings, the thicknesses are enlarged in order to clearly express various layers and regions. Like reference numerals are attached to similar parts throughout the specification. When a part such as a layer, film, region, plate, etc. is said to be "on" another part, this includes not only the case where the other part is "directly above", but also the case where there is another part in the middle. Conversely, when one part is "directly above" another part, it means that there is no other part in the middle.

Cell capture separation filter and manufacturing method

In performing a liquid biopsy necessary for effective diagnosis and prognosis related to cancer treatment, blood cancer cells are used as important biomarkers. The blood cancer cells are present in a very small amount in the blood and have a size of several µm to 15 µm, so that they overlap with red blood cells of about 6 to 7 µm, white blood cells of about 7 to 9 µm, and platelets of less than 5 µm. Has. Accordingly, there is a need for a method of selectively separating only blood cancer cells. Unless otherwise specified below, the cells are blood cancer cells.

1 is a schematic diagram showing a cell capture separation filter according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view of FIG. 1.

Referring to FIG. 1, the cell capture and separation filter 1 has a plurality of penetrations 3 formed in the thickness direction of the sheet 5 in order to separate the cells to be separated from the fluid sample.

The sheet 5 includes a photosensitive resin cured material, specifically a photosensitive resin cured film, among polymer materials. This material is distinguished from a conventional metal filter or a polymer material such as polyethylene, and prevents deformation and damage of blood cancer cells due to the use of the metal filter.

In addition, by controlling the shape of the penetrating part 3, the problem of size similarity of cells such as hemocytosis cells, white blood cells, red blood cells, and platelets present in the blood is solved.

Referring to FIG. 2, the through part 3 is designed in a tapered shape having a narrow structure in which the width W1 of the lower opening is narrow compared to the width W2 of the upper opening through which the fluid sample is introduced. In the case of cells (eg, platelets) having a diameter narrower than the width W1 of the lower opening, the filter 1 is easily passed, but hemocancer cells, white blood cells, and red blood cells having a larger size cannot pass through. The blood cancer cells, white blood cells, and red blood cells reach the upper opening by fluid movement, and cells with high deformability due to pressure or gravity applied from the outside are pushed by the pressure and the penetrating part of the filter 1 due to elastic deformation ( 3) pass.

On the other hand, in the case of cancer cells, such as cancer cells, which have significantly less elasticity than normal cells, the deformation does not occur sufficiently and thus the penetrating portion 3 cannot be passed through. As a result, a very small amount of cancer cells in the blood remain on the upper side of the penetrating part 3 by using the difference in size between blood cells and the difference in mechanical deformability.

The opening shape of the through part 3 is preferably at least one shape selected from the group consisting of a circle, an ellipse, a rounded rectangle, a rectangle, and a square when viewed from the front (top). In order to increase the separation efficiency of cancer cells, the width (W2) of the upper opening of the penetrating part 3 is 8.5 µm to 18 µm, 9 µm to 17 µm, and 10 µm to 15 µm, and the width (W1) of the lower opening is 3 µm. It is formed to be 8.4㎛, 3.5㎛ to 8.2㎛, 4㎛ to 8㎛.

If an opening is formed in the shape of a rectangular or rounded rectangle whose length direction is longer than that of the width direction, cells with weak deformability among cells are removed while preventing clogging by other cells, and cells with less deformability are filtered out and the cells can be recovered without damage. I can. At this time, the length of the upper opening (L2) is 10㎛ to 70㎛, 20㎛ to 60㎛, 30㎛ to 50㎛, and the length (L1) of the lower opening is 20㎛ to 60㎛, 25㎛ to 50㎛, 30㎛ It is formed in -40 µm.

According to a preferred embodiment, the through part 3 is manufactured in a rounded rectangle including two long sides of the same length and two semicircles, as shown in FIG. 1, but the width W2 of the upper opening is 10 μm. It is designed to have a range of ∼15 µm, a length L2 of 30 µm to 50 µm, a width W1 of a lower opening of 4 µm to 8 µm, and a length L1 of 30 µm to 40 µm.

According to a preferred embodiment, when W2/W1 has a range of 2 to 3, and L2/L1 has a range of 1 to 1.5, high separation efficiency can be achieved.

