KR102528787B1 - Separating filter for capturing cell, separating apparatus and method using it - Google Patents

Abstract

The present invention relates to a cell trapping separation filter using a cured photosensitive resin film as a filter material, and a cell separation device and method using the same, while preventing deformation and damage of CTCs by controlling the shape of a penetrating portion where separation occurs.

Description

Cell capture separation filter, cell separation device and method using the same {Separating filter for capturing cell, separating apparatus and method using it}

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

Recently, cancer has emerged as a major cause of death worldwide, and the social and economic costs accordingly are greatly increasing. Accordingly, the need to develop technologies for effective diagnosis and prognosis prediction associated with cancer treatment is emerging. As a result, it is important to secure technology related to liquid biopsy that enables non-invasive sample collection.

Liquid biopsy refers to a test that obtains cancer information from cancer-related biomarkers such as blood and urine, and uses it for personalized prognosis prediction and diagnosis, such as cancer diagnosis, treatment effect analysis, and prediction of metastasis and recurrence.

Representative biomarkers used in the liquid biopsy include circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and nano-endosomes.

Circulating tumor cells (CTCs, hereinafter referred to as circulating tumor cells) refer to cancer cells that separate from a primary cancer and circulate in the blood. The CTCs are present in the blood in extremely small amounts of 1 to 10 per 1 billion blood cells, and their size overlaps with the size of leukocytes by several μm to 15 μm. Therefore, it is a great challenge to develop a physical separation method and apparatus for the separation because it must have high sensitivity for the separation of CTCs.

Separation methods that are currently being widely studied include a method using antibodies that react to specific cancer cells, a separation method using physical filters or centrifugal force using size differences, a method using optical specificity, and various other microfluidic-based or electric or magnetic fields. Several methods are presented.

Korean Patent Publication No. 10-2014-0074321 discloses that a metal filter made of copper having a plurality of through holes can efficiently capture cancer cells circulating in the blood, and Korean Patent Publication No. 10-2015-0020290 A cancer cell-trapping metal filter in which the opening of the hole has a corrugated shape and the surface of the metal sheet is gold-plated is presented.

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

Korean Patent Publication No. 10-2017-0042685 mentions materials such as polyethylene terephthalate, polycarbonate, polyimide, polytetrafluoroethylene, polyurethane, polylactic acid, and parylene as filter materials. However, there is only mention of these polymer materials, and no consideration is given to manufacturing methods or advantages using them.

Republic of Korea Patent Publication No. 10-2014-0074321 (2014.06.17), Manufacturing method of metal filter Republic of Korea Patent Publication No. 10-2015-0020290 (2001.02.25), Cancer cell trapping metal filter, cancer cell trapping metal filter sheet, cancer cell trapping device and manufacturing method thereof Republic of Korea Patent Publication No. 10-2017-0042685 (2019.04.19), cell trapping method, manufacturing method of specific cell trapping device, and manufacturing method of specific cell-containing solution

The present inventors prevented the transformation or damage of CTCs, a problem that occurred in existing metal filters, and manufactured a new type of filter by designing a process that drastically simplified the complexity of the manufacturing process, and separated CTCs using it It was confirmed that the separation efficiency was high and high reliability of the obtained results could be secured.

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

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 trapping separation filter formed with a plurality of penetrating portions in the thickness direction of a sheet to separate cells to be separated from a fluid sample.

At this time, the sheet includes a material in which a photosensitive resin is cured.

The penetrating portion has a tapered structure in which a width of a lower opening is gradually narrowed compared to a width of an upper opening through which a fluid sample is introduced.

At this time, the width W2 of the upper opening of the through portion 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 is 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 disposing a photomask on the uncured photosensitive resin film;

a developing step for removing uncured regions of the uncured photosensitive resin film; and

Separating the cured film on which the penetrating portion is formed from the substrate;

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

In addition, a step of further performing a hard baking process after the developing process is included.

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

Applying a photosensitive resin composition on a substrate and drying it;

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

a developing step to remove uncured regions in the cured film; and

Separating the cured film on which the penetrating portion is formed from the substrate;

At this time, a step of forming a sacrificial layer on the substrate before application of the photosensitive resin composition is further included.

