KR20240073414A - Method and apparatus for separating fetal cells based centrifugal force - Google Patents

Abstract

The centrifugal force-based fetal cell separation method according to an embodiment of the present invention performs centrifugation of whole blood collected from the mother, using density gradient media (DGM) and white blood cells (WBC). Using micro beads that recognize cells, nucleated red blood cells (nRBCs) and trophoblasts of fetal cells are simultaneously separated and captured from the whole blood.

Description

Method for separating fetal cells based on centrifugal force and device therefor {METHOD AND APPARATUS FOR SEPARATING FETAL CELLS BASED CENTRIFUGAL FORCE}

The present invention relates to a centrifugal force-based fetal cell separation method and device for the same. More specifically, the nucleated red blood cells and trophoblasts of fetal cells can be simultaneously obtained from the mother's blood in an automated manner in the absence of a single platform, and the maternal It relates to a centrifugal force-based fetal cell separation method and device for the same that can minimize the loss of fetal cells in the process of separating blood.

Figure 1 is a schematic diagram showing an example of a method for separating fetal cells according to the prior art.

As shown in Figure 1, the prior art fetal cell separation method uses antibodies specific for trophoblast cells and nucleated red blood cells to separate trophoblast cells, which are fetal cells (10) present in the mother's blood, and fetal-derived nucleated red blood cells. This is a technology that separates trophoblast cells and nucleated red blood cells in the mother's blood by fixing specific antibodies on a microfluidic chip (20).

The prior art states that two types of fetal cells 10 are separated simultaneously, but in reality, one microfluidic chip 20 has a structure in which antibodies specific for one type of fetal cell are immobilized, so two different microfluidic chips are separated. This corresponds to a technology for separating trophoblast cells and nucleated red blood cells using the chip 20. Therefore, it is difficult to simultaneously separate two types of fetal cells 10 (eg, trophoblasts and nucleated red blood cells) using the same blood.

Therefore, in the existing prior art, blood is divided to separate two types of fetal cells 10, such as trophoblasts and nucleated red blood cells, and the fetal cells 10 are individually separated on two different microfluidic chips 20. There are limits to progress.

In addition, the overall work process for separating nucleated red blood cells and trophoblasts is very complicated and difficult to use in the actual field, and since it is performed manually in different spaces, there is a high risk of loss of fetal cells, so it is difficult to secure a sufficient amount. There are also difficulties in doing so.

Embodiments of the present invention can easily obtain nucleated red blood cells and trophoblasts of fetal cells from the mother's blood in an automated manner in the absence of a single platform, and minimize the loss of fetal cells in the process of separating the mother's blood. Provides a centrifugal force-based fetal cell separation method and a device for the same.

In addition, embodiments of the present invention include micro beads, density gradient media (DGM), and lysis buffer that recognize white blood cells (WBC) during centrifugation of maternal whole blood. Provides a centrifugal force-based fetal cell separation method and device for securing two types of fetal cells simultaneously by applying a lysis buffer to gradually remove substances other than nucleated red blood cells and trophoblasts. .

According to one embodiment of the present invention, centrifugation of whole blood collected from a mother is performed, and microbeads (DGM) that recognize density gradient media (DGM) and white blood cells (WBC) are used. A centrifugal force-based fetal cell separation method is provided, characterized in that nucleated red blood cells (nRBC) and trophoblasts of fetal cells are simultaneously separated and captured from the whole blood using micro beads.

Preferably, the centrifugal force-based fetal cell separation method according to an embodiment of the present invention includes the steps of injecting the whole blood and the first density gradient material into a blood chamber, and using the first density gradient material to Separating whole blood into plasma, peripheral blood mononuclear cells (PBMC), and red blood cells, flowing the plasma from the blood chamber to the plasma chamber, transferring the peripheral blood mononuclear cells to the blood chamber flowing into a first mixing chamber, mixing leukocytes contained in the peripheral blood mononuclear cells with first microbeads previously stored in the first mixing chamber and attaching the first microbeads to the leukocytes, the peripheral Flowing the trophoblast cells contained in blood mononuclear cells and the leukocytes to which the first microbeads are attached from the first mixing chamber to the first depletion chamber, the trophoblast cells and the leukocytes to which the first microbeads are attached. It may include separating the second density gradient material previously stored in the first depletion chamber, and flowing the trophoblast cells separated in the first depletion chamber into a first collection chamber and storing them.

Preferably, the centrifugal force-based fetal cell separation method according to an embodiment of the present invention includes the steps of supplying a lysis buffer to the blood chamber by a buffer chamber to lyse the red blood cells, Flowing the normal blood cells lysed by the lysis buffer from the blood chamber to the waste chamber, and flowing the nucleated red blood cells and the leukocytes not lysed by the lysis buffer among the red blood cells from the blood chamber to a second mixing chamber. A step of flowing, mixing the nucleated red blood cells and the leukocytes with second microbeads previously stored in the second mixing chamber and attaching the second microbeads to the leukocytes, attaching the nucleated red blood cells and the second microbeads flowing the leukocytes from the second mixing chamber to the second depletion chamber, the nucleated red blood cells and the leukocytes to which the second microbeads are attached to a third density gradient material previously stored in the second depletion chamber. The method may further include separating the nucleated red blood cells separated in the second depletion chamber and storing them in a second collection chamber.

Here, the blood chamber, the first and second waste chambers, the buffer chamber, the first and second mixing chambers, the first and second depletion chambers, and the first and second collection chambers are attached to a rotatable disk member. Each groove may be formed in a different position.

