JP2014168450A - Method for producing basal plate for cell array, and method for producing cell array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To correctly arrange a cell on a given position of a basal plate, and to correctly and efficiently insert a cell insertion member into a target cell.SOLUTION: A method comprises, in a pillar array which has multiple pillar shaped bodies with almost the same length on a main face, the steps for contacting BAM solution to edge faces of the pillar shaped bodies, and positioning the pillar array so that the edge faces of pillar shaped bodies are opposite to the main face of a basal plate and transferring the BAM solution attached to the edge face to the main face of the basal plate.

Description

  The present invention relates to a method for producing a cell array substrate in which a plurality of cells can be arranged on a substrate in a desired arrangement, and to produce a cell array in which a plurality of cells are arranged in a desired arrangement using the cell array substrate. Regarding the method.

  As a cell separation technique, FACS (Fluoresence activated cell sorting) is currently used. This means uses fluorescently labeled antibodies to label only the target cells in the cell sample. Fluorescence emitted from individual cells in the cell sample by irradiating laser light while flowing the cell sample as a liquid flow Is used to identify the target cells and to sort only the target cells.

  However, this means targets an antigen on the cell surface, and does not identify and separate cells using an intracellular substance such as a protein produced inside the cell as an index. In FACS, it is possible to use the protein in the cell as an index by a method of expressing a fusion protein with a fluorescent protein by genetic recombination, but for the purpose of separating natural cells for the purpose of transplantation therapy etc. Genetically modified cells are not applicable to. In addition, FACS requires a very precise control mechanism for linking the fluorescence measurement results of individual cells and sorting, and must be extremely expensive, and requires the use of fluorescently labeled antibodies. Also had a point.

  On the other hand, the present inventor has developed a method for observing the response of a cell by inserting a nano-level needle having a gene or the like fixed at the tip thereof (see Patent Document 1). In addition, a technique for lifting and separating only the target cells after inserting the needle-like material has already been presented. This method selectively binds to the marker substance by inserting a needle-like substance, for example, an antibody or other substance immobilized, that selectively binds to the intracellular marker substance, into each cell on the cell culture substrate. After binding the marker substance in the cell to the needle-like substance through the substance to be collected, the needle-like substance is pulled up, and only the cells containing the marker substance are selected and picked up and separated (Patent Document 2). reference).

  In this method, the binding force between a cell and a needle based on the binding between a marker substance in a cell and a substance that selectively binds to the marker binding substance is greater than the adhesion force of the cell to the substrate, and the antibody The non-specific adsorption force between the immobilized needles and cells that do not express the marker substance is set to be smaller than the adhesion force of the cells to the substrate, thereby identifying only specific cells that contain the marker substance. At the same time, the target cells can be picked up by the needles.

  Also, in this method, sample cells arranged on the substrate can be picked up sequentially by using a single needle-like object, or a plurality of sample cells can be picked up simultaneously by using a plurality of needle-like objects. it can. As the plurality of needle-like objects, for example, an example in which 100 × 100 needle-like objects having a diameter of about 200 nm and a length of about 20 μm are arranged at intervals of 100 μm is disclosed. Furthermore, although not described in Patent Document 2, a member that does not damage the cells to be picked up and is excellent in insertion efficiency is being studied, and is not limited to the needle-like material disclosed in Patent Document 2. Shaped cell insertion members can be used in this method.

  As described above, the cell array formed by adhering cells on the substrate is a use form in which nano-level needles having genes fixed at the tip are inserted into the cells, and marker substances by fixing the marker binding substance at the tip. It is utilized for the usage pattern etc. which pick up the cell containing sucrose.

JP 2003-325161 A JP 2011-182761 A

  However, in various usage forms of the cell array as described above, in order to insert a cell insertion member such as a needle-like object into a target cell accurately and efficiently, it is important to accurately arrange the cells on the substrate. It becomes. Therefore, the present invention provides a method for producing a cell array substrate that can accurately place cells at a predetermined position on the substrate, and can accurately and efficiently insert a cell insertion member into a target cell, and An object of the present invention is to provide a method for producing a cell array.

  As a result of intensive studies by the present inventors to achieve the above-described object, a region in which the solution is transferred by transferring the cell membrane modifier solution to one main surface of the substrate using a pillar array having a plurality of columnar bodies A method for allowing cells to adhere to each other has been developed, and it has been found that a plurality of cells can be accurately arranged at predetermined positions on the substrate, and the present invention has been completed. The present invention includes the following.

  (1) In a pillar array having a plurality of columnar bodies having substantially the same length on one main surface, a step of bringing a cell membrane modifier (Biocompatible Anchor for Membrane) solution into contact with the end surface of the columnar body; The pillar array is positioned so that the end faces of the columnar bodies face each other, and the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution attached to the end faces is transferred to one main surface of the substrate. Method.

