JP2006081482A - Method and device for fractionating and culturing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-incubation chip enabling many cells to be simultaneously incubated or cultured; to provide a method for fractionating the cells for the cell incubation chip; and to provide a device therefor. <P>SOLUTION: The device for fractionating the cells has a pipet capable of inserting a solution containing a plurality of the cells and having a tip opening diameter suitable for allowing the cell or a cell aggregation having a prescribed size to pass through, a means for observing the cell in the interior of the pipet, a means for forming a droplet at the tip part of the pipet by extruding the solution containing the cells from the interior of the pipet, and a means for detecting the presence or absence of the cell in the droplet and for discriminating the droplet containing the cell and the droplet not containing the cell, and drops and arranges the droplets on prescribed positions on a substrate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell incubation chip capable of simultaneously incubating or culturing a large number of cells, a method for sorting cells into a cell incubation chip, and an apparatus therefor. That is, the present invention relates to a cell culture chip and a cell culture system capable of arranging a predetermined number of cells in an array and culturing a large number of cells simultaneously.

The basis for studying cells is the separation of target cells and individual culture of the separated cells. Separation techniques can be broadly classified as follows.
(1) A method of separating cells by difference in specific gravity,
(2) A method based on information stained with fluorescently labeled antibodies or visual information,
(3) A method using a cell sorter.
Either method can separate the target cells from the cell suspension and is used according to the purpose.

  The method of (1), for example, has two types of sperm cells with X chromosomes and Y chromosomes, and should be used in the family with severe genetic illness to distinguish between male and female. There is. When separation is performed using a slight difference in specific gravity of sperm cells, separation is performed by a velocity sedimentation method. Although this technology is versatile, the target cell is exposed to a reagent that adds specific gravity, and because it is exposed to high acceleration by centrifugation, the state of cells such as gene expression in normal cells fluctuates. There is a possibility. Furthermore, since it takes a long time for separation, the target cells are usually exposed to a low temperature of about 4 ° C. for a long time.

  The method (2) is used when there is almost no difference in specific gravity of cells, such as distinguishing between unsensitized immune-presenting cells and sensitized immune cells. It is necessary to separate, and the burden on the worker required for separation is large. Furthermore, since the target cell is stained with a fluorescently labeled antibody, the state of the target cell may vary.

  The method (3) is one of techniques for separating cells without using specific gravity. The cell sorter isolates and drops the cells after fluorescent staining in a charged droplet in units of one cell, and based on the presence or absence of fluorescence in the droplet and the amount of light scattering, In the process of falling, by applying a high electric field in any direction in the normal plane direction to the falling direction, the falling direction of the droplet is controlled and fractionated into a plurality of containers placed underneath It is technology to collect. Details of this technique are reported in Non-Patent Document 1.

  In order to culture or incubate the cells separated in this way for a specified period of time, first place a predetermined number of cells in a microplate, culture dish or culture flask to maintain the pH constant. It is common to incubate in the presence of moderate carbon dioxide. In order to dispense a predetermined number of cells, the number of cells in the cell suspension is counted in advance using a counting board or cell counter, and the volume corresponding to the desired number of cells is calculated using a microplate It is common to use a pipette.

  As a culture container, a method using a microplate is suitable for culturing a large number of cells. Culture dishes are suitable for culturing relatively few types of cells, and culture flasks are suitable for mass culture. In general, cells are put into these containers by operating a pipette while a researcher or medical worker judges. In addition, cells have an unknown community effect, and isolated cells are difficult to grow, and some cells must exist in the culture system.

Kamarck, M.E, Methods Enzymol., Vol. 151, pp. 150-165 (1987)

  The prior art does not always satisfy researchers and medical professionals as a method and apparatus for culturing separated cells simultaneously. In general, only a large number of culture flasks are arranged side by side, and the labor and cost of the culture become enormous. In addition, the number of cells to be treated for each culture is counted in advance by counting the number of cells contained in the cell suspension in advance, and then calculating the volume calculated in reverse. For this reason, for example, in order to carry out the culture with each cell as a start, it is necessary to take a method such as selecting only those that have been grown by seeding a cell suspension diluted infinitely on a microplate or dish.

