KR102302776B1 - A DISK SEPARATING PBMCs USING PHOTOSENSITIVE HYDROGEL - Google Patents

Abstract

The present invention provides a PBMC separation disk having a disk shape, comprising: a chamber into which a sample collected from a user is injected through an injection hole disposed on an upper surface of the PBMC separation disk; a rotation driving unit mounted in a mounting groove disposed at the lower end of the central portion of the PBMC separation disk and rotating the sample injected into the chamber in one direction; And when the PBMC separation disk is rotated by the rotation driving unit, different components are located in the first chamber region, the second chamber region, and the third chamber region constituting the chamber, and the peripheral blood mononuclear cells (Peripheral blood mononuclear cells) are located in the second chamber region. When a mononuclear cell (PBMC) is located, it provides a PBMC separation disk using a photocurable hydrogel, configured to include a laser for curing both sides of the second chamber area.

Description

PBMC separation disc using photocurable hydrogel {A DISK SEPARATING PBMCs USING PHOTOSENSITIVE HYDROGEL}

The present invention relates to a PBMC separation disk using a photocurable hydrogel, and more particularly, to a PBMC separation disk using a photocurable hydrogel capable of high-purity separation of PBMCs from blood by improving the PBMC separation method.

Peripheral blood mononuclear cells (PBMC) are used for various diagnostic/research purposes. For example, in order to confirm in vitro whether a therapeutic antibody exerts a cytotoxic effect on cancer cells, PBMCs are isolated from peripheral blood to check whether there is an abnormality in the immune system, or hematopoietic stem cell transplantation And in adoptive immune therapy, which is being used to treat solid cancer, using PBMCs, natural killer cells (NK cells), or dendritic cells (DC) to check whether the patient's immune system is restored The separation technology of PBMC is being used for various purposes such as Alternatively, PBMC is being used in various studies such as genome analysis through DNA and RNA extraction.

Conventional PBMC separation technology employs a centrifugation method using a density gradient material such as Ficoll from the peripheral blood of a patient. In this case, a blood collection vessel such as a CPT tube, ACD tube, or EDTA tube was placed in a centrifuge to separate PBMCs. However, the PBMC separation technology using the prior art, that is, the separation technology using a blood collection vessel and a centrifuge, requires a high level of skill of the researcher and proceeds through a complicated process. Therefore, there is a need for a separation technology capable of obtaining simple and high-quality PBMC.

The description underlying the invention has been prepared to facilitate understanding of the invention. It should not be construed as an admission that the matters described in the background technology of the invention exist as prior art.

US 10-1436168 B1

The problem to be solved by the present invention is to provide a PBMC separation disk using a photocurable hydrogel in order to solve the problems as described above. That is, the problem to be solved by the present invention is to provide a PBMC separation disk, a system and a method using a photocurable hydrogel so that the peripheral mononuclear blood cells (PBMC) can be easily separated even by a non-skilled person.

In addition, the problem to be solved by the present invention is to provide a PBMC separation disk, a system and a method using a photocurable hydrogel that can increase work efficiency by simplifying the process of separating peripheral mononuclear blood cells using centrifugation of the disk. will be.

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

In order to solve the above problems, the PBMC separation system using a photocurable hydrogel according to an embodiment of the present invention is a first chamber region, a second chamber region and a third that are sequentially arranged on a concentric circle with respect to a central axis. a chamber including a chamber area and into which a mixture of a sample collected from a user and a photocurable hydrogel is injected through an injection hole; and a mounting groove; a PBMC (Peripheral Blood Mononuclear Cell) separation disk comprising; a rotation driving unit coupled to the mounting groove and rotating the PBMC separation disk so that the sample injected into the chamber rotates in one direction; and when the sample is centrifuged into the first chamber area, the second chamber area, and the third chamber area by the rotation of the rotary driving part, and the PBMC component of the sample is separated in the second chamber area, the PBMC component is removed from the first chamber area and the second chamber area In order to prevent movement to any one of the regions, a laser beam for curing the photocurable hydrogel in the second chamber region may be irradiated to a partial region of the second chamber region.