Separating the whole blood into the plasma, peripheral blood mononuclear cells, and red blood cells, attaching the first microbead to the leukocyte, separating the trophoblast and the leukocyte to which the first microbead is attached. , in any one of the steps of lysing the red blood cells, attaching the second microbead to the leukocyte, or separating the nucleated red blood cell and the leukocyte to which the second microbead is attached, the disc member Centrifugation can be performed by spinning.

In the step of separating the whole blood into the plasma, the peripheral blood mononuclear cells, and the red blood cells, the first density gradient material having a higher density than the plasma and the peripheral blood mononuclear cells and a lower density than the red blood cells is used. Thus, the plasma, peripheral blood mononuclear cells, and red blood cells can be separated.

In addition, in the step of separating the trophoblast cells and the leukocytes to which the first microbeads are attached, the second density gradient material, which has a higher density than the trophoblast cells and a lower density than the leukocytes to which the first microbeads are attached, is used. The trophoblast cells and the white blood cells attached to the first microbead can be separated.

In addition, in the step of separating the nucleated red blood cells and the white blood cells to which the second microbeads are attached, the third density gradient material having a higher density than the nucleated red blood cells and a lower density than the white blood cells to which the second microbeads are attached is used. The nucleated red blood cells and the white blood cells attached to the second microbead can be separated.

Additionally, in the step of attaching the first microbead to the leukocyte, CD45 beads, which have a higher density than the trophoblast cells, can be used as the first microbead to attach to the leukocyte. In the step of attaching the second microbead to the leukocyte, CD45 beads, which have a higher density than the nucleated red blood cell, can be used as the microbead to attach to the leukocyte.

According to another aspect of the present invention, a disc member is selectively rotated for centrifugation of fetal cells, formed at a first position of the disc member, and rotated when the disc member is rotated after injecting the first density gradient material and whole blood. A blood chamber that separates the whole blood into plasma and peripheral blood mononuclear cells and red blood cells through a first density gradient material, formed at a second position of the disc member to be connected to a first portion of the blood chamber, and in the blood chamber A plasma chamber for receiving and storing plasma, formed at a third position of the disc member to be connected to a second part of the blood chamber, and mixing the peripheral blood mononuclear cells with first microbeads stored in advance after receiving them from the blood chamber. A first mixing chamber for attaching the first microbead to the leukocytes included in the peripheral blood mononuclear cells, formed at the fourth position of the disk member to be connected to the first mixing chamber and included in the peripheral blood mononuclear cells A first depletion chamber for receiving trophoblast cells and leukocytes to which the first microbeads are attached from the first mixing chamber and then separating the trophoblast cells through a pre-stored second density gradient material, the first depletion chamber A first collection chamber formed at the fifth position of the disk member to be connected to the chamber and receiving and storing trophoblast cells separated by the second density gradient material from the first depletion chamber, a third of the blood chamber A buffer chamber formed at a sixth position of the disc member to be connected to the site and supplying lysis buffer to the blood chamber from which the plasma and peripheral blood mononuclear cells are removed to lyse the red blood cells, a fourth portion of the blood chamber Nucleated red blood cells and white blood cells, which are formed at the 7th position of the disc member to be connected to the site and have not been lysed by the lysis buffer among the red blood cells, are transferred from the blood chamber and mixed with the second microbead stored in advance to form the white blood cells. A second mixing chamber for attaching the second microbead to the second mixing chamber, formed at an eighth position of the disk member to be connected to the second mixing chamber, and mixing the nucleated red blood cells and the leukocytes to which the second microbead is attached with the second mixing chamber. a second depletion chamber that separates the fluid red blood cells through a third density gradient material previously stored after being delivered from the chamber, and is formed at a ninth position of the disc member to be connected to the second depletion chamber, 3. It provides a centrifugal force-based fetal cell separation device including a second collection chamber that receives and stores the humoral red blood cells separated by the density gradient material from the second depletion chamber.

Preferably, the centrifugal force-based fetal cell separation device according to an embodiment of the present invention is formed at the tenth position of the disc member to be connected to the fifth part of the blood chamber and separates the lye from the red blood cells in the blood chamber. It may further include a waste chamber for receiving and storing general blood cells dissolved by the cis buffer.

Preferably, the first position of the blood chamber may be set to be radially spaced further from the rotation center of the disk member than the sixth position of the buffer chamber. The second position of the plasma chamber may be set to be radially spaced further from the rotation center of the disk member than the first position of the blood chamber. The third and seventh positions of the first and second mixing chambers may be set to be radially spaced further from the rotation center of the disk member than the first position of the blood chamber. The fourth and eighth positions of the first and second depletion chambers may be set to be radially spaced further from the rotation center of the disk member than the third and seventh positions of the mixing chamber. The fifth and ninth positions of the first and second collection chambers may be set to be spaced further apart in the radial direction from the rotation center of the disk member than the fourth and eighth positions of the first and second depletion chambers. You can. The tenth position of the waste chamber may be set to be radially spaced further from the rotation center of the disk member than the first position of the blood chamber.

Preferably, the first density gradient material may be provided as a material that has a higher density than the plasma and the peripheral blood mononuclear cells and a lower density than the red blood cells. The second density gradient material may be provided as a material that has a higher density than the trophoblast cells and a lower density than the white blood cells to which the first microbeads are attached. The third density gradient material may be provided as a material that has a higher density than the nucleated red blood cells and a lower density than the white blood cells to which the second microbeads are attached. The first and second microbeads may be provided as CD45 beads that adhere to the leukocytes while having a higher density than the trophoblasts and nucleated red blood cells.

