Cell separation chip and cell culturing method using the same

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view schematically showing one example of a system structure of a cell separation and culturing apparatus according to the present invention;

FIG. 2 is a view illustrating the manner in which a cell-containing buffer solution in a microscopic flow channel 204 is merged with a buffer solution in microscopic flow channels 205 and 205′ to flow down a microscopic flow channel 240, and is further merged with a buffer solution in a microscopic flow channel 204′ immediately before a cell detection area 221 to flow down a microscopic flow channel 247;

FIG. 3 is a view illustrating the cell distribution in the post-merging flow channel 240 as a result of the buffer solution flowing down the flow channel 204 being pushed to the center of the flow channel 240 by the buffer solution flowing down the flow channels 205 and 205′;

FIG. 4(A) and FIG. 4(B) are cross-sectional views showing a problem caused by increasing the width of a flow channel and an example of means for solving the problem;

FIG. 5(A) is a cross-sectional view taken along line (A)-(A) in FIG. 1 and seen in the direction of the arrows thereof for illustrating a reservoir 203, openings 201 and 201′, and the flow channels 204 and 204′ described with reference to FIG. 4(A) and FIG. 4(B) in more detail; and FIG. 5(B) is a cross-sectional view taken along line (B)-(B) in FIG. 1 and seen in the direction of the arrows thereof for illustrating culturing tanks 213 and 214, openings 211 and 212, flow channels 218 and 219, a semipermeable membrane 280, and a reservoir 285 on the flow channel exit side in more detail;

FIG. 6 is a plan view showing a system structure of a cell separation and culturing apparatus with a different structure of gel electrode section;

FIG. 7 illustrates an algorithm for recognizing cells from an image captured by a CCD camera and numbering and identifying each of the cells;

FIG. 8 is a plan view schematically showing one example of a system structure of a cell separation and culturing apparatus, which has a special arrangement for the introduction of sample cells as compared to the structure shown in FIG. 1;

FIG. 9(A), FIG. 9(B) and FIG. 9(C) are partial cross-sectional views illustrating the arrangement of the sample cell introduction section in FIG. 8;

FIG. 10 illustrates a flow of processing for specifically labeling cell surface antigen CD4-presenting cells with a P-phycoerythrin-modified RNA aptamer and separating the cells by a cell separation and culturing apparatus;

FIG. 11 shows an examination result of the influence on the fluorescence intensity of β-phycoerythrin as an identifying substance exerted by addition of nuclease;

FIG. 12 shows that the cell surface antigen CD4-presenting cells obtained by removing the β-phycoerythrin-modified RNA aptamer are culturable;

FIG. 13 is a view schematically showing an example of a cell culturing device for culturing the cells collected in the culturing tanks 213 and 214;

FIG. 14(A) through FIG. 14(C) are views showing an overview of processing for cutting off the culturing tanks 213 and 214 together with the chip substrate from the cell separation and culturing apparatus; and

FIG. 15 is an overall conceptual view of an optical system in the cell detection area 221.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
(System Structure of the Cell Separation and Culturing Apparatus)

FIG. 1 is a plan view schematically showing one example of a system structure of a cell separation and culturing apparatus according to the present invention. A cell separation and culturing apparatus 100 includes a chip substrate 101. The cell separation and culturing apparatus 100 includes flow channels formed in a bottom surface thereof and openings communicating to the flow channels in a top surface thereof. The openings act as supply openings for samples and necessary buffer solutions (mediums). The cell separation and culturing apparatus 100 also includes reservoirs for supplying a sufficient amount of buffer solution and adjusting the flow rate in each flow channel. The flow channels can be formed by so-called injection molding, by which a plastic material such as PMMA or the like is injected into a mold. The chip substrate 101 has an overall size of 20×30×1 mm (t). In order to shape the grooves or through-holes formed in the bottom surface of the chip substrate 101 into flow channels or wells, a 0.1 mm thick laminate film is attached to the bottom surface having the grooves by thermal pressurization. Using an objective lens having a numerical aperture of 1.4 and a magnification of ×100, cells flowing in the flow channels can be observed through the 0.1 mm thick laminate film. In the case where the plastic material is highly light-transmissive, such cells can be observed also from above the chip substrate 101.

In the top surface of the chip substrate 101, holes 201 for introducing a cell-containing sample buffer solution to the microscopic flow channels, holes 201′, 202 and 202′ for introducing a buffer solution not containing cells, and a reservoir 203 for surrounding the holes 201, 201′, 202 and 202′, are formed. Accordingly, when a sufficient amount of buffer solution is supplied to the reservoir 203, the holes 201, 201′, 202 and 202′ are communicated with one another via the buffer solution. Thus, flow channels 204 and 204′ respectively communicated with the holes 201 and 201′ are supplied with the buffer solution at an equal level of liquid surface. Therefore, where the flow channels 204 and 204′ have an equal width (when having an equal height), or have a substantially equal cross-sectional area or length, the flow channels 204 and 204′ can provide substantially the same flow rate. Similarly, flow channels 205 and 205′ respectively communicated with the holes 202 and 202′ are supplied with the buffer solution at an equal level of liquid surface, and the flow rate of the buffer solution flowing in the flow channels 205 and 205′ can be adjusted to be a predetermined ratio to the flow rate of the buffer solution in the flow channel 204.

Around the hole 201 for introducing the cell-containing buffer solution, a wall 250 is provided for preventing the cell-containing buffer solution from diffusing. The wall 250 is lower than the wall of the reservoir 203, and the reservoir 203 is filled with the buffer solution up to a level higher than the wall 205.

The cell-containing buffer solution introduced to the hole 201 flows in the microscopic flow channel 204 (width: 20 μm, depth: 15 μm) and is introduced to a cell detection area 221 and a cell separation area 222. In the microscopic flow channel 204, a filter 230 directly built in the chip as a microscopic element is optionally provided in order to prevent the microscopic flow channel 204 from clogging. Meanwhile, the buffer solution not containing cells which is introduced to the holes 202 and 202′ flows in the flow channels 205 and 205′ (width: 12 μm; depth: 15 μm) and is merged with the cell-containing buffer solution in the microscopic flow channel 204. Reference numeral 240 represents a microscopic flow channel formed by merging the buffer solutions, which is introduced to the cell detection area 221. The microscopic flow channel 240 is further introduced to the cell separation area 222.

The buffer solution not containing cells which is introduced to the hole 201′ flows in the microscopic flow channel 204′ (width: 20 μm; depth: 15 μm) and is introduced to the cell separation area 222 to be merged with the microscopic channel 240. The width of the post-merging flow channel will be described later. The post-merging flow channel is separated at an exit of the cell separation area 222 into a microscopic flow channel 218 (width: 20 μm; depth: 15 μm) and a microscopic flow channel 219 (width: 20 μm; depth: 15 μm).

Reference numerals 206, 206′, 207 and 207′ represent holes for introducing an electrolyte-containing gel. The gel introduced to the holes 206 and 207 are respectively sent to the holes 206′ and 207′ via microscopic elements 208 and 209 (each is a bent groove of 200 μm (width)×15 μm (height)) which are formed in the bottom surface of the chip substrate 101. Therefore, the microscopic elements 208 and 209 are filled with the electrolyte-containing gel. Connection sections 241 and 242 are liquid junctions formed between the bent portions of the microscopic elements 208 and 209, and the microscopic flow channels 204 and 204′. The connection sections 241 and 242 each have a length of about 20 μm. Owing to this, in the cell separation area 222, the gel can be in direct contact with the buffer solution flowing in a flow channel 247 (FIG. 2) formed by merging the microscopic flow channels 240 and 204′. The gel and the buffer solution are in contact with each other in an area of about 15 μm (length along the flow channel)×15 μm (height). The holes 206 and 207 for introducing the gel each have an electrode represented with the black circle inserted therein. These electrodes are connected to a power supply 215 and a switch 216 via lines 106 and 107. The switch 216 is turned on only for applying a voltage to the buffer solution flowing in the flow channel 247 formed by merging the microscopic flow channels 240 and 204′.

The connection sections 241 and 242 which allow the gel to contact the buffer solution flowing in the flow channel 247 in the cell separation area 222 are structured such that the connection section 241 is located upstream with respect to the connection section 242 as shown in FIG. 1. Owing to this structure, when the electrode in the hole 206 is supplied with a positive voltage and the electrode in the hole 207 is supplied with a negative voltage, the cells flowing in the microscopic flow channel 240 can be efficiently moved to the flow channel 218. The reason is that when an electric current flows, an electrophoretic force acts on the cells charged negative, and this force and a vector received from the buffer solution are combined to form a synthetic vector. As result, in the structure of FIG. 1, as compared with the case where the connection sections 241 and 242 are located at the same position with respect to the flow (located symmetrically with respect to the flow line), the electric field is usable more efficiently and thus cells can be moved to the microscopic flow channel 218 or 219 more stably at a lower voltage.