In this case, the lower surface of the first chamber area and the lower surface of the third chamber area may be lower than the lower surface of the second chamber area.

In addition, during centrifugation by the rotary driving unit, centrifugation may be performed such that the first chamber region receives the plasma component, the second chamber region receives the PBMC component, and the third chamber region receives the red blood cell component.

In addition, the PBMC separation disk may further include an extraction port for extracting the PBMC component in the second chamber area.

In addition, at least one inlet and outlet may be formed on the upper surface of the PBMC separation disk, respectively.

In addition, the laser locally hardens the vicinity of the edge of the second chamber region, and the photocurable hydrogel in the second chamber region may be cured in a straight line.

In addition, the laser can determine the photocurable position of the photocurable hydrogel by a linear actuator.

In addition, it further includes a camera, the laser senses the interface of the layer of the PBMC component in the image taken by the camera, and after moving to the interface sensed by the linear actuator It can be irradiated with laser light to the photocurable hydrogel .

In addition, the mixture further includes a density gradient material, and the PBMC component may move to the second chamber area after centrifugation by the density gradient material.

In addition, the PBMC separation disk may further include a density gradient material inlet for replenishing the density gradient material.

On the other hand, the PBMC separation method according to an embodiment of the present invention comprises: injecting a mixture of a sample collected from a user and a photocurable hydrogel through an injection hole disposed on the top of the PBMC separation disk; centrifuging the sample by rotating the PBMC separation disk by a rotary driving unit mounted in a mounting groove disposed at the lower end of the central portion of the PBMC separation disk; When the PBMC component separated from the sample is located in one area of the chamber included in the PBMC separation disk, irradiating a laser light to the photocurable hydrogel located in the one area to separate one area of the chamber; and extracting the PBMC component located between the interfaces separated by the laser, wherein the chamber is a first chamber region, a second chamber region and a third chamber sequentially arranged concentrically with respect to the central axis of the PBMC separation disk. In the step of centrifuging, the sample is centrifuged into the first chamber region, the second chamber region, and the third chamber region by the rotation of the rotation driving unit, and the step of separating one region of the chamber is in the second chamber region. When the PBMC component in the sample is separated, irradiating laser light to harden the photocurable hydrogel in a portion of the second chamber area so that the PBMC component does not move to the first chamber area or the second chamber area have.

On the other hand, the PBMC separation disk according to the embodiment of the present invention includes a first chamber area, a second chamber area and a third chamber area which are respectively arranged in a concentric circle with respect to the central axis, and collected from the user through the inlet. a chamber into which a mixture of a sample and a photocurable hydrogel is injected; and a mounting groove; but, when the rotational driving unit mounted in the mounting groove is coupled with the mounting groove to rotate the chamber, centrifugation is performed to accommodate the PBMC component in the second chamber region, and the PBMC component is removed from the first chamber region or the second chamber region. The laser light may be irradiated to harden a part of the photocurable hydrogel inside the second chamber area without moving to the chamber area.

On the other hand, the effect according to the present invention is not limited by the contents exemplified above, and more various effects are included in the present specification.

1 is a schematic diagram for explaining the components of a PBMC separation disk according to an embodiment of the present invention.
FIG. 2 is an exemplary view illustrating a cross-section taken along II-II′ of FIG. 1 according to an embodiment of the present invention.
3 is a flowchart illustrating a PBMC separation process using a photocurable hydrogel according to an embodiment of the present invention.
4 is an exemplary view for explaining a process of irradiating a laser to the photocurable hydrogel according to an embodiment of the present invention.
5 to 6 are exemplary views for explaining a process of separating PBMCs using a photocurable hydrogel according to an embodiment of the present invention.
7 and 8 are exemplary views for explaining a PBMC separation process according to another embodiment of the present invention.