  This method is effective for cloning of single-cell organisms such as E. coli and yeast, but has a problem of poor efficiency in cells derived from multicellular organisms. Furthermore, as described above, when the community effect between cells is cut off, it can be easily estimated that the behavior differs from the cells in the population. For example, every 10 cells are dispensed into a microplate well or dish. In practice, however, there is a problem that it cannot be dispensed accurately.

  An object of the present invention is to propose a method of arranging a predetermined number of cells in an array and a cell culture chip capable of simultaneously culturing a large number of cells.

  In the present invention, hydrophilic regions are independently formed at predetermined intervals on the surface of the cell culture chip so that a predetermined number of cells or cell masses can be arranged at predetermined positions on the cell culture chip. A drop of an appropriate size having the required number of cells is dropped from the tip of the pipette that sucked up the cell suspension into the hydrophilic region. At this time, the tip of the pipette is monitored by an optical system to monitor and control the size of the droplet and the number of cells.

  According to the present invention, it is possible to automatically create a droplet containing a certain number of cells in a certain minute volume and place it at a predetermined position on the cell culture chip. Therefore, a predetermined cell can be incubated in a minute volume space. Furthermore, being small is advantageous in terms of scale that allows a large number of cells to be incubated in an array in a limited area.

Example 1
FIG. 1A is a plan view of a cell culture chip 100 suitable for Example 1, and FIG. 1B is a cross-sectional view when viewed in the direction of the arrow at the AA position of the plan view. Reference numeral 1 denotes a silicon substrate, for example, having a thickness of 1 mm and a size of 20 mm × 20 mm. Reference numeral 2 denotes a wall which is made of a silicon substrate and has a thickness of, for example, 1 mm and a height of 0.5 mm. A region surrounded by the wall 2 is a hydrophobic region 3 in which hydrophilic regions 4 are periodically arranged. The size of the hydrophilic region 4 is determined by the size or number of cells accommodated in one of the regions, but is about 400 μm × 400 μm. The spacing between the hydrophilic regions 4 is set so that the droplets containing cells do not come into contact with each other and are preferably about 2000 to 4000 μm in view of handling convenience. Reference numeral 5 denotes a positioning marker, which is formed on one surface of the silicon substrate 1.

As a method of creating the hydrophilic region and the hydrophobic region, for example, the upper surface of the hydrophobic silicon substrate 1 is oxidized, and the entire region is once converted into a hydrophilic SiO ₂ thin film. Thereafter, the hydrophobic region may be created by dissolving and removing the SiO ₂ thin film in the region to be made hydrophobic with hydrofluoric acid.

(Example 2)
FIG. 2A is a conceptual diagram illustrating a system configuration of Example 2 in which cells are distributed to the cell culture chip 100. FIG. 2B is a diagram in which cells are distributed to the hydrophilic region 4 of the cell culture chip 100. It is sectional drawing which shows a result.

  In Example 2, the cells 12 are distributed to the hydrophilic region 4 of the cell culture chip 100 while optically monitoring a droplet formed at the tip of the pipette 11 for distributing the cells 12. In FIG. 2A, 19 is a stage driven in the XY directions, and 27 is a drive device for the stage 19. A heater 22 for controlling the temperature of the cell culture chip 100 is provided on the upper surface of the stage 19, and the cell culture chip 100 is placed on this upper surface. On the upper part of the cell culture chip 100, a pipette 11 in which a suspension 13 containing cells 12 to be distributed is sucked and held in advance is arranged. A syringe pump 31 is provided at the base of the pipette 11 via a tube 30, and a drive device 32 is attached to the syringe pump 31. When the syringe pump 31 is driven by the driving device 32, the suspension 13 in the pipette 11 is pushed out together with the cells 12. Note that the pipet 11 is shown as being separated from the base part of the pipette 11 and the tube 30 in order to enlarge and display the pipette 11 and is not separated.