Recovery holes 211 and 212 for recovering the cells separated in the cell separation area 222 are respectively formed downstream with respect to the microscopic flow channels 218 and 219. Culturing tanks 213 and 214 for accommodating the recovered cells are respectively provided around the holes 211 and 212. The culturing tanks 213 and 214 are surrounded by a reservoir 285. The reservoir 285 is located at an exit of the above-mentioned flow channels. The reservoir 285 is filled with the buffer solution to some level by the introduction thereof before the separation, but this level is lower than the level of the buffer solution in the reservoir 203 on the entrance side of the flow channels.

The level of the buffer solution in the reservoir 203 is higher than that in the reservoir 285. This level difference is used as a driving force for moving the buffer solution flowing in the flow channels and creates a stable flow with no pulsation. As long as a sufficient amount of buffer solution is accumulated in the reservoir 285, the cell-containing buffer solution introduced to the hole 201 can entirely flow to the flow channel 204. By putting a lid on the reservoir 203 to pressurize the space with air, the driving force for moving the buffer solution can be increased to raise the throughput.

The cells determined to fulfill a predetermined condition in the cell detection area 221 are separated from the other cells in the cell separation area 222 and collected in the culturing tank 213 after flowing down the flow channel 218. The cells determined not to fulfill the predetermined condition are separated in the cell separation area 222 and collected in the culturing tank 214 after flowing down the flow channel 219. The culturing tanks 213 and 214 are covered with a semipermeable membrane 280 at a top surface thereof in order to prevent the culturing tanks 213 and 214 from being contaminated with foreign substances during the cell separation. During the cell separation operation, the semipermeable membrane 280 is provided for protecting the culturing tanks 213 and 214 from the contamination. During the cell culturing operation performed in a culturing device after the flow channels 218 and 219 communicating with the culturing tanks 213 and 214 are closed and the culturing tanks 213 and 214 are cut off from the cell separation and culturing apparatus 100, the semipermeable membrane 280 acts as a membrane for supplying the cells with a medium as described later. When the cells not fulfilling the predetermined condition do not need to be cultured, the culturing tank 214 may be omitted.

FIG. 2 illustrates the manner in which the cell-containing buffer solution in the microscopic flow channel 204 is merged with the buffer solution in the microscopic flow channels 205 and 205′ and flows in the obtained microscopic flow channel 240 to reach the cell detection area 221, and is further merged with the buffer solution in the microscopic flow channel 204′ and flows in the obtained microscopic flow channel 247 to reach the cell separation area 222.

Now, the reason why the buffer solution not containing the cells which flows in the flow channels 205 and 205′ is merged with the cell-containing buffer solution flowing in the microscopic flow channel 204 at an upstream position with respect to the cell detection area 221 will be described. As described above, the flow channel 204 in which the cell-containing buffer solution flows is merged with the flow channels 205 and 205′ in which the buffer solution not containing the cells flows at an upstream position with respect to the cell detection area 221. The holes 201, 202 and 202′ provided at upstream ends of the flow channels are in the common reservoir 203 having a uniform liquid level. Because the flow channels 204, 205 and 205′ have an equal height, the flow rate of the buffer solution flowing in each of the flow channels 204, 205 and 205′ is in proportion to the width thereof. The width of the post-merging flow channel 240 is made substantially equal to that of the flow channel 204 for the cell-containing buffer solution. The term “substantially equal” means being equal in consideration of processing errors, and does not mean being strictly equal. Owing to this structure, the buffer solution flowing from the flow channel 204 is pushed to the center of the flow channel 240 at a constant ratio by the buffer solution flowing in the flow channels 205 and 205′. As a result, the cells, which flow in the flow channel 204 in contact with the side walls thereof, do not contact the side walls of the post-merging flow channel 240.

In the microscopic flow channel 247 in the cell separation area 222, the buffer solution from the flow channel 240 and the buffer solution from the flow channel 204′ flow while keeping the layers thereof, i.e., as if keeping the widths thereof, as represented with the dashed line, and flow down the flow channels 218 and 219. In the cell detection area 221, the cells fulfilling the predetermined condition are detected in the flow channel 240 and separated in the cell separation area 222 by an electric field acting by the function of the connection sections 241 and 242 in which the gel contacts the buffer solution flowing in the flow channels. Namely, when the electric field does not act, the cell-containing buffer solution flowing in the flow channel 240 flows down the flow channel 219. By contrast, when the electric field acts in the cell separation area 222, the cells in this location are pushed to the buffer solution flowing down the flow channel 218. In FIG. 2, the black circles represent the cells which do not fulfill the predetermined condition, and the stars represent the cells which fulfill the predetermined condition.

In FIG. 1 and FIG. 2, there are two routes, i.e., a route for cells selected as fulfilling the predetermined condition, and a route for non-selected cells. Alternatively, there may be three or more routes. In this case, flow channels are provided downstream with respect to the cell separation area 222, in addition to the flow channels 218 and 219 shown in FIG. 2. The number of the additional flow channels depends on the number of the routes. Cell information obtained in the cell separation area 222 is evaluated in accordance with the criterion prepared for the route determination, and the level of electric field to act on the cells by the connection sections 241 and 242 in the cell separation area 222 is controlled in accordance with the evaluation result. In consequence, the cells are controlled while flowing in the cell separation area 222 to reach the entrance of the route which is determined in accordance with the level of electric field and flow down the determined route. In this case, an independent culturing tank is provided at a downstream end of each of the additional route, needless to say.

Alternatively, a plurality of stages of cell separation areas may be provided in cascades in the route for the non-selected cells (flow channel 219) downstream with respect to the cell separation area 222. The route is separated into two at each stage, so that the cells in the flow channel 219 can be moved to the route for the selected cells (flow channel 218) at each stage. At which stage the cells are to be moved to the route for the selected cells is controlled in accordance with the cell information obtained in the cell detection area 221. In this case, a flow channel corresponding to the flow channel 204′ needs to be provided in each stage, and the structure is complicated.

FIG. 3 illustrates the cell distribution in the post-merging flow channel 240 after the buffer solution flowing down the flow channel 204 is pushed to the center of the flow channel 240 by the buffer solution flowing down the flow channels 205 and 205′ at an upstream position with respect to the cell detection area 221. In FIG. 3, reference numeral 255 represents side walls of the flow channel. FIG. 3 shows the manner in which the cell-containing buffer solution flowing down the flow channel 204 having a width of 20 μm is pushed to the center of the flow channel 240 having a width of 20 μm by the buffer solution flowing down the flow channels 205 and 205′ each having a width of 12 μm. The horizontal axis represents the position in the flow channel 204, and the vertical axis represents the appearing frequency of the cells. Curve 301 indicates that in the case where the amount of the buffer solution flowing down each of the flow channels 205 and 205′ is about half of the amount of the buffer solution flowing down the flow channel 204, i.e., in the case where the width of each of the flow channels 205 and 205′ is about half of the width of the flow channel 204, the cells are distributed in a central area, having a width of an about 10 μm, of the flow channel 240. Curve 302 shows the cell distribution in the case where the flow channels 205 and 205′ have a smaller width, and curve 303 shows the cell distribution in the case where the flow channels 205 and 205′ are not provided. As is clear from curve 301, by appropriately setting the width of the flow channels 205 and 205′, the cells can be substantially allowed to flow with a certain distance from the side walls of the flow channels and thus can be substantially prevented from contacting the side walls.

Although not described with reference to FIG. 3, the flow channels 240 and 204′ are merged together in the cell separation area 222. Therefore, the characteristic shown in FIG. 3 tends to be slightly expanded toward the flow channel 204′ side, but no significant change occurs because the buffer solution in the flow channel 240 and the buffer solution in the flow channel 204′ substantially maintain the respective layers as represented with the dashed line in FIG. 2. Namely, the width of a part of the flow channel 247 corresponding to the flow channel 240 tends to be slightly expanded in the cell separation area 222 but there is no substantial change.

In FIG. 1, the flow channels 204, 204′, 205 and 205′ each have the same width throughout the length thereof. In order to decrease the resistance in the flow channel, the width of the flow channel may be expanded in the vicinity of the reservoir 203. In order to obtain an appropriate cell distribution, it is sufficient that a flow channel has a predetermined width over a length of, for example, about 100 μm. Therefore, the area having a larger width may be extended in the length direction in consideration of the resistance in the flow channel. For example, the flow channel 205 which forms a side flow for the buffer solution with no cells may be made wider than the flow channel 204 for the cell-containing buffer solution, and a portion of the flow channel 205 having the predetermined width may be shortened. In this case, the resistance in the flow channel 205 is smaller than that of the flow channel 204. As a result, the buffer solution from the flow channel 204 is pushed more to the center of the post-merging flow channel 240. Namely, cell distribution curve in FIG. 3 is made more acute as shown by curve 301.