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

In addition, the present invention provides a cell separation device equipped with the cell capture separation filter, inserts a fluid sample into a 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. A cell capture method is provided.

The cell trapping separation filter according to the present invention is made of a polymer material and controls the shape of the penetrating portion where separation occurs, thereby preventing CTC cells from being deformed or damaged, and at the same time increasing the separation efficiency and reliability.

In particular, as the photosensitive resin is used as the filter material, there is an advantage in that the filter can be manufactured by 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.
Figure 2 is a cross-sectional view of Figure 1;
3 is a schematic view showing an exemplary shape of a penetrating portion of the cell trapping 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 a cell capture separation filter according to a second embodiment of the present invention.
6 is a view showing a manufacturing process of a cell trapping separation filter according to a third embodiment of the present invention.
7 is a view showing a manufacturing process of a cell capture separation filter according to a fourth embodiment of the present invention.
8 is a schematic diagram showing a separation device equipped with a cell trapping 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 skilled in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

In the drawings, the thickness is shown enlarged to clearly express the various layers and regions. Like reference numerals have been assigned to like parts throughout the specification. When a part such as a layer, film, region, or plate is said to be "on" another part, this includes not only the case where it is "directly on" the other part, but also the case where there is another part in between. Conversely, when a part is said to be "directly on" another part, it means that there is no other part in between.

Cell capture separation filter and manufacturing method

CTCs are used as important biomarkers in performing liquid biopsies required for effective diagnosis and prognosis associated with cancer treatment. The CTCs exist in extremely small amounts in the blood and have a size of several μm to 15 μm, overlapping with about 6-7 μm red blood cells, about 7-9 μm white blood cells, and less than 5 μm platelets. have Accordingly, a method for selectively isolating only CTC cells is required. Hereinafter, cells are CTCs unless otherwise specified.

1 is a schematic diagram showing a cell trapping 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 trapping separation filter 1 is formed with a plurality of penetrating portions 3 in the thickness direction of the sheet 5 in order to separate cells to be separated from the fluid sample.

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

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

Referring to FIG. 2 , the penetrating portion 3 is designed in a tapered shape having a structure in which the width W1 of the lower opening is narrower than the width W2 of the upper opening through which the fluid sample flows. Cells (eg, platelets) having a narrower diameter than the width W1 of the lower opening pass through the filter 1 easily, but CTCs, leukocytes, and red blood cells having a larger size cannot pass through the filter 1 . The CTCs, leukocytes, and red blood cells reach the upper opening by fluid movement, and the highly deformable cells are pushed by the pressure and elastically deformed by pressure or gravity applied from the outside to pass through the filter 1 ( 3) pass.

On the other hand, in the case of cancer cells, which are remarkably less elastic than normal cells, such as cancer cells, they cannot pass through the penetrating portion 3 because they are not sufficiently deformed. As a result, a very small amount of blood cancer cells remain on the upper side of the penetrating portion 3 by using the difference in size and mechanical deformability between blood cells.

The shape of the opening of the penetrating portion 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, or 10 μm to 15 μm, and the width W1 of the lower opening is 3 μm. to 8.4 μm, 3.5 μm to 8.2 μm, and 4 μm to 8 μm.

If the opening is formed in a rectangular or rounded rectangle shape where the length direction is longer than the width direction, cells with weak deformability can escape while preventing clogging by other cells, and cells with low deformability will be filtered out so that the cells can be recovered without damage. can At this time, the length L2 of the upper opening is 10 μm to 70 μm, 20 μm to 60 μm, and 30 μm to 50 μm, and the length L1 of the lower opening is 20 μm to 60 μm, 25 μm to 50 μm, and 30 μm. It is formed to ~40 μm.

According to a preferred embodiment, as shown in FIG. 1, the penetrating portion 3 is made of a round rectangle including two long sides of the same length and two semicircular shapes, but the width W2 of the upper opening is 10 μm. 15 μm, the length L2 is 30 μm to 50 μm, the width W1 of the lower opening is 4 μm to 8 μm, and the length L1 is designed to be in the range of 30 μm to 40 μm.

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

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

The cross section of the through portion 3 may have various shapes as shown in FIG. 3 . As shown in (a) of FIG. 3, the cross-sectional shape in the thickness direction of the sheet region between two adjacent through portions is semicircular.