In the tapered structure as shown in FIG. 1, the taper angle is preferably 10° or less, 8° or less, 7° or less, 5° or less, and 4° or less.

The cross section of the through part 3 may have various shapes as shown in FIG. 3.

In addition, the arrangement of the through parts 3 may be arranged on the sheet 1 at regular intervals as shown in FIG. 1, or they may be arranged in a staggered manner, and if necessary, the arrangement may be arranged in a radial shape such as a spiral. It is possible.

The thickness of the cell capture separation filter 1 according to the present invention is formed to a thickness of 70 µm to 250 µm, 85 µm to 230 µm, 90 µm to 215 µm, and 100 µm to 200 µm. The thickness is appropriately determined in consideration of the amount of a fluid sample such as blood applied to the cell trapping and separating filter 1, the diameter of the penetrating portion 3, physical factors such as time, flow rate, and internal pressure, operability, cost, and the like.

If it is formed in a thin thickness, when the cell capture separation filter 1 is bent due to the pressure during the cell separation process, the separation efficiency decreases, or the through part 3 is deformed due to the bending and passes through it or Damage to the cells in contact with it may occur. Conversely, when the thickness is thicker than the above range, it is not easy to control the shape of the through part 3 in the manufacturing process, and the time required for separation may be lengthened.

The thickness is related to the taper angle, and when the thickness is increased, the width (W2) of the upper opening is widened (at least 15 μm to 23 μm) when targeting an angle of 4°, so that the location where the blood cancer cells are captured is inward. It can go in and make it difficult to identify the cells. On the contrary, if you target the length (W2/W1 = 2~3), the angle becomes smaller and other cells are also captured, so it may be difficult to select.

In addition, the size of the cell capture separation filter 1 according to the present invention is preferably 150 mm 2 to 850 mm 2, 170 mm 2 to 750 mm 2, 180 mm 2 to 650 mm 2, and 200 mm 2 to 500 mm 2. If the size of the cell trapping metal filter is formed smaller than the above range, the time required for separation may be lengthened. Conversely, if the size of the cell trapping metal filter exceeds the above range, the dead space increases. Therefore, it is appropriately adjusted within the above range. The size is related to the size of the housing of the device (220 in FIG. 8), which will be described later, when the size of the housing when viewed from the top has a size of 5 mm×5 mm to 50 mm×50 mm, and more When designing a larger or smaller housing, it is possible to manufacture it below or above the above range.

The penetrating portion 3 of the cell trapping and separating filter 1 having such a size is designed in consideration of the thickness and fluid velocity of the filter 1 and the pressure applied to the filter 1. Preferably, when the fluid velocity is 25 ml/h and the thickness of the filter 1 is 200 μm (100 μm), the number of penetrating portions 3 is 700 at a level in which the filter 1 is not damaged. It can form ㎟∼2000 pieces/㎟, 800 pieces/㎟∼1800 pieces/㎟, 900 pieces/㎟∼1700 pieces/㎟, and 1000 pieces/㎟∼1500 pieces/㎟.

The aperture ratio of the filter 1 varies depending on the size and number of the through portions 3, but ranges from 10% to 50%, 10% to 45%, and 10 to 40%. The larger the aperture ratio, the greater the fluid throughput, and thus the separation time may be shortened, but it causes the strength of the filter 1 itself to decrease. Conversely, an aperture ratio of too low a level increases the separation time and may block the flow of fluid during the separation process, resulting in clogging.

As already mentioned, the filter 1 according to the present invention is made of a polymer material different from that of a conventional metal filter, and a photolithography process can be applied to regularly form the penetrating portion 3 having a fine pattern. Resins are used, and they are made of a cured photosensitive resin cured film.

As the photosensitive resin, a'negative photosensitive resin' that is cured by light irradiation is used, and the material of the filter 1 is made of a'photocurable photosensitive resin' in which a curing reaction is caused by light.

The negative photosensitive resin includes 25 to 55% by weight of a photocurable resin, 25 to 60% by weight of a reactive diluent, and 1 to 15% by weight of a photoinitiator, and may further include a solvent and various additives.

The photosensitive resin is selected from polyester acrylate, epoxy acrylate, urethane acrylate, polyether acrylate, polyacrylate, silicone acrylate, unsaturated polyester, epoxy, and polyimide containing photocurable organic functional groups. One or more polymer resins are possible, and double epoxy acrylates are preferred.

