JP2003274924A - Method and apparatus for separating cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve problem that a conventional apparatus for separating cells has disadvantages: having a large size, being expensive and damaging cells a great deal by applying an electric field during separation of the cells. <P>SOLUTION: In the apparatus for separating the cells a cell inlet, a channel, a branched channel and pumping means are formed on an integrated chip to make the apparatus small and inexpensive. Especially, an electroosmotic flow pump which does not directly apply the electric field to the cells is used as the pumping means. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention utilizes T-cells such as lymphocytes contained in blood by utilizing the difference in electrophoretic velocity and specific gravity in order to judge the health condition, perform immunodiagnosis and immunotherapy. The present invention relates to a separating means and a device for distinguishing into subpopulations such as cells and B cells, counting the number, or separating into subpopulations. In particular, some of the necessary functions and structures are integrated in a single plate-shaped chip, and only a small amount of the required sample is needed, which is characterized by portability, immediacy, disposableness, and low cost. TAS, Lab-o
It relates to the field called na-Chip.

[0002]

2. Description of the Related Art Lymphocytes are the center of the body responsible for immune functions and are functionally and systematically different cell populations, such as B cells, killer T cells, helper T cells, and suppressor T cells. It is composed of subgroups. Needless to say, lymphocytes have various information regarding the immune response of an individual, and an immunodiagnosis can be made by sorting a subgroup and examining it in detail, or by simply measuring the ratio of lymphocyte subgroups in peripheral blood. You can get useful information for. Alternatively, the distinction / separation of lymphocyte subgroups has become an important and general basic also in immunotherapy in which a certain kind of lymphocytes are cultured outside the body and returned to the body.

Usually, for the measurement of such a lymphocyte subgroup ratio and the sorting of a specific subgroup, lymphocytes to which a fluorescent antibody associated with the specific subgroup has acted are contained in fine water droplets from a nozzle. Flow cytometry method is used, in which a subgroup is extruded into the air in a shape, and the subgroups are distinguished and counted from each fluorescence and scattering, and the orbit of only water droplets containing a specific subgroup is bent and sorted by electrostatic force. However, the equipment is large and expensive, and the maintenance is difficult, so it was not easy to use when needed. As a simple method, a method of separating by passing through a column filled with a special fibrous or bead-like resin such as antibody immobilized on the surface so that only specific subgroups are adsorbed However, this has a problem in quantification because it damages lymphocytes, it takes time to condition the column, and the adsorption rate varies depending on conditions such as the dropping rate. In addition, a method of separating T cells and B cells based on the difference in surface charge concentration using a cell electrophoresis apparatus or a free flow electrophoresis apparatus was often used before. Although it is being replaced by the metric method, the advantage of this method is that there is little cell damage because it does not use an antibody, and it is still used for applications such as immunotherapy.

In recent years, microTAS, Lab-on-
The technical field called a-Chip is starting up, and by integrating the conventional analysis technology and chemical synthesis method on one chip, downsizing of the system, reduction of the amount of necessary reagents and specimens, and low cost It realizes high speed, high speed and high functionality. An example in which T and B cells of lymphocytes are separated on such a chip is already known. (Japanese Patent Application 2001-
304178) According to this method, T and B cells can be separated by electrophoresing lymphocytes arranged in a band at one location while suppressing the pH change of the buffer solution. Also,
It has been shown that these cells can be similarly separated by simultaneously performing centrifugation and electrophoresis.

A blood analysis chip, such as a healthcare device, has recently been developed for the purpose of performing one-chip blood analysis necessary for human health management from one drop of blood at home or in an emergency medical field. Has been done. (JP 2001-
258868) If cells can be separated on one chip, immunodiagnosis can provide useful information for health care.

[0006]

As described above, since the flow cytometry device currently used for cell separation is large-scale and expensive, it provides the benefits of immunodiagnosis and immunotherapy to all. It is one of the major barriers to If such cell separation can be realized on a chip, it can be applied to daily immunodiagnosis, health management, etc. by the advantages of small size, low cost, simple, and immediate chip, and contribute to people's well-being. It's big. Further, in the case of the flow cytometry method, since an electric field is applied to cells during cell separation, there is a problem that cells are damaged in no small amount.

