WO2006001196A1 - Cell separator and cell separating method - Google Patents

Abstract

Fluid containing magnetic beads and specified cells is divided to the right and left by using a first fluid dividing means provided in a micromixer, and the fluid divided to the right and left is then divided upward by using a second fluid dividing means provided in the micromixer. Subsequently, the fluid divided to the right and left is divided downward by using a third fluid dividing means provided in the micromixer. The fluid divided upward and the fluid divided downward are joined by using a fluid joining means provided in the micromixer. Thereafter, magnetic beads in the fluid after passing through the micromixer and the cells adhering the magnetic beads are separated from the remaining part of the fluid by means of a separator.

Description

 Specification

 Cell separation device and cell separation method

 Technical field

 The present invention relates to a cell separation device and a cell separation method.

 Background art

 [0002] As a conventional cell separation apparatus using magnetic beads, for example, US5536475 is intended to extract a large amount of sample force and desired hematopoietic cells. In addition to mixing the cells, sufficient incubation time was required for the magnetic beads and the cells to be joined by molecular diffusion.

 [0003] On the other hand, since stem cells and the like are capable of self-proliferation, if even a very small sample force can be extracted, it is sufficient to observe subsequent growth. However, in a system that extracts target cells from a minute sample, the Reynolds number of the flow decreases, so the mixing of magnetic beads and cells is suppressed, and the target cells cannot be extracted. there were.

 Disclosure of the invention

 Problems to be solved by the invention

[0004] The present invention provides a cell separation method using magnetic beads, regardless of the Reynolds number, sufficiently mixing the magnetic beads and cells, attaching the cells to the magnetic beads, and separating the cells. The purpose is to carry out simply and reliably.

 Means for solving the problem

In order to achieve the above object, the present invention provides:

 A first fluid dividing means for dividing a fluid containing magnetic beads and predetermined cells into left and right; a second fluid dividing means for dividing the fluid divided into the left and right upward; A micromixer having third fluid dividing means for dividing the divided fluid downward, and fluid merging means for joining the fluid divided upward and the fluid divided downward When,

The magnetic beads in the fluid after passing through the micromixer and the front A separator for separating the cells attached to the magnetic beads from the remainder of the fluid;

 It is related with the cell separation apparatus characterized by comprising.

 [0006] The present invention also provides:

 Using a first fluid dividing means provided in the micromixer, a first step of dividing a fluid containing magnetic beads and predetermined cells into right and left;

 Using the second fluid dividing means provided in the micromixer, the second step of dividing the fluid divided into the left and right upwards;

 A third step of dividing the fluid divided into the left and right using a third fluid dividing means provided in the micromixer; and

 A fourth step of joining the fluid divided above and the fluid divided below using a fluid joining means provided in the micromixer;

 A fifth step for separating the residual force of the fluid by a separator between the magnetic beads in the fluid after passing through the micromixer and the cells attached to the magnetic beads;

 It is related with the cell separation method characterized by comprising.

[0007] According to the present invention, the fluid containing the magnetic beads and the predetermined cells is divided in the left-right direction, and further, the divided fluid is divided in the vertical direction, and then the divided fluids are joined. I am doing so. In this process, the cross section of the fluid divided in the vertical direction can be rotated by a predetermined angle with respect to the cross section of the first fluid, and the fluid divided in the vertical direction is joined. In this case, it can be rotated again by a predetermined angle with respect to the cross section of the first fluid. Therefore, through the above process, the first fluid is divided, and the divided fluid is rotated by a predetermined angle, and then joined, so that the fluid is sufficiently stirred. Will be mixed.

[0008] Therefore, the magnetic beads and the cells contained in the fluid are also sufficiently agitated and mixed with each other, so that the cells are sufficiently attached to the magnetic beads. Magnetic separator with high efficiency due to the separator provided at the rear Separated.