In addition, the reactive diluent may correspond to one or more polyfunctional acrylates, methacrylates, glycidyl ethers, epoxy, vinyl ethers, and styrenes (i.e., 1 type or 2 or more types). have.

Photoinitiators can be used regardless of their type as long as they can absorb light in the ultraviolet region having a wavelength of 100 nm to 400 nm and form radicals by decomposition of molecules. Alternatively, a phosphine oxide-based photoinitiator or the like may be used.

As the additives, known additives such as crosslinking agents, pressure-sensitive adhesives, viscosity modifiers, leveling agents, waxes, antifoaming agents, anti-aging agents, stabilizers, coloring agents, particulate fillers, light stabilizers, ultraviolet absorbers, pigments, dispersants, wetting agents, and curing accelerators are used. do.

The crosslinking agent includes amine compounds such as urea, melamine and glycolurea, and amino plasters, and specifically, urea-formaldehyde oligomers, melamine-formaldehyde oligomers, benzoguanamine-formaldehyde oligomers, glycoluril-formaldehyde oligomers And hexa(methoxymethyl) melamine oligomers.

The solvent varies depending on the composition, and known organic solvents such as water, alcohol-based solvent, ether-based solvent, ketone-based solvent, amide-based solvent, and sulfone-based solvent may be used.

The method of manufacturing the filter 1 using the negative photosensitive resin can be performed by various methods as described below by laminating the resin using a film state or coating it in a solution state.

(Example 1)

4 is a view showing a manufacturing process of the cell capture separation filter 1 according to the first embodiment of the present invention.

The production of the cell capture separation filter 1 according to the first embodiment of FIG. 4

a) providing a substrate 30;

b) laminating an uncured photosensitive resin film 10a on the substrate 30;

c) an exposure step after placing the photomask 40 on the uncured photosensitive resin film 10a;

d) a developing step for removing the uncured region of the uncured photosensitive resin film 10; And

e) separating the cured film 10 on which the penetrating portion is formed from the substrate 30;

The substrate 30 in step a) may be a silicon substrate, a glass substrate, a quartz substrate, a plastic substrate, or a metal substrate.

The photosensitive resin film 10a is a film in which the negative photosensitive resin composition is present in an uncured state (ie, in a dry state), and may be provided in the form of a sheet. It can be manufactured directly or purchased commercially and used. Considering that the thickness of the filter 1 to be produced lasts (70 µm to 300 µm) and shrinks after curing, the photosensitive resin film 10 a is preferably at most 300 µm or less, preferably at least 50 µm or more. .

The lamination process in step b) is a process of attaching the photosensitive resin film 10a on the substrate 30, so that the space between the substrate 30 and the photosensitive resin film 10a is eliminated compared to the method using a conventional roll. The adhesion is increased, thereby minimizing the occurrence of holes or craters due to air existing in the space.

In step c), a photomask 40 for pattern formation is faced or laminated on the photosensitive resin film 10a, and then light is irradiated to perform photocuring of the photosensitive resin film 10a.

As a light source for light irradiation, in addition to rays such as far ultraviolet, ultraviolet, near ultraviolet, and infrared rays, electromagnetic waves such as X-rays and γ-rays, electron beams, proton rays, and neutron rays, etc. can be used, but curing speed, acquisition of irradiation devices It is advantageous to cure by light irradiation because of its ease of use and cost.

As an exposure method, a method of irradiating an active light onto an image through a negative or positive mask pattern called artwork (mask exposure method) is exemplified. In addition, a method of irradiating active light into an image shape may be employed by a direct drawing exposure method such as a laser direct imaging (LDI) exposure method or a digital light processing (DLP) exposure method.

A known light source can be used as the light source of the active light, for example, a carbon arc lamp, a mercury vapor arc lamp, a high pressure mercury lamp, a xenon lamp, a gas laser such as an argon laser, a solid laser such as a YAG laser, and an ultraviolet ray such as a semiconductor laser. , Effective emission of visible light, etc. is used.

As the wavelength (exposure wavelength) of the active light, it is preferably in the range of 350 to 410 nm, and more preferably in the range of 390 to 410 nm.