Advantages of the invention, and methods of achieving them, will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

The shapes, sizes, proportions, angles, numbers, etc. disclosed in the drawings for explaining the embodiments of the present invention are illustrative and the present invention is not limited to the illustrated matters. In addition, in describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. When 'including', 'having', 'consisting', etc. mentioned in this specification are used, other parts may be added unless 'only' is used. When a component is expressed in a singular, the case in which the plural is included is included unless otherwise explicitly stated.

In interpreting the components, it is interpreted as including an error range even if there is no separate explicit description.

Each feature of the various embodiments of the present invention may be partially or wholly combined or combined with each other, and technically various interlocking and driving are possible, as will be fully understood by those skilled in the art, and each embodiment may be independently implemented with respect to each other, It may be possible to implement together in a related relationship.

Hereinafter, a configuration of a PBMC separation disk according to an embodiment of the present invention will be described with reference to FIGS. 1 to 2 .

1 is a schematic diagram for explaining the components of a PBMC separation disk according to an embodiment of the present invention. FIG. 2 is an exemplary view showing a cross-section taken along II-II′ of FIG. 1 .

A mounting groove 110 having the shape of a cylindrical groove is disposed at the upper end of the central portion of the PBMC separation disk 100 , and the rotational driving unit 200 may be disposed at the lower end of the central portion. The rotation driving unit 200 generates centrifugal force for movement and centrifugation of blood by rotating the PBMC separation disk 100 . Accordingly, the PBMC separation disk 100 may be rotated clockwise by the rotation driving unit 200 . However, the rotation direction of the PBMC separation disk 100 is not limited thereto. Here, it is assumed that the rotation driving unit 200 is operated by a control unit connected to the PBMC separation disk 100 .

1 and 2 , the PBMC separation disk 100 includes a chamber 120 . The chamber 120 includes a first chamber region C1 , a second chamber region C2 , and a third chamber region C3 formed around the mounting groove 110 . The first chamber area C1 is formed along the inside of the PBMC separation disk 100 , and the third chamber area C3 is formed along the outside of the PBMC separation disk 100 . In addition, the second chamber region C2 is disposed between the first chamber region C1 and the third chamber region C3 . That is, the first chamber area C1 , the second chamber area C2 , and the third chamber area C3 are sequentially arranged on the outside with respect to the central axis of the PBMC separation disk 100 . When observing the upper surface of the PBMC separation disk 100 , the first chamber area C1 , the second chamber area C2 , and the third chamber area C3 are in order based on the central axis of the PBMC separation disk 100 . arranged in the form of concentric circles.

In addition, lower surfaces of the first chamber area C1 and the third chamber area C3 located on both sides with respect to the second chamber area C2 are lower than the lower surfaces of the second chamber area C2 (ie, PBMC). The lower surface of the third chamber area C3 may be formed close to the lower surface of the separation disk. In this shape, the PBMC occupies only a small area of the entire sample, and the PBMC is formed only in the second chamber area C2, so it may be easier to extract the PBMC 301 from the extraction port h2. .

In the present specification, a sample refers to a biological sample in which peripheral mononuclear blood cells (PBMC) may be present. For example, the sample may be selected from the group consisting of a biopsy sample, a tissue sample, a cell suspension in which isolated cells are suspended in a liquid medium, a cell culture, and combinations thereof. In addition, the sample may be selected from the group consisting of blood, bone marrow fluid, saliva, tear fluid, urine, semen, mucosal fluid, and combinations thereof. In addition, the sample may include a plurality of components (eg, red blood cells, plasma, PBMCs, etc.) different from each other. In the present invention, in order to isolate PBMC, it is assumed that blood is used as a sample.