FIG. 4(A) is a cross-sectional view showing a problem caused by increasing the width of a flow channel, and FIG. 4(B) is a cross-sectional view showing an example of means for solving the problem. In FIG. 4(A) and FIG. 4(B), reference numeral 101 represents a substrate, reference numeral 260 represents a groove formed in the substrate 101, and reference numeral 410 represents a laminate film covering the groove 260. The flow channel 205 is formed of the groove 260 and the laminate film 410 covering the groove 260. As shown in FIG. 4(A), in the case where the width of the flow channel 205 is increased, when the laminate film 410 is attached to the chip substrate 101 by thermal pressurization, the laminate film 410 drops into the groove 260 and thus closes the flow channel 205. By contrast, in FIG. 4(B), a beam 400 is provided in a wide portion of the groove 260 and prevents the laminate film 410 from dropping into the groove 260. Thus, the flow channel 205 is not closed.

FIG. 5(A) is a cross-sectional view taken along line (A)-(A) in FIG. 1 and seen in the direction of the arrows thereof. FIG. 5(A) shows the reservoir 203 on the flow channel entrance side, the openings 201 and 201′, and the flow channels 204 and 204′ in more detail. FIG. 5(B) is a cross-sectional view taken along line (B)-(B) in FIG. 1 and seen in the direction of the arrows thereof. FIG. 5(B) shows the culturing tanks 213 and 214, the openings 211 and 212, the flow channels 218 and 219, the semipermeable membrane 280, and the reservoir 285 on the flow channel exit side in more detail.

In the bottom surface of the chip substrate 101, grooves corresponding to the microscopic flow channels 204 and 204′ are formed and are covered with the laminate film 410. Thus, the flow channels 204 and 204′ are formed. The hole 201 for introducing the cell-containing sample buffer solution to the microscopic flow channel 204 is provided at an upstream end of the flow channel 204, and the hole 201′ for introducing the buffer solution with no cells to the microscopic flow channel 204′ is provided at an upstream end of the flow channel 204′. The wall or the reservoir 250 is provided to surround the hole 201 in order to prevent the cell-containing sample buffer solution injected to the hole 201 from being diffused.

In addition to the holes 201 and 201′ and the wall or the reservoir 250 for preventing the cell-containing sample buffer solution injected to the hole 201 from being diffused, the holes 202 and 202′ not shown in FIG. 5(A) are also provided in the reservoir 203. The reservoir 203 is filled with a buffer solution 200 and the buffer solution 200 is supplied to all the holes in the reservoir 203. Therefore, the buffer solution flowing in the microscopic flow channels 204 and 204′ can have a substantially equal flow rate. The buffer solution flowing in the microscopic flow channels 205 and 205′ can also have a substantially equal flow rate. The flow rate of the buffer solution in the microscopic flow channel 204 and that of the buffer solution in the microscopic flow channel 205 can be maintained at a predetermined ratio stably. The hole 201 is cone-shaped to ensure that the sample cells flow to the flow channel 204. The wall 250 may be a low, small reservoir located within the reservoir 203 as shown in FIG. 1, or may be like a simple partitioning plate. A membrane filter 231 covering the hole 201 on the top surface of the chip substrate 101 is provided for removing dust from the sample. The wall 250 prevents the cells from being diffused, and therefore it is not necessary to directly inject the cell-containing sample buffer solution to the hole 201.

The structure of providing a common reservoir at an upstream end of the flow channels is one of the core elements of the cell separation and culturing apparatus according to the present invention. Because the flow channels have a common liquid surface level owing to the common reservoir, the buffer solution can be sent to the plurality of flow channels at the same pressure. This is the simplest liquid delivery system which can be incorporated to the substrate. In order to distinguish the liquids in the flow channels from one another, a partitioning plate lower than the liquid surface level is provided. Owing to this structure, different types of buffer solutions can be flown to the different flow channels at the same pressure. For the buffer solution to be separated by the partitioning plate, a buffer solution having a larger specific gravity than that of the buffer solution forming the common liquid surface level is preferably used. Then, the different types of buffer solutions are not mixed together. The cells basically cause no problem because the cells have a greater specific gravity as they are and precipitate in the container. For chemotactic cells, the partitioning plate is formed to have a height that the cells cannot go beyond. For example, nerve cells cannot go beyond a wall (partitioning plate) having a height of several tens of micrometers. In the case of cells such as E. coli, a sponge-like membrane through which the buffer solution can pass freely but not the cells may be provided over the wall 250. Thus, the cells are prevented from entering different flow channels.

As is clear from FIG. 5(B), the culturing tanks 213 and 214 are covered with the semipermeable membrane 280 at a top surface thereof for protecting the culturing tanks 213 and 214 against contamination during the separation operation. The reservoir 285 is provided to surround the culturing tanks 213 and 214. The reservoir 285 is sufficiently higher than the culturing tanks 213 and 214. The buffer solution put into the reservoir 203 in a sufficient amount on an initial stage of the separation is moved toward the reservoir 285 as the separation proceeds. FIG. 5(B) schematically shows a state where the separation has proceeded to some extent and the culturing tanks 213 and 214 are precipitated in the buffer solution (medium) 200. Reference numeral 287 represents a layer of collagen, polylysine or fibronectin applied on a bottom surface of the culturing tanks 213 and 214. Instead of applying such a substance, the bottom surface of the culturing tanks 213 and 214 may be treated to be hydrophobic. To apply the above-mentioned substances to the bottom surface or to treat the bottom surface to be hydrophobic is convenient to culture cells which form colonies on an agar medium. Hence, in the case where the cells to be separately cultured are bacteria or others which do not form colonies on the agar medium, such application or treatment is not necessary. In FIG. 5(B), the black circles and the stars on the collagen layer 287 applied on the culturing tanks 213 and 214 represent cells separated as described above with reference to FIG. 2.

Next, the gel electrodes will be practically described. A gel electrode section includes holes 206, 206′, 207 and 207′, the microscopic element 208 connecting the holes 206 and 206′, the microscopic element 209 connecting the holes 207 and 207′, and the connection sections 241 and 242 acting as liquid junctions in the cell separation area 222 shown in FIG. 1. On the stage where the chip substrate 101 is produced by injection molding and the cell separation and culturing apparatus 100 is produced by covering the grooves with the laminate film 410, the gel electrode section does not accommodate any gel. In the following example, agarose gel containing an electrolyte is used as an electrolyte solution.

On the negative side of the gel electrode section, i.e., on the side of the microscopic element 209 and the connection section 242, a gel having a composition of 1% agarose, 0.25 M NaCl, and 0.296 M sodium phosphate (pH 6.0) buffer solution is provided. On the positive side, i.e., the side of the microscopic element 208 and the connection section 241, a gel having a composition of 1% agarose, 0.25 M NaCl, and 0.282 M sodium phosphate (pH 8.0) buffer solution is provided. The pH values are made different in order to avoid the phenomenon that bubbles are generated by electrolysis when an electric current flows. Hydrogen ions generated on the positive side are neutralized by the buffer solution having a high pH value before becoming hydrogen molecules. Hydroxy ions generated on the negative side are neutralized by the buffer solution having a low pH value and thus inhibit the generation of oxygen molecules.

The gel electrode is preferably formed of a gel substance containing sugar. In this case, the sugar preferably contains 3% to 50% of nonreducible disaccharide, 1% to 50% of trehalose, 5% to 30% of glycerol, 5% to 40% of ethyleneglycol, or 5% to 30% of dimethylsulfoxide.

Now, it is assumed that gel is injected from the holes 206 and 207 formed in the chip substrate 101 to complete the production of the gel electrode-equipped cell separation and culturing apparatus 100, and then the apparatus 100 is left without being used. The gel is in contact with the air in the openings of the holes 206, 206′, 207 and 207′ and in the flow channels and the connection sections 241 and 242 as liquid junctions in the cell separation area 222. Therefore, the gel starts drying from these areas. In order to store the gel electrode-equipped cell separation and culturing apparatus produced above, the following needs to be done. In order to prevent the gel from drying in the openings of the holes 206, 206′, 207 and 207′, the holes 206, 206′, 207 and 207′ are sealed until immediately before the apparatus 100 is used. In order to prevent the gel from drying in the flow channels and the connection sections 241 and 242 in the cell separation area 222, the apparatus 100 is stored in a sealed container together with a water-containing sheet, such that the gel is not dried. Thus, the apparatus 100 can be easily stored at 4° C. for about 3 months. As the sealed container, a laminate pack is suitable in order to minimize the air space.

In order to prevent the gel from drying and store the apparatus 100 for an extended period of time, gel is supplied with a humectant. As the humectant, for example, about 1% to 10% of disaccharide such as trehalose or sucrose, or oligosaccharide, or about 5% to 10% of glycerin is effective to prevent drying.

For long-time storage, it is preferable to freeze the gel electrode-equipped cell separation and culturing apparatus 100 in a laminate pack. In this case, a problem occurs that ice crystals are generated at the time of freezing and melting and destroy the gel structure. When ice crystals are generated in a gel electrode formed in a tiny area such as in the cell separation and culturing apparatus, the portion in which the ice crystals are generated becomes hollow after the gel electrode is melted. Then, when an electric field is applied to the electrode, the cells enter the hollow portion or such cells unnecessarily flow out to the flow channels of the cell separation and culturing apparatus 100.

