JP2012255720A - Plane development method of cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a development method capable of densely aggregating biological materials on a filter without impairing lateral capillary force for clumping the biological materials together, even when a solvent contains salt such as physiological saline in which particulate biological materials such as cells or the like are dispersed and suspended, and a development device applied in the method.SOLUTION: A development method of biological materials includes: a solution supplying step for using a solution supplying mechanism to supply a solution (hereinafter called "sample solution") containing particulate biological materials, on one surface (hereinafter called "front surface") of a filter through which water content in the sample solution permeates but the biological materials do not permeate; and a water content exhausting step for using a water content exhausting mechanism to exhaust from the other surface (hereinafter called "reverse surface") of the filter, the water content in the sample solution supplied onto the front surface of the filter, while acting a force towards one end in a plane direction of the filter.

Description

  The present invention relates to a method for developing a particulate biological material and a developing device used in the method. More specifically, the present invention relates to a method for developing a particulate bio-related substance that can close-pack cells and the like on a single layer, and a developing device used in the method.

  In modern medicine, much information obtained from cell morphology plays an extremely important role in the prevention, diagnosis and treatment of diseases. These cytology include exfoliation cytology and abrasion cytology, as well as puncture cytology that more actively collects lesion cells.

  In these cytodiagnosis, cells are collected by rubbing, puncturing, etc., and then applied to the slide glass mainly by the smearing method (procedure) or the auto smear method (using a centrifugal force to attach the specimen on the slide glass). Create a splash surface. Then, the smeared specimen is immersed in each solution such as alcohol, staining liquid, and distilled water stored in a container such as a staining vat, and after the specimen is stained, the mounting medium and the cover glass are covered. A preparation (specimen) is prepared by performing a series of operations described above, and it is microscopically examined with a microscope or the like to diagnose a lesion.

  In Patent Document 1, in the capture staining apparatus shown in FIG. 16, a membrane filter (1) having countless fine straight holes is used to filter a solution containing a specimen such as a cell, and the specimen is removed from the membrane filter (1). A capture staining method is disclosed in which various solutions are sequentially injected onto the membrane filter (1) and filtered after being captured on the top.

  However, in this method, cells overlap and accumulate on the membrane filter, and there is a possibility that the target cells may be overlooked when performing microscopic examination.

Japanese Patent Laid-Open No. 2-129531

  The present invention is a solvent containing salts such as physiological saline in which particulate biological substances such as cells are dispersed and suspended, without weakening the lateral capillary force that aggregates the biological substances, An object of the present invention is to provide a developing method and a developing device used in the method, which can be densely agglomerated while suppressing multilayering of the biological substance on the filter.

  As a result of intensive studies to solve the above problems, the present inventors have absorbed moisture containing salt through a filter into a moisture absorbing member, thereby preventing particles such as cells without disturbing the transverse capillary force. The present invention has been completed by finding that the living body-related substance in the form of agglomerates on the surface of the filter.

  That is, in the method for developing a particulate biological material according to the present invention, a solution containing the particulate biological material (hereinafter referred to as “sample solution”) is converted into moisture in the sample solution using a solution supply mechanism. Supplying a solution on one side of the filter (hereinafter referred to as “surface”) that does not permeate the biological material, but is supplied onto the surface of the filter by using a moisture discharge mechanism. A moisture discharge step of discharging moisture in the sample solution from the other surface of the filter (hereinafter referred to as “back surface”) while applying a force toward one end in the planar direction of the filter. To do.

It is preferable that the moisture discharging mechanism includes a moisture absorbing member brought into contact with the back surface of the filter and / or a suction port communicating with the moisture suction device as the moisture discharging member.
Even if the solution supply member provided in the solution supply mechanism and / or the water discharge member provided in the water discharge mechanism is moved relative to the filter in a direction parallel to the surface of the filter. Good.

  While supplying the sample solution in a state where the tip of the solution supply member provided in the solution supply mechanism is not in contact with the surface of the filter but in contact with the sample solution supplied to the surface of the filter, the surface of the filter In a direction parallel to the filter, the filter may be moved relative to the filter.