  On the other hand, the tip of another pipette 20 for supplying a culture solution to the tip of the pipette 11 is disposed at the tip of the pipette 11. A syringe pump 35 is provided at the base of the pipette 20 via a tube 34, and a drive device 36 is attached to the syringe pump 35. When the syringe pump 35 is driven by the drive device 36, the culture solution in the syringe pump 35 is pushed out from the pipette 20.

  In addition, a pipette vertical movement drive device 37 is provided for transferring a droplet formed on the tip of the pipette 11 to the hydrophilic region 4 of the cell culture chip 100. Here, it is assumed that the vertical movement drive device 37 is linked to the pipette 11. When the user gives a signal to lower the pipette 11 to the vertical movement drive device 37, the pipette 11 moves downward, and the droplet formed on the tip of the pipette 11 is transferred to the hydrophilic region 4 of the cell culture chip 100. Transfer. When the user gives a signal for restoring the pipette 11 to the vertical movement drive device 37, the pipette 11 returns to the position shown in the figure. The recovery of the pipette 11 to the position shown in the drawing may be performed in a time sequential manner by the personal computer 26 from the lowering operation. A one-point difference line 39 means linkage between the vertical movement drive device 37 and the pipette 11.

  Further, a light source 16 and a condensing lens 17 constituting an optical system for monitoring the size of droplets formed in and near the tip of the pipette 11 are provided, and at positions facing this. A collimating lens 18 and a monitor 25 are provided below the cell culture chip 100. Therefore, the cell culture chip 100, the temperature controller 22 and the stage 19 need to be optically transparent. A so-called personal computer 26 is a control signal obtained from a predetermined program stored in advance according to an input signal from the monitor 25, and an operation input signal given by the user while viewing the display screen of the monitor 25. 28, the necessary signals are given to the driving devices 27, 32, 36 and 37. Although not shown here, it is convenient to display the same display on the monitor of the personal computer 26 as the screen detected by the monitor 25. Then, the monitor 25 can be a small CCD camera. The operation input signal 28 is given via an input device of the personal computer 26.

  Here, the size of the pipette 11 is considered as follows. The pipette 11 needs to be able to form an appropriately sized droplet having the required number of cells at its tip. On the other hand, in the pipette 11, the suspension containing the cells is used after being sucked up by the pipette. However, it is necessary that the monitor 25 can detect the cells passing through the tip of the pipette 11 without error when forming a droplet. It is. Therefore, the diameter of the tip of the pipette 11 allows one cell or a lump of a predetermined number of cells to pass, but it is assumed that cells that cannot be counted cannot pass at once. In other words, it is not as thick as the culture pipette currently used for general purposes, it is transparent and the tip diameter is 20 to 100 μm for general animal cells, and for microorganisms such as bacteria. The thickness is preferably about 5 μm.

  The operation for distributing the cells 12 to the hydrophilic region 4 of the cell culture chip 100 will be described below. First, when the system is activated, the user positions the cell culture chip 100 at a predetermined activation position while paying attention to the marker 5 described with reference to FIG. Next, the stage 19 is operated by the drive device 27 in response to an operation input signal 28 for moving the initial distribution position of the cells 12 to a position corresponding to the tip of the pipettes 11 and 20. When the cell culture chip 100 reaches a predetermined position, an operation of discharging the cell suspension 13 inside the pipette 11 together with the cells 12 is performed. At this time, the outside of the tip portion of the pipette 11 and the inside in the vicinity of the tip portion are monitored by an optical system including the light source 16 and the monitor 25. The output of the monitor 25 can be taken into the personal computer 26 and the drive device 32 can be operated based on the image calculation result of the personal computer 26 to control the liquid feeding of the syringe pump 31.