The centrifugal force-based fetal cell separation method and device for the same according to an embodiment of the present invention can simultaneously obtain nucleated red blood cells and trophoblasts of fetal cells from the mother's blood in an automated manner in the absence of a single platform, and the maternal blood The loss of fetal cells can be minimized during the separation process.

In addition, the centrifugal force-based fetal cell separation method and device for the same according to an embodiment of the present invention apply microbeads, density gradient material, and lysis buffer that recognize leukocytes in the process of centrifuging maternal whole blood to whole blood. Two types of fetal cells can be smoothly obtained by gradually removing substances other than the nucleated red blood cells and glazed membrane cells, thereby significantly increasing the accuracy of genetic testing of fetal cells.

In addition, the centrifugal force-based fetal cell separation method and device for the same according to an embodiment of the present invention include a blood chamber, a plasma chamber, a waste chamber, a buffer chamber, first and second mixing chambers, and a first, 2 Because it is a structure that appropriately provides whole blood, microbeads, density gradient material, and lysis buffer to the depletion chamber and the first and second collection chambers, the mother's whole blood is collected in one go using the centrifugal force caused by the rotation of the disc member. It can be centrifuged through a process, and in the process, nucleated red blood cells and trophoblasts of fetal cells contained in whole blood can be stably obtained at the same time. In particular, in this embodiment, two types of fetal cells are sufficiently secured through a single process of centrifuging the mother's whole blood, so they can be very conveniently used in medical settings that perform genetic testing and diagnosis of the fetus, and the nucleated nucleus of the fetal cells By securing sufficient red blood cells and trophoblasts, the accuracy and efficiency of genetic testing can be improved.

Figure 1 is a schematic diagram showing an example of a method for separating fetal cells according to the prior art.
Figure 2 is a diagram showing a centrifugal force-based fetal cell separation device according to an embodiment of the present invention.
Figure 3 is a diagram illustrating the fetal cell separation method for each process using the centrifugal force-based fetal cell separation device shown in Figure 2.

이하에서, 본 발명에 따른 실시예들을 첨부된 도면을 참조하여 상세하게 설명한다. 그러나, 본 발명이 실시예들에 의해 제한되거나 한정되는 것은 아니다. 각 도면에 제시된 동일한 참조 부호는 동일한 부재를 나타낸다.

Hereinafter, embodiments according to the present invention will be described in detail with reference to the attached drawings. However, the present invention is not limited or limited by the examples. The same reference numerals in each drawing indicate the same members.

Figure 2 is a diagram showing a centrifugal force-based fetal cell separation device 100 according to an embodiment of the present invention, and Figure 3 is a diagram showing fetal cell separation using the centrifugal force-based fetal cell separation device 100 shown in Figure 2. This is a drawing showing the method by process.

Referring to Figures 2 and 3, the centrifugal force-based fetal cell separation device 100 according to an embodiment of the present invention includes a disk member 110, a blood chamber 120, and a plasma chamber. (130), waste chamber (140), buffer chamber (150), first mixing chamber (160), first depletion chamber (170), It may include a first collection chamber 180, a second mixing chamber 190, a second depletion chamber 200, and a second collection chamber 210.

The centrifugal force-based fetal cell separation device 100 according to this embodiment uses micro beads (MB) that recognize white blood cells (WBC) contained in the mother's blood and maintains an appropriate density gradient. Using density gradient media (DGM), nucleated red blood cells (nRBC) and trophoblasts (T) of fetal cells can be separated and captured at the same time.

In addition, the fetal cell separation device 100 of this embodiment uses plasma (P) such as plasma and peripheral blood mononuclear cells (PBMC) and red blood cells through the first density gradient material (DGM1). (RBC: red blood cell) can be separated into layers, and after combining the first microbead (MB1) with white blood cells (WBC) contained in peripheral blood mononuclear cells (PBMC), the second density gradient material (DGM2) is added. By removing white blood cells (WBC), trophoblast cells (T) contained in peripheral blood mononuclear cells (PBMC) can be separated and captured.

In addition, after removing all but the red blood cells (RBCs), nucleated red blood cells (nRBCs) and some white blood cells (WBCs) can be obtained by removing only general blood cells through a lysis process using lysis buffer (LB). You can. Then, the nucleated red blood cells (nRBC) can be separated and captured by binding the second microbead (MB2) to the corresponding white blood cell (WBC) and then removing the white blood cell (WBC) through the third density gradient material (DGM3). .

At this time, nucleated red blood cells (nRBC) are a type of fetal cell distributed in the mother's blood and can be used for genetic testing of the fetus because they are red blood cells derived from the fetus. On the other hand, normal blood cells are non-nucleated red blood cells found in normal adults and cannot be used for genetic analysis of the fetus because they correspond to the mother's red blood cells. Meanwhile, trophoblast cells (T) are peripheral blood mononuclear cells (PBMC), which correspond to fetal cells and can be used for genetic testing of the fetus.

In addition, unlike the prior art, the fetal cell separation device 100 of this embodiment separates two types of fetal cells, trophoblasts (T) and nucleated red blood cells (nRBC), present in the same blood of the mother, in a single platform member. can do. Therefore, compared to the conventional method of dividing the blood in half and separating the two types of fetal cells, this embodiment can prevent the loss of fetal cells and obtain more fetal cells.