In order to prevent this, the gel in the gel electrodes is supplied with a substance for suppressing the crystal growth of ice so as to store the cell separation and culturing apparatus 100 in a frozen state for an extended period of time. This is one of the most important points of the present invention. As the substance for suppressing the crystal growth, substantially the similar substances to those for the humectant are usable. It is most effective to mix a disaccharide such as trehalose or sucrose, or oligosaccharide during the production of gel. Trehalose has a very small function to general animal cells and thus is very effective. The concentration of trehalose may be as low as 1%, and about 50% at the highest. Sucrose is also effective, but is biofunctional to animal cells and may be inappropriate depending on the purpose. By replacing a part of the hydroxyl group of a sugar chain with a sulfuric acid group, the freezing prevention capability can be maintained to reduce the biochemical influence. It is preferable to introduce a sulfuric acid group to the hydroxyl group of these disaccharides. Other sugars such as glycerin and ethyleneglycol are also effective. Dimethylsulfoxide is also effective. It should be considered that ethyleneglycol and dimethylsulfoxide may have a problem of cell toxicity in some cases, but dimethylsulfoxide or the like elutes to a cell sorting flow channel generally in a tiny amount and thus is ignorable.

Practical examples will be described. A cathode electrolyte solution and an anode electrolyte solution having the following compositions are heated and melted in a microwave oven and made into buffer solutions. Separately, the chip substrate 101 is heated on a hot plate heated to 60° C. The cathode electrolyte solution and the anode electrolyte solution in a buffer solution state are respectively injected to the holes 206 and 207 using a syringe and suctioned from the holes 206′ and 207′ to fill the microscopic elements 208 and 209 and the connection sections 241 and 242. Melted gel enters the connection sections 241 and 242 by the capillary phenomenon. After being left at room temperature for 10 minutes, the buffer solutions in the microscopic elements 208 and 209 and the connection sections 241 and 242 are gelated. The flow channel 247 has a larger cross-section than that of the connection 241 and 242, and thus the gelated buffer solution does not go into the flow channel 247.

The improved gel composition will be shown below.

Negative electrolyte solution: 1% trehalose, 0.25 M NaCl, 0.296 M sodium phosphate (pH 6.0), 1% agarose

Positive electrolyte solution: 1% trehalose, 0.25 M NaCl, 0.282 M sodium phosphate (pH 8.0), 1% agarose

The surface of the gel electrode-equipped cell separation and culturing apparatus 100 prepared as above is sealed with an adhesive tape. “Plas-Chamois”, which is a porous plastic towel, is immersed with water, and a 2 cm×2 cm squeezed piece of “Plas-Chamois” is put in a plastic bag having a size of 30 mm×40 mm together with the gel electrode-equipped cell separation and culturing apparatus 100. The plastic bag is sealed with a sealer.

The plastic bag is stored at 4° C. or −20° C. in this state.

The state of the electrode section of the chip is observed with a microscope immediately after the chip is produced but before frozen, and after the chip is stored at 4° C. and −20° C. for 1 month, 3 months, and 6 months. In addition, the reservoir 203 is supplied with a culturing solution to fill the flow channels 204, 205 and 205′, and the hole 201 is supplied with erythrocytes. An application of an electric field to the gel electrodes is turned on and off, so that it is confirmed that the cells are separated to the flow channels 218 and 219. In the chip immediately after being produced, the microscopic elements 208 and 209 and the connection sections 241 and 242 are filled with the gel, with no external damages such as cracks or drying. When an electric field is applied to the gel electrodes, the erythrocytes flowing in the flow channel in the cell separation area 222 are moved to the flow channel 218 and accumulated in the culturing tank 213 via the hole 211. Without the electric field, the erythrocytes are moved to the flow channel 219 and accumulated in the culturing tank 214 via the hole 212. Even in the case where the chip is frozen for six months before use, the cells can be collected in the culturing tank 213 by applying an electric field to the gel electrodes, and in culturing tank 214 by turning off the electric field, similarly to immediately after the production of the chip. In the case where the chip is stored at 4° C., the gel is retracted to the connection sections 241 and 242 in three months according to an observation with a microscope. However, when the cells are caused to flow, the cells can be separated similarly to immediately after the chip is produced.

FIG. 6 is a plan view of the cell separation and culturing apparatus 100 in which the gel electrode section has a different structure from that shown in FIG. 1. In this example, the lines 106 and 107 and the electrodes inserted to the holes for introducing the gel in FIG. 1 are formed of a conductive film which is vapor-deposited on the laminate film 410 applied to the bottom surface of the chip substrate 101. Since the laminate film 410 is applied to the bottom surface of the chip substrate 101, the electrodes and the like formed of the conductive film are not actually seen in the plan view of the chip substrate 101. In FIG. 6, however, the electrodes and the like are also shown in order to clarify the relationship thereof with the other elements.

With the structure shown in FIG. 6, pentagonal microscopic elements 208 and 209 are used in place of the microscopic elements 208 and 209 shown in FIG. 1. The pentagonal microscopic elements 208 and 209 are respectively in communication with the openings 206 and 207 formed in the chip substrate 101. The gel is injected through the openings 206 and 207. The openings 206′ and 207′ are respectively in communication with the microscopic elements 208 and 209 for discharging air. The gel injection may be terminated when the openings 206′ and 207′ are overflown with the gel. The connection sections 241 and 242 are projected from the pentagonal microscopic elements 208 and 209, and thus the gel can be in contact with the buffer solution flowing in the flow channel 247. In order to obtain a sufficient electric connection between the gel injected to the pentagonal microscopic elements 208 and 209 and the conductive films 106 and 107 replacing the lines 106 and 107, the conductive films 106 and 107 are vapor-deposited at positions where the gel injected to the pentagonal microscopic elements 208 and 209 are in contact with ends of the conductive films 106 and 107 in an appropriate area. The other ends of the conductive films 106 and 107 act as terminals to be connected to the power supply 215.

Since the laminate film 410 is applied to the chip substrate 101, the terminals at the other ends of the conductive films 106 and 107 are hidden by the chip substrate 101. Although not shown in FIG. 6, an element connected to the terminals for making the terminals connectable with the power supply 215 on the top surface of the chip is provided.

Also with the structure shown in FIG. 6, cells determined to fulfill a predetermined condition in the cell detection area 221 are separated from the other cells in the cell separation area 222, flow down the flow channel 218 and are collected in the culturing tank 213. Cells determined not to fulfill the predetermined condition are collected in the culturing tank 214. The culturing tanks 213 and 214 are covered with the semipermeable membrane 280 at a top surface thereof for protecting the culturing tanks 213 and 214 from contaminants during the cell separation. During the cell separation operation, the semipermeable membrane 280 is provided for protecting the culturing tanks 213 and 214 from contaminants. During the cell culturing operation performed in a culturing device after the flow channels 218 and 219 communicating with the culturing tanks 213 and 214 are closed and the culturing tanks 213 and 214 are cut off from the separation and culturing apparatus 100, the semipermeable membrane 280 acts as a membrane for supplying the cells with a medium as described later. The reservoir 285 is provided to surround the culturing tanks 213 and 214. When the cells not fulfilling the predetermined condition do not need to be cultured, the culturing tank 214 may be omitted. Instead of providing two routes, i.e., a route for cells selected as fulfilling the predetermined condition and a route for non-selected cells as described above with reference to FIG. 1 and FIG. 2, there may be three or more routes.

Using the cell separation and culturing apparatus 100 in FIG. 1, an algorithm for cell recognition and separation which is based on image recognition will be described. As described above with reference to FIG. 1, a cell suspension is injected to the hole 201. The wall 250 is provided to surround the hole 201 in order to prevent the cell suspension from being diffused. The wall 250 is provided within the reservoir 203, and the liquid surface level in the hole 201 is equal to the liquid surface level in the reservoir 203. The cells flow from the hole 201 to the flow channel 204 and are merged with the buffer solution in the flow channel 205, which forms a side flow, before the cell detection area 221. Thus, the cells are pushed to the center of the flow channel (FIG. 3).

The cells passing through the cell detection area 221 from the flow channel 204 are imaged by a CCD camera. A CCD camera capable of capturing images at, for example, 200 frames per second is used. With such an imaging capability, each of the cells can be recognized even when the flow rate of the buffer solution passing through the cell detection area 221 is about 1 mm/sec.