On the other hand, an injection hole (h1) and an extraction hole (h2) may be formed at the upper end of the PBMC separation disk 100 . In this specification and drawings, the inlet h1 and the outlet h2 are shown as open, but during actual rotation, the rotation proceeds in a state in which the sample is blocked with a stopper (not shown) in order to prevent leakage of the sample.

Here, the injection hole h1 is formed in at least a partial region of the upper end of the first chamber region C1. Although the injection hole h1 may be formed in any part, it is preferable that the injection hole h1 is formed in the first chamber region C1 in consideration of the centrifugation direction.

The outlet h2 is formed in at least a partial area of the upper end of the second chamber area C2. However, the extraction port may be formed on the lower surface of the PBMC separation disk 100 for easier extraction. In addition, one or more inlet (h1) and outlet (h2) may be formed. A more detailed description of the outlet h2 will be described later with reference to FIG. 4 .

According to an embodiment of the present invention, the mixture of the sample and the photocurable hydrogel is injected into the first chamber region C1 of the PBMC separation disk 100 through the injection hole h1. Thereafter, when the PBMC separation disk 100 is rotated in one direction (eg, clockwise), a centrifugal force acts on the mixture so that RBC, PBMC and plasma components in the sample inside the PBMC separation disk 100 are PBMC according to the mass. The separation disk 100 moves in the outer direction (the third chamber C3 direction).

In addition to the photocurable hydrogel, a density gradient material may also be added to this mixture. The density gradient material includes, for example, Histopaque-1077, Ficoll, and the like.

Here, the photocurable hydrogel is a network structure in which a water-soluble polymer forms a three-dimensional cross-linkage by physical and chemical bonds such as hydrogen bonds and hydrophobic interactions. It is a material with a three-dimensional network structure that can be And, when light of a certain energy or more is irradiated, it is solidified in a solid form.

In the case of a sample, it is known that the area of the buffy coat containing PBMCs excluding plasma components and red blood cells is less than 1%, and the sum of components including red blood cells and white blood cells is known to be about 45%. Therefore, preferably, it is assumed that the photocurable hydrogel is uniformly mixed with the sample, and the volume of (photocurable hydrogel + sample) is 45 in the third chamber area (C3) so that the buffy coat area containing PBMC is not included. The volume of the third chamber area C3 may be designed such that % is substantially equal to or greater than the volume of the third chamber area C3. Alternatively, the amount of the sample and the hydrogel may be adjusted so that 45% of the volume of (photocurable hydrogel + sample) is substantially equal to or larger than the volume of the third chamber region C3.

On the other hand, when the density gradient material is included in the third chamber region C3, 45% of the volume of the density gradient material + (the photocurable hydrogel and the sample) volume is substantially the same as the volume of the third chamber region C3 Alternatively, the volume of the third chamber region C3 may be designed to be large or 45% of the volume of the density gradient material + (the photocurable hydrogel and the sample) volume is substantially the same as the volume of the third chamber region C3 Alternatively, the amount of the density gradient material, the sample and the hydrogel may be adjusted to be larger.

Accordingly, the sample is injected into the first chamber area C1 of the PBMC separation disk 100 through the injection port h1, and peripheral blood mononuclear cells separated by the PBMC separation disk 100 through the extraction port h2. Alternatively, the buffy coat containing peripheral blood mononuclear cells may be extracted. At this time, the configuration for extracting the sample through the extraction port h2 is a vacuum pump (Vacuum pump, 600).

Hereinafter, a separation process using the PBMC separation disk 100 according to an embodiment of the present invention will be described with reference to FIGS. 3 to 6 .

3 is a flowchart illustrating a PBMC separation process using a photocurable hydrogel according to an embodiment of the present invention. 4 is an exemplary view for explaining a process of irradiating a laser to the photocurable hydrogel according to an embodiment of the present invention. 5 to 6 are exemplary views for explaining a process of separating PBMCs using a photocurable hydrogel according to an embodiment of the present invention.