  The sample solution supplied to the surface of the filter may be developed according to gravity by inclining the filter, or the sample solution may be stored on one side of the surface of the filter.

  The relative movement and / or inclination of the solution supply member provided in the solution supply mechanism with respect to the filter causes the sample solution to be developed on the surface of the filter, and the relative relationship between the moisture discharge member provided in the moisture discharge mechanism with respect to the filter. It is preferable that the trajectory due to a simple movement is substantially the same.

  In the solution supplying step, the sample solution is supplied to one end of the surface of the filter in a state where the filter is overlaid on the substrate, and in the moisture discharging step, the filter is sequentially separated from the substrate from the other end of the filter. Alternatively, the moisture may be discharged by adsorbing or holding the moisture on the substrate.

The supply amount of the sample solution per unit time is preferably the same as or larger than the amount of water discharged per unit time.
You may perform a solution supply process and a water | moisture-content discharge | release process simultaneously in parallel.

The biological substance is preferably a cell.
Further, the developing device of the present invention includes a solution supply mechanism and a water discharge mechanism; a filter arrangement portion for arranging a filter; a jig for holding the solution supply mechanism or the water discharge mechanism above and below the filter arrangement portion; and A moving mechanism capable of relatively moving the solution supply mechanism and / or the moisture discharging mechanism in a direction parallel to the filter surface and adjusting the moving speed, and the jig is attached to the jig with the solution supply mechanism or It is characterized by being used in the above-described developing method, in which the distance between the solution supply mechanism and the water discharge mechanism and the filter arrangement portion can be adjusted in a state where the water discharge mechanism is held.

  According to the method for developing a particulate biological material of the present invention, for example, blood cells can be densely aggregated and developed while suppressing multilayer lamination of the filter surface. And the area which can be expand | deployed is not limited to small areas, such as a slide glass, for example, It can apply also to the desired large area. That is, it is possible to continuously produce a large amount of a membrane made of cells or the like.

  In addition, when the particulate biological material is a cell, the solvent is usually physiological saline. When the solvent is evaporated, the salt precipitates and hinders the lateral capillary force. Since a solvent such as saline can be quickly discharged, such a problem can be solved.