FIG. 7 shows an algorithm for recognizing cells from an image captured by the CCD camera and numbering and identifying each of the cells. In FIG. 7, the images captured one after another are represented as “frame 1”, “frame 2”, . . . , “frame N”. In each frame, cells are displayed. In frame 1, only one cell represented with a black circle is displayed. This cell represented with the black circle is recognized as an image and numbered as No. 421. In frame 2, a cell represented with a white circle and a cell represented with a star are displayed in addition to cell 421. Frame 2 is the first frame in which the cell represented with the white circle and the cell represented with the star appear, and these cells appear simultaneously. These cells are recognized as images and numbered. The cell which is seen downstream with respect to the other, i.e., which is detected first, is assigned with a smaller number. Here, the cell represented with the white circle appears downstream with respect to the cell represented with the star. Thus, the cell represented with the white circle is numbered as No. 422, and the cell represented with the star is numbered as No. 423. In frame 3, no new cell is recognized. It is understood by comparing frame 2 and frame 3 that cell 422 is moved faster than cell 421 and cell 423. In frame 4, no new cell is recognized. Cell 422 is moved fast and thus is barely seen in frame 4. Cell 421 and cell 423 are seen as moving almost at the same speed. The cells can be recognized in up to frame 8. The image recognition is performed using, as indices, the luminance center of gravity, area size, circumferential length, longer diameter, and shorter diameter.

Based on the moving velocity of each cell recognized as an image and numbered, the time necessary for the respective cell to reach the cell separation area 222 (more strictly, the connection section 241 or 242) is found. The cells are divided into the cells sent to the recovery hole 211 and the cells sent to the recovery cell 212 by applying a negative electric field or no electric field to the gel electrode in the connection 241 and applying a positive electric field or no electric field to the gel electrode in the connection 242. In other words, the moving velocity (V) of each of the cells numbered based on the images captured at an interval of a predetermined time period is calculated, and the cells are separated by applying a voltage at a timing of (L/V) to (L/V+T). The length (L) and the application time (T) are input in advance in relation to the cell moving velocity (V).

FIG. 8 is a plan view schematically showing one example of a system structure of a cell separation and culturing apparatus according to the present invention. As compared to the apparatus in FIG. 1, the apparatus in FIG, 8 has a special arrangement for the introduction of sample cells. FIG. 9(A), FIG. 9(B) and FIG. 9(C) are partial cross-sectional views illustrating the arrangement of the sample cell introduction section. The elements bearing the identical reference numerals as those in FIG. 1 are identical elements thereto or act in an identical manner thereto.

As is clear from a comparison between FIG. 8 and FIG. 1, in the cell separation and culturing apparatus shown in FIG. 8, the flow channel 204 is extended to a further upstream position and an opening 290 is provided for introducing a buffer solution. Except for this, the apparatus in FIG. 8 is the same as the apparatus in FIG. 1. As understood from FIG. 9(A), the flow channel 204 is in communication with the reservoir 250 via the opening 201, and is further extended to be in communication with the opening 290. In FIG. 1 and FIG. 2, there are two routes, i.e., a route for cells selected as fulfilling the predetermined condition and a route for non-selected cells. Alternatively, there may be three or more routes.

FIG. 9(B) schematically shows the buffer solution and the cells flowing from the openings 201 and 290 to the flow channel 204. As understood from this, in this embodiment, the layer of the buffer solution flowing from the opening 290 to the flow channel 204 prevents the buffer solution and the cells flowing from the opening 201 to the flow channel 204 from contacting the laminate film 410. Namely, the buffer solution and the cells flowing from the opening 201 to the flow channel 204 flow on the layer of the buffer solution flowing from the opening 290 to the flow channel 204. Owing to this arrangement, the cells are prevented from contacting the laminate film 410, and the resultant jamming of cells is avoided.

FIG. 9(C) schematically shows that the cells flowing from the opening 201 to the flow channel 204 contacts the laminate film 410 and thus are jammed. Once one of the cells contacts the laminate film 410 and stays there, the other cells are caught by the first cell and stay there one after another. Finally, the flow of the cells is stopped.

The structure in FIG. 8 is described as being realized by extending the flow channel 204 in FIG. 1 to an further upstream position. Clearly, the flow channel 204 in FIG. 6 may be similarly extended. The point is that the buffer solution is introduced upstream with respect to the position of cell introduction, so that a flow layer of the buffer solution is formed before the cells are introduced. Thus, the cells are prevented from contacting the bottom surface of the flow channel.

(Example of Cell Modification)

In the following, cells are modified with a fluorescent dye, gold microparticles or non-gold nanoparticles, and aptamer is used to detect the cells with fluorescence or scattered light. In order to identify or separate cells, some distinguishing index is needed. In the following example, a substance decomposable under a mild condition is used for labeling a surface antigen, and the labeling substance for the surface antigen is decomposed and thus removed under a physiological condition with no influence on the cells. Practically, polynucleotide capable of forming various steric structures is used as the labeling substance. Here, polynucleotide is used as an element generally conceived as an aptamer. For example, various types of synthetic polynucleotides as follows are prepared: the total length is 80 bases; 20 bases on the 3′ terminus side and 20 bases on the 5′ terminus side are of a regulated known basic sequence; and 40 bases at the center are of a random sequence. These synthetic polynucleotides are passed through a column, on an inner surface of which a surface antigen of the cell to be separated is immobilized. As a result, a polynucleotide having a sequence with affinity to the surface antigen of the cell to be separated is captured on the inner surface of the column. This column is alkali-treated to separate and thus recover the captured polynucleotide. The recovered polynucleotide is PCR-amplified. Thus, a polynucleotide specifically bound to the cell surface antigen is obtained. Namely, an aptamer as a surface antigen labeling substance which is decomposable under a mild condition is obtained.

In order to obtain an aptamer (polynucleotide) having a higher specificity and binding strength, an evolutionary engineering means of intentionally lowering the fidelity at the time of the PCR amplification to change the sequence and repeating the affinity purification may be additionally used. In some cases, the binding strength may be increased by modifying and thus charging a base portion bound to the surface antigen. Alternatively, the binding strength may be increased by using nucleotide in which the sugar chain portion of the bases is modified.

The backbone structure of the obtained structure-recognizable polynucleotide may be of a ribonucleotide type or a deoxyribonucleotide type. In general, the ribonucleotide type is more advantageous as being usable for various structures, but may occasionally be difficult to use because RNase in the periphery thereof causes unpredictable decomposition. The deoxyribonucleotide type is more easily usable because there are not many DNase outside the cells and deactivation is easily done.

The structure-recognizable polynucleotide (aptamer) obtained in this manner as the labeling substance is modified with a fluorescent substance, or gold or magnetic nanoparticles as an identifying substance, thus to produce an identifying element. The identifying element is mixed with the sample cells to identify the cells having a site bound to the labeling substance and to separate such cells by the cell separation and culturing apparatus based on the identification information.

After the separation, the cells are treated with nuclease to decompose and thus remove the labeling polynucleotide bound to the surface antigen. In the case where the labeling polynucleotide is of the ribonucleotide type, RNase is used for decomposition. In the case where the labeling polynucleotide is of the deoxyribonucleotide type, DNase is used for decomposition. When a modified nucleotide is used in order to increase the stability, it is important that the modified nucleotide should not entirely inhibit the decomposition by the nuclease. The nucleotide structure which has a possibility of inhibiting the effect of the nuclease should be introduced to only a part of the aptamer molecule, if introduced. With such an arrangement, the aptamer molecule is decomposed to be of a sufficiently low molecular weight when considered as a whole, although nuclease may not work for a part of the aptamer.

By this method, the structure-recognizable polynucleotide (aptamer) as the labeling substance for the cell surface antigen can be easily removed with nuclease. Since RNase and DNase cannot pass through the cell membrane, the RNAs or DNAs in the cell are not damaged. Since the RNAs or DNAs are not considered to be exposed to the cell surface, it is considered that the cell itself is not influenced by the nuclease due to the structure-recognizable polynucleotide (aptamer) bound to the cell surface antigen. Therefore, the cells are prevented from being denatured due to the treatment performed to separate the cells.

Preparation of an aptamer for the cell surface antigen CD4 will be described. This aptamer is one of aptamers useful as a labeling substance.

As the aptamer as a labeling substance, the aptamer for the cell surface antigen CD4 described in “Staining of cell surface human CD4 with 3′-F-pyrimidine-containing RNA aptamers for flow cytometry”, Nucleic Acids Research 26, 3915-3924 (1998) is used. This aptamer is of a ribonucleotide, i.e., is an RNA aptamer. In the above-mentioned article, the aptamer is made identifiable with fluorescence by introducing GDP-β-S as an identifying substance to the 5′ terminus of the RNA aptamer by in vitro transcription. Namely, at this point, a thiophosphoric acid group is inserted to the 5′ terminus of the RNA aptamer. The thiophosphoric acid group is reacted with biotin, to which an iodoacetyl group is introduced, and thus a 5′ biotinated RNA aptamer is obtained.