First, a mixture in which a photocurable hydrogel is mixed with a sample taken from a user is injected through the injection port h1 (S100). Then, the sample is centrifuged by rotating the PBMC separation disk 100 by the rotation driving unit 200 (S200). Then, after the PBMC 301 is separated in the second chamber region C2, the laser 300 is irradiated (S300). Then, after the interface 320 of the second chamber region C2 is hardened by the laser (S400), the PBMC 301 separated by the interface 320 is extracted with the vacuum pump 600 (S500).

Specifically, when the PBMC separation disk 100 is rotated, each component of the sample located in the chamber moves in the direction of the arrow as shown in FIG. 4 by centrifugal force. That is, each component of the sample moves outward according to the mass from the center of the PBMC separation disk 100 . Here, the sample includes a buffy coat containing PBMC 301, plasma 302, and red blood cells (RBC, 303), and includes a photocurable hydrogel, and preferably further includes a density gradient material.

However, when using a density gradient material including Ficoll, it is injected separately from the sample through the inlet h3 of the third chamber area C3. That is, it is preferable to inject the density gradient material in advance so as not to mix with the sample.

At this time, as each component of the sample rotating in one direction in the PBMC separation disk moves toward the edge of the chamber 120 by centrifugal force, the red blood cells 303 are located in the third chamber region C3, and the PBMC 301 ) area is located in the second chamber area C2.

As shown in FIG. 4 , when the buffy coat including the PBMC 301 is located in the second chamber area C2 through centrifugal force, the laser light 310 is emitted from the second chamber area C2 through the laser device. The interface 320 is irradiated to locally harden between the chamber regions C1, C2, and C3. Here, the laser 300 may utilize a UV laser.

The laser irradiation position according to an embodiment of the present invention may be defined as the interface between the buffy coat layer including the PBMC 301 separated by centrifugal force and the plasma 302 and the interface between the buffy coat layer and the RBC 303. . In this case, it is preferable to design the volume of the second chamber region C2 to be substantially the same as the volume of the buffy coat layer. For example, since the buffy coat layer is known to be about 1% of the sample, the volume of the second chamber region (C2) is adjusted to be about 1% of the volume of the sample + the photocurable hydrogel based on the generally used sample amount (10ml). can be designed

In this case, after the PBMC 301 is positioned in the second chamber region C2, the laser beam ( 300) can be investigated (see FIG. 5).

Accordingly, when the laser-irradiated photocurable hydrogel 310 cures the interface 320 between the chamber regions, each chamber region C1, C2, C3 may be separated by the interface 320. have. Accordingly, as shown in FIG. 6 , only the PBMC 301 separately separated in the second chamber region C2 can be extracted through the extraction port h2 using the vacuum pump 600 .

In general, PBMCs can be isolated using various centrifugation methods. Conventionally, a gradient centrifugation method has been generally used. Specifically, it was separated from the patient's peripheral blood by using the Ficoll-Paque (Amersham Biosciences AB) centrifugation method or using a blood collection vessel such as a CPT tube, ACD tube, or EDTA tube.

However, the PBMC separation technology using the prior art, that is, the separation technology using a blood collection vessel and a centrifuge, requires a high level of skill of the researcher and proceeds through a complicated process, but according to the present invention, it is automatically With this method, the buffy coat layer containing PBMC can be easily extracted.

On the other hand, the PBMC separation disk 100 using the photocurable hydrogel according to an embodiment of the present invention has the effect of easily separating high-purity PBMCs. Accordingly, there is an effect of effectively separating target cells from blood to obtain a large amount of high-purity sample.

7 is an exemplary view for explaining a PBMC separation process according to another embodiment of the present invention. Unlike FIGS. 4 to 6 , since the rest of the configuration is substantially the same except that the laser irradiation position is adjustable, a redundant description will be omitted.