  (2) The arrangement of the plurality of columnar bodies is a cell insertion member in which a plurality of needle-like objects are arranged on a support, and is close to a cell array formed by adhering cells to a cell array substrate. The method for producing a cell array substrate according to (1), which corresponds to the arrangement of the plurality of needles in a cell insertion member capable of inserting the needles into cells.

  (3) The pillar array is obtained by patterning a plurality of substantially circular resist films on one main surface of a substrate made of an IV type semiconductor material, and forming the plurality of columnar bodies by dry etching. (1) The method for producing a cell array substrate according to (1).

  (4) The cell array substrate according to (1), wherein a bovine serum albumin film is formed in advance on one main surface of the substrate to which the cell membrane modifier (Biocompatible Anchor for Membrane) solution is transferred. Manufacturing method.

  (5) The method for producing a cell array substrate according to (1), wherein the cell adhesive force is adjusted by the concentration of the cell membrane modifying agent contained in the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution.

  (6) The method for producing a cell array substrate according to (1), wherein the cell membrane modifier (Biocompatible Anchor for Membrane) solution contains glycerin.

  (7) In a pillar array having a plurality of columnar bodies having substantially the same length on one main surface, a step of bringing a cell membrane modifier (Biocompatible Anchor for Membrane) solution into contact with the end surface of the columnar body; The pillar array is positioned so that the end surfaces of the columnar bodies face each other, and the cell membrane modifier (Biocompatible Anchor for Membrane) solution attached to the end surfaces is transferred to one main surface of the substrate; And a step of adhering cells to a region where the cell membrane modifier (Biocompatible Anchor for Membrane) solution is transferred by contacting the cell suspension.

  (8) In the step of adhering the cells, after the cell suspension is brought into contact with one main surface of the substrate, a process of removing cells that have not adhered to the substrate from the substrate is performed ( 7) The manufacturing method of the cell array of description.

  (9) The arrangement of the plurality of columnar bodies is a cell insertion member in which a plurality of needle-like objects are arranged on a support, and the needle-like objects are placed on a substrate by being close to the cell array. (7) The method for producing a cell array according to (7), which corresponds to the arrangement of the plurality of needles in a cell insertion member that can be inserted into the adhered cells.

  (10) The pillar array is obtained by patterning a plurality of substantially circular resist films on one main surface of a substrate made of an IV type semiconductor material, and forming the plurality of columnar bodies by dry etching. (7) The method for producing a cell array according to (7).

  (11) The production of the cell array according to (7), wherein a bovine serum albumin film is formed in advance on one main surface of the substrate onto which the cell membrane modifier (Biocompatible Anchor for Membrane) solution is transferred. Method.

  (12) The method for producing a cell array according to (7), wherein the cell adhesive force is adjusted by the concentration of the cell membrane modifying agent contained in the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution.

  (13) The method for producing a cell array according to (7), wherein the cell membrane modifier (Biocompatible Anchor for Membrane) solution contains glycerin.

  The method for producing a cell array substrate according to the present invention uses a pillar array having a plurality of columnar bodies to transfer a cell membrane modifier (Biocompatible Anchor for Membrane) solution to a predetermined position on one main surface of the substrate. Thereby, according to the manufacturing method of the substrate for cell arrays which concerns on this invention, the substrate for cell arrays which can arrange | position a cell in the predetermined position in a board | substrate can be manufactured.

  The cell array manufacturing method according to the present invention uses a pillar array having a plurality of columnar bodies to transfer a cell membrane modifier (Biocompatible Anchor for Membrane) solution to a predetermined position on one main surface of the substrate. Thereby, according to the manufacturing method of the cell array which concerns on this invention, the cell array which has arrange | positioned the cell in the predetermined position in a board | substrate can be manufactured.