A conjugate of phycobiliprotein and streptoadipine as a fluorescent colorant is reacted with the above-obtained aptamer, and a phycobiliprotein-modified RNA aptamer is obtained through a biotin-adipine reaction. Among phycobiliproteins, β-phycoerythrin is a fluorescent protein type fluorescent substance having a high absorbance of 2.41×10⁶M⁻¹cm⁻¹and a high quantum efficiency of 0.98 and thus is suitable for high sensitivity detection, but has problems of a molecular weight which is as high as 240 K Dalton, and the non-specific adsorption and instability because of being protein. Here again, a phycobiliprotein-modified RNA aptamer is usable as a practical example, but this is equivalent to using particles of about 10 nm as an identifying substance in terms of size because the phycobiliprotein-modified RNA aptamer has a molecular weight of as great as 240 K Dalton. Therefore, in addition to phycobiliprotein, fluorescent colorant-containing particles having a diameter of 10 nm, gold nanoparticles having a diameter of 10 nm, and magnetic particles having a diameter of 10 nm are also used as an identifying substance.

In this example, an identifying element using phycobiliprotein or nanoparticles as an identifying substance will be described.

(i) Phycobiliprotein-modified RNA aptamer: The method described in the above-mentioned article may be used, but another method is used in this example. A synthetic RNA aptamer can be obtained with certainty by chemical synthesis. An amino group is introduced to the 5′ terminus of the synthetic RNA aptamer at the time of chemical synthesis thereof. The amino group introduced to the 5′ terminus is reacted with a bivalent reagent such as N-(8-maleimidocapryloxy)sulfosuccinimide, and a maleimido group reactable with an SH group is introduced to the 5′ terminus of the RNA aptamer. Separately, β-phycoerythrin having an SH group introduced thereto is prepared. For introducing the SH group, the amino group of the β-phycoerythrin is modified with 2-iminothiorane. The RNA aptamer having the maleimido group introduced thereto, and the β-phycoerythrin having the SH introduced thereto by modification with 2-iminothiorane, are mixed together at pH 7, and thus a β-phycoerythrin-modified RNA aptamer is obtained.

(ii) Gold nanoparticle-modified RNA aptamer: A method for preparing gold nanoparticle-modified RNA aptamer referring to the method described in Tonya M. Herne and Michael J. Tarlov, J. Am. Chem. Soc. 1997, 119, 8916-8920 and the method described in James J. Storhoff, J. Am. Chem. Soc. 1998, 120, 1959-1964 will be described. To a gold nanoparticle (20 nmφ) suspension, a synthetic RNA aptamer having an SH group at the 5′ terminus and 6-mercapto-1-hexanol are added, and left for 1 hour. The molar ratio of the synthetic RNA aptamer and 6-mercapto-1-hexanol is 1:100. In the case where the gold nanoparticles coagulate or in the case where the synthetic RNA aptamer and the gold nanoparticles are not bound together, the molar ratio may be optionally varied to find an optimum condition. Since the gold nanoparticles easily coagulate, the synthetic RNA aptamer is added while stirring the suspension, such that the concentration gradient of the potassium carbonate buffer solution or the concentration gradient of the synthetic RNA aptamer is not generated. The reaction is caused at the molar ratio of the gold nanoparticles and the synthetic RNA aptamer of 1:100. Namely, the reaction occurs where the number of the gold nanoparticles and the number of the synthetic RNA aptamer molecules are at the ratio of 1:1000. The synthetic RNA aptamer having an SH group is obtained by chemical synthesis. After the reaction, the resultant substance is centrifuged at 8000 G for 1 hour to remove the supernatant. The resultant substance is suspended again in a 10 mM potassium carbonate buffer solution (pH 9) containing 0.1 M NaCl, centrifuged again to remove the supernatant, and suspended in a 10 mM potassium phosphate buffer solution (pH 7.4) containing 0.1 M NaCl. The resultant substance is used as a stock.

(iii) Non-gold nanoparticle-modified RNA aptamer: For example, nanoparticles such as quantum dots are generally formed of inorganic nanoparticles. A product covered with polyethyleneglycol having biotin introduced thereto is already commercially available under the trade name of, for example, EviFluor from Evident Technologies. Nanoparticles with biotin may be used with a streptoadipine-bound RNA aptamer. A method for preparing an RNA aptamer bound to streptoadipine will be described. The RNA aptamer having a maleimido group introduced to the 5′ terminus, and streptoadipine having an SH introduced thereto by modification with 2-iminothiorane, are mixed together at pH 7 by the method described in (i) above, and thus a streptoadipine-bound RNA aptamer is obtained. The streptoadipine-bound RNA aptamer and the nanoparticles with biotin are mixed together, and thus a nanoparticle-labeled RNA aptamer is obtained as an identifying element.

When nanoparticles having a carboxylic group introduced thereto is used, a nanoparticle-labeled RNA aptamer as an identifying element can be obtained by a well known method of active-esterifying the carboxylic group with carbodiimide and reacting the active ester with 5′ aminized RNA aptamer.

So far, methods for preparing nanoparticle-modified RNA aptamers have been described. Also in the case of a DNA aptamer formed of deoxyribonucleotide, an SH group or an amino group can be introduced to the 5′ terminus when synthesizing a DNA aptamer with a synthesizing apparatus, like in the case of the above-described RNA aptamers. Therefore, with deoxyribonucleotide, a phycobiliprotein-modified DNA aptamer, a gold nanoparticle-modified DNA aptamer and a non-gold nanoparticle-modified DNA aptamer can be prepared in a similar manner.

An RNA aptamer may be produced by an established method other than the above-described synthesis methods. According to the established method, a single chain DNA having T7 promoter at the 5′ terminus is synthesized, and then is transcribed to an RNA using RNA polymerase.

Now, an identifying element using an RNA aptamer as a labeling substance for labeling the cell surface antigen CD4 and using β-phycoerythrin as an identifying substance for separating and recovering cells having the RNA aptamer bound thereto will be described. The cell surface antigen CD4-presenting cells are specifically labeled with the above-mentioned β-phycoerythrin-modified RNA aptamer, and separated using a cell separation and culturing apparatus including the plastic chip substrate 101 as shown in FIG. 1, FIG. 6 or FIG. 8.

FIG. 10 illustrates a flow of processing for specifically labeling the cell surface antigen CD4-presenting cells with a β-phycoerythrin-modified RNA aptamer and separating the cells by a cell separation and culturing apparatus. A top part of FIG. 10 shows two types of cells 3 and 4 mixed in a sample 10. The cells 3 each have a cell surface antigen CD4 represented with black triangles and reference numeral 1. The cells 4 each have a non-CD4 cell surface antigen 2 represented with black circles. This sample 10 is mixed with a β-phycoerythrin-modified RNA aptamer 11 as described above. The RNA aptamer is represented with reference numeral 5, and β-phycoerythrin is represented with reference numeral 6. The concentration of the labeling substance 11 is 100 nM. A reverse-Y-shaped double headed arrow is shown below the top part of FIG. 1, and a leftward arrow is directed to the common part of the reverse-Y-shaped double headed arrow. The leftward arrow indicates that the β-phycoerythrin-modified RNA aptamer 11 is mixed with the sample 10.

As a result, to the CD4 antigen 1 existing on the surface of the cells 3, the β-phycoerythrin-modified RNA aptamer as the labeling substance is bound. The labeling substance RNA aptamer is not bound to the surface antigen 2 other than CD4. The β-phycoerythrin as an identifying substance for modifying the labeling substance RNA aptamer, when excited by second harmonic of 532 nm from a YAG laser, emits strong fluorescence having a wavelength close to 575 nm. Utilizing this, the cell separation and culturing chip can separate the CD4-presenting cells from the other cells by detecting the fluorescence. Below the reverse-Y-shaped double headed arrow, reference numeral 12 represents a group of cells 3 bound to the labeling substance RNA aptamer, and reference numeral 13 represents a group of cells 4 not bound to the labeling substance RNA aptamer.

Next, the CD4-presenting cells collected in the culturing tank 213 of the cell separation and culturing apparatus 100 are cut off from the cell separation and culturing apparatus 100 while being contained in the culturing tank 213 and put into an arbitrary culturing device. Immediately after this, nuclease 14 is put into the culturing device and introduced to the culturing tank 213 via the semipermeable membrane 280, so that the nuclease 14 acts on the CD4-presenting cells. The RNA aptamer has a steric structure and therefore in some cases is not sufficiently decomposed only with such a type of nuclease as ribonuclease A for decomposing a single chain RNA. Therefore, it is effective to use an enzyme for decomposing both a single chain RNA and a double chain RNA. In this example, an enzyme having the trade name Benzonase (registered trademark; European Patent No. 0229866, U.S. Pat. No. 5,173,418) obtained by mass-producing the nuclease derived from Serratia marcescens described in The Journal of Biological Chemistry 244, 5219-5225 (1969) in a genetic engineering manner is used. This enzyme acts at 37° C. and is usable in a neutral area of pH 6 to 9, and thus is easily usable for cells. The enzymatic activity is lost by highly concentrated phosphoric acid or monovalent metal ions. Therefore, in this example, a non-phosphoric acid-system buffer solution, for example, 10 mM HEPES (pH 7.4) containing 0.15 M NaCl, 2 mM MgCl₂and 1 mg/ml BSA is used. When it is unavoidable to use a phosphoric acid-system buffer solution, such a buffer solution is used under the conditions that the concentration of potassium phosphate/sodium is limited to 5 mM and that 0.15 M NaCl, 2 mM MgCl₂and 1 mg/1 ml BSA are contained. Benzonase (registered trademark) is used in an amount of 10 to 100 u/ml. Alternatively, a mixture of ribonuclease A and ribonuclease T1 is usable, but nuclease derived from Serratia marcescens is more generally usable.