FIG. 1 is a diagram schematically showing some of the modes in which only the solution supply mechanism moves in the developing method of the present invention, and shows (a) lower part discharge and (b) end face discharge. FIGS. 2A and 2B are diagrams schematically showing some of the modes in which only the moisture discharging mechanism moves in the developing method of the present invention, and show (a) an inclined structure and (b) a shutter structure. FIG. 3 is a view schematically showing some of the modes in which the solution supply mechanism and the water discharge mechanism move together in the developing method of the present invention, and shows (a) lower discharge and (b) end face discharge. FIG. 4 is a diagram schematically showing some of the modes in which the filter moves in the developing method of the present invention, in which (a) lower discharge, (b) take-up filter structure, (c) disk structure, And (d) shows a dip structure, which can be said to be a mode in which both the solution supply mechanism and the water discharge mechanism move. FIG. 5 shows a schematic diagram of one embodiment of a cell stacking device including a deploying device (cell stacking unit in the figure) used in the deploying method of the present invention. FIG. 6 is a diagram schematically showing the configuration (a) of the solution supply mechanism and the perspective view (b) of the solution supply mechanism used in the examples. FIG. 7 is a view schematically showing the developing method of the present invention used in Example 1 (a mode in which neither the filter nor the solution supply mechanism / moisture discharge mechanism moves). FIG. 8 is a diagram schematically showing the developing method of the present invention (a mode in which both the solution supply mechanism and the water discharge mechanism move) used in Example 2. FIG. 9 is a diagram schematically showing the development method of the present invention (a mode in which only the moisture discharging mechanism moves) used in Example 3, and the solution was placed in a state where the filter was leveled as in (b). After feeding, the entire filter is tilted as shown in (a). FIG. 10 is a diagram schematically showing the developing method of the present invention (a mode in which only the solution supply mechanism moves) used in Example 4. FIG. 11 is a diagram schematically showing the developing method of the present invention (a mode in which only the moisture discharging mechanism moves) used in Example 5, and the filter indicated by the dotted line in the figure is peelable. FIGS. 12A and 12B are diagrams (a) is a perspective view and (b) is a vertical cross-sectional view schematically showing a deployment method of the present invention (a mode in which only the moisture discharging mechanism moves) used in Example 6. FIG. , (A), the filter is supported by the housing member, and the moisture absorbing member can be pressed from the lower end. FIGS. 13A and 13B show the results of the images of Example 1 and Comparative Example 1, respectively. FIGS. 14A and 14B show the results of the image of Example 2, respectively. FIGS. 15A and 15B show the results of the images of Example 4, respectively. FIG. 16 shows a front view of the capture staining apparatus disclosed in Patent Document 1. As shown in FIG. More specifically, the injection unit (3) includes a plurality of syringes (14), a pressure body (7) for injecting a solution by pressing the syringe head (13), and a transfer body for holding the syringe (14) ( 6) and a rotating shaft for fixing and assembling the transfer body (6) at its center. The same solution is stored in these syringes (14), transferred to the holder (17) by the transfer body (6), and then injected into the solution holder (17) by the pressure body (7). The Then, after a solution containing a specimen such as a cell is injected into the holder (17), the solution is suction filtered by the filtration section (4), and the specimen is captured on the membrane filter (1). Next, after a solution for dyeing treatment is injected into the holder (17), the solution is suction filtered after a lapse of a certain time necessary for dyeing. The filtration unit (4) is composed of a suction pump (18) and a waste liquid tank (19), and sucks the solution in the holder (17) with the suction pump (18) and discharges it into the waste liquid tank (19). It is said.

Next, the developing method of the particulate biological material and the developing device used in the developing method of the present invention will be described in detail.
In the present invention, a solution containing a particulate biological material may be referred to as a “sample solution”. In addition, one surface of the filter which is the side where the sample solution is supplied and the cells are finally developed is “surface”, and the other side (the other) of the filter which is the side which discharges the moisture of the supplied sample solution. The surface is sometimes referred to as the “back surface”.

<Deployment method>
The development method of the present invention includes at least the following steps:
Solution supply step: a step of supplying a solution containing a particulate biological material using a solution supply mechanism onto the surface of a filter that allows moisture in the sample solution to permeate but does not penetrate particulate biological material; And moisture discharging step: a step of discharging moisture in the sample solution supplied on the surface of the filter from the back surface of the filter while applying a force toward one end in the planar direction of the filter by using a moisture discharging mechanism.

  As one preferred embodiment of the development method of the present invention, for example, as shown in FIG. 8, a part of the filter is bent (tilted), and a moisture absorbing member such as a filter paper is inserted below the folded filter, A mode in which the solution supply member and the water absorption member are moved together while supplying cells suspended in PBS (phosphate buffered saline) from the solution supply member can be mentioned. Only PBS is absorbed by the filter paper, and the cells are fixed from the left hand side of the inclined filter. Starting from the first fixed cells, the cells are sequentially accumulated by the lateral capillary force derived from surface tension and the force (laminar flow force) caused by the flow of liquid in the wetting film. Cells suspended in PBS repel each other to form an electric double layer and are dispersed. However, the repulsive force gradually weakens as they are deposited, and can therefore aggregate in two dimensions.