It is a perspective view which shows the outline of a pillar array. It is a manufacturing method of the substrate for cell arrays, and is a perspective view showing the process of dropping a cell membrane modifier solution on a substrate. It is a manufacturing method of the substrate for cell arrays, and is a perspective view which shows the process of spin-coating the dripped cell membrane modifier solution. It is a manufacturing method of the substrate for cell arrays, and is a perspective view showing the process of forming the coat of the cell membrane modifier solution on the substrate. It is a manufacturing method of the substrate for cell arrays, and is a perspective view showing the process of making the pillar array face the coating of the cell membrane modifier solution. It is a manufacturing method of the substrate for cell arrays, and is a perspective view which shows the process which made the pillar array contact the film of a cell membrane modifier solution. It is a manufacturing method of the cell array substrate, and is a perspective view showing a process in which a pillar array in a state where a cell membrane modifying agent solution is held on an end face of a columnar body is opposed to a cell culture dish. It is a manufacturing method of the cell array substrate, and is a perspective view showing a process in which an end surface of a columnar body holding a cell membrane modifier solution is brought into contact with a cell culture petri dish. It is a perspective view of the cell array substrate manufactured by the manufacturing method of the cell array substrate. It is a manufacturing method of a cell array, Comprising: It is a perspective view which shows the process of dripping a cell suspension on the cell array substrate. It is a manufacturing method of a cell array, Comprising: It is a perspective view which shows the process of making a cell adhere to a cell array substrate. It is a perspective view of the cell array manufactured by the manufacturing method of a cell array. It is a perspective view of the board | substrate used for a micro pillar array. It is a perspective view which shows the state which formed the resist film into the board | substrate. It is a perspective view which shows the state which positioned the photomask so as to oppose a resist film. It is a perspective view which shows the state which formed the pattern corresponding to a photomask in the resist film. It is a perspective view which shows the state which formed the resist film of the predetermined shape on the board | substrate by developing. It is a perspective view which shows the state which formed the micro pillar by dry etching. It is a perspective view of the micro pillar array produced by removing a resist film. It is a conceptual diagram showing the aspect of the pick-up separation of the target cell using a cell insertion member. It is a characteristic view showing the result of measuring the average adhesion force of cells to a substrate when using three types of BAM ink liquids with BAM concentrations of 0.5, 1, and 10 mM. It is a SEM photograph of a micro pillar array. The top is a micro pillar array with a diameter of 2 μm, a length of 20 μm and a pitch of 30 μm, and the bottom is a micro pillar array with a diameter of 5 μm, a length of 20 μm and a pitch of 30 μm. It is the scanning electron micrograph which image | photographed the cell insertion member inclining 30 degrees from horizontal. The top image is 300 times and the bottom image is 15000 times. The scale bars are 100 μm and 3 μm, respectively.

  A method for producing a cell array substrate and a method for producing a cell array according to the present invention will be described in detail with reference to the drawings.

  The cell array substrate means a substrate to which cells can be adhered, and the cell array means a substrate in which cells are adhered to the cell array substrate. That is, the cell array substrate is different from a cell array in which cells are not adhered yet and in a state where cells are adhered. Here, the cells that adhere to the cell array substrate are not particularly limited, and examples thereof include animal cells derived from mammals such as humans, mice, rats, and monkeys.

  As shown in FIG. 1, the method for manufacturing a cell array substrate according to the present invention uses a pillar array 2 having a plurality of columnar bodies 1 having substantially the same length on one main surface. In addition, arrangement | positioning of the some columnar body 1 in the pillar array 2 respond | corresponds to arrangement | positioning of the cell adhesion area | region in the board | substrate for cell arrays. For example, when the shape of the columnar body 1 is a cylinder having a diameter of approximately 1 to 4 μm, the interval between adjacent columnar bodies 1 is, for example, 25 to 150 μm, preferably 28 to 100 μm, and more preferably 30 to 50 μm. be able to. That is, the columnar bodies 1 are arranged in a 10 mm square region, for example, 4,000 to 160,000, preferably 10,000 to 120,000, more preferably 40,000 to 110,000, and aligned in the XY direction. Can do. However, the number and arrangement of the plurality of columnar bodies 1 constituting the pillar array 2 are not limited at all.

  Note that setting the interval between adjacent columnar bodies 1 to 30 μm, for example, means that the interval between cells adhered to the cell array substrate is approximately 30 μm. Usually, the diameter of the floating somatic cell is about 10 μm, and if the distance between the cells adhering to the cell array substrate is about 30 μm, adhesion between adjacent cells can be reliably prevented.

  In the pillar array 2, since the plurality of columnar bodies 1 have substantially the same length from one main surface, the flat plate can be brought into contact with the end surfaces of these columnar bodies 1 with almost no gap. In addition, the end surface of the columnar body 1 may be a flat surface, may be subjected to a rough surface treatment so as to have a predetermined roughness, or a groove having a predetermined shape may be formed. Moreover, the planar shape of the end surface of the columnar body 1, that is, the cross-sectional shape of the columnar body 1 is not particularly limited, and may be a circle or a polygon such as a triangle or a rectangle.

  In the method for producing a cell array substrate according to the present invention, first, a cell membrane modifier (Biocompatible Anchor for Membrane) solution is brought into contact with the end face of the columnar body 1 in the pillar array 2 shown in FIG. As a result, a predetermined amount of the cell membrane modifier solution is held on the end surfaces of the plurality of columnar bodies 1. Next, one main surface of the substrate is brought into contact with the end surface of the columnar body 1 while the cell membrane modifier solution is held on the end surface of the columnar body 1. Thereby, the cell membrane modifier solution held on the end face of the columnar body 1 can be transferred to one main surface of the substrate. In this manner, a plurality of regions to which the cell membrane modifier solution is transferred corresponding to the arrangement of the plurality of columnar bodies 1 constituting the pillar array 2 are formed on one main surface of the substrate. Through at least the above steps, a cell array substrate in which a plurality of regions having the cell membrane modifier solution transferred thereon is formed on one main surface of the substrate can be produced. The substrate used as the base of the cell array substrate is not particularly limited. For example, a petri dish used for cell culture, other glass substrates, and a resin substrate can be appropriately used.