Optionally, serum is usable instead of a buffer solution. In this case, nuclease inhibitor in the serum may have an influence. Therefore, it may be necessary to adjust the amount of Benzonase (registered trademark) nuclease for each lot of serum. Generally when serum is used, a good result is obtained with an amount of Benzonase of 100 to 400 u/ml.

In FIG. 10, nuclease is shown with a downward arrow below the group 12 of cells 3 bound to the labeling substance RNA aptamer. This arrow indicates the treatment of adding nuclease, and this treatment is represented with reference numeral 14. Owing to the action of nuclease, the labeling substance RNA aptamer 11 bound to the CD4 antigen 1 on the surface of the cells 3 is decomposed. In FIG. 10, the decomposed RNA aptamer is shown as a collection of dots and represented with reference numeral 7. Reference numeral 15 represents a mixture of the cells 3, the decomposed labeling substance RNA aptamer 7, and the identifying substance β-phycoerythrin 6.

The aptamer introduced to the culturing tank 213 via the semipermeable membrane 280 and decomposed in the culturing tank 213 by the action of nuclease is then discharged via the semipermeable membrane 280. The culturing device accommodating the culturing tank 213 is preferably of a shaking type in order to promote the introduction of the nuclease to the culturing tank 213 via the semipermeable membrane 280, the decomposition of the aptamer in the culturing tank 213, and the discharge of the decomposed aptamer and the identifying substance β-phycoerythrin from the culturing tank 213. Reference numeral 18 represents a collection of the cells 3 remaining in the culturing tank 213 and recovered as still having the CD4 antigen 1 on the surface thereof. Here, the cells are represented with 3′ and the CD4 antigen is represented with 1′ in order to indicate that the cells and the CD4 antigen are not exactly the same before and after the action of nuclease, because the cells and the CD4 antigen may possibly be influenced by the nuclease even though slightly.

FIG. 11 shows an examination result of the time-wise change of the fluorescence intensity of the identifying substance β-phycoerythrin bound to the cell surface, the change being caused by the addition of nuclease. In this example, a cell is put on a plate and the cell surface is observed with a fluorescent microscope. The accumulated value of the fluorescence intensity obtained from the entire cell is found. When the aptamer portion is decomposed by nuclease, the identifying substance β-phycoerythrin is diffused from the cell surface and becomes undetectable. Utilizing this phenomenon, how nuclease decomposes the aptamer portion can be observed by tracing the fluorescence intensity at the cell surface. In FIG. 11, the horizontal axis represents the time, and the vertical axis represents the fluorescence intensity per cell, which is the accumulated fluorescence intensity from one cell. Namely, using the cell surface antigen CD4-presenting cells to which the β-phycoerythrin-modified RNA aptamer decomposed by the cell separation and culturing apparatus is bound (the group of cells 3 to which the labeling substance RNA aptamer 12 is bound in FIG. 10), the fluorescence intensity at the cell surface is traced in accordance with the time by the fluorescent microscope (exciting wavelength: 532 nm; fluorescence wavelength: 575 nm; a bandpass filter is used). In order to avoid discoloration by the fluorescence, the time of radiation of the exciting light is minimized. For example, the fluorescence intensity is measured while radiating light for 1 second at an interval of 1 minute.

Curve 22 represents the time-wise change of the fluorescence intensity. Arrow 21 represents the timing at which Benzonase (registered trademark) is added. Even if the time of radiation of the exciting light having a wavelength of 532 nm is short, it is difficult to completely avoid discoloration. Even without using Benzonase (registered trademark) (time zone 23), the fluorescence intensity is slightly decreased as the time passes. When Benzonase (registered trademark) nuclease is added at time 21, the fluorescence intensity detectable from the cell is rapidly decreased as shown in time zone 24, although being slightly delayed.

This result indicates the following: on the stage of separating the cell surface antigen CD4-presenting cells to which the β-phycoerythrin-modified RNA aptamer is bound using the cell separation and culturing apparatus, there is no problem with the function of β-phycoerythrin as the identifying substance; when nuclease is added, the RNA aptamer portion (reference numeral 5 in FIG. 10) of the β-phycoerythrin-modified RNA aptamer bound to the cell surface is decomposed and the β-phycoerythrin 6, which is a fluorescent substance, is diffused to the solution.

FIG. 12 shows that the cell surface antigen CD4-presenting cells obtained by removing the β-phycoerythrin-modified RNA aptamer are culturable in the culturing tank 213. The horizontal axis represents the time, and the vertical axis represents the number of cells. Based on the characteristic shown in FIG. 11, the time at which the β-phycoerythrin-modified RNA aptamer is considered to be removed to a sufficient level as a result of the addition of Benzonase (registered trademark) nuclease is evaluated in advance.

In this manner, the labeling substance RNA aptamer is bound to the cells to recognize the surface antigen, and the aptamer is decomposed and removed with ribonuclease when cell labeling becomes unnecessary. Thus, the cells can be returned to a pre-separation natural state in which the cells can be divided. In addition, according to the present invention, the cells obtained by the cell separation performed using the cell separation and culturing apparatus are cultured while being accommodated in the culturing tank used for collecting the cells. Therefore, the cells can be prevented from being contaminated, with certainty.

In the following example, the aptamer as the labeling substance is of an RNA type binding to EpCAM, and the identifying substance is magnetic particles (diameter: about 100 nm). The purpose is to separate and detect cancer-derived cells circulating in blood and having EpCAM as a surface antigen.

An RNA aptamer bound to EpCAM is prepared as follows. A 26-base sequence (SEQ. ID. NO:1) containing a T7 promoter sequence is introduced to the 5′ terminus of a single chain DNA having a random sequence of 40 bases, and a 24-base PCR priming site (SEQ. ID. NO:2) is introduced to the 3′ terminus of the single chain DNA. As a result, a sequence of 90 bases in total is synthesized. The sequence to be introduced to the 5′ terminus of the random sequence of 40 bases is shown as SEQ. ID. NO:1. TAATACGACT CACTATAGGG AGACAA (SEQ. ID. NO:1)

The sequence to be introduced to the 3′ terminus of the random sequence of 40 bases is shown as SEQ. ID. NO:2. NTTCGACAGG AGGCTCACAA CAGG (SEQ. ID. NO:2)

The obtained 90-base sequence is transcribed to an RNA with RNA polymerase using the T7 sequence. For the transcription to the RNA, 100 μl of T7 polymerase is caused to act on 100 μmol of DNA at a scale of 500 μl. As the substrate, 3 mM of each of 2′-F-CTP and 2′-F-UTP and 1 mM of each of ATP and GTP are used. The transcription is performed at 25° C. for 10 hours. After the transcription to the RNA, the DNA is decomposed with DNaseI, and the RNA transcription product is recovered with electrophoresis. The recovered RNA transcription product is thermally denatured, and then passed through an EpCAM immobilized sepharose CL4B column in PBS (pH 7.4) containing 2 mM of MgCl₂. A bound transcribed RNA component is eluted with a solution containing 7 M urea. The obtained transcribed RNA component is reserve-transcribed, and PCR-amplified with a primer pair having a complementary sequence to the known sequence portions at both ends. The obtained PCR product is again transcribed with the T7 promoter, and captured with an EpCAM immobilized sepharose CL4B column in a similar manner. Then, the bound transcribed RNA component is recovered. The steps of transcription—capturing—recovery—PCR amplification are repeated 15 times, and thus an RNA aptamer specifically reactive with EpCAM is obtained.

To the 5′ terminus of the obtained aptamer, a thiophosphoric acid group is inserted with in vitro transcription described in “Staining of cell surface human CD4 with 3′-F-pyrimidine-containing RNA aptamers for flow cytometry”, Nucleic Acids Research 26, 3915-3924 (1998). The thiophosphoric acid group is reacted with biotin having an iodoacetyl group introduced thereto, and thus a 5′ biotinated RNA aptamer is obtained. The 5′ biotinated RNA aptamer is reacted with streptoadipine conjugate magnetic beads, and thus an RNA aptamer which has magnetic particles as the identifying substance and is specifically reactive with EpCAM is obtained.