In addition, in this embodiment, the condensation process of general blood cells and nucleated red blood cells (nRBC) and the lysis process of general blood cells can proceed automatically, and the above processes as well as the separation process of white blood cells (WBC) are all performed in the absence of a single platform. As it progresses, reaction stability and loss of fetal cells can be improved. That is, in this embodiment, aggregation and lysis of red blood cells occur in the absence of a single platform, and since each step is not performed manually, the loss of fetal cells that may occur at each step can be minimized.

Referring to Figures 2 and 3, the disc member 110 of this embodiment can be selectively rotated for centrifugal separation of fetal cells. The disk member 110 as described above is formed in the form of a thin disk substrate, and can be rotated at high speed for centrifugal separation by centrifugal force. Meanwhile, the disk member 110 may be sufficiently capable of not only being rotated but also shaking in directions other than rotation.

Here, the disk member 110 includes a blood chamber 120, a plasma chamber 130, a waste chamber 140, a buffer chamber 150, a first mixing chamber 160, a first depletion chamber 170, The first collection chamber 180, the second mixing chamber 190, the second depletion chamber 200, and the second collection chamber 210 are each groove-shaped at different positions on the upper surface of the disk member 110. can be formed.

And, the plasma chamber 130, the waste chamber 140, the buffer chamber 150, the first mixing chamber 160, and the second mixing chamber 190 are 1, 2, 3, and 4 in the blood chamber 120. , Can be connected to five chamber flow paths (L1, L2, L3, L4, L5), respectively, and the first and second depletion chambers (170, 200) are connected to the first and second mixing chambers (160, 190) with the sixth and second depletion chambers (170, 200). They can be connected to 7-chamber flow paths (L6, L7), respectively, and the first and second collection chambers (180, 210) are connected to the first and second depletion chambers (160) with the 8th and 9th chamber flow paths (L8, L9). Each can be connected. The first to ninth chamber passages L1 to L9 as described above may each be formed in the disk member 110 in the shape of a fine channel.

Meanwhile, valves (V) for controlling flow may be disposed in the first to ninth chamber passages (L1 to L9), respectively. For example, the valve V may be provided as a micro-flow valve. In other words, the valve V may be provided as a normally closed valve structure that closes the first to ninth chamber passages (L1 to L9) so that fluid cannot flow until it is opened by receiving energy from the outside. The valve V as described above includes a valve material that is solid at room temperature, and the valve material exists in a solid state on the flow path, thereby blocking the first to ninth chamber flow paths (L1 to L9). However, the valve material may melt at a high temperature and solidify again while moving into the space within the first to ninth chamber passages (L1 to L9) and leaving the passages open. External energy irradiated to the valve material includes electromagnetic waves, laser beams, visible light, or infrared light.

Referring to FIGS. 2 and 3, the blood chamber 120 of this embodiment is configured to inject the first density gradient material (DGM1) and whole blood (WB) and then generate the first density gradient material (DGM1) when the disk member 110 rotates. Through DGM1), whole blood (WB) can be separated into plasma (P), peripheral blood mononuclear cells (PBMC), and red blood cells (RBC). The blood chamber 120 as described above may be formed in a groove shape at a first position close to the rotation center O of the disk member 110. The first position of the blood chamber 120 may be set to be further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the sixth position of the buffer chamber 150, which will be described later.

The first density gradient material (DGM1) is used for centrifugation through the density difference between red blood cells (RBC) relative to plasma (P) and peripheral blood mononuclear cells (PBMC). It can be provided as a material that is dense but less dense than red blood cells (RBCs). For example, in this embodiment, a density gradient material in which the density of the first density gradient material (DGM1) is selected to be 1.07 g/mL may be used.

Plasma (P) may include plasma of whole blood (WB) and some white blood cells. Peripheral blood mononuclear cells (PBMC) may include trophoblasts (T) and some white blood cells. Red blood cells (RBC) may include general blood cells (eg, anucleated red blood cells) and nucleated red blood cells (nRBC).

Referring to FIGS. 2 and 3 , the plasma chamber 130 of this embodiment can receive plasma P from the blood chamber 120 and store it. The plasma chamber 130 may be connected in communication with the blood chamber 120 through the first chamber flow path L1.

The plasma chamber 130 as described above is formed in a groove shape at the second position of the disk member 110 and may be connected to the first part of the blood chamber 120. The second position of the plasma chamber 130 may be set to be further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the first position of the blood chamber 120.

Referring to Figures 2 and 3, the waste chamber 140 of this embodiment can receive and store normal blood cells dissolved by lysis buffer (LB) among red blood cells (RBCs) in the blood chamber 120. The waste chamber 140 may be connected in communication with the blood chamber 120 through the second chamber passage L2.

The waist chamber 140 as described above is formed in a groove shape at the tenth position of the disk member 110, and may be connected to the fifth position of the blood chamber 120. The tenth position of the waste chamber 140 is set to be farther apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the first position of the blood chamber 120, but the plasma chamber 130 It may be set to be spaced further apart in the radial direction (R) from the rotation center (O) of the disk member 110.