In the deployment method of the present invention, the relative positions of the solution supply member and / or the moisture discharge member with respect to the filter in the planar direction of the filter may be fixed or may be varied. At this time, the relative position of the solution supply member with respect to the moisture discharging member in the plane direction of the filter may be fixed or may be changed. The positions as described above can be freely designed according to conditions such as the form of the solution supply member, the solution discharge member and the filter used, and the development area of the particulate biological material on the filter. For example,
A mode in which all of the solution supply member, the water discharge member and the filter are fixed A mode in which the water discharge member and the filter are fixed and only the solution supply member is moved (FIG. 1)
A mode in which the solution supply member and the filter are fixed and only the water discharge member is moved (FIG. 2).
A mode in which only the filter is fixed and the solution supply member and the moisture discharge member are moved (FIG. 3)
A mode in which the solution supply member and the water discharge member are fixed and only the filter is moved (FIG. 4).
Etc. When the relative positions of the solution supply member and the moisture discharge member with respect to the filter are changed, it is preferable that the locus drawn by the solution supply member on the filter and the locus drawn by the moisture discharge member on the filter are substantially the same.

Further, the solution supply step and the water discharge step may be performed sequentially or simultaneously,
-Even after the completion of the solution supply process,
-The aspect which performs a water | moisture-content discharge | emission process may be sufficient as performing a solution supply process.

further,
・ Linear development (FIGS. 1 to 3 other than FIG. 3 (c) “disc structure”)
Even
-Mode of unfolding (Fig. 3 (c) "Disk structure")
It may be.

The obtained development surface is rectangular in the former case and circular in the latter case.
As a specific mode (development in a straight line) in the deployment method of the present invention,
A mode in which all of the solution supply mechanism, the water discharge mechanism and the filter are fixed and the solution is discharged after the supply (FIG. 7).
A mode in which only the filter is fixed, the solution supply mechanism and the moisture discharge mechanism are moved, and the solution is discharged while being supplied (FIG. 8).
A mode in which the filter and the solution supply mechanism are fixed, only the moisture discharge mechanism is moved, and the solution is discharged after being supplied (FIG. 9).
A mode in which the water discharge mechanism and the filter are fixed, only the solution supply mechanism is moved, and the solution is discharged while supplying the solution (FIG. 10).
Etc. are preferable.

(Solution containing particulate biological material)
The particulate biological material has a diameter in the same range as that of the colloid, specifically 100 nm or more and 100 μm or less, and examples thereof include all kinds of cells and viruses, and the present invention is not particularly limited. . However, it is a condition that the diameter of the target particulate bio-related substance to be developed on the filter surface is larger than the pore diameter of the filter to be used.

  Examples of the solution (sample solution) containing particulate biological materials include PBS in which cells are suspended. The medium for suspending or dispersing the particulate biological material (referred to as “water” in the present invention) is not particularly limited, and can be appropriately selected by those skilled in the art. In addition, the density of the biological substance in the sample solution can be appropriately adjusted according to the discharge capacity by the solution discharge mechanism.

(filter)
If the filter has an infinite number of pores whose diameter is smaller than the diameter of the target particulate bio-related substance (water in the sample solution can pass through), the material, size, thickness, etc. are those skilled in the art. Can be selected as appropriate.

A commercially available product can be used as such a filter. For example, “New Clipore Membrane” manufactured by GE Healthcare Japan, Inc. is preferable.
Further, as shown in FIG. 1, the filter can be used by being laminated with a support having an infinite number of pores. Such a support may be made of metal, glass, or resin.

  The filter may be arranged so that the whole is horizontal, or may be arranged so that a part or the whole is inclined. For example, as shown in FIG. 9, when the sample solution is supplied to the filter and then tilted, the formed droplets flow down along the tilted direction due to gravity, so the sample solution was supplied while moving the solution supply member. In the same state as the case, it is possible to develop the particulate biological material on the filter. Further, as shown in FIG. 2 (a), the filter is tilted after being surrounded by a frame member, the sample solution is supplied and stored therein, and the moisture discharging member is moved from the upstream side of the tilt toward the downstream side. You may make it make it. The inclination angle may be set so that the development state of the particulate biological substance is appropriate while taking into consideration the water discharging ability and movement speed of the water discharging member, and the inclination angle can be freely changed. It is preferable to adjust appropriately using an angle adjusting means.