  While monitoring the tip of the pipette 11 with the monitor 25, the driving device 32 is operated, the syringe pump 31 is moved, the suspension 13 containing the cells 12 is discharged from the tip of the pipette 11, and the droplet 21 is applied to the tip of the pipette. Form. At this time, the personal computer 26 recognizes that a predetermined number of cells have been inserted into the droplet 21 through the monitor 25, issues a stop command to the drive device 32, and stops the syringe pump 31.

  Hereinafter, in order to simplify the description, the number of cells 12 inserted into the droplet 21 will be described as one, but the number of cells may be arbitrarily determined by the user according to the purpose. For example, 10 may be sufficient. The cell 12 may be recognized by directly detecting the cell 12 present in the droplet 21 at the tip of the pipette 11, but more efficiently, the cell 12 moving inside the pipette 11 is monitored by the monitor 25. Then, the position and moving speed of the cells in the pipette may be calculated by the personal computer 26, and the syringe pump 31 may be controlled by predicting the time when the cells are discharged from the tip of the pipette 11 into the droplet 21. If the latter recognition method is used, it is advantageous when only one cell is put in the droplet when, for example, a plurality of cells are moving in the pipette at short intervals.

  Here, when the cell concentration of the cell suspension 13 is low, the droplet 21 starts to form immediately before it exits from the tip of the pipette 11, and if the droplet formation is stopped after a predetermined time, the size of the droplet 21 is increased. The thickness can be made constant. When it is not desired to form droplets, for example, the liquid coming out from the tip of the pipette 11 may be blown off with a blower. Alternatively, a drain may be provided outside the substrate 1 and discharged there.

  On the other hand, when the cell concentration of the cell suspension 13 is high, the amount of liquid discharged from the pipette 11 varies. That is, since the frequency of discharging the cells 12 discharged from the pipette 11 increases, if the time for discharging the liquid is fixed at a predetermined time, the next cell enters the liquid 21 within that time. There is a possibility. In such a case, the pipette 20 is used. In the pipette 20 and the syringe pump 35 connected thereto, only the culture solution or the cell dilution solution is placed. That is, when the personal computer 26 confirms that the cell 12 enters the droplet 21 through the monitor 25, a stop command is issued to the driving device 32 to stop the syringe pump 31, and the droplet 21 is formed by this time. The volume of the droplet 21 at that time is determined from the feed amount of the syringe pump 31 that is driven. The personal computer 26 calculates the difference between this volume and the desired volume of the droplet 21. In accordance with the calculation result, an operation signal is sent from the personal computer 26 to the driving device 36 to drive the syringe pump 35 so that the culture solution or the cell dilution solution is added to the droplet 21 formed at that time with the pipette 20. The liquid is added using the pipette 20 until the volume of the droplet 21 reaches a predetermined value.

  At this time, it is preferable that the tip of the pipette 20 has a size that prevents passage of cells, for example, 0.2 μmφ so that the cells in the droplets do not flow back into the pipette 20. Alternatively, it is preferable to have a filter structure with a tip of 0.2 μm.

  The droplet 21 containing one cell thus prepared is brought into contact with the hydrophilic region 4 on the substrate 1 placed on the stage 19 by the vertical movement drive device 37 of the pipette 11, and the liquid The droplet 21 moves to the hydrophilic region 4 of the substrate 1. When it is confirmed that the droplet 21 containing the cells 12 has moved to the hydrophilic region 4 of the substrate 1, that is, the hydrophilic region 4 of the cell culture chip 100, the user gives an operation signal 28, and the stage driving device 10 is moved, and the cell culture chip 100 is moved so that the pipette tip is located at the position where the next droplet is placed. This movement can be automatically performed by the personal computer 26 if information on the arrangement of the hydrophilic region 4 is given to the personal computer 26. Then, at this new position, a new droplet is formed at the tip of the pipette 11 as described above and moved to the hydrophilic region 4 of the cell culture chip 100. By repeating this, a droplet is placed on a necessary portion of the hydrophilic region 4 of the cell culture chip 100. All these operations are performed in a humid atmosphere to prevent drying. When the arrangement of the droplets 15 is completed, the silicon oil 38 is spread over the entire area surrounded by the wall 2.