In addition, the arrangement of the penetrating parts 3 may be arranged spaced apart from each other at regular intervals as shown in FIG. 1 on the sheet 1, or may be arranged with their positions staggered. possible.

The thickness of the cell trapping separation filter 1 according to the present invention is 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 separation 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 formed with a thin thickness, if the cell capture separation filter 1 is bent due to the pressure during the cell separation process, the separation efficiency is lowered or the penetrating portion 3 is deformed due to the bending to pass through or Cells in contact with it may be damaged. Conversely, when the thickness is greater than the above range, it is not easy to control the shape of the penetrating portion 3 in the manufacturing process, and the time required for separation may be increased.

The thickness is related to the taper angle, and when the thickness is increased, when the angle 4° is targeted, the width (W2) of the upper opening widens (at least 15 μm to 23 μm), so that the position at which CTC cells are captured is inward It can enter and make cell identification difficult. Conversely, if the length is targeted (W2/W1 = 2~3), the angle becomes smaller and other cells are captured, making it difficult to sort.

In addition, the size of the cell trapping 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 increase, and on the contrary, if the size exceeds the above range, the dead space increases, so it is appropriately adjusted and used within the above range. The size is related to the size of the housing (220 in FIG. 8) of the device to be described later, and the size of the housing when viewed from above has a size of 5 mm × 5 mm to 50 mm × 50 mm, more than this When designing a large or small housing, it is possible to manufacture it below or above the above range.

The penetration part 3 of the cell capture separation filter 1 having this size is designed considering the thickness of the filter 1, the fluid velocity, 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 through portions 3 is 700 / It is possible to form mm2 to 2000 pieces/mm2, 800 pieces/mm2 to 1800 pieces/mm2, 900 pieces/mm2 to 1700 pieces/mm2, and 1000 pieces/mm2 to 1500 pieces/mm2.

The aperture ratio of the filter 1 varies depending on the size and number of the through portions 3, but has a range of 10% to 50%, 10% to 45%, and 10% to 40%. As the aperture ratio increases, the fluid throughput increases and the separation time can be shortened, but it causes a decrease in the strength of the filter 1 itself. Conversely, if the aperture ratio is too low, the separation time becomes long, and the flow of the fluid is blocked during the separation process, which may cause clogging.

As already mentioned, the filter 1 according to the present invention is made of a polymer material different from conventional metal filters, and has photosensitivity to which a photolithography process can be applied to regularly form the penetrating portion 3 having a fine pattern. Resin is used, and it consists of a cured photosensitive resin film.

The photosensitive resin uses a 'negative photosensitive resin' that is cured by light irradiation, and the material of the filter 1 is made of a 'photocurable photosensitive resin' that is cured 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. More than one polymeric resin is possible, of which epoxy acrylate is preferred.

In addition, the reactive diluent may correspond to one or more (ie, one or two or more) of monofunctional, bifunctional or more polyfunctional acrylates, methacrylates, glycidyl ethers, epoxies, vinyl ethers, and styrenes. there is.

The photoinitiator can be used regardless of its type as long as it can absorb light in the ultraviolet region having a wavelength of 100 nm to 400 nm and form radicals by decomposition of molecules, and benzoin-based, hydroxy ketone-based, amino ketone-based Alternatively, a phosphine oxide-based photoinitiator may be used.

As the additives, known additives such as crosslinking agents, adhesives, viscosity modifiers, leveling agents, waxes, antifoaming agents, anti-aging agents, stabilizers, colorants, fine particle 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 glycol urea, and amino plast, specifically, urea-formaldehyde oligomer, melamine-formaldehyde oligomer, benzoguanamine-formaldehyde oligomer, glycoluril-formaldehyde oligomer , Hexa (methoxymethyl) melamine oligomer, etc. are mentioned.

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

The manufacturing method of the filter 1 using the negative photosensitive resin may be performed by using the resin in a film state and laminating it or by coating it in a solution state in various ways as set forth below.

(First Embodiment)

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

Manufacturing of the cell trapping separation filter 1 according to the first embodiment of FIG. 4

a) providing a substrate 30;

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

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

d) a developing step for removing uncured regions of the uncured photosensitive resin film 10; and

e) separating the cured film 10 having the penetrating portion 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, a dry state), and may be provided in a sheet form. It can be manufactured directly or purchased and used commercially. Considering the thickness of the final filter 1 (70 μm to 300 μm) and shrinkage after curing, the photosensitive resin film 10a preferably has a thickness of at most 300 μm or less, preferably at least 50 μm or more. .