  As shown in FIG. 4, when only the filter (or the filter and the support) is moved, that is, when the relative position of the solution supply member and / or the moisture discharge member with respect to the filter is moved, the filter is moved. For example, the movement of the support (a) and the device for moving the filter in a device in which a stainless plate having a micropore (for example, 1 μmφ) through which moisture can pass is used as a support and the filter is fixed on the stainless steel plate. It is necessary to rotate the support (c) configured as a disk, or rotate the roll (b) with the filter suspended between a pair of rolls, or rotate the take-up roller that winds the filter (d). is there.

(Solution supply mechanism)
The solution supply mechanism may be in any form as long as it can supply the sample solution onto one surface (surface) of the filter. As typical embodiments, a solution supply member (or simply a liquid supply unit), a sample solution storage unit (or simply a water storage unit) communicating with the liquid supply unit, and an apparatus for moving the liquid supply unit as necessary And a system including a flow path and the like.

  If the amount of the particulate biological material to be developed is small, a fixed amount of the sample solution may be supplied at a time without moving the solution supply member to complete the solution supply step. On the other hand, if the amount of the particulate biological material to be developed is large, a predetermined amount of the sample solution may be supplied per unit time continuously or intermittently while moving the solution supply member.

  The sample solution can be supplied from above the filter or from the side of the filter. When the solution supply member is moved relative to the filter, the tip (supply port) of the solution supply member is not in contact with the surface of the filter but is kept in contact with the sample solution supplied to the surface of the filter. Preferably, the solution supply member is moved by a moving mechanism described later, or the filter is moved.

  More specifically, examples of the solution supply mechanism that can supply the sample solution as described above include a blade coater, a dip coater, an applicator, and an ink jet.

  In the solution supply step, the supply amount per unit time of the sample solution supplied onto the filter surface by the solution supply mechanism is the unit of moisture in the sample solution discharged from the back surface of the filter in the solution discharge step by the solution discharge mechanism. The amount of discharge per hour may be the same or more. If the supply amount and the discharge amount are the same, it is possible to discharge moisture while keeping the volume of the liquid pool of the supplied sample solution constant, so the solution supply step and the moisture discharge step proceed simultaneously. Can be made. On the other hand, if the supply amount is larger than the discharge amount, the volume of the liquid reservoir of the supplied sample solution increases to some extent. Therefore, it is necessary to complete or suspend the solution supply step before reaching the allowable limit.

(Moisture drainage mechanism)
The moisture discharging mechanism applies a force directed to one end in the planar direction of the filter while applying the solution containing the particulate biological material supplied on one surface (surface) of the filter to the other surface of the filter. Any mode may be used as long as it can be discharged from the surface (back surface). A typical embodiment includes a system including a moisture discharging member (or simply a discharging unit) and means for moving the discharging unit as required. If the ability to discharge the solution from the discharge part is sufficiently high, the lateral capillary force can be applied to the particulate bio-related substance at a distant position without moving the discharge part relative to the filter. It is possible to achieve the purpose of the period.

  On the other hand, the sample solution is supplied to one end of the surface of the filter in the solution supply step in a state where the filter is overlaid on the support, and in the moisture discharge step, the filter is sequentially separated from the support from the other end of the filter, For example, the moisture may be discharged by adsorbing or holding moisture on the support, that is, the solution may be discharged by holding the solution on the support using capillary force. The support used here may be composed of any material, but the surface of the support needs to be sufficiently hydrophilic.

  Further, as shown in FIG. 2B, the shutter structure, a shutter is sandwiched between the filter and the moisture absorbing member, and a discharge portion is formed by moving the shutter portion, and the solution is discharged from one end of the back surface of the filter. The aspect which performs is also mentioned.

  It is preferable to use a moisture absorbing member as the moisture discharging member. By bringing the moisture absorbing member into contact with the back surface of the filter from one end side of the sample solution (liquid reservoir) supplied to the filter surface, a force directed toward the one end is applied to the particulate biological material and moisture. While the water can be discharged. As such a moisture absorbing member, for example, filter paper or the like can be used. When the moisture absorbing member is brought into contact with the back surface of the filter, a part of the filter may be lifted (folded) so that the moisture absorbing member is sandwiched between the filter and the substrate, or the entire filter can be kept flat. The support may be inclined, and the moisture absorbing member may be sandwiched between the filter and the support.