It is performed by irradiating exposure energy of 100mJ/cm2 to 850mJ/cm2, 150mJ/cm2 to 700mJ/cm2, and 200mJ/cm2 to 400mJ/cm2 using a light source having the above exposure wavelength.

Incidentally, the irradiation time varies depending on the type of light source, the distance between the light source and the coating film, the thickness of the coating film, and other conditions, but may usually be several seconds to several tens of seconds, and in some cases, a few seconds.

Only the light-irradiated portion of the photosensitive resin film 10a is selectively cured by the light irradiation.

In particular, the filter 1 of the present invention is designed in a tapered shape in which the width (W1) and the length (L2) of the lower opening are narrow, respectively, compared to the width (W2) and the length (L2) of the upper opening. This can be achieved through control of the amount of light and time.

Since active light is irradiated from the top of the photomask 40, the photosensitive resin film 10a on the upper side is easily cured under the influence of the active light, and the photosensitive resin film 10a on the lower side is cured compared to the upper side. It's hard to be. As a result, the cured photosensitive resin film 10 has a different degree of curing as it faces from the top to the bottom, so that a tapered shape can be obtained after the development process.

In step d), a developing process for removing the uncured region is performed to pattern the cured film 10 in which the penetrating portion (black display region) is formed.

As the developing method, a method using a dip method, a battle method, a spray method, brushing, slapping, scraping, oscillating immersion, and the like can be exemplified. From the viewpoint of improving the resolution, a high-pressure spray method is most suitable. These can also develop by combining two or more types of methods.

Examples of the developer include an alkaline aqueous solution, an aqueous developer, and an organic solvent-based developer. When the alkaline aqueous solution is used as a developer, it is safe and stable and has good operability. Examples of the base of the alkaline aqueous solution include alkali metal hydroxides such as lithium, sodium, or potassium hydroxides; Carbonate or bicarbonate salts of lithium, sodium, potassium or ammonium; Alkali metal phosphates such as potassium phosphate and sodium phosphate; Alkali metal pyrophosphates such as sodium pyrophosphate and potassium pyrophosphate are used.

As the alkaline aqueous solution, a dilute solution of 0.1 to 5% by weight sodium carbonate, a dilute solution of 0.1 to 5% by weight potassium carbonate, a dilute solution of 0.1 to 5% by weight sodium hydroxide, a dilute solution of 0.1 to 5% by weight sodium tetraborate, etc. are preferable. .

The pH of the alkaline aqueous solution is preferably in the range of 9 to 11, and the temperature is adjusted according to the alkali phenomenon of the photosensitive resin composition layer. In the alkaline aqueous solution, a surface active agent, an antifoaming agent, and a small amount of an organic solvent for accelerating development may be mixed.

In step e), the cured film 10 having the penetrating portion is peeled from the substrate 30, and then turned over to manufacture the cell capture separation filter 1 having the penetrating portion 3 formed thereon.

(2nd implementation example)

The surface of the cell capture separation filter 1 may damage cells, and for a smoother surface, it should be easy to peel from the substrate 30. Accordingly, in the present invention, a release film is further formed between the substrate 30 and the photosensitive resin film to perform the manufacturing process of the filter 1.

5 is a view showing a manufacturing process of the cell capture separation filter 1 according to the second embodiment of the present invention.

Looking at step a), it can be seen that a release film is formed on the substrate 30, and a photosensitive resin film is formed on the release film in step (b). Lamination of these two films is performed by the laminating process mentioned in the first embodiment.

As the release film 32, polyethylene (PE), polytetrafluoroethylene film, polypropylene film, surface-treated paper, or the like can be used. Due to the lamination of the release film 32, peeling of the substrate 30 in step e) and the cured film in which the penetrating portion is formed easily occurs. At this time, when peeling the release film 32, it is preferable that the adhesion between the substrate 30 and the release film 32 is higher than the adhesion between the photosensitive resin film and the release film 32.

In addition to the first to second embodiments, a hard baking process is performed using a heating device such as an oven before step d), which is a developing process, to increase the degree of crosslinking of the photosensitive resin film, and a more dense film can be produced. .

The hard baking process may be performed at a temperature of 50°C to 150°C and 60°C to 90°C. In addition, in order to further cure before the hard baking, additional exposure may be performed using ultraviolet rays or the like.

6 is a view showing a manufacturing process of the cell capture separation filter 1 according to the third embodiment of the present invention.