  A more detailed method for producing a cell array substrate will be described with reference to FIGS. First, as shown in FIG. 2, a cell membrane modifier solution containing SUNBRIGHT OE-020CS (manufactured by NOF Corporation) having a succinimide group, isopropanol, glycerin and water as a cell membrane modifier is dropped onto the glass substrate 10. Here, the cell membrane modifier solution is particularly preferably a composition containing glycerin. When the cell membrane modifier solution contains glycerin, the cell membrane modifier solution can be prevented from drying in the treatment described below. In addition, the dry prevention of the cell membrane modifier solution is not limited to glycerin, and various compounds having a water retention effect such as other polysaccharides such as hyaluronic acid and betaine can be used. The cell membrane modifying agent solution contains fluorescein, which is a fluorescent reagent, so that the pattern when transferred to the cell array substrate can be confirmed based on the fluorescein.

  Next, as shown in FIG. 3, a coating 11 of the cell membrane modifier solution is formed on the glass substrate 10 by a so-called spin coating method (FIG. 4). Next, as shown in FIG. 5, the end surface of the columnar body 1 in the pillar array 2 is opposed to the surface of the glass substrate 10 on which the coating film 11 of the cell membrane modifier solution is formed. Next, as shown in FIG. 6, the pillar array 2 and / or the glass substrate 10 are moved in the approaching direction, and the end surface of the columnar body 1 is brought into contact with the coating 11 of the cell membrane modifier solution. The pillar array 2 and / or the glass substrate 10 may be moved until the end face of the columnar body 1 comes into contact with the glass substrate 10. Through the above steps, the cell membrane modifier solution can be held on all end faces of the plurality of columnar bodies 1.

  Next, as shown in FIG. 7, the end surface of the columnar body 1 in the pillar array 2 is opposed to the cell culture petri dish 12 serving as the base of the cell array substrate, with the cell membrane modifier solution held on the end surface of the columnar body 1. Let In addition, in the petri dish for cell culture 12, one main surface facing the end face of the columnar body 1 is previously filled with a 1 mg / mL bovine serum albumin BSA solution to form a BSA coating. Next, as shown in FIG. 8, the pillar array 2 and / or the cell culture petri dish 12 is moved in the approaching direction, and the end surface of the columnar body 1 is brought into contact with the BSA coating formed on the cell culture petri dish 12. The pillar array 2 and / or the cell culture petri dish 12 may be moved until the end surface of the columnar body 1 contacts the cell culture petri dish 12. Then, by separating the pillar array 2 and the cell culture petri dish 12, the cell membrane modifier solution held on the end surface of the columnar body 1 can be transferred to one main surface of the cell culture petri dish 12. Thereafter, the succinimide group in the cell membrane modifying agent and BSA can be reacted by allowing to stand at room temperature for about 60 minutes.

  As described above, a cell array substrate having a plurality of cell adhesion regions 13 having the cell membrane modifier solution transferred onto one main surface of the cell culture petri dish 12 as shown in FIG. 9 can be produced. The manufactured cell array substrate can adhere cells to a plurality of cell adhesion regions 13 by, for example, processes shown in FIGS. In other words, a cell array can be manufactured according to the process shown in FIGS. 10-12 using the manufactured cell array substrate.

  Specifically, first, as shown in FIG. 10, the cell suspension is brought into contact with one main surface of the cell array substrate on which the cell adhesion region 13 is formed. For example, when the cell culture petri dish 12 is used as the cell array substrate, the cell suspension is filled in the cell culture petri dish 12. Then, for example, shake at 10 rpm for 10 minutes. Thereby, as shown in FIG. 11, the cells 14 can be adhered to the cell adhesion region 13 of the cell array substrate. Then, as shown in FIG. 12, by removing the cells 14 that have not adhered to the cell adhesion region 13 of the cell array substrate, a plurality of cells 14 are adhered according to the arrangement of the cell adhesion region 13, and the cell array 15 can be manufactured.

  In the cell array 15 manufactured as described above, the number of cells to be adhered can be adjusted by the size of the cell adhesion region 13. For example, when it is desired to adhere one cell, the diameter of the region 13 is, for example, 1 to 5 μm, preferably 1.5 to 4 μm, more preferably 2 to 3 μm.

  In the above-described example, since the BSA coating is formed on the cell culture dish 12, the BSA coating is exposed in regions other than the cell adhesion region 13 to which the cell membrane modifier solution has been transferred. Since BSA can suppress cell adhesion, in the above-described example, it is possible to prevent cell adhesion to other than the cell adhesion region 13 to which the cell membrane modifier solution is transferred. In the above-described example, for example, even after 24 hours of incubation, the cells adhered to the cell adhesion region 13 can be prevented from moving to a region other than the cell adhesion region 13.