Also, when cells are separated by using electrophoresis on a chip, it is considered that the cells are damaged due to the application of an electric field to the cells. This is a major problem that significantly narrows the application of cell separation. Is.

[0008]

A downflow type electroosmotic flow pump is used for cell separation without applying an electric field that adversely affects cells. The pump is described in detail in a known example (Japanese Patent Application No. 2001-116091), which will be briefly described with reference to FIG. Flow path means 101,
Upstream channel 102, small cross-sectional area channel 103, downstream channel 104
And the liquid reservoir 110 is filled with the electrolyte solution in advance,
Two gel electrodes 105 and 1 are provided in the middle of these channels.
Platinum electrodes 106 and 108 are connected via 07. Further, 109 is also a flow path means, which is connected to various upstreams. Now, if it is desired to transport the electrolytic solution in the direction of the arrow in the figure, a positive voltage is applied to the platinum electrode 106 on the upstream side, and the platinum electrode 108 on the downstream side is grounded. As a result, due to the electroosmotic flow generated by the voltage applied between the electrodes 106 and 108, the electrolyte liquid in the flow path moves in the direction of the arrow in the figure. As a result, a negative pressure (suction force) is generated in the flow path means 109 on the upstream side, which means that the mechanism shown in FIG. 1 has a function as a pump. Here, the voltage is applied through the gel electrode is
The reason is to suppress the generation of hydrogen and oxygen and the pH fluctuation of the electrolyte solution due to the electrode reaction, and the reason for having the small cross-sectional area channel 103 is to obtain a higher pumping force. What should be emphasized here is that no electric field is applied to the electrolytic solution on the upstream side of the electroosmotic pump shown in FIG. That is, when the pump is formed on a chip and used for cell separation, any cell can be transported in any direction without directly applying an electric field to the cell. Therefore, combine this with cell detection means. Thus, a plurality of types of cells can be sorted into arbitrary cell reservoirs.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 2 shows a cell separation chip produced according to the present invention and its peripheral configuration diagram. Reference numeral 201 denotes a substrate constituting a chip, on which a cell reservoir 202 for a sample, which is a sample, and a flow channel 203 are provided, and the flow channel branches in a Y-shape downstream thereof, and a branch flow channel A204 and a branch flow channel, respectively. There is B205. An electroosmotic flow pump A206 and an electroosmotic flow pump B207 are arranged downstream of the respective branch flow paths. Now, a plurality of two types of cells are put in the liquid reservoir 202 to fill the liquid reservoir and the channel with a liquid such as a buffer solution. At this time, a fluorescent antibody was previously attached to one type of cells. (In this case, the cells to which the fluorescent antibody is attached are referred to as cells A, and the cells to which the fluorescent antibody is not attached are referred to as cells B). Then, the cells are guided downstream by a pressure flow or an electroosmotic pump on the chip. At this time, light is emitted from the light source 210 in the middle of the flow path 203, and this light emits fluorescence when there is a fluorescent antibody on the cell surface, and this fluorescence is detected by the detector 211. At the same time, the cells passing through this point are also observed by the CCD camera 212. These data are sent to the control computer 213, and the on / off timing of the electroosmotic flow pumps A and B is calculated from the flow velocity of the cells and the distance from the observation point to the Y-shaped branch point. That is, if a cell that emits fluorescence (that is, cell A) is detected, the electroosmotic flow pump A is turned on and the electroosmotic flow pump B is turned off immediately before the cell reaches the Y-shaped bifurcation, and the cell A is branched. It is drawn into the flow path A. In the case of cells B that do not emit fluorescence, on the contrary, the electroosmotic flow pump A is turned off, the electroosmotic flow pump B is turned on, and the electroosmotic flow pump B is drawn into the branch channel B. By repeating such a process, two types of cells can be separated only by the flow of liquid by the electroosmotic pump without applying an electric field or the like. In the case of a cell B that does not emit fluorescence, the CCD camera 212 confirms the cell.

In the above case, the two types of cells are distinguished by the presence or absence of fluorescence, but the cells can be similarly distinguished by a method such as distinguishing from the difference in the shape of other cells.