The cell capture separation filter 1 according to FIG. 6 is

a) providing a substrate 30;

b) laminating an uncured photosensitive resin film 10a on the substrate 30;

c) an exposure step after placing the photomask 40 on the uncured photosensitive resin film 10a;

d) a developing step for removing the uncured area in the cured film 10;

e) hard baking step; And

f) separating the cured film 10 on which the penetrating portion is formed from the substrate 30;

This step is advantageous when the plastic substrate 30 is used as the substrate 30. That is, due to the flexibility of the material of the plastic substrate 30, the substrate 30 may be slightly bent and peeled off from the cured film 10 in which the penetrating portion is formed. At this time, by performing a hard baking process on the film, the strength of the film is increased, so that it can sufficiently withstand peeling through the bending of the substrate 30.

The laminating method using the photosensitive resin film has an advantage in that the process is easy, and for ease of thickness control, the following method of applying the composition is used.

(Example 4)

7 is a view showing a manufacturing process of the cell capture separation filter 1 according to the seventh embodiment of the present invention.

The production of the cell capture separation filter 1 according to the fourth embodiment of FIG. 7

a) forming a sacrificial layer 134 on the substrate 130;

b) drying after applying a photosensitive resin composition on the sacrificial layer (134);

c) an exposure step after disposing the photomask 140 on the obtained uncured photosensitive resin film 110a;

d) a developing step for removing the uncured region in the cured film 110; And

f) the step of separating the cured film 110 in which the penetrating portion is formed from the substrate 130;

In step a), a sacrificial layer 134 is formed on the substrate 130.

For the formation of the sacrificial layer 134, a photosensitive resin or a non-photosensitive resin that does not harden or decompose by light may be used, and a composition containing them is applied on the substrate 30 and dried to form a sacrificial layer in a film state. You get (134). The formation of the sacrificial layer 134 is optional and is not an essential process for manufacturing the cured film 110.

Application may use conventional methods and apparatus known to be able to be used to apply the composition, for example spin coaters, comma coaters, blade coaters, lip coaters, rod coaters, squeeze coaters, reverse coaters, transfer roll coaters, A graiba coater, a spray coater, or the like can be used.

The sacrificial layer 134 film has a thickness of 1 µm to 100 µm, 5 µm to 70 µm, and 10 µm to 50 µm.

In step b), a photosensitive resin composition is applied on the sacrificial layer 134 and then dried to form an uncured photosensitive resin film 110a.

As the photosensitive resin composition, the negative photosensitive resin composition as described above may be used, and a photosensitive resin film in a film state may be obtained through drying after the coating process as mentioned above.

At this time, the thickness of the uncured resin film 110a may be the same as, or thicker or thinner than the thickness of the photosensitive resin film 110 mentioned in the first embodiment.

Thereafter, steps c) to e) perform the same process as mentioned in the first embodiment to fabricate the cell capture separation filter 1 having the penetrating part 3 formed thereon.

The cell capture and separation filter 1 manufactured by the above-described method is applied to a blood cancer cell separation device to separate blood cancer cells.

Cell capture and separation device and method

8 is a schematic diagram showing an apparatus 200 for separating blood cancer cells according to an embodiment of the present invention.

The blood cancer cell separation apparatus 200 according to FIG. 8 includes a housing 220 having at least one fluid inlet 221 and a fluid outlet 223, respectively; And a filter 1 installed in the housing 220 for separating cells. At this time, the filter 1 is equipped with the above-described cell capture and separation filter 1.

The housing 220 has a structure capable of introducing a liquid into the interior and discharging the liquid that has passed through the filter 1 to the outside, and may be an open housing 220 and a closed housing 220. Further, in the housing 220, an introduction region having a predetermined volume is provided so that the liquid level of the liquid remaining on the filter 1 can be maintained at a predetermined height.

The fluid inlet 221 is a region into which a fluid sample is introduced, and its upper side is open, so that it can be directly introduced. As a medium for introducing a liquid, it is preferable to use a device for automatically introducing a certain amount of liquid and a case where the measurement is performed by hand technique of a measuring person. As a device for introducing a certain amount of liquid, a semi-automatic device such as a burette, or a mechanically controlled automatic device is preferable.