The lamination process of step b) is a process of attaching the photosensitive resin film 10a on the substrate 30, so that there is no space between the substrate 30 and the photosensitive resin film 10a compared to the conventional method using a roll. Adhesion is increased, and generation of holes or craters due to air existing in the space is minimized.

In step c), after facing or stacking the photomask 40 for pattern formation on the photosensitive resin film 10a, light is irradiated to perform photocuring of the photosensitive resin film 10a.

As a light source for light irradiation, 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 can be used, but the curing speed and availability of irradiation devices Curing by light irradiation is advantageous in terms of ease of use, cost, and the like.

As an exposure method, a method of irradiating an image with actinic light through a negative or positive mask pattern called artwork (mask exposure method) is exemplified. Further, a method of irradiating actinic rays in an image form by a direct drawing exposure method such as a laser direct imaging (LDI) exposure method or a digital light processing (DLP) exposure method may be employed.

A known light source can be used as the light source of the actinic 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-state laser such as a YAG laser, and an ultraviolet ray such as a semiconductor laser. , those that effectively emit visible light or the like are used.

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

It is performed by irradiating exposure energy of 100 mJ/cm 2 to 850 mJ/cm 2 , 150 mJ/cm 2 to 700 mJ/cm 2 , and 200 mJ/cm 2 to 400 mJ/cm 2 using a light source having the exposure wavelength.

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 is usually several seconds to several tens of seconds, and in some cases, a fraction of a second.

By the light irradiation, only the light-irradiated portion of the photosensitive resin film 10a is selectively cured.

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

Since actinic 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 actinic light, and the photosensitive resin film 10a on the lower side is cured compared to the upper side. hard to be As a result, the curing degree of the cured photosensitive resin film 10 varies 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 uncured regions is performed to pattern the cured film 10 having penetration portions (black display regions) formed thereon.

As the developing method, a method using a dip method, a battle method, a spray method, brushing, slapping, scraping, rocking dipping, or the like is exemplified, and a high-pressure spray method is most suitable from the viewpoint of resolution improvement. These may develop by combining two or more types of methods.

Examples of the developing solution include an alkaline aqueous solution, an aqueous developing solution, and an organic solvent based developing solution. When an alkaline aqueous solution is used as a developing solution, it is safe and stable and has good operability. As the base of the alkaline aqueous solution, alkali metal hydroxides such as hydroxides of lithium, sodium or potassium; carbonates or bicarbonates 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, etc. are used.

As the alkaline aqueous solution, a 0.1 to 5 wt% dilute solution of sodium carbonate, a 0.1 to 5 wt% dilute solution of potassium carbonate, a 0.1 to 5 wt% dilute solution of sodium hydroxide, a 0.1 to 5 wt% dilute solution of 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 development of the photosensitive resin composition layer. In the alkaline aqueous solution, a surface active agent, an antifoaming agent, a small amount of an organic solvent for accelerating development, and the like may be incorporated.

In step e), after peeling the cured film 10 having the penetrating part from the substrate 30, the cell trapping filter 1 having the penetrating part 3 is fabricated by turning it over.

(Second embodiment)

The surface of the cell trapping separation filter 1 may damage cells, and should be easily separated from the substrate 30 for a smoother surface. 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 trapping separation filter 1 according to the second embodiment of the present invention.

In 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, separation between the substrate 30 in step e) and the cured film in which the penetrating portion is formed occurs easily. At this time, when the release film 32 is peeled off, it is preferable that the adhesive force between the substrate 30 and the release film 32 is higher than the adhesive force between the photosensitive resin film and the release film 32 .

In addition, in the first to second embodiments, a hard baking process is performed using a heating device such as an oven before step d) of the developing process to increase the degree of crosslinking of the photosensitive resin film and to produce a film with higher density. .

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

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

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

a) providing a substrate 30;

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

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

d) a developing step to remove uncured regions in the cured film 10;

e) hard baking step; and

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

This step is advantageous when a 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 to separate the cured film 10 having the penetrating portion. 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 bending of the substrate 30 .