Referring to Figures 2 and 3, the buffer chamber 150 of this embodiment can lyse red blood cells (RBC) by supplying lysis buffer (LB) to the blood chamber 120 from which the plasma (P) has been removed. there is. The buffer chamber 150 may be connected in communication with the blood chamber 120 through the third chamber passage L3,

The buffer chamber 150 as described above may be formed in a groove shape at the sixth position of the disk member 110 to be connected to the third part of the blood chamber 120. The sixth position of the buffer chamber 150 may be set to be closer to the rotation center O of the disk member 110 in the radial direction (R) than the first position of the blood chamber 120.

Referring to Figures 2 and 3, the first mixing chamber 160 of this embodiment receives peripheral blood mononuclear cells (PBMC) from the blood chamber 120 and mixes them with the first microbeads (MB1) stored in advance. The first microbead (MB1) can be attached to white blood cells (WBC) included in peripheral blood mononuclear cells (PBMC). The first mixing chamber 160 may be connected in communication with the blood chamber 120 through the fourth chamber flow path L4.

The first mixing chamber 160 as described above may be formed in a groove shape at the third position of the disk member 110 to be connected to the second part of the blood chamber 120. The third position of the first mixing chamber 160 may be set to be further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the first position of the blood chamber 120.

The first microbead (MB1) may be provided as a CD45 bead that has a higher density than trophoblast cells (T) and adheres to white blood cells (WBC). Therefore, the white blood cells (WBC) to which the first microbead (MB1) is attached have a higher density than the trophoblast cells (T).

Referring to Figures 2 and 3, the first depletion chamber 170 of this embodiment is a white blood cell (WBC) to which trophoblast cells (T) and the first microbead (MB1) are attached to the first mixing chamber (160). After receiving from ), trophoblast cells (T) can be separated through the second density gradient material (DGM2) stored in advance. The first depletion chamber 170 may be connected in communication with the first mixing chamber 160 through the sixth chamber passage L6.

The first depletion chamber 170 as described above may be formed at the fourth position of the disk member 110 to be connected to the first mixing chamber 160. The fourth position of the first depletion chamber 170 is set to be spaced further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the third position of the first mixing chamber 160. You can.

The second density gradient material (DGM2) has a higher density than the trophoblast cells (T) and contains the first micro beads ( MB1) can be provided as a material with a lower density than attached white blood cells (WBC). For example, in this embodiment, a density gradient material (DGM2) whose density is selected to be 1.05 to 1.13 g/mL may be used.

Referring to Figures 2 and 3, the first collection chamber 180 of this embodiment transfers the trophoblast cells (T) separated by the second density gradient material (DGM2) from the first depletion chamber 170. You can receive and save it. The first collection chamber 180 may be connected to the first depletion chamber 170 through an eighth chamber passage L8.

The first collection chamber 180 may be formed at the fifth position of the disk member 110 to be connected to the first depletion chamber 170. The fifth position of the first collection chamber 180 will be set to be spaced further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the fourth position of the first depletion chamber 170. You can.

Referring to Figures 2 and 3, the second mixing chamber 190 of the present embodiment contains nucleated red blood cells (nRBC) that are not lysed by lysis buffer (LB) and some white blood cells (WBC) among red blood cells (RBC). After being delivered from the blood chamber 120, the second microbead (MB2) can be attached to white blood cells (WBC) by mixing with the pre-stored second microbead (MB2). The second mixing chamber 190 may be connected in communication with the blood chamber 120 through the fifth chamber passage L5.

The second mixing chamber 190 as described above may be formed in a groove shape at the seventh position of the disk member 110 to be connected to the fourth portion of the blood chamber 120. The seventh position of the second mixing chamber 190 may be set to be radially spaced further from the rotation center of the disk member 110 than the first position of the blood chamber 120.

The second microbead (MB2) may be provided as a CD45 bead that has a higher density than nucleated red blood cells (nRBC) and is attached to white blood cells (WBC). Therefore, white blood cells (WBC) to which the second microbead (MB2) is attached have a higher density than nucleated red blood cells (nRBC).

Referring to Figures 2 and 3, the second depletion chamber 200 of the present embodiment is a second mixing chamber 190 for nucleated red blood cells (nRBC) and white blood cells (WBC) to which the second microbead (MB2) is attached. ), nucleated red blood cells (nRBCs) can be separated through pre-stored third density gradient material (DGM3). The second depletion chamber 200 may be connected to the second mixing chamber 190 through a seventh chamber passage L7.

The second depletion chamber 200 as described above may be formed at the eighth position of the disk member 110 to be connected to the second mixing chamber 190. The eighth position of the second depletion chamber 200 will be set to be spaced further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the seventh position of the second mixing chamber 190. You can.

The third density gradient material (DGM3) has a higher density than the nucleated red blood cells (nRBC) and contains the second micro beads (MB2) to centrifuge the nucleated red blood cells (nRBC) and the white blood cells (WBC) to which the second micro beads (MB2) are attached by density difference. MB2) can be provided as a material with a lower density than the attached white blood cells (WBC). For example, in this embodiment, a density gradient material (DGM3) whose density is selected to be 1.10 to 1.15 g/mL may be used.

Referring to Figures 2 and 3, the second collection chamber 210 of this embodiment transfers nucleated red blood cells (nRBCs) separated by the third density gradient material (DGM3) from the second depletion chamber 200. You can receive and save it. The second collection chamber 210 may be connected to the second depletion chamber 200 through a seventh chamber passage L7.

The second collection chamber 210 may be formed at the ninth position of the disk member 110 to be connected to the second depletion chamber 200. The ninth position of the second collection chamber 210 will be set to be spaced further apart in the radial direction (R) from the rotation center (O) of the disk member 110 than the ninth position of the second depletion chamber 200. You can.