  On the other hand, a coating for promoting cell adhesion may be formed on one main surface of the cell culture petri dish 12 instead of the BSA coating. Examples of the coating that promotes cell adhesion include a fibronectin coating, a collagen coating, and a polylysine coating. By forming such a coating that promotes cell adhesion, the cells are temporarily transferred to regions other than the cell adhesion region 13 with weaker adhesion than the cell adhesion region 13 to which the cell membrane modifier solution is transferred. Can be glued. For example, culture cells can form a normal adhesion state in a region other than the cell adhesion region 13 by performing incubation for 24 hours after the cells are adhered to the cell adhesion region 13.

  The shape and size of the cell adhesion region 13 can be understood from the above-described manufacturing process of the cell array substrate, and the shape and size of the end surface of the columnar body 1 in the pillar array 2 and the cell membrane modifier solution. Depends on viscosity etc. Therefore, by adjusting the shape and size of the end face of the columnar body 1 and the viscosity of the cell membrane modifier solution, the cell adhesion region 13 can be made to have a desired shape and size.

  The arrangement of the plurality of cell adhesion regions 13 in the cell array substrate corresponds to the arrangement of the plurality of columnar bodies 1 in the pillar array 2 as can be understood from the manufacturing process of the cell array substrate described above. Therefore, the positions of the plurality of cell adhesion regions 13 on the cell array substrate, that is, the cells 14 adhered to the cell array substrate can be adjusted by the arrangement of the plurality of columnar bodies 1 in the pillar array 2.

  The cell array 15 formed by adhering a plurality of cells 14 is not particularly limited. For example, the cell array 15 can be used in a cell separation device disclosed in Japanese Patent Application Laid-Open No. 2011-182761. That is, the cell separation device disclosed in Japanese Patent Application Laid-Open No. 2011-182761 includes a plurality of needle-like objects on which a marker binding substance is immobilized, and inserts needle-like objects into a plurality of cells adhered to a substrate, respectively. To do. In this separation device, when a needle-like object is inserted into a cell having a marker substance, the marker substance and the marker-binding substance are bound to each other, but the adhesive force of the cell to the substrate is the above needle-like shape when the marker substance is bound. It is set smaller than the binding force between objects and cells. Therefore, in this separation apparatus, after inserting the needle-like object into the cell, the needle-like object is pulled up, whereby only the cell having the marker substance can be peeled from the substrate and separated from the cell not having the marker substance.

  The cell array 15 described above can be used in this separation apparatus. In particular, the cell separation efficiency can be greatly improved by adhering the cells to positions corresponding to the arrangement of the plurality of needles provided in the separation device. That is, it is preferable to use the pillar array 2 used when producing the cell array substrate, which has a plurality of columnar bodies 1 arranged so as to coincide with the arrangement of the plurality of needle-like objects provided in the separation device. . Thereby, the cell array can adhere | attach the cell 14 so that it may correspond with arrangement | positioning of the some acicular object with which a separation apparatus is equipped. For example, when a needle-like object having a diameter of 200 nm is produced from the pillar array 2 by a method to be described later, it is possible to position the cell and the needle-like object adhered to the cell array substrate very accurately. For example, when a needle-like object having a diameter of 200 nm is produced from the pillar array 2 by the method described later, the position of the center of the cell adhered to the cell array substrate and the tip of the needle-like object is within a range of about 5 μm which is the radius of the cell. Can fit in.

  The cell array 15 described above is not limited to the cell separation device disclosed in Japanese Patent Application Laid-Open No. 2011-182761, but may be applied to a separation device of a type in which needles inserted into cells are improved. it can. As an example, a separation apparatus using a nanoneedle array in which a large number of needles having a characteristic shape are arranged can be mentioned.

  Here, the nanoneedle (needle) of the nanoneedle array is integrally processed with the support, and the arrangement density of the nanoneedle can be variously changed according to the arrangement state of the cells on the culture substrate. In the case where one nanoneedle is inserted into each animal cell, for example, the number is 10,000 to 50,000 in a 10 mm square support. Each nanoneedle has a length (height from the support surface) of 10 to 50 μm, and a diameter of 200 to 300 nm in a ½ length portion of the needle-like material. The shape of this nanoneedle is a bat-like shape of a thick baseball, and the maximum diameter of the nanoneedle is 10 to 30% larger than the diameter of the ½ length of the needle-like object. Yes.

  Efficient cell insertion is determined by the diameter and length of the needle. When the diameter of the needle-like material is 100 to 400 nm and the length is 10 μm or more, a high insertion efficiency of 80% or more can be obtained for somatic cells derived from various animals such as humans, mice, and rats. The maximum diameter of the needle is 400 nm or less, and high insertion efficiency can be achieved. Note that the insertion efficiency refers to the achievement rate of the needle-like material penetrating through the cell membrane with respect to the number of trials of the needle-like object approaching the cell.