  Further, as the water discharge member, a suction device capable of actively and continuously sucking water, for example, a suction port communicating with a decompression device such as a pump can be used. Such a supply port may be used on the back surface of the filter in combination with such a suction port and the moisture absorbing member as required.

<Deployment device>
The developing device of the present invention includes a solution supply mechanism and a moisture discharge mechanism; a filter arrangement portion for arranging a filter; a jig for holding the solution supply mechanism or the moisture discharge mechanism above and below the filter arrangement portion; A moving mechanism capable of moving the mechanism and / or the moisture discharging mechanism relatively in a direction parallel to the filter surface and adjusting the moving speed, and the jig is connected to the solution supply mechanism or the moisture discharging mechanism. The present invention is characterized in that the distance between the solution supply mechanism and the water discharge mechanism and the filter arrangement portion can be adjusted while the mechanism is held.

  For example, as a more specific deployment device, the device shown in FIG. The filter is wound between two rotating rolls, and the filter can be moved in a certain direction by rotating the two rolls in the same direction at a constant speed. Place and fix the moisture absorption member in contact with the filter, and place and fix the solution supply member (FIG. 6) whose tip on the solution supply side is flat on the side opposite to the filter moving direction with respect to the moisture absorption member. In addition, the cell suspension can be supplied from the solution absorbing member simultaneously with the rotation of the roll.

  Such a deployment device can be incorporated into a cell accumulation device as shown in FIG. This cell accumulating apparatus is, for example, a cell accumulating section including a developing apparatus shown in FIG. 4B (a developing apparatus suitable for a developing method in which the solution supply mechanism and the water discharge mechanism are fixed and only the filter is moved), It comprises a staining section, an observation section, and a drive section that winds up the filter. The steps of developing cells on the filter, staining the cells, and observing (photographing) the stained cells can be performed continuously.

Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.
Preparation of the cell suspension and preparation of the cell development plane and the solution supply member were performed as follows.

(Preparation of cell suspension)
Jurkat cells were suspended and fixed in PBS containing paraformaldehyde 4%, and then cell nuclei were stained with “Hoechst 33342” (Invitrogen). The cells were washed with PBS, adjusted to a concentration of 8 × 10 ⁶ cells / mL, and used as a cell suspension.

(Preparation of cell expansion plane)
On a slide glass (“S091120” manufactured by Matsunami Glass Industry Co., Ltd .; 76 mm × 52 mm) as a glass support, a filter (“New Clipore Membrane” manufactured by GE Healthcare Japan Co., Ltd .; pore diameter 2 μm) is used. These were stacked and used as a cell expansion plane.

(Production of solution supply member)
As shown in FIG. 6 (a), two pieces of stainless steel plates having a thickness of 0.7 mm and a width of 5 mm are used as spacers at both ends of a cover glass (“C218181” manufactured by Matsunami Glass Industry Co., Ltd .; 18 mm × 18 mm). A solution supply member having such a tip on the solution supply side was produced by laminating a cover glass.

Moreover, the cell expansion | deployment apparatus used in Example 2, 4 is comprised as follows.
The jig of the solution supply mechanism is provided on the upper part of the filter arrangement part, and the height of the solution supply mechanism can be adjusted with the jig holding the solution supply mechanism. A moving mechanism (moving speed adjustable) that moves in a direction parallel to the plane is provided.

  As the supply of the cell suspension by the solution supply mechanism, the solution supply mechanism is held by the jig, and 100 μL of the cell suspension is stored in the water storage section of the solution supply mechanism. The stored cell suspension can be supplied from the tip of the solution supply member placed on the upper part of the cell development plane.