As described above, in this embodiment, the blood chamber 120, the plasma chamber 130, the waste chamber 140, the buffer chamber 150, the first mixing chamber 160, and the first depletion chamber 170. , the first collection chamber 180, the second mixing chamber 190, the second depletion chamber 200, and the second collection chamber 210 are based on the rotation center (O) of the disk member 110. It will be described as being arranged in a direction away from the rotation center (O) along the radial direction (R) of the disk member 110. Accordingly, when the disk member 110 rotates, various fluids flow into the buffer chamber 150, the blood chamber 120, the waste chamber 140, the first and second mixing chambers 160, according to the centrifugal force of the disk member 110. 190), the first and second depletion chambers 170 and 200, and the first and second collection chambers 180 and 210, respectively.

The centrifugal force-based fetal cell separation method according to an embodiment of the present invention configured as described above is as follows.

Referring to Figure 3, the centrifugal force-based fetal cell separation method according to this embodiment performs centrifugation of whole blood (WB) collected from the mother, and uses a microscopic device that recognizes density gradient material (DGM) and white blood cells (WBC). Using beads (MB), nucleated red blood cells (nRBCs) and trophoblasts (T) of fetal cells can be simultaneously separated and captured from whole blood (WB).

For example, the centrifugal force-based fetal cell separation method according to an embodiment of the present invention includes the step of injecting whole blood (WB) and first density gradient material (DGM1) into the blood chamber 120 ((a in FIG. 3) ), a step of separating whole blood (WB) into plasma (P), peripheral blood mononuclear cells (PBMC), and red blood cells (RBC) using the first density gradient material (DGM1) (see (a) of Figure 3). ), flowing plasma (P) from the blood chamber 120 to the plasma chamber 130 (see (a) and (b) of Figure 3), peripheral blood mononuclear cells (PBMC) in the blood chamber 120 In the step of flowing into the first mixing chamber 160 (see Figures 3 (a) and (c)), white blood cells (WBC) contained in peripheral blood mononuclear cells (PBMC) are pre-stored in the first mixing chamber 160. A step of attaching the first microbead (MB1) to white blood cells (WBC) by mixing with the first microbead (MB1) (see (c) of FIG. 3), trophoblast cells (T) contained in peripheral blood mononuclear cells (PBMC) ) and flowing the white blood cells (WBC) to which the first microbead (MB1) is attached from the first mixing chamber 160 to the first depletion chamber 170 (see (c) and (d) of FIG. 3 ), separating the white blood cells (WBC) to which the trophoblast cells (T) and the first microbeads (MB1) are attached with the second density gradient material (DGM2) previously stored in the first depletion chamber 170 (FIG. 3 (d)), and flowing the trophoblast cells (T) separated in the first depletion chamber 170 into the first collection chamber 180 and storing them ((d) and (e) of FIGS. 3) reference), the buffer chamber 150 supplies lysis buffer (LB) to the blood chamber 120 to lyse red blood cells (RBCs) (see Figures 3 (a) and (f)), red blood cells ( A step of flowing general blood cells dissolved by lysis buffer (LB) among RBCs from the blood chamber 120 to the waste chamber 140 (see (a) and (g) of FIG. 3), red blood cells (RBC) Among them, flowing nucleated red blood cells (nRBCs) and white blood cells (WBCs) that have not been dissolved by lysis buffer (LB) from the blood chamber 120 to the second mixing chamber 190 ((a) and (h) in FIGS. 3 ) Reference), nucleated red blood cells (nRBC) and white blood cells (WBC) are mixed with the second micro beads (MB1) previously stored in the second mixing chamber 190 to attach the second micro beads (MB2) to the white blood cells (WBC). Step (see (h) of FIG. 3), flowing nucleated red blood cells (nRBC) and white blood cells (WBC) attached with microbeads (MB) from the second mixing chamber 190 to the second depletion chamber 200. Step (see (h) and (i) of FIG. 3), nucleated red blood cells (nRBC) and white blood cells (WBC) to which the second microbead (MB2) is attached are stored in the third depletion chamber 200 in advance. Separating with a density gradient material (DGM3) (see (i) in FIG. 3), and flowing the nucleated red blood cells (nRBCs) separated in the second depletion chamber 200 into the second collection chamber 210 for storage. It may include steps (see (i) and (j) of FIG. 3).

Here, the step of separating whole blood (WB) into plasma (P), peripheral blood mononuclear cells (PBMC), and red blood cells (RBC) (see (a) of FIG. 3), the first microbead (MB1) is added to the white blood cells (WBC). ) (see (c) in Figure 3), separating white blood cells (WBC) to which the trophoblast (T) and the first microbead (MB1) are attached (see (d) in Figure 3), red blood cells Step of lysing cells (RBC) (see Figure 3 (a) and (f)), attaching the second microbead (MB2) to white blood cell (WBC) (see Figure 3 (h)), nucleated red blood cell In one of the steps of separating (nRBC) and white blood cells (WBC) to which the second microbead (MB2) is attached (see (i) in FIG. 3), centrifugation is performed by rotating the disk member 110. You can.