  In addition, one nanoneedle may be arranged for each cell on the culture substrate, but two or three nanoneedles are arranged corresponding to the arrangement interval of each cell. May be. In particular, when three are arranged, it is preferable to arrange them so as to form a triangle, preferably an equilateral triangle, with respect to the culture substrate surface.

  According to such an arrangement of nanoneedles, it is possible to simultaneously measure data at a plurality of different locations within the same cell as a probe for cell analysis, and to exhibit a function of preventing lateral shift during cell insertion operation. In addition, when the target cells are lifted and separated, the nanoneedle also contributes to an increase in cell retention.

  Such a nanoneedle array is obtained by a manufacturing method including the following steps a) to d).

a) Photoresist Coating Step First, as shown in FIG. 13, a substrate 20 made of an IV type semiconductor material is prepared. Next, as shown in FIG. 14, one main surface of the substrate 20 is coated with a photoresist to form a resist film 21. As the photoresist (photosensitive resin), either a positive type or a negative type can be used, and examples of the IV type semiconductor substrate 20 include those made of inorganic materials such as Si, SiC, and Ge.

b) Mask Pattern Development Step Next, as shown in FIG. 15, a photomask 22 having a mask pattern printed thereon is placed on the substrate 20 coated with the above-described photoresist and irradiated with light. The photomask 22 has an independent circular repetitive pattern 23 having a diameter of 1 to 4 μm on a transparent material such as synthetic quartz glass, and the position of each circular portion in the pattern 23 depends on the cell on the culture substrate to be used. What is necessary is just to correspond to a position, and although it is not decided, what is necessary is just to set the space | interval of each circular part to 50-100 micrometers normally.

  As the photomask 22, for example, one containing chromium, chromium oxide, emulsion or the like can be used. When a positive photoresist is used, the mask agent is applied to each circular portion of the photomask. When a negative photoresist is used, the mask agent is applied to other regions while leaving each circular portion.

  As the light irradiation means, a mask aligner, a stepper, or the like can be used. By irradiating the resist film 21 with light through the photomask 22, only the region 24 corresponding to the pattern 23 is cured as shown in FIG. Thereafter, when the resist film 21 is developed using a developer, only the cured region 24 remains on the substrate 20 as shown in FIG. Here, as the developer, an alkaline solution such as TMAH (tetramethylammonium hydroxide) is used when a positive photoresist is used, and an organic solvent is used when a negative photoresist is used. With such a developer, in the case of the positive resist film 21, the masked circular portion remains and other areas are removed, and in the case of the negative resist film 21, the masked area is removed and the mask is removed. The circular part that was not done remains. In any case, an independent circular repetitive pattern (region 24) is drawn by the photoresist remaining on the silicon substrate 20.

c) Dry Etching Step The substrate 20 on which this repetitive pattern (region 24) is drawn digs up a region other than the portion corresponding to the circular portion drawn by the photoresist, as shown in FIG. 18, by dry etching. In dry etching, for example, an inductively coupled plasma etching apparatus is used to store the substrate on which the pattern is drawn in the apparatus, and etching gas and protective gas are repeatedly introduced into the apparatus alternately (for example, 60 to 120 cycles). This is done by etching. As the etching gas, for example, an etching gas such as SF ₆ , IF ₅ , CHF ₃ , or CF ₄ is used, and C ₄ H ₈ is used as the protective gas for protecting the etching sidewall. Other dry etching apparatuses that can be used include an inductively coupled plasma (ICP) dry etching apparatus.

  Next, the resist film 21 is removed by an asher from the obtained micro-apillar. Thereby, as shown in FIG. 19, the micro pillar array 26 which has the several pillar 25 with a diameter of 2-3 micrometers can be produced. In addition, it is preferable to use the micro pillar array 26 produced here as the pillar array 2 at the time of manufacturing the substrate for cell arrays mentioned above. That is, the plurality of columnar bodies 1 in the pillar array 2 described above correspond to the pillars 25 in the micro pillar array 26 shown in FIG.

d) Wet etching step After removing the resist film 21 as described above, it is thermally oxidized under wet conditions of 1100 ° C. or more for 10 to 20 hours to form an oxide layer, and then a hydrofluoric acid-based wet etching agent is used. By performing wet etching, a nanoneedle array having the above-described characteristic structure can be manufactured. Here, as the hydrofluoric acid-based wet etching agent, for example, hydrofluoric acid or buffered hydrofluoric acid in which hydrofluoric acid and ammonium fluoride are mixed can be used. The above-mentioned unique tapered shape of the nanoneedle is obtained for the first time by adding such a wet etching process.

  The nanoneedle array produced as described above should be used to detect and measure the expression state of genes in each cell, for example, by immobilizing genes or substances involved in gene expression to each nanoneedle. Can do. The above-mentioned genes are fixed to each nanoneedle of the nanoneedle array, and the nanoneedle array is aligned and lowered so that each nanoneedle matches the position of each cell adhering to the cell array. Needles can be inserted into individual cells simultaneously.