[0011]

EXAMPLES [First Example] Using the cell separation chip shown in FIG. 2, an attempt was made to separate CD4 and CD8 from T cells of lymphocytes. Inexpensive polyethylene terephthalate (PET) is used as the chip substrate, and the pattern is transferred to the chip substrate by pressing the flow path and the liquid storage pattern against a quartz mold formed by the light exposure method or the dry etching method.
Formed. By examining the number ratio of CD4 and CD8, it is possible to diagnose acquired immunodeficiency syndrome (AIDS) and hepatitis B. These cells are previously treated with a fluorescent antibody that adheres only to CD4 and introduced into the liquid reservoir 102. At this time, the flow path and the entire liquid reservoir are filled with HBSS solution (HE
PES buffered Hanks' balan
ced salt solution). After that, the cells are allowed to flow downstream, the presence or absence of fluorescence is detected during the process, and the data is used to separate the cells by turning the electroosmotic flow pump on and off. As a result, only CD4 that fluoresces in the branch channel A and CD8 that does not fluoresce in the branch channel B
It was confirmed that only those were separated.

[Second Embodiment] FIG. 3 shows a modification of the cell separation chip shown in FIG. In the figure, the same reference numerals as those in FIG. 2 are used as they are in FIG. In this case, an electroosmotic pump is installed in the middle of the two branch flow paths, and a cell reservoir A301 and a cell reservoir B302 for storing the sorted cells are installed downstream of the electroosmotic pump. By doing so, when a large number of cells were sorted in the case of FIG. 1, the cells gathered on the pumps of the respective branch flow paths, which acted as a resistance, and the pumping capacity had been reduced. As described above, the electroosmotic pump is installed on the way, and the liquid reservoir is further provided downstream thereof, whereby the sorted cells can be separated while being stored in the liquid reservoir. Actually, when the separation of CD4 and CD8 was attempted in the same manner as in the first example, they could be separated without a decrease in the capacity of the electroosmotic flow pump.

[0013]

As described above, in the cell separation device according to the present invention, the types of a plurality of cells are judged from the fluorescence and the cell shape, and the electroosmotic pump is controlled from the result to separate the cells. . Since the electroosmotic pump does not directly apply an electric field to cells, undamaged cell separation can be realized. In the present invention, two types of cells were separated, but it is easily possible to simultaneously separate more types of cells by increasing the number of branch channels or arranging them in multiple stages. You can guess.
Further, since the cell separation device of the present invention can be constructed in a smaller size and at a lower cost than conventional devices, it has become possible to provide immunodiagnosis and immunotherapy to many people.

[Brief description of drawings]

FIG. 1 is a diagram illustrating an electroosmotic pump.

FIG. 2 is a diagram illustrating the configuration of the cell separation device of the present invention.

FIG. 3 is a diagram illustrating the configuration of the cell separation device of the present invention.

[Explanation of symbols]

101 flow path means 102 upstream flow path 103 Small cross-section area 104 downstream flow path 105 gel electrode 106 Platinum electrode 107 gel electrode 108 Platinum electrode 109 flow path means 110 liquid reservoir 201 substrate 202 Sample inlet liquid reservoir 203 flow path 204 Branch flow path A 205 Branch flow path B 206 Electroosmotic Pump A 207 Electroosmotic pump B 208 cells A 209 cells B 210 light source 211 detector 212 CCD camera 213 Control computer 301 Cell Reservoir A 302 Cell Reservoir B

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hiroyuki Axa             1-18-20 Seiwadai, Kita-ku, Kobe-shi, Hyogo (72) Inventor Yasuhiro Horiike             3-2-12 Higashifushimi, Nishi-Tokyo, Tokyo F-term (reference) 4B029 AA23 AA27 BB11 CC01                 4B065 AA90X BA30 CA44 CA46

Claims (3)

[Claims] 1. An apparatus for separating a plurality of types of cells, comprising a cell inlet, a flow path for moving the cells, a plurality of branch flow paths for guiding the separated cells, and a plurality for transporting the cells. Of the present invention is characterized in that the pump means is integrally formed on a single plate or rod, and when the pump means is operated, no electric field is directly applied to the cells, and the cells are identified. A cell separation device, characterized in that, based on a signal from a detection means, each pump means is activated or deactivated to introduce cells of a desired type into a desired branch flow path for cell separation. 2. A cell separation device, characterized in that the pumping means according to claim 1 is in particular an electroosmotic pump. 3. A cell separation method, wherein the pump means according to claim 1 is an electroosmotic pump in particular.