 [0009] As is apparent from the above description, in the present invention, the fluid is stirred and mixed by dividing the fluid and rotating the fluid cross section! Therefore, regardless of the Reynolds number of the fluid, stirring and mixing of the magnetic beads and the cells can be sufficiently promoted to attach the cells to the magnetic beads.

 The invention's effect

 [0010] As described above, according to the present invention, the magnetic beads and cells are sufficiently mixed for cell separation using magnetic beads, regardless of the Reynolds number. The cells can be attached to the beads and the cells can be separated easily and reliably. Brief Description of Drawings

 FIG. 1 is a block diagram showing an example of a cell separation device of the present invention.

 FIG. 2 is an exploded configuration diagram showing an example of a micromixer in the cell separation apparatus shown in FIG. 1.

 FIG. 3 is a diagram showing a state of fluid when flowing in the micromixer shown in FIG. 2.

 4 is a diagram showing the state of fluid when flowing in the micromixer shown in FIG.

 FIG. 5 is an enlarged view showing a peripheral portion including a separator in the cell separation device shown in FIG. 1.

 FIG. 6 is an enlarged view showing a peripheral portion including the purification separation device in the cell separation device shown in FIG. 1.

 FIG. 7 is a configuration diagram showing a modification of the cell separation device shown in FIG. 1.

 Explanation of symbols

[0012] 10 Cell separation device

 11, 12 entrance

 13, 14

 15, 16 Exit

 20 Micromixer section

21 Micromixer 30 Separator section

 31 Separator

 40 flow path

 51 Purification separator

 60 Bypass channel

 61 pump

 S1, S2 multilayer fluid

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] Details of the present invention, as well as other features and advantages, will be described in detail below based on the best mode.

 FIG. 1 is a configuration diagram showing an example of the cell separation device of the present invention. A cell separation apparatus 10 shown in FIG. 1 includes a micromixer unit 20 in which a plurality of micromixers 21 are connected in series, a separator unit 30 in which a plurality of separators 31 are arranged in series, and magnetic beads and cells. A flow path 40 is provided for flowing a fluid containing the fluid and passing it through the micromixer section 20 and the separator section 30 so that the fluid is subjected to cell separation according to the steps described in detail below.

 In the cell separation device 10 shown in FIG. 1, for example, a fluid containing magnetic beads is introduced from the inlet 11, and a fluid containing stem cells such as bone marrow fluid or blood, or blood cells is introduced from the inlet 12 to flow. Mix in path 40. Next, the fluid obtained by mixing is introduced into the micromixer part 20 and stirred and mixed in each micromixer 21 as described in detail below.

 FIG. 2 is an exploded configuration diagram showing an example of the micromixer 21 in the cell separation device 10 shown in FIG. A micromixer 21 shown in FIG. 2 includes a first plate-like member 22, a second plate-like member 23 and a fourth plate-like member 25 that are sequentially provided above the first plate-like member 22, A third plate-like member 24 and a fifth plate-like member 26 are sequentially provided below the first plate-like member 22.

[0017] The first plate-like member 22 is formed with a T-shaped first opening 221 and an I-shaped fourth opening 222, and ends 221A and 222A of these openings are respectively First plate member 22 Are opened at opposite side ends.

 The second plate-like member 23 is formed with an L-shaped second opening 231, and one end 231 A thereof is the first opening in the first plate-like member 22. The end of the upper side opening of 221 is continuous with 11B. Further, the other end 231 B of the second opening 231 is continuous with the front end 222 B of the fourth opening 222 in the first plate-like member 22.

 The third plate-like member 24 is similarly formed with an L-shaped third opening 241, and one end 241 A thereof is the first opening 221 in the first plate-like member 22. The edge of the upper side opening 2 is continuous with 21C. The other end 241 B of the third opening 241 is continuous with the front end 222 B of the fourth opening 222 in the first plate-like member 22.

 [0020] Note that the fourth plate member 25 and the fifth plate member 26 are provided as lids so as to seal the first opening 221 to the fourth opening 222, respectively. This is to make the micromixer shown in Fig. 2 function as an actual device.