  In particular, by using the micro-pillar array 26 in the middle of producing the nanoneedle array as the pillar array 2 described above, it is possible to produce a cell array substrate and a cell array that are optimal for the nanoneedle array. Further, in order to produce a cell array substrate and cell array that are optimal for a nanoneedle array, a pillar array 2 is separately produced using the photomask 22 used for producing the nanoneedle array. A cell array substrate and a cell array may be produced by using them.

  Examples of immobilization of genes or substances involved in gene expression include physical adsorption, use of avidin biotin bonds using immobilized avidin, etc. For example, when the material is silicon-based, the surface of the nanoneedle After the oxidation treatment, a functional group can be formed using various silanizing agents, and the functional group can be used to immobilize with a covalent bond.

  Detection of the gene expression state in each cell may be performed separately for each cell on the substrate after the nanoneedle array is pulled up. For detection and measurement of the gene expression state, for example, a molecular beacon is used. If a functional probe is modified on the surface of a needle-like object, gene expression in the cell is measured while the needle-like object is inserted into the cell, and a large number of cells are analyzed simultaneously over time. be able to.

  The molecular beacon is a DNA having a stem-and-loop structure having a fluorescent substance and a quencher at both ends, and has a sequence capable of hybridizing with a gene to be analyzed in the loop portion.

  The immobilized gene is, for example, a cancer growth-suppressing gene. If a cell insertion test is performed in the presence of a test drug and the immobilized marker gene is expressed, the drug can be evaluated as having an effect. . In addition, each gene involved in differentiation can be immobilized on a nanoneedle, and the change in the expression state of each gene involved in differentiation can be measured in the presence of a test drug. Similarly, for example, when a substance involved in the expression of a differentiation gene is immobilized, how the expression state of the differentiation gene is changed by the substance can also be detected and measured.

  For modification of molecular beacons, for example, biotinylated molecular beacons are used. The needle-like surface is biotinylated by chemical modification using a silane coupling reaction, and the biotinylated molecular beacon is immobilized by binding via streptavidin. When a molecular beacon-modified needle is inserted into a cell, if the mRNA that is the target of the molecular beacon is expressed in the cell, the fluorescence on the surface of the needle increases due to hybridization with the molecular beacon. By capturing and analyzing this fluorescence using a confocal fluorescence microscope, it is possible to detect and measure intracellular mRNA.

  On the other hand, the nanoneedle array described above is particularly useful as a cell separation member for selectively separating target cells from a large number of cells.

  An overview of cell separation using these is shown in FIG. When a nanoneedle array is used as a cell separation member, a substance that selectively binds to a marker substance in a cell, such as an antibody, is immobilized on each nanoneedle. The immobilization method is as described above. Next, after positioning the nanoneedle array so that each nanoneedle corresponds to each cell on the cell array, the nanoneedle array is lowered and inserted into each nanoneedle and each cell on the substrate, and the marker substance After the intracellular marker substance is bound to the nanoneedle via the selectively binding substance, the nanoneedle array is pulled up, and only the cells containing the marker substance are selected and separated. The insertion of the nanoneedle into these many cells and the lifting of the target cell are performed by once lowering and raising the nanoneedle array.

  The binding force between the nanoneedle and the cell through the binding between the marker substance and the marker binding substance is based on the increase in the amount of the marker binding substance immobilized, the selection of the immobilizing agent, or the nanoneedle residence time after cell insertion. It is possible to adjust to 5 nN or more by adjusting. Furthermore, when a plurality of nanoneedles are arranged for each cell described above, it is possible to increase the holding power for the cells during lifting.

  In general, the adhesive force between the cell and the substrate is usually 20 to 40 nN, and the non-specific adsorption force between the needle-like material and the cell not expressing the marker substance is usually about 200 pN. The lower limit of the adhesive strength between the cell culture substrate and the cell culture substrate is more than 200 pN, preferably 500 pN or more, and the upper limit is at least less than 5 nN, preferably 3 nN or less, more preferably 2 nN or less. The regulation of this adhesive force can be achieved by proteolytic enzymes such as trypsin, ROCK (Rho-associated coiled-coil forming kinase) inhibitors, butyryl oxazolidinone compounds, collagenase inhibitors, elastase inhibitors, fibronectin binding inhibitors, etc. Can be achieved by treating cells with an inhibitor of adhesion protein expression. Moreover, the method of coating a culture substrate with the film formation agent which consists of a mixture of BSA (bovine serum albumin) and BAM (Biocompatible Anchor for Membrane) -BSA complex containing a chelating agent can also be adopted. In this film-forming agent, the cell and the substrate can be adjusted to a desired adhesive force by changing the mixing ratio of BSA and BSA-BAM complex.