By increasing the diameter or number of the fluid outlet 223 compared to the fluid inlet 221, the discharge rate of the filtrate that has passed through the filter 1 can be increased, and the blockage of the filter 1 can be suppressed to improve the separation precision. have.

In the separation method using the blood cancer cell separation device 200 of FIG. 8, a fluid sample is introduced into the fluid inlet 221 of the cell separation device and passed through the cell capture separation filter 1, and the cells to be separated from the fluid sample Selectively capture.

The fluid sample can be applied to both blood cancer cells, particularly to body fluids containing cells with seeded and untransferred CTCs. Specifically, there are lymphatic fluid, urine, sputum, ascites, effusion fluid, amniotic fluid, aspiration fluid, organ fluid, intestinal fluid, lung fluid, bronchial fluid, bladder fluid, feces and especially bone marrow and blood. The cell-containing body fluid may be directly filtered to concentrate the cells, but first, the cell-containing body fluid is used as a preparation step, and components that do not contain cells can be removed in advance by a density gradient centrifugation method or the like.

The moving speed of the fluid sample is preferably in the range of 50 to 1000 μL/min, more preferably in the range of 200 to 800 μL/min. By setting the movement speed in the above range, it is possible to increase the recovery efficiency of specific cells on the filter 1 without damaging specific cells.

The target material such as CTC separated through the cell capture separation filter 1 may be recovered through an aspirator such as a pipette. If necessary, suction can be performed after applying constant vibration to the housing using a mixer or shaker.

1: Cell capture separation filter
3: through 5: sheet
10, 110: photosensitive resin cured film
10a, 110a: uncured photosensitive resin film
30, 130: substrate
32: release film
40, 140: photomask
134: sacrificial layer
200: blood cancer cell separation device
220: housing
221: fluid inlet
223: fluid outlet

Claims (13)

In order to separate the cells to be separated from the fluid sample, a plurality of penetrations are formed in the thickness direction of the sheet,
The sheet is a cell capture separation filter comprising a photosensitive resin cured material.
The method of claim 1,
The through portion has a tapered structure in which the width of the lower opening is gradually narrowed compared to the width of the upper opening through which the fluid sample is introduced.
The method of claim 1,
The width (W2) of the upper opening of the through portion is 8.5㎛ to 8㎛, the width (W1) of the lower opening is 3㎛ * ㎛, cell capture separation filter.
The method of claim 1,
The length (L2) of the upper opening of the through portion is 10㎛ ~ 70㎛, the length (L1) of the lower opening is 20㎛ ~ 60㎛, cell capture separation filter.
The method of claim 1,
The photosensitive resin is a negative photosensitive resin, cell capture separation filter.
The method of claim 1,
The cells are blood cancer cells, cell capture and separation filter.
Providing a substrate
Laminating an uncured photosensitive resin film on a substrate;
An exposure step after placing a photomask on the uncured photosensitive resin film;
A developing step for removing the uncured region of the uncured photosensitive resin film; And
Separating the cured film in which the penetrating portion is formed from the substrate; The method of manufacturing a cell capture separation filter according to any one of claims 1 to 6, including.
The method of claim 7,
A method of manufacturing a cell capture separation filter comprising the step of further performing a process of attaching a release film to a substrate before the lamination.
The method of claim 7,
A method of manufacturing a cell capture separation filter comprising the step of further performing a hard baking process after the developing process.
Drying after coating the photosensitive resin composition on a substrate;
An exposure step after disposing a photomask on the obtained uncured photosensitive resin film;
A developing step for removing the uncured region in the cured film; And
Separating the cured film in which the penetrating portion is formed from the substrate; The method of manufacturing a cell capture separation filter according to any one of claims 1 to 6, including.
The method of claim 10,
A method of manufacturing a cell capture separation filter further comprising the step of forming a sacrificial layer on the substrate before applying the photosensitive resin composition.
A housing each having at least one fluid inlet and a fluid outlet; And
It includes a filter for separating cells installed in the housing,
The filter is a cell capture separation filter according to any one of claims 1 to 6, a cell separation device.
To prepare a cell separation device equipped with a cell capture separation filter according to claim 12,
Injecting a fluid sample into the fluid inlet of the cell separation device and passing through the cell capture separation filter,
A cell capture method for selectively capturing the cells to be separated in a fluid sample.

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