  FIG. 2B shows that the cells were distributed to the hydrophilic region 4 of the cell culture chip 100 by the system of Example 2 that distributes the cells to the cell culture chip 100 described with reference to FIG. It is sectional drawing which shows a result. Cells 12 and droplets 15 that wrap around the cells 12 are arranged in the hydrophilic region 4 in the region surrounded by the wall 2 on the silicon substrate 1. Silicon oil 38 is stretched over the entire area surrounded by the wall 2. Since the droplet 15 is about 0.2 to 2 μl, if the silicon oil 38 is put inside the wall 2 having a height of 0.5 mm, the droplet 15 is protected from drying.

  Here, the reason for using silicon oil is that silicon oil is excellent in gas permeability. Thereby, oxygen can always be supplied to the cells 12 in the droplets 15, and the cells 12 can be kept in a very small amount of culture solution. It is better that the thickness of the silicon oil is thin, and it is good that the droplet 15 is buried in the silicon oil. For example, the silicon oil is gently poured so that the depth becomes 0.5 mm. Thus, although it depends on the type and state of the cells, for example, epithelial cells can usually be observed for several hours. The cell observation may use the monitor 25 or may be transferred to another observation device.

(Example 3)
In order to incubate and observe the cells for a longer period of time, it is not sufficient to ensure oxygen permeability, and it is necessary to replace the droplets 15 enclosing the cells 12 with a new culture solution.

  FIG. 3 is a conceptual diagram for explaining the system configuration of the third embodiment focusing on the function of exchanging the droplet 15 enclosing the cells 12 with a new culture solution in the system configuration of the second embodiment. As an entity, since the pipette 20 having the system configuration of the second embodiment and the tube 34, the syringe pump 35, and the driving device 36 linked to the pipette 20 can be used, an unrelated configuration will be described with reference to FIG. To do. Of course, it is needless to say that the pipette 20, the tube 34 linked to the pipette 20, and the syringe pump 35 may be replaced with new ones from the viewpoint of preventing contamination.

  The stage 19 is moved so that the tip of the pipette 20 is at the position of the droplet 15 to be replaced with a new culture solution, and the droplet 15 of interest is monitored by the monitor 25. While monitoring the droplet 15 and the tip of the pipette 20 with the monitor 25, the pipette 20 is inserted into the droplet 15. Here, it is assumed that the vertical movement drive device 37 is linked to the pipette 20. When a signal for lowering the pipette 20 is given to the vertical movement drive device 37 by the user, the pipette 20 moves downward and the tip of the pipette 20 is inserted into the droplet 15.

  If it can be confirmed through the monitor 25 that the tip of the pipette 20 has entered the droplet 15, the user gives a signal 28 to the personal computer 26 to replace the culture medium. The personal computer 26 drives the syringe pump 36 in response to a signal 28 indicating that the culture medium is to be replaced if information on the size of the droplet 15 and the number and size of the cells contained therein is given. Then, a predetermined amount of the old culture solution is discharged (suctioned and discarded outside), and an operation for supplying a new culture solution containing a substrate or a growth factor can be automatically and time-sequentially performed. . In this case, it is important that the cells 12 contained in the droplet 15 should not be discharged together with the old culture solution, and that unnecessary bacteria are not allowed to be mixed with the new culture solution.

  For this reason, it is preferable that the tip of the pipette 20 has an inner diameter at which cells are not sucked, for example, 0.2 μm. Alternatively, it may have a filter structure with a tip of 0.2 μm. In addition, the hygiene management of the pipette 20 and the tube 34 and syringe pump 35 associated therewith is sufficiently performed.

Example 4
An operation of incubating the cells for a predetermined time, ending the observation by the monitor 25, and collecting only the predetermined cells will be described.