  The shape of this nanoneedle is a bat-like shape of a thick baseball, and the maximum diameter of the nanoneedle is 10 to 30% larger than the diameter of the ½ length of the needle-like object. Yes. Nanoneedle with such a shape is more difficult to drop off cells when lifting cells than normal tapered needles, and the amount of marker immobilization can be increased. Can be suppressed. In addition, the size of animal cells is usually 10 to 30 μm, and there is almost no increase in damage to the cells when the diameter on the tip side is increased.

  As described above, it is possible to pick up cells by using nanoneedles, but in particular, it can also be used for transporting / introducing substances to cells adhered to a cell array substrate. . That is, a substance (for example, a functional molecule such as DNA or protein) is attached to the tip of each nanoneedle, and the nanoneedle is inserted into a plurality of cells adhered to the cell array substrate. It is also possible to transport and introduce the substance at the same time. In this case, it is preferable that the cells adhered to the cell array substrate are adhered so as not to be lifted from the substrate when the inserted needle is pulled out. That is, in this case, it is preferable to set the adhesive force between the cell array substrate and the cells to be stronger than that in the case where the cells are picked up. In other words, when the cells are adhered to the cell array substrate, it is desirable to control the adhesive force to an arbitrary level according to the usage form.

  In addition, as described above, by attaching cells in an accurate arrangement to the cell array substrate, preferably by attaching cells with an appropriate adhesive force, only cell operations using needles can be performed. It can also be used as a cell array for other cell analysis and cell manipulation such as imaging cytometry.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the technical scope of this invention is not limited to a following example.

  In this example, first, a cell culture dish was filled with a 1 mg / mL BSA solution to prepare a BSA coating. For cell membrane modification (BAM), SUNBRIGHT OE-020CS (manufactured by NOF Corporation) having a succinimide group was used. A BAM ink solution for contact printing was prepared using a mixed solution of isopropanol, glycerin, water and DMSO (dimethyl sulfoxide) as a solvent. In this example, the BAM ink liquid contained a fluorescent reagent fluorescein for pattern confirmation. That is, in this example, a BAM ink liquid was prepared so as to have the composition shown in Table 1. That is, in this example, three types of BAM ink solutions having BAM concentrations of 0.5, 1, and 10 mM were prepared.

  Next, the prepared BAM ink liquid was spin-coated on a glass substrate to produce an ink liquid thin film. A micro pillar array was brought into contact with this BAM ink liquid thin film, and the ink liquid was applied only to the tip of the micro pillar. The BAM ink solution was transferred by bringing the micropillar array into contact with a petri dish for cell culture on which a BSA coating was formed. Due to the reaction between the succinimide group of BAM and BSA, it was left at room temperature for 60 minutes after the transfer. Thereafter, the cell suspension was filled in a petri dish and shaken at 10 rpm for 10 minutes to form a cell array in which cells were arranged on a dot pattern. Excess floating cells that were not arranged were removed. In this example, mouse fibroblast NIH3T3 was used as the cell.

  For three types of BAM ink solutions (BAM concentration: 0.5, 1, 10 mM), the adhesion of the arrayed cells was measured. Specifically, the maximum value of the force curve when the cells were forcibly detached from the substrate was measured as an adhesive force using an AFM probe (NanoWorld) processed into a saddle shape. JPK NanoWizard II was used for the measurement. The measurement results are shown in FIG. The average adhesive strengths of the three types of BAM ink solutions having BAM concentrations of 0.5, 1, and 10 mM were 11 nN, 36 nN, and 200 nN, respectively.

  In addition, the micro pillar array used in this example was manufactured as follows. First, HMDS was spin-coated as a photoresist adhesion promoter on a silicon wafer having a diameter of 4 inches and a thickness of 0.4 mm, and then a positive photoresist TSMR-V90 was spin-coated. Using a photomask in which circular mask patterns (chrome) having a diameter of 2 μm were arranged on a glass substrate at intervals of 30 μm, a pattern was formed on the resist by light irradiation with a mask aligner and development with 2.38% NMD-3.

Thereafter, by removing the portion where the photoresist has been removed by dry etching using an inductively coupled plasma (ICP) dry etching apparatus MUC-21 (STS), pillars having a diameter of 2 to 3 microns are arranged. A fabricated substrate (micropillar array) was produced (for example, the upper photograph in FIG. 22). As the conditions of the introduced gas in the etching, etching SF ₆ (450 standard cc / min) for 3 seconds and protective C ₄ F ₈ (135 standard cc / min) for 2 seconds were repeated 60 to 120 cycles.

  A nanoneedle array can be produced from the micropillar array produced in this way as follows. That is, after removing the photoresist with an asher, the micro pillar array is thermally oxidized at 1100 ° C. for 20 hours under wet conditions to produce an oxide layer, and then wet etching with 20% buffered hydrofluoric acid is performed. Was removed. by this. A nanoneedle array in which 10,000 or more nanoneedles having a diameter