 Next, a fluid mixing method using the micromixer 21 shown in FIG. 2 will be described.

 3 and 4 are diagrams showing the state of the fluid when flowing in the micromixer 21 shown in FIG. In this example, a case of a multilayer fluid in which a fluid containing magnetic beads and a fluid containing cells form layers in the upward and downward directions will be described.

 First, the multilayer fluid S 1 is introduced into the first opening 221 in the first plate-like member 22 of the micromixer 21. At this time, the multilayer fluid S1 is divided in the left-right direction at the upper side of the first opening 221. Next, the multilayer fluid S1 divided in the left-right direction is continuous with the first opening 221 and the second opening 231 in the second plate member 23 and the second fluid in the third plate member 24, respectively. It is introduced into the opening 241 of 3, and as a result, it is divided in the vertical direction. At this time, since the multilayer fluid S1 is rotated 90 degrees with respect to the flow direction, the cross section thereof is rotated 90 degrees.

[0023] Next, the multilayer fluid S1 is introduced into the fourth opening 222 of the first plate-like member 22 that is continuous with the second opening 231 and the third opening 241 and is divided in the vertical direction. The multilayer fluid S1 joins to become multilayer fluid S2. At this time, each of the divided multilayer fluids S1 is further rotated by 90 degrees with respect to the flow direction, so that the cross section is rotated by 90 degrees. Therefore, the cross section of the multilayer fluid S2 is 180 ° compared to the multilayer fluid S1. The stacking order of the fluid composing each layer is reversed and the number of stacks is doubled.

As a result, the multilayer fluid S1 is divided in the cross-sectional direction by passing through the micromixer 21 shown in FIG. 2, and the cross-section itself is rotated 180 degrees. In the multilayer fluid S 2, each layer is more mixed and uniform than the multilayer fluid S 1. Therefore, the mixing and stirring of the magnetic beads and cells contained in each layer is promoted, and the cells adhere to the magnetic beads with high efficiency.

 [0025] It is preferable that an antigen as a surface marker is attached to the surface of the cell, and an antibody that causes an antigen-antibody reaction with the antigen is attached to the surface of the magnetic bead. In this case, the cells can be efficiently and firmly attached to the magnetic beads through the antigen-antibody reaction.

 FIG. 5 is an enlarged view showing a peripheral portion including the separator 31 in the cell separation device 10 shown in FIG. As is clear from FIG. 3, the separator 31 includes a pair of magnets 311 and 312 arranged to face each other with the flow path 40 interposed therebetween. However, the magnets 311 and 312 need not be arranged so as to sandwich the flow path 40, but may be disposed close to the flow path 40. In FIG. 3, arrows indicate the direction of fluid flow, black circles indicate magnetic beads, and scaly members indicate cells.

 When the fluid including the magnetic beads and cells that have passed through the micromixer unit 20 reaches the separator unit 30, it is affected by the magnetic field B between the magnets 311 and 312. At this time, the magnetic beads and the cells attached to the magnetic beads are attracted to the right by the magnetic field B and separated and removed from the rest of the fluid.

 [0028] Thereafter, the separated magnetic beads and adherent cells reach the valve 13 and are taken out through the outlet 15. On the other hand, the remaining portion of the fluid reaches the valve 14 and is taken out through the outlet 16. Therefore, through the above steps, only desired cells are attached to the magnetic beads, and the cells can be easily and reliably separated.

Although not shown in FIG. 1, in the cell separation device shown in FIG. 1, a pure water separation device can be provided on the downstream side of the separator portion 30. Figure 6 shows the cell separation device shown in Figure 1. 3 is an enlarged view of a peripheral portion including a pure water separator 51 in the apparatus 10; FIG. As is clear from FIG. 6, the purification / separation device 51 includes a pair of magnets 511 and 512 arranged to face each other with the flow path 40 interposed therebetween. In FIG. 6, arrows indicate the direction of fluid flow, black circles indicate magnetic beads, and scaly members indicate cells.