In addition, in the step of separating whole blood (WB) into plasma (P), peripheral blood mononuclear cells (PBMC), and red blood cells (RBC) (see (a) of Figure 3), plasma (P) and peripheral blood mononuclear cells ( Plasma (P) and peripheral blood mononuclear cells (PBMC) can be separated from red blood cells (RBC) using the first density gradient material (DGM1), which has a higher density than PBMC and a lower density than red blood cells (RBC). In fact, plasma (P) and peripheral blood mononuclear cells (PBMC) are located on the upper side of the first density gradient material (DGM1), and red blood cells (RBC) are located on the lower side of the first density gradient material (DGM1).

In addition, in the step of attaching the first and second microbeads (MB1, MB2) to white blood cells (WBC) (see (c) and (h) of Figure 3), the density is higher than that of nucleated red blood cells (nRBC) and trophoblasts (T). High-density CD45 beads can be used as the first and second microbeads (MB1, MB2) to attach to white blood cells (WBC). Therefore, white blood cells (WBC) to which the first and second microbeads (MB1, MB2) are attached can be formed at a higher density than nucleated red blood cells (nRBC) or trophoblasts (T).

In addition, in the step of separating the trophoblast cells (T) and the white blood cells (WBC) to which the first micro beads (MB1) are attached (see (d) of FIG. 3), the first micro beads have a higher density than the trophoblast cells (T). The trophoblast cells (T) and the white blood cells (WBC) to which the first microbead (MB1) is attached can be separated using the second density gradient material (DGM2), which has a lower density than the white blood cell (WBC) to which (MB1) is attached. . In fact, the trophoblast cells (T) are located on the upper side of the second density gradient material (DGM2), and the white blood cells (WBC) to which the first microbeads (MB1) are attached are located on the lower side of the second density gradient material (DGM2).

In addition, in the step of separating the nucleated red blood cells (nRBC) and the white blood cells (WBC) to which the second micro beads (MB2) are attached (see (i) in FIG. 3), the density of the second micro beads is higher than that of the nucleated red blood cells (nRBC). Nucleated red blood cells (nRBC) and white blood cells (WBC) attached to the second microbead (MB2) can be separated using a third density gradient material (DGM3) that has a lower density than the white blood cells (WBC) to which (MB2) is attached. . In fact, nucleated red blood cells (nRBC) are located on the upper side of the third density gradient material (DGM3), and white blood cells (WBC) to which the second microbead (MB2) is attached are located on the lower side of the third density gradient material (DGM3).

As described above, the embodiments of the present invention have been described with specific details such as specific components and limited examples and drawings, but this is only provided to facilitate a more general understanding of the present invention, and the present invention is limited to the above embodiments. This does not mean that various modifications and variations can be made from this description by those skilled in the art. Accordingly, the spirit of the present invention should not be limited to the described embodiments, and all claims that are equivalent or equivalent to the claims as well as the following claims fall within the scope of the present invention.

Claims (10)