[Example 1]
First, a 0.5 mm thick water absorption member (GE Healthcare Japan Co., Ltd. blotting filter paper square type (GB005)) is placed between a horizontally installed cell spreading plane filter and a slide glass. Inserted and fixed. Next, 10 μL of the cell suspension was dropped at a location about 5 mm away from the tip of the moisture absorbing member to form a droplet (FIG. 6). After 30 seconds, the cell image in the droplet was photographed with an inverted fluorescence microscope (“Observer D1” manufactured by Carl Zeiss Co., Ltd.).

  As a result, as shown by the white circles in FIG. 13A, a collective arrangement in the direction of the water absorbing member by Jurkat cells could be confirmed. In addition, the place shown with the square in this figure is a location which has arrange | positioned the moisture absorption member.

[Comparative Example 1]
In Example 1, cell images were taken in the same manner as in Example 1 except that the water absorbing member was not used. That is, 10 μL of the cell suspension was dropped on a filter on the cell development plane placed horizontally to form a droplet, and then a cell image in the droplet was photographed with a fluorescence microscope.
As a result, as shown in FIG. 13 (b), no assembly sequence by Jurkat cells was observed.

[Example 2]
As shown in FIG. 8, first, the tip of the solution supply member was placed about 2 mm above the horizontally placed cell development plane to supply the cell suspension. Next, the moisture absorbing member used in Example 1 was inserted between the filter on the cell development plane and the slide glass, and approached from the lateral direction of the droplet at a speed of about 1 mm / sec. It was moved about 1 cm at the same speed. The state of cell accumulation after migration was photographed over time with a fluorescence microscope.

  The results are shown in FIGS. 14 (a) and 14 (b). (A) is an image 30 seconds after the movement of the moisture absorbing member / solution supply member is started, and (b) is an image 50 seconds after the movement is started.

[Example 3]
As shown in FIG. 9A, 100 μL of a cell suspension was added to form a droplet on a cell development plane placed at an inclination angle of 15 degrees with respect to the horizontal plane. Next, the water absorbing member used in Example 1 is inserted between the filter on the cell development plane and the slide glass so as to synchronize with the drop of the liquid droplet on the lower slope (speed: about 0.2 mm / second). A cell accumulation plane was prepared by bringing the droplet close to the lateral direction.

[Example 4]
As shown in FIG. 10, the water absorbing member used in Example 1 was inserted and fixed between a horizontally installed filter on a cell development plane and a slide glass. Next, a meniscus of about 2 mm is formed at a location about 5 mm away from the tip of the moisture absorbing member in the same manner as in Example 2, and the solution is moved away from the tip of the moisture absorbing member at a speed of about 1 mm / second. A cell accumulation plane was prepared by moving the supply member about 1 cm.
The results are shown in FIGS. 15 (a) and 15 (b). (A) is an image 5 seconds after the movement of the solution supply member is started, and (b) is an image 20 seconds after the movement is started.

[Example 5]
A filter housing (Advantech's “filter holder for vacuum filtration” (KGS-90)) is used to sandwich the filter on the cell development plane, add 100 μL of the cell suspension to form droplets, and then filter the polypropylene film. Pressed from the bottom. Next, as shown in FIG. 11, the moisture absorbing member used in Example 1 is inserted between the filter on the cell development plane and the slide glass, and is brought close to the droplet lateral direction at a speed of about 1 mm / second. A cell accumulation plane was prepared.

[Example 6]
As shown in FIG. 12 (a), a filter on the cell development plane is sandwiched between filter housings (“filter holder for vacuum filtration” (KGS-90) manufactured by Advantech), and 100 μL of cell suspension is added to the solution. Drops formed. Next, the moisture-absorbing member used in Example 1 was pressed against the lower part of the filter on the cell development plane, and the cell accumulation plane was prepared by approaching it from the lateral direction of the droplet at a speed of about 1 mm / sec (FIG. 12B). .

  In Examples 1 to 6 described above, the moisture absorbing member having a thickness of 0.5 mm was used. However, the thickness of the moisture absorbing member does not have to be uniform, and the thickness of the tip of the moisture absorbing member that contacts the filter. If the thickness is about 0.5 mm, it is suitable for insertion into the lower portion of the filter, and the tip may be further thinned to make insertion easier, or the shape may gradually increase from the tip. Further, the moisture absorption amount can be increased by increasing the thickness of the moisture absorbing member except for the portion inserted so as to be in contact with the filter (for example, the rear end side of the moisture absorbing member).