 [0030] The magnetic beads and the adherent cells that have been separated and removed by passing through the separator 30 are introduced into the pure water separator 51 together with the buffer solution. Since the buffer solution has an action of separating the cells from the magnetic beads, when the mixed solution is affected by the magnetic field B between the magnets 511 and 512, the magnetic beads are independent of whether or not the cells are separated. It is drawn to the right by the influence of magnetic field B, and is taken out through the nozzle 13 and the outlet 15. On the other hand, the cells separated from the magnetic beads are taken out through the valve 14 and the outlet 16 without being affected by the magnetic field B.

 Thus, by providing the pure soot separating device 51 on the downstream side of the separator portion 30, the magnetic beads can be easily recovered and only the cells to be separated can be easily extracted. .

 FIG. 7 is a configuration diagram showing a modification of the cell separation device shown in FIG. Components that are the same as or similar to the components shown in FIG. 1 are denoted by the same reference numerals. In the cell separation device 10 shown in FIG. 7, a bypass channel 60 is provided between the inlets 11 and 12 and the micromixer unit 20 and between the separator unit 30 and the valves 13 and 14, and is separated and removed by the separator unit 30. The fluid containing the magnetic beads is circulated by the pump 61 to the upstream side of the micromixer section 20. According to such a configuration, the magnetic beads to be adsorbed with cells are always circulated and reused, so that the total amount of magnetic beads used for cell separation can be reduced.

[0033] In the micromixer 21, the first opening 221 and the fourth opening 222 provided in the first plate-like member 22, and the second opening provided in the second plate-like member 23. The corners can be chamfered in at least one of the portion 231 and the third opening 241 provided in the third plate-like member 24. If there are sharp corners in these openings, the flow velocity decreases in the corners as the multilayer fluid S1 flows through those openings. As a result, the multilayer fluid SI may not be sufficiently mixed. Therefore, in order to suppress the occurrence of these problems, it is preferable to chamfer the corners of the opening as described above.

 [0034] Each plate-like member can be formed of any material. However, as long as the above-described fluid mixing method of the present invention can be realized, a force such as resin, metal, glass, etc. can be formed. . Therefore, preparation of each plate-like member and processing for each plate-like member are facilitated, and the formation of the above-described opening, that is, the formation of the micromixer itself can be easily performed.

 As described above, the present invention has been described in detail based on the embodiments of the present invention with specific examples. However, the present invention is not limited to the above contents, as long as it does not depart from the scope of the present invention. Any modification or change is possible.

 For example, in the above specific example, the case where the magnetic beads and the cells constitute a multilayer fluid has been described. However, the present invention is not limited to the case where the magnetic beads and the like constitute a multilayer fluid. Even when it is a fluid of a layer, it can be preferably used. In the above specific example, the micromixer unit 20 is composed of a plurality of micromixers 21 and the separator unit 30 is composed of a plurality of separators 31. A single separator can also be constructed.

 [0037] Further, in the micromixer 21, the multilayer fluid S1 is caused to flow backward from the fourth opening 222 to the first opening 221 through the second opening 231 and the third opening 241. You can also. In this case, the multilayer fluid S1 divides the multilayer fluid S1 upward at the second opening 231, divides the multilayer fluid S1 downward at the third opening 241 and upwards at the first opening 221. It is also possible to join the divided multilayer fluid S 1 and the multilayer fluid S 1 divided below. Even in this case, the multilayer fluid S1 can be sufficiently mixed in the manner shown in FIGS. 3 and 4 to obtain the multilayer fluid S2.