Referring to FIG. 7 , two interfaces are formed in the PBMC separation disk 100 by a laser. Here, the laser may be irradiated to a position variable by the actuator 710 .

The linear actuator 710 is configured to move the laser 300 to the interface between the PBMC component 301 and the plasma component 302 or the PBMC component and the RBC 303 in the chamber. In this case, the interface between the PBMC component 301 and the plasma component 302 or the PBMC component and the RBC 303 may be determined by analyzing an image captured by the camera 700 . At this time, since the PBMC component 301 , the plasma component 302 , and the RBC 303 have different colors, the boundary surface may be set using the color difference.

Specifically, as shown in FIG. 7 , in order to prevent the PBMC component 301 among the samples injected from the first chamber region C1 from moving to the third chamber region C3, the second chamber region C2 and the third The laser 300 moves to the interface between the PBMC component and the RBC 303 between the chamber regions C3 and the laser beam is irradiated to the interface between the PBMC component and the RBC 303 .

In addition, in order to prevent the PBMC component 301 from moving into the first chamber region C1, the PBMC component 301 and the plasma component 302 are located between the second chamber region C2 and the first chamber region C1. The laser 300 moves to the interface of , and the laser beam is irradiated to the interface between the PBMC component and the RBC 303 .

Although both sides of the boundary surface 720 of the PBMC 301 formed in the chamber are variable in FIG. 7 , one side of the boundary surface 720 of the PBMC 301 may be fixed and the other side may be variable.

8 is an exemplary view for explaining a PBMC separation process according to another embodiment of the present invention. 8 is substantially the same except for having an additional density gradient material injection hole in FIGS. 4 to 6 , so a redundant description thereof will be omitted.

Referring to FIG. 8 , the PBMC separation disk according to an embodiment of the present invention may further include a density gradient material injection hole h3 . When the amount of the sample, hydrogel, or density gradient material is small in the course of the experiment, the density of the density gradient material and the red blood cells (RBC) is substantially the same as the volume of the third chamber area C3. It is possible to supplement the density gradient material through the gradient material inlet (h3).

For example, when there is a camera sensor as shown in FIG. 7 , when the PBMC layer is formed in the third chamber region C3 instead of in the second chamber region C2, the density is additionally added to the density gradient material injection hole h3. After replenishment of gradient material, spin again. In this case, when the PBMC layer is formed in the second chamber region C2 , the laser may be irradiated to the boundary of the PBMC layer after sensing it by the camera 700 .

Therefore, according to the present invention, PBMCs can be easily detected in a blood sample even without a highly skilled person.

Although the embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be made within the scope without departing from the technical spirit of the present invention. . Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

100: PBMC separation disk 110: mounting groove
120: chamber 200: rotational driving unit
300: laser 301: PBMC
302: plasma 303: red blood cells (RBC)
310: laser light 320: interface
600: vacuum pump C1, C11, C12: first chamber area
C2, C21, C22: second chamber area C3, C31, C32: third chamber area
700: camera 710: linear actuator

Claims (18)