  FIG. 4 is a conceptual diagram illustrating the system configuration of the fourth embodiment focusing on the function of collecting the cells from the droplets 15 enclosing the predetermined cells 12 in the system configuration of the second embodiment. As the substance, since the pipette 11 having the system configuration of the second embodiment and the tube 30, the syringe pump 31, and the driving device 32 linked to the pipette 11 can be used, an unrelated one is deleted using FIG. To do. Of course, it goes without saying that the pipette 11, the tube 30 linked to this, and the syringe pump 31 may be replaced with new ones from the viewpoint of preventing contamination and the like. Further, in consideration of cell recovery, a pipette 11 having a larger size may be used.

  The stage 19 is moved so that the position of the droplet 15 containing the cell to be collected is reached, and the target droplet 15 is monitored by the monitor 25. While monitoring the droplet 15 and the tip of the pipette 11 with the monitor 25, the pipette 11 is inserted into the droplet 15. Here, it is assumed that the vertical movement drive device 37 is linked to the pipette 11. When a signal for lowering the pipette 11 is given to the vertical movement drive device 37 by the user, the pipette 11 moves downward, the tip of the pipette 11 is inserted into the droplet 15, and the cell 12 in the droplet 15 is inserted. Is sucked into the pipette 11 and collected.

  When it can be confirmed through the monitor 25 that the tip of the pipette 11 has entered the droplet 15, the user gives a signal 28 to the personal computer 26 to aspirate the cells 12 in the droplet 15. The personal computer 26 drives the syringe pump 31 in response to a signal 28 indicating that the cells 12 are sucked if information on the size of the droplet 15 and the number and size of the cells contained therein is given. Then, the operation of sucking the cells 12 together with the culture solution into the pipette 11 can be automatically and time-sequentially executed. Here, since the pipette 11 is inserted into the droplet 15 containing the cells 12 and sucked up through the silicon oil 38, some silicon oil 38 is sucked up together, but there is no problem even if ignored. It is only about the amount.

  The cells 12 sucked by the pipette 11 are discharged into a predetermined collection container, and the target cells are collected.

  When recovering the cells 12 from another droplet 15 after recovering the target cells, the stage 19 is set so that the position of the droplet 15 containing the cells to be newly recovered can be monitored by the monitor 25. The cell 12 is sucked into the pipette 11 according to the above-described procedure while the newly focused droplet 15 is monitored by the monitor 25, and discharged into a predetermined collection container to collect a new target cell.

  Here, it is as follows when the size of the pipette 11 suitable for Example 4 is examined. When creating the droplets of Example 2, the diameter of the tip of the pipette 11 should be 20 to 100 μm for general animal cells and about 5 μm for microorganisms such as bacteria. In Example 4, in consideration of taking out the cells after incubation for a predetermined time, it is necessary to have a diameter sufficient to suck up the cells that have been divided into cell clusters. Specifically, it is about 100 to 400 μm.

(Example 5)
FIG. 5A is a plan view showing another configuration example of the cell culture chip 100 of Example 5 suitable for the implementation of the present invention, and FIG. 5B is a plan view taken in the direction of the arrow at the AA position. FIG. 5C is a diagram for explaining a droplet forming method. As is clear by comparing FIG. 5A and FIG. 1A, the cell culture chip 100 of Example 5 has the same planar structure as that of Example 1. The same applies to the material, size, and manufacturing method. The cross-sectional structure of the cell culture chip 100 of Example 5 is different from that of Example 1. That is, the region surrounded by the wall 2 is a hydrophobic region 3, and the hydrophilic region 4 is formed as a well, although it does not change in that the hydrophilic region 4 is periodically arranged therein. This size is a diameter of 400 μm (or 400 μm × 400 μm) and a depth of 100 μm.