Claims

The scope of the claims  [1] A first fluid dividing means for dividing a fluid containing magnetic beads and predetermined cells into left and right, a second fluid dividing means for dividing the fluid divided into the left and right upwards, Third fluid dividing means for dividing the fluid divided into the left and right downwards, and fluid merging means for joining the fluid divided upward and the fluid divided downward A micromixer having  A separator for separating the magnetic beads in the fluid after passing through the micromixer and the cells attached to the magnetic beads from the rest of the fluid;  A cell separation device comprising: [2] In the micromixer, the first fluid dividing means includes a T-shaped first opening having one end opened at an end of the first plate-like member. The cell separation device according to claim 1. [3] In the micromixer, the second fluid dividing means is an L-shaped continuous member in the second plate member that is continuous with the left end or the right end of the upper opening of the first opening. The cell separation device according to claim 2, further comprising a second opening. [4] In the micromixer, the third fluid dividing means is a third plate member, wherein the second opening is continuous with the first opening and the upper opening. 4. The cell separation device according to claim 3, further comprising an L-shaped third opening continuous with the left end or the rest of the right end. [5] In the micromixer, in the first plate member, the fluid merging means includes a distal end portion of the second opening portion and a third opening portion behind the first opening portion. 5. The cell separation device according to claim 4, further comprising an I-shaped fourth opening that is continuous with the distal end of the cell. [6] In the second fluid dividing means of the micromixer, the cross section of the fluid is rotated by 90 degrees as compared to the time when the fluid flows into the first fluid dividing means. The cell separation device according to any one of claims 1 to 5. [7] In the third fluid dividing means of the micromixer, the cross section of the fluid is The cell separation device according to any one of claims 1 to 6, wherein the fluid is rotated by 90 degrees compared to when the fluid flows into the first fluid dividing means.  [8] The fluid merging means of the micromixer is characterized in that the cross section of the fluid is rotated by 180 degrees as compared to when the fluid flows into the first fluid dividing means. The cell separation device according to any one of claims 1 to 7.  [9] The cell separation device according to any one of [1] to [8], wherein the fluid is a multilayer fluid in which a fluid containing the magnetic beads and a fluid containing the cells are laminated. .  [10] When the multilayer fluid is divided by the first fluid dividing means, the second fluid dividing means, and the third fluid dividing means, and then joined by the fluid merging means, 10. The cell separation device according to claim 9, wherein the stacking order is reversed and the stacking number is doubled.  [11] The micromixer according to claim 1, wherein corner chamfering is performed in at least one of the first opening force and the fourth opening. The cell separation device according to any one of the above.  [12] In the micromixer, at least one of the first fluid dividing means, the third fluid dividing means, and the fluid merging means is made of resin, metal, or glass. The cell separation device according to any one of claims 1 to 11.  [13] In the micromixer, the fluid is caused to flow backward from the fluid merging means to the first fluid dividing means via the second fluid dividing means and the third fluid dividing means, and The fluid is divided upward by the second fluid dividing means, the fluid is divided downward by the third fluid dividing means, and the fluid divided by the first fluid dividing means and the downward The cell separation device according to any one of claims 1 to 12, wherein the cell separation device is configured to join the fluid divided into two.  [14] The cell separation device according to any one of [1] to [13], wherein a plurality of the micromixers are connected in series.  [15] The cell separation device according to any one of [1] to [14], wherein the separator includes a magnet provided in the flow path of the fluid. [16] Adhering to the magnetic beads and the magnetic beads after passing through the micromixer And a purification / separation device for mixing and removing only the cells from the fluid containing the cells adhering to the magnetic beads by mixing a buffer solution with the fluid containing the cells. Item 16. The cell separation device according to any one of Items 1 to 15. [17] The purification separation device includes a magnet provided in the fluid flow path. The cell separation device according to claim 16. [18] The antigen as a surface marker is attached to the surface of the cell, and an antibody that causes an antigen-antibody reaction with the antigen is attached to the surface of the magnetic bead. The cell separator according to any one of 17 above. [19] The present invention is characterized by comprising circulation means for circulating and reusing the magnetic beads. The cell separation device according to any