A chamber and a mounting groove into which a mixture of a sample collected from a user and a photocurable hydrogel is injected through an injection hole, including a first chamber region, a second chamber region, and a third chamber region, which are sequentially arranged in a concentric circle with respect to the central axis PBMC (Peripheral Blood Mononuclear Cell) separation disk comprising a;
a rotation driving unit coupled to the mounting groove and rotating the PBMC separation disk so that the sample injected into the chamber rotates in one direction; and
When the sample is centrifuged in the first chamber region, the second chamber region, and the third chamber region by the rotation of the rotary driving unit, and the PBMC component of the sample is separated in the second chamber region, the PBMC component is removed from the sample In order to prevent movement to any one of the first chamber area and the second chamber area, a laser beam for curing the photocurable hydrogel in the second chamber area is irradiated to a partial area of the second chamber area. including,
and a lower surface of the first chamber area and a lower surface of the third chamber area are formed to be lower than a lower surface of the second chamber area.
delete According to claim 1,
Upon centrifugation by the rotary driving unit, the first chamber region receives the plasma component, the second chamber region receives the PBMC component, and the third chamber region is centrifuged to receive the red blood cell component. separation system. According to claim 1,
The PBMC separation disk further comprises an outlet for extracting the PBMC component in the second chamber area. 5. The method of claim 4,
The inlet and the outlet are each formed at least one on the upper surface of the PBMC separation disk, PBMC separation system using a photocurable hydrogel. According to claim 1,
The laser locally cures the vicinity of the edge of the second chamber area, and the photocurable hydrogel in the second chamber area is cured in a straight line. 7. The method of claim 6,
The laser determines the photocurable position of the photocurable hydrogel by a linear actuator, PBMC separation system. 8. The method of claim 7,
Further comprising a camera, wherein the laser senses the interface of the layer of the PBMC component in the image taken by the camera, moves to the interface sensed by the linear actuator, and then applies laser light to the photocurable hydrogel Investigating, PBMC Isolation System. According to claim 1,
The mixture further comprises a density gradient material, and the PBMC component moves to the second chamber area after centrifugation by the density gradient material. 10. The method of claim 9,
The PBMC separation disk further comprises a density gradient material inlet capable of replenishing the density gradient material. injecting a mixture of a sample collected from a user and a photocurable hydrogel through an inlet disposed on the top of the PBMC separation disk;
centrifuging the sample by rotating the PBMC separation disk by a rotary driving unit mounted in a mounting groove disposed at the lower end of the central portion of the PBMC separation disk;
When the PBMC component separated from the sample is located in one region of the chamber included in the PBMC separation disk, irradiating laser light to the photocurable hydrogel located in the one region to separate one region of the chamber; and
Comprising the step of extracting the PBMC component located between the interface separated by the laser,
The chamber includes a first chamber area, a second chamber area and a third chamber area sequentially arranged in a concentric circle with respect to the central axis of the PBMC separation disk, and in the centrifugal separation step, the The sample is centrifuged in the first chamber region, the second chamber region, and the third chamber region;
The step of separating the one region of the chamber may include: when the PBMC component of the sample is separated from the second chamber region, the PBMC component does not move to the first chamber region or the second chamber region of the second chamber region. Including the step of irradiating a laser light to cure the photocurable hydrogel in a part of the inside, PBMC separation method. 12. The method of claim 11,
In the centrifugation step, the first chamber region contains a plasma component.
and wherein the second chamber region contains the PBMC component, and the third chamber region is centrifuged to receive the red blood cell component. 12. The method of claim 11,
The laser determines the photocurable position of the photocurable hydrogel by a linear actuator, PBMC separation method. 14. The method of claim 13,
The laser senses the interface of the layer of the PBMC component in the image taken by the camera, and after moving by the linear actuator to the sensed interface, irradiating laser light to the photocurable hydrogel, PBMC separation method. 12. The method of claim 11,
The mixture further comprises a density gradient material, and the PBMC component moves to the second chamber area after centrifugation by the density gradient material. 16. The method of claim 15,
The PBMC separation disk further comprises a density gradient material inlet capable of replenishing the density gradient material, PBMC separation method. a chamber unit including a first chamber region, a second chamber region, and a third chamber region, each arranged in a concentric circle with respect to the central axis, and into which a mixture of a sample collected from a user and a photocurable hydrogel is injected through an inlet; and
mounting groove; including,
When the rotary driving unit mounted on the mounting groove rotates the chamber, centrifugation is performed to accommodate the PBMC component in the second chamber area, and the PBMC component does not move to the first chamber area or the second chamber area. Laser light is irradiated to harden the photocurable hydrogel in a portion of the inside of the second chamber area,
and a lower surface of the first chamber area and a lower surface of the third chamber area are formed lower than a lower surface of the second chamber area.
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