  As shown in FIG. 5C, in the fifth embodiment, silicon oil 38 is applied in advance to the region surrounded by the wall 2. The pipette 11 and the pipette 20 of Example 2 described with reference to FIG. 2 are inserted through the layer of the silicone oil 38, and the cell suspension 13 is pipetted from the pipette 11 and the diluted solution is pipetted 20 in the well 51. The droplet 21 is directly formed in the well of the hydrophilic region 4. By causing the tips of the pipettes 11 and 20 to contact the wall of the well of the substrate 1, the formed droplet 21 can be automatically formed in the well to form the droplet 15 in the second embodiment. .

  Also in the fifth embodiment, the well region where the droplet 21 is formed and the tips of the pipettes 11 and 20 need to be controlled while being monitored by the monitor 25, but can be easily understood from the description of the second embodiment. , Simplified illustration and description.

(Example 6)
In each of the above-described embodiments, one pipette is described as having one function. In the sixth embodiment, an example in which two functions are provided in one pipette will be described.

  FIG. 6A shows an example in which the tip of the pipette 81 has two flow paths by the partition plate 82, and FIG. 6B shows two flow paths as a structure having a pipette 89 inside the pipette 87. FIG.

  In the structure shown in FIG. 6A, the first flow path 83 is made sufficiently larger than the second flow path 84, and the cell suspension and the second flow path 84 are changed from the first flow path 83. By adopting a structure capable of supplying the diluent from the pipette 11, the pipette 11 and the pipette 20 described in the second embodiment can be integrated. Needless to say, each flow path is controlled by an independent fringe pump.

  In the structure shown in FIG. 6B, the inner pipette 89 has an inner diameter of 50 μm, and the distance from the outer pipette 87 to the inner wall is 8 μm at the maximum. Thus, the cell suspension is supplied from the inner pipette 87 and the diluent is supplied from the outer pipette 89, so that the pipette 11 and the pipette 20 described in the second embodiment can be integrated. Needless to say, each flow path is controlled by an independent fringe pump.

  In any case, the pipette 11 and the pipette 20 described in Example 2 are integrated with each other by setting the size on the side for supplying the diluent so that the cells do not wrap around from the side for supplying the cell suspension. Can be.

(Other)
In any of the above-described embodiments, the lower surface of the substrate 1 has means 22 for controlling the substrate temperature when cells are incubated. Since the incubation is basically performed while observing the cells with a microscope, the substrate 1 itself needs to be transparent. The heater 22 for controlling the temperature also needs to be transparent, and an ITO element is preferably used. In the case where the ITO element is not used, for example, a structure in which a transparent circulating liquid has a temperature-controlled solution flowing therein may be used. In this case, the optical system of the monitor 25 may be restricted, but this can be solved by using a long focus objective lens.

  For counting cells that move through the pipette tip, for example, a pair of electrodes is placed on the pipette tip to capture electrical changes as the cell exits the pipette, or to irradiate the tip with a laser or the like. It is also possible to detect light scattering when the cell passes and confirm that the cell is discharged from the pipette and count.

  The function of exchanging the droplet 15 enclosing the cell 12 with a new culture solution has been described with reference to FIG. 3, but if this function is used, various substances that affect the cell, for example, cell culture Substrates, growth factors, cytokines, and chemical substances such as endocrine disrupting substances can be injected into the droplets to evaluate the effects on the cells.