  When cells are observed using the development method of the present invention, when the cell suspension is, for example, blood (10 mL) collected from a subject, a large number of cells are developed at a high density within a narrow field of view of a microscope. Therefore, it is possible to efficiently find a cell having a small number such as a cancer cell.

DESCRIPTION OF SYMBOLS 1 .... Membrane filter 2 .... Filter holding part 3 .... Injection part 4 .... Filtering part 5 .... Housing 6 ... ... Transfer 7 ... Pressure 12 ... Rotating shaft 13 ... Syringe head 14 ... Syringe 15 ... Holding plate 16 ··· Support rod 17 ··· Holder 18 ··· Suction pump 19 ··· Waste liquid tank 20 ··· Control panel 26 ···・ Fixed shaft

Claims (11)

Using a solution supply mechanism, a solution containing particulate biological substances (hereinafter referred to as “sample solution”) is one side of a filter that allows moisture in the sample solution to pass through but does not pass through the biological substances. (Hereinafter referred to as “surface”) solution supply step to be supplied on; and using the moisture discharge mechanism, moisture in the sample solution supplied on the surface of the filter is directed to one end in the planar direction of the filter. A method for developing the biological material, comprising a step of draining water from the other surface (hereinafter referred to as “back surface”) of the filter while applying a force.   The expansion | deployment method of Claim 1 with which the said water | moisture-content discharge | release mechanism is provided with the suction opening connected to the water | moisture-content absorption member contacted with the back surface of the said filter, and / or a water | moisture-content suction apparatus as a water | moisture-content discharge | emission member.   The solution supply member provided in the solution supply mechanism and / or the water discharge member provided in the water discharge mechanism are moved relative to the filter in a direction parallel to the surface of the filter. Deployment method described in 1.   While supplying the sample solution in a state where the tip of the solution supply member provided in the solution supply mechanism is not in contact with the surface of the filter but in contact with the sample solution supplied to the surface of the filter, the surface of the filter The expansion | deployment method in any one of Claims 1-3 moved relatively with respect to this filter in the direction parallel to.   The sample solution supplied to the surface of the filter is developed according to gravity by tilting the filter, or the sample solution is stored on one side of the surface of the filter. Deployment method described in 1.   The relative movement and / or inclination of the solution supply member provided in the solution supply mechanism with respect to the filter causes the sample solution to be developed on the surface of the filter, and the relative relationship between the moisture discharge member provided in the moisture discharge mechanism with respect to the filter. The development method according to claim 1, wherein trajectories due to simple movement are substantially the same. In a state where the filter is overlaid on the support,
In the solution supply step, a sample solution is supplied to one end of the surface of the filter,
2. The developing method according to claim 1, wherein in the moisture discharging step, the filter is sequentially separated from the support from the other end of the filter, and the moisture is discharged by adsorbing or holding the moisture on the support. .   The developing method according to any one of claims 1 to 7, wherein the supply amount of the sample solution per unit time is the same as or greater than the discharge amount of water per unit time.   The expansion | deployment method in any one of Claims 1-8 which perform the said solution supply process and the said water | moisture-content discharge | release process simultaneously in parallel.   The development method according to claim 1, wherein the biological substance is a cell. A solution supply mechanism and a water discharge mechanism; a filter arrangement portion for disposing a filter; a jig for holding the solution supply mechanism or the water discharge mechanism above and below the filter arrangement portion; and a solution supply mechanism and / or a water discharge mechanism. A moving mechanism capable of moving the filter relatively in a direction parallel to the filter surface and adjusting the moving speed,
The jig can adjust the distance between the solution supply mechanism and the water discharge mechanism and the filter arrangement portion in a state where the jig holds the solution supply mechanism or the water discharge mechanism.
A deployment apparatus, which is used in the deployment method according to claim 1.