Centrifugation of whole blood collected from the mother is performed, and microbeads that recognize density gradient media (DGM) and white blood cells (WBC) are used to separate the whole blood from the whole blood. A centrifugal force-based fetal cell separation method characterized by simultaneously separating and capturing nucleated red blood cells (nRBC) and trophoblasts of fetal cells.
According to paragraph 1,
Injecting the whole blood and the first density gradient material into a blood chamber;
Separating the whole blood into plasma, peripheral blood mononuclear cells (PBMC), and red blood cells using the first density gradient material;
flowing the plasma from the blood chamber to the plasma chamber;
Flowing the peripheral blood mononuclear cells from the blood chamber to a first mixing chamber;
mixing the leukocytes contained in the peripheral blood mononuclear cells with first microbeads previously stored in the first mixing chamber and attaching the first microbeads to the leukocytes;
Flowing the trophoblast cells contained in the peripheral blood mononuclear cells and the leukocytes to which the first microbeads are attached from the first mixing chamber to a first depletion chamber;
separating the trophoblast cells and the leukocytes to which the first microbeads are attached using a second density gradient material previously stored in the first depletion chamber; and
Flowing the trophoblast cells separated from the first depletion chamber into a first collection chamber and storing them;
A centrifugal force-based fetal cell separation method including.
According to paragraph 2,
supplying a lysis buffer to the blood chamber to lyse the red blood cells;
flowing general blood cells dissolved by the lysis buffer among the red blood cells from the blood chamber to the waste chamber;
flowing the nucleated red blood cells and the white blood cells that are not lysed by the lysis buffer among the red blood cells from the blood chamber to a second mixing chamber;
mixing the nucleated red blood cells and the white blood cells with second micro beads previously stored in the second mixing chamber and attaching the second micro beads to the white blood cells;
flowing the nucleated red blood cells and the white blood cells to which the second microbeads are attached from the second mixing chamber to a second depletion chamber;
separating the nucleated red blood cells and the white blood cells to which the second microbeads are attached using a third density gradient material previously stored in the second depletion chamber; and
flowing the nucleated red blood cells separated in the second depletion chamber into a second collection chamber and storing them;
A centrifugal force-based fetal cell separation method further comprising:
According to paragraph 3,
The blood chamber, the first and second waste chambers, the buffer chamber, the first and second mixing chambers, the first and second depletion chambers, and the first and second collection chambers are groove-shaped in the rotatable disk member. are formed in different locations,
Separating the whole blood into the plasma, peripheral blood mononuclear cells, and red blood cells, attaching the first microbead to the leukocyte, separating the trophoblast and the leukocyte to which the first microbead is attached. , in any one of the steps of lysing the red blood cells, attaching the second microbead to the leukocyte, or separating the nucleated red blood cell and the leukocyte to which the second microbead is attached, the disc member A centrifugal force-based fetal cell separation method characterized by centrifugation by rotation.
According to paragraph 3,
In the step of separating the whole blood into the plasma, the peripheral blood mononuclear cells, and the red blood cells, the first density gradient material having a higher density than the plasma and the peripheral blood mononuclear cells and a lower density than the red blood cells is used. Separating plasma from the peripheral blood mononuclear cells and the red blood cells,
In the step of separating the trophoblast cells and the leukocytes to which the first microbeads are attached, the second density gradient material, which has a higher density than the trophoblast cells and a lower density than the leukocytes to which the first microbeads are attached, is used to Separating cells and white blood cells to which the first microbead is attached,
In the step of separating the nucleated red blood cells and the white blood cells to which the second microbeads are attached, the nucleated red blood cells are separated using the third density gradient material, which has a higher density than the nucleated red blood cells and a lower density than the white blood cells to which the second microbeads are attached. A centrifugal force-based fetal cell separation method characterized by separating red blood cells and white blood cells to which the second microbead is attached.
According to paragraph 3,
In the step of attaching the first microbead to the leukocyte, CD45 beads, which have a higher density than the trophoblast cells, are used as the first microbead to attach to the leukocyte,
In the step of attaching the second microbead to the leukocyte, a centrifugal force-based fetal cell separation method characterized in that CD45 beads, which have a higher density than the nucleated red blood cell, are used as the microbead to attach to the leukocyte.
A disc member that is selectively rotated for centrifugation of fetal cells;
It is formed at the first position of the disc member, and after injecting the first density gradient material and whole blood, the whole blood is separated into plasma, peripheral blood mononuclear cells, and red blood cells through the first density gradient material when the disc member is rotated. blood chamber;
a plasma chamber formed at a second position of the disk member to be connected to a first part of the blood chamber, and receiving and storing the plasma from the blood chamber;
It is formed at a third position of the disc member to be connected to the second part of the blood chamber, and the peripheral blood mononuclear cells are mixed with the first microbeads stored in advance after being delivered from the blood chamber and included in the peripheral blood mononuclear cells. a first mixing chamber for attaching the first microbead to the white blood cells;
It is formed at the fourth position of the disc member to be connected to the first mixing chamber, and the trophoblasts contained in the peripheral blood mononuclear cells and the leukocytes to which the first microbeads are attached are received in advance from the first mixing chamber. a first depletion chamber that separates the trophoblast cells through a stored second density gradient material;
A first collection chamber formed at a fifth position of the disk member to be connected to the first depletion chamber, and receiving and storing trophoblast cells separated by the second density gradient material from the first depletion chamber. ;
A buffer chamber formed at a sixth position of the disc member to be connected to a third part of the blood chamber, and supplying lysis buffer to the blood chamber from which the plasma and peripheral blood mononuclear cells have been removed to lyse the red blood cells. ;
A second portion is formed at the seventh position of the disc member to be connected to the fourth portion of the blood chamber, and is pre-stored after receiving nucleated red blood cells and white blood cells that have not been lysed by the lysis buffer among the red blood cells from the blood chamber. a second mixing chamber that mixes with microbeads and attaches the second microbeads to the white blood cells;
It is formed at the eighth position of the disk member to be connected to the second mixing chamber, and after receiving the nucleated red blood cells and the leukocytes to which the second microbeads are attached from the second mixing chamber, a pre-stored third density gradient material is used. a second depletion chamber that separates the red blood cells through a second depletion chamber; and
A second collection chamber formed at the ninth position of the disk member to be connected to the second depletion chamber, and receiving and storing the fluid red blood cells separated by the third density gradient material from the second depletion chamber. ;
A centrifugal force-based fetal cell separation device comprising a.
In clause 7,
a waste chamber formed at a tenth position of the disc member to be connected to a fifth part of the blood chamber, and receiving and storing normal blood cells dissolved by the lysis buffer among the red blood cells in the blood chamber;
A centrifugal force-based fetal cell separation device further comprising:
According to clause 8,
The first position of the blood chamber is set to be radially spaced further from the rotation center of the disk member than the sixth position of the buffer chamber,
The second position of the plasma chamber is set to be radially spaced further from the rotation center of the disk member than the first position of the blood chamber,
The third and seventh positions of the first and second mixing chambers are set to be radially spaced further from the rotation center of the disk member than the first position of the blood chamber,
The fourth and eighth positions of the first and second depletion chambers are set to be radially spaced further from the rotation center of the disk member than the third and seventh positions of the mixing chamber,
The fifth and ninth positions of the first and second collection chambers are set to be radially spaced further from the rotation center of the disk member than the fourth and eighth positions of the first and second depletion chambers. ,
A centrifugal force-based fetal cell separation device, characterized in that the tenth position of the waste chamber is set to be radially spaced further from the rotation center of the disc member than the first position of the blood chamber.
In clause 7,
The first density gradient material is provided as a material that has a higher density than the plasma and the peripheral blood mononuclear cells and a lower density than the red blood cells,
The second density gradient material is provided as a material that has a higher density than the trophoblast cells and a lower density than the white blood cells to which the first microbeads are attached,
The third density gradient material is provided as a material that has a higher density than the nucleated red blood cells and a lower density than the white blood cells to which the second microbeads are attached,
The first and second microbeads are centrifugal force-based fetal cell separation devices, characterized in that the CD45 beads are attached to the leukocytes while having a higher density than the trophoblasts and nucleated red blood cells.