(A) is a top view of the cell culture chip | tip 100 suitable for Example 1, (b) is sectional drawing when it sees in the arrow direction in the AA position of a top view. (A) is a conceptual diagram explaining the system configuration of Example 2 in which cells are distributed to the cell culture chip 100, and (b) is a cross section showing a result of cells being distributed to the hydrophilic region 4 of the cell culture chip 100. FIG. It is a conceptual diagram explaining the system configuration | structure of Example 3 which paid its attention to the function which replaces | exchanges the droplet 15 which has wrapped the cell 12 for a new culture solution among the system configurations of Example 2. FIG. It is a conceptual diagram explaining the system configuration | structure of Example 4 which paid its attention to the function which collect | recovers cells from the droplet 15 which has wrapped the predetermined cell 12 among the system configurations of Example 2. FIG. (A) is a top view which shows the other structural example of the cell culture chip | tip 100 of Example 5 suitable for implementation of this invention, (b) is a cross section when it sees to an arrow direction in the AA position of a top view. FIG. 4C is a diagram for explaining a droplet forming method. (A) is a figure which shows the example by which the front-end | tip of the pipette 100 was made into two flow paths by the partition plate 101, (b) is the example made into two flow paths as a structure which has the pipette 111 inside the pipette 110. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Silicon substrate, 2 ... Wall, 3 ... Hydrophobic area | region, 4 ... Hydrophilic area | region, 5 ... Marker for arrangement determination, 12 ... Cell, 11, 20 ... Pipette, 13 ... Suspension containing the cell 12, 16 DESCRIPTION OF SYMBOLS ... Light source, 17 ... Condensing lens, 18 ... Collimating lens, 19 ... Stage, 22 ... Heater, 25 ... Monitor, 26 ... Personal computer, 27, 32, 36, 37 ... Drive apparatus, 28 ... Operation input signal, 30, 34 ... Tube, 31, 35 ... Syringe pump, 38 ... Silicone oil, 39 ... Line indicating linkage between driving device and pipette, 100 ... Cell culture chip.

Claims (9)

A pipette capable of holding a solution containing a plurality of cells and having a tip opening diameter suitable for passing a cell or cell mass of a predetermined size;
Means for observing cells at the tip of the pipette;
Means for extruding a solution containing cells at the tip of the pipette from inside the pipette to form droplets;
Means for determining that a predetermined cell is contained in the droplet and the droplet has a predetermined size;
A cell sorting device, wherein the droplets formed at the tip of the pipette are dropped and arranged at predetermined positions on the substrate.   Means for forming droplets containing a predetermined number of cells; means for controlling the size of the droplets; a substrate for arranging the droplets in an array on the substrate; A cell culture system comprising a solvent layer having a specific gravity smaller than that of a solvent of a droplet and substantially not mixed with the droplet.   Means for forming droplets containing a predetermined number of cells; means for controlling the size of the droplets; a substrate on which the droplets are arranged in an array; and a solvent for the droplets formed on the substrate. A solvent layer having a lower specific gravity and substantially not mixed with the solvent of the droplet, means for exchanging the solvent of the droplet on the substrate, means for controlling the temperature of the droplet during the cultivation of the cells, on the substrate A cell culture system comprising means for observing cells in droplets arranged in an array, and means for collecting the cells after culturing for a predetermined time.   A substrate in which one surface of a substrate having a predetermined size is made a hydrophobic region and a plurality of independent hydrophilic regions are formed in the hydrophobic region at predetermined intervals, and a substrate surrounding the plurality of hydrophilic regions A cell formed on the hydrophilic region, arranged in the hydrophilic region, and having a specific gravity smaller than the solvent of the droplet and substantially having a solvent of the droplet. A cell culture chip covered with a solvent layer that is not mixed.   The cell culture chip according to claim 4, wherein after the solvent layer is first provided, cells contained in a predetermined droplet are arranged in a plurality of hydrophilic regions of the substrate.   The means for forming a droplet containing a predetermined number of cells is arranged in such a manner that the tip of a pipette that supplies a suspension containing cells and the tip of a pipette that supplies a culture solution are attached to each other. The cell sorting apparatus according to claim 1, wherein the liquid size is controlled to control the size of the droplet.   The means for forming a droplet containing a predetermined number of cells is arranged in such a manner that the tip of a pipette that supplies a suspension containing cells and the tip of a pipette that supplies a culture solution are attached to each other. The cell culture system according to claim 2 or 3, wherein the liquid size is controlled to control the size of the droplet.   The cell sorting apparatus according to claim 6, wherein flow paths corresponding to the two pipettes are formed in one pipette. The cell culture system according to claim 7, wherein flow paths corresponding to the two pipettes are formed in one pipette.

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