one of claims 1 to 18. [20] using a first fluid dividing means provided in the micromixer, a first step of dividing a fluid containing magnetic beads and predetermined cells into right and left;  Using the second fluid dividing means provided in the micromixer, the second step of dividing the fluid divided into the left and right upwards;  A third step of dividing the fluid divided into the left and right using a third fluid dividing means provided in the micromixer; and  A fourth step of joining the fluid divided above and the fluid divided below using a fluid joining means provided in the micromixer;  A fifth step for separating the residual force of the fluid by a separator between the magnetic beads in the fluid after passing through the micromixer and the cells attached to the magnetic beads;  A cell separation method comprising the steps of: [21] In the first step, the first fluid dividing means in the first step includes a T-shaped first opening having one end opened at an end of the first plate-like member. , Claim 2 The cell separation method according to 0. [22] In the second plate, the second fluid dividing means in the second step is an L-shaped continuous member in the second plate member, which is continuous with the left end or the right end of the upper side opening of the first opening. First The cell separation method according to claim 21, comprising two openings. [23] In the third plate, the third fluid dividing means in the third step is the third plate member, wherein the second opening is continuous in the upper opening of the first opening. 23. The cell separation method according to claim 22, comprising an L-shaped third opening that is continuous with the left end or the rest of the right end.  [24] In the first plate member, the fluid merging means in the fourth step includes a tip end portion of the second opening portion and the third opening portion behind the first opening portion. 24. The cell separation method according to claim 23, further comprising an I-shaped fourth opening that is continuous with the tip of the part.  [25] In the second step, the second fluid dividing means rotates the cross section of the fluid by 90 degrees as compared to the time when the fluid flows into the first fluid dividing means. 25. The cell separation method according to any one of claims 20 to 24, wherein:  [26] In the third step, the third fluid dividing means rotates the cross section of the fluid by 90 degrees as compared to the time when the fluid flows into the first fluid dividing means. 26. The cell separation method according to any one of claims 20 to 25, wherein:  [27] In the fourth step, the fluid merging means rotates the cross section of the fluid by 180 degrees compared to when the fluid flows into the first fluid dividing means. 27. The cell separation method according to any one of claims 20 to 26.  [28] The cell separation method according to any one of [20] to [27], wherein the fluid is a multilayer fluid in which the fluid containing the magnetic beads and the fluid containing the cells are laminated. Law.  [29] When the multi-layer fluid is divided by the first fluid dividing means, the second fluid dividing means, and the third fluid dividing means, and then merged by the fluid merging means, 29. The cell separation method according to claim 28, wherein the stacking order is reversed and the number of stacks is doubled. [30] The chamfering of the corner of at least one of the first opening force and the fourth opening in the first fluid dividing means to the third fluid dividing means and the fluid merging means. 30. The cell separation method according to any one of claims 20 to 29, wherein the method is performed. [31] At least one of the first fluid dividing means, the third fluid dividing means, and the fluid merging means is also made of grease, metal, or glass force. Any one of  The cell separation method described.  [32] The cell separation method according to any one of [20] to [31], wherein the cycle of the first step is performed a plurality of times, with the fifth step as one cycle. [33] The method according to any one of claims 20 to 32, wherein, in the fifth step, the separation is performed using a magnetic force by a magnet provided so as to sandwich the fluid flow path. The cell separation method according to any one of the above. [34] After passing through the micromixer, a buffer solution is mixed with the magnetic beads and the fluid containing the cells attached to the magnetic beads, and from the fluid containing the cells attached to the magnetic beads, The cell separation method according to any one of claims 20 to 33, further comprising a step of separating and removing only cells. [35] The cell separation method according to [34], wherein the separation / removal is performed by using a magnetic force by a magnet provided so as to sandwich the fluid flow path. [36] The method comprising the steps of attaching an antigen as a surface marker to the surface of the cell, and attaching an antibody that causes an antigen-antibody reaction with the antigen to the surface of the magnetic bead. 36. The cell separation method according to any one of -35. [37] The cell separation method according to any one of [20] to [36], further comprising a step of circulating and reusing the magnetic beads by a predetermined circulation means.