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

(4th embodiment)

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

Manufacturing of the cell trapping separation filter 1 according to the fourth embodiment of FIG. 7

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

b) coating a photosensitive resin composition on the sacrificial layer 134 and then drying it;

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

d) a developing step to remove uncured regions in the cured film 110; and

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

In step a), the 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 is not cured or decomposed by light may be used, and a composition containing these may be applied on the substrate 30 and then dried to form a film-like sacrificial layer. (134) is obtained. The formation of the sacrificial layer 134 is optional and is not an essential process for manufacturing the cured film 110 .

The application can use conventional methods and apparatus known to be able to be used to apply the composition, for example, a spin coater, a comma coater, a blade coater, a lip coater, a rod coater, a squeeze coater, a reverse coater, a transfer roll coater, Graiva coaters, spray coaters and 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), the 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 described above.

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

After that, in steps c) to e), the same process mentioned in the first embodiment is performed to manufacture the cell trapping separation filter 1 having the penetrating portion 3 formed thereon.

The cell trapping separation filter 1 manufactured by the above-described method is applied to a CTC cell separation device to separate CTCs.

Apparatus and method for capturing and separating cells

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

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

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

The fluid inlet 221 is an area to introduce a fluid sample, and since the upper side is open, direct introduction is possible. As the medium for introducing the liquid, it is preferable to use a device for automatically introducing a certain amount of liquid, or a case where the measurer's hand technique is used. As a device for introducing a certain amount of liquid, a semi-automatic device such as a burette and an automatic device mechanically controlled are preferable.

By increasing the diameter or number of fluid outlets 223 compared to the fluid inlet 221, the rate of discharge of the filtrate that has passed through the filter 1 is increased, and the filter 1 is prevented from clogging to improve separation precision. there is.

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 to separate target cells from the fluid sample. is selectively captured.

Fluid samples can be applied to both bodily fluids containing CTCs, especially cells with seeded and unmetastatic CTCs. Specifically, there are lymph, urine, sputum, ascites, exudate, amniotic fluid, aspiration fluid, organ lavage fluid, intestinal lavage fluid, lung lavage fluid, bronchial lavage fluid, bladder lavage fluid, feces, and particularly bone marrow and blood. Although such a cell-containing bodily fluid can be directly filtered to concentrate the cells, the cell-containing bodily fluid can be used as a preparation step beforehand to remove cell-free components by density gradient centrifugation 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 within the above range, the recovery efficiency of the specific cells on the filter 1 can be increased without damaging the specific cells.

Target substances such as CTCs 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: penetration part 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 (11)

A cell trapping separation filter formed with a plurality of penetrating portions in the thickness direction of a sheet in order to separate cells to be separated from a fluid sample,
The frontal shape of the opening of the through portion is rectangular, and the cross-sectional shape in the thickness direction of the sheet region between two adjacent through portions is semicircular,
The width of the lower opening of the through portion is narrower than the width of the upper opening through which the fluid sample flows,
The width W2 of the upper opening is 8.5 μm to 18 μm, and the width W1 of the lower opening is 3 μm to 8.4 μm,
The length L2 of the upper opening is 10 μm to 70 μm, and the length L1 of the lower opening is 20 μm to 60 μm,
The W2 / W1 is 2 to 3, and the L2 / L1 is 1 to 1.5, the cell capture separation filter. delete delete delete delete According to claim 1,
A cell capture separation filter having a thickness of the filter of 70 to 250 μm. According to claim 1,
The cell capture separation filter having an aperture ratio of 10% to 50% of the filter. According to claim 1,
The cell capture separation filter, wherein the sheet includes a material in which a photosensitive resin is cured. According to claim 1,
The cell capture separation filter, wherein the cell is a blood cancer cell. a housing each having at least one fluid inlet and one or more fluid outlets; and
It includes a filter for cell separation installed in the housing,
The cell separation device, wherein the filter is the cell trapping separation filter according to any one of claims 1 and 6 to 9. Preparing a cell separation device equipped with the cell capture separation filter according to claim 10,
Injecting a fluid sample into the fluid inlet of the cell separation device to pass through a cell capture separation filter;
A cell capture method for selectively capturing separation target cells in a fluid sample.

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