The reaction of RNA aptamer-labeled magnetic particles with EpCAM-positive cancer cells will be described. 10 ml of blood is suspended in 5 times the volume of culturing solution, and RNA aptamer-labeled magnetic particles corresponding to EpCAM are added and stirred mildly for 30 minutes. The resultant suspension is put to a tube having an inner diameter of 2 mm, and the magnetic particles in the tube are captured by neodymium-system magnet arrays located along the tube at an interval of 1 cm. The cells to which the recovered magnetic particles are bound are separated from the magnetic particles as shown in FIG. 10, washed with the culturing solution, and the cells are separated by the cell separation and culturing apparatus 100 in FIG. 1. Whether each cell is to be separated or not is determined by the shape recognition based on an image. As described above, the cells separated and collected in the culturing tank 213 by the cell separation and culturing apparatus 100 are cut off from the cell separation and culturing apparatus 100 while being contained in the culturing tank 213 and put into an arbitrary culturing device. Immediately after this, Benzonase (registered trademark) nuclease is added to the culturing device to decompose the aptamer, and thus biological cells are obtained. The aptamer introduced to the culturing tank 213 via the semipermeable membrane 280 and decomposed in the culturing tank 213 by the action of nuclease is discharged via the semipermeable membrane 280. The culturing device accommodating the culturing tank 213 is preferably of a shaking type in order to promote the introduction of the nuclease to the culturing tank 213 via the semipermeable membrane 280, the decomposition of the aptamer in the culturing tank 213, and the discharge of the decomposed aptamer and the identifying substance β-phycoerythrin from the culturing tank 213. Cancer cells are durable against long-time culturing, and some cells start to be divided soon in the culturing tank 213.

In general, biological cells circulating in the blood are mostly derived from cancer cells, except for hemopoietic cells. In the blood, cells are not peeled off while being alive from the endothelial surface of the blood vessel; and even if peeled off, the cells are decomposed in the blood owing to the protection mechanism. By contrast, cancer cells are peeled off while being alive, exhibit resistance even in the blood, and circulate in the blood vessel while being alive. However, the cancer cells are existent in a small quantity and are not suitable for biopsy. If the cancer-derived cells circulating in the blood can be concentrated and cultured for a certain time period, it can be found whether a lesion exists somewhere in the body although the site of cancer cannot be not specified.

In the case where the identifying substance of the identifying element is particles or magnetic particles, an image of the particles, scattered light detection, or magnetic detection of the identifying substance for the identifying element is usable to identify the cells to which the labeling substance for the identifying element is bound.

(Example of Cell Culturing Device)

FIG. 13 schematically shows an example of cell culturing device. Reference numeral 350 represents the culturing device. FIG. 13 shows the culturing tanks 213 and 214 together with the chip substrate 101 after being cut off from the cell separation and culturing apparatus and put into the culturing device 350. The culturing tanks 213 are put on a rack 351 and placed in the culturing device 350. In FIG. 13, each rack 351 has five stages of tables. The culturing device 350 includes an air supply pipe 354 for supplying air containing 5% of CO₂and a supply pipe 355 for supplying a medium 352. Each pipe has an open/close valve. The rack 351 is not absolutely necessary to culture the cells separated and put into the culturing tanks 213 and 214 which are accommodated in the culturing device 350, and the culturing tanks 213 and 214 may be appropriately located in the culturing device 350.

FIG. 14(A) through FIG. 14(C) show an example of processing for cutting the culturing tanks 213 and 214 together with the chip substrate 101 from the cell separation and culturing apparatus 100. FIG. 14(A) is a plan view showing only the culturing tanks 213 and 214 of the cell separation and culturing apparatus 100 and the reservoir 285 surrounding the culturing tanks 213 and 214. One-dot chain line 300 represents the cutting line along which the culturing tanks 213 and 214 are cut off together with the chip substrate 101. FIG. 14(B) is a cross-sectional view taken along line C-C of FIG. 14(A) and seen in the direction of the arrows thereof during the cutting operation. FIG. 14(C) is a cross-sectional view taken along line D-D of FIG. 14(A) and seen in the direction of arrows thereof during the cutting operation.

Before cutting off the culturing tanks 213 and 214, it is preferable that the buffer solution (medium) in the reservoir 285 is removed. In this case, the buffer solution (medium) in the culturing tanks 213 and 214 is in the same state as that after the separation operation is finished; i.e., the culturing tanks 213 and 214 accommodates the separated and collected cells and also the buffer solution (medium). Reference numeral 289 represents cutting teeth, which are formed such that the cutting tips thereof match the cutting line represented with the one-dot chain line in FIG. 14(A). The cutting tip of each cutting tooth 289 is formed to have a relatively large angle of about 60° to 130°. This is because the chip substrate 101 is cut as if being crushed with the cutting teeth 289 heated to about 80° C. to 110° C. In other words, the operation of cutting off the culturing tanks 213 and 214 also needs to close the flow channels 218 and 219 in communication with the culturing tanks 213 and 214. As shown in FIG. 14(C) which is the cross-sectional view taken along line D-D in FIG. 14(A) and seen in the direction of the arrows thereof, the flow channel 218 appears to have a lengthy opening (on the right). When the tip of the cutting tooth 289 is pushed to the chip substrate 101 at this position, the laminate film 410 is pushed up along the slanted face of the tip of the cutting tooth 289 (the right cutting tooth 289 in FIG. 14(C)) and thus closes the flow channel 218. In a portion with no opening such as the flow channel 218 or 219, the chip substrate 101 is cut off by the tips of the cutting teeth 289 as shown in FIG. 14(B) and in the left part of FIG. 14(C).

(Example of Optical System)

FIG. 15 is an overall conceptual view of an optical system in the cell detection area 221. A light source 25 for radiating light to a cell, and a filter 26, are provided on the top surface of the chip substrate 101. On the bottom surface of the chip substrate 101, a detection system for detecting light radiated to the cell is provided. FIG. 15 shows a cross-sectional view from the cell detection area 221 through the cell separation area 222 to the flow channel 218, along the flow direction of the flow channel 218 as shown in FIG. 2. The cells are modified with a fluorescent dye, gold particles or nanoparticles as shown in FIG. 10.

The cell flowing in the flow channel 218 in the cell detection area 221 is irradiated with light from the light source 25 through the filter 26. An image of the cell irradiated with the light is detected by an objective lens 44, and is captured as an image by a CCD camera 48 via a dichroic mirror 45, a filter 46, and a lens 47. The image data obtained by the CCD camera 48 is transferred to a computer 60 having an image processing function. The image data of the cell is checked against the prepared image data on the cell to be detected. When determining that the image data obtained by the CCD camera 48 has a predetermined relationship with the prepared image data on the cell to be detected, the computer 60 outputs a signal 70 to turn on the switch 216 of the cell separation and culturing apparatus 100. Thus, in the cell separation area 222, a voltage is applied to the buffer solution flowing in the flow channel 247 obtained by merging the microscopic flow channel 240 and the microscopic flow channel 204′, and thus a force is caused to act on the cell. Needless to say, the moving velocity of the cell flowing down the flow channel (the flow rate of the buffer solution in the flow channel 247) is separately detected, so that the voltage is applied at the timing when the cell evaluated in the cell detection area 221 passes the cell separation area 222.

When the cell is modified with a fluorescent dye, the fluorescence at the cell irradiated with the light is detected by the objective lens 44, passes through the dichroic mirror 45, and is captured by a photomultiplier 54 as a light spot via a reflective mirror 51, a fluorescent filter 52 and a lens 53. Alternatively, when the cell is modified with gold microparticles or nanoparticles, the scattering light from the cell irradiated with the laser light is detected by the objective lens 44. In this case, the fluorescent filter 52 is removed. The light detected by the objective lens 44 passes the dichroic mirror 45, and captured by the photomultiplier 54 as a light spot via the reflective mirror 51 and the lens 53. The light spot obtained by the photomultiplier 54 is sent to the computer 60 having a light processing function, and the computer 60 determines whether the cell has been modified as predetermined. When determining that the light spot is from the cell which has been modified as predetermined, the computer 60 outputs a signal 70 to turn on the switch 216 of the cell separation and culturing apparatus 100. Thus, in the cell separation area 222, a voltage is applied to the buffer solution flowing in the flow channel 247 obtained by merging the microscopic flow channel 240 and the microscopic flow channel 204′, and a force is caused to act on the cell. Needless to say, the moving velocity of the cell flowing down the flow channel (the flow rate of the buffer solution in the flow channel 247) is separately detected, so that the voltage is applied at the timing when the cell evaluated in the cell detection area 221 passes the cell separation area 222.

In the case where the CCD camera 48 is a photon counter or a photomultiplier, the intensity of the scattering light from the cell or the gold microparticles or nanoparticles bound to the cell may be continuously measured and sent to the computer 60 in accordance with the intensity change. The computer 60, having the light processing function, determines whether or not the cell has been modified as predetermined. In the case where the photomultiplier 54 is an optoelectric double speed camera, which area of the cell is labeled with fluorescence may be captured by images, and such information may be sent to the computer 60. The computer 60, having the light processing function, checks the obtained image data against the prepared image data on the cell to be detected. In this manner, it can be determined whether or not the cell has been modified as predetermined more precisely.

Needless to say, the image processing and the fluorescence or scattering light processing may be used together. The image data captured by the camera 48 may be displayed on a computer monitor such that the user can observe the cell.

Cell separation chip and cell culturing method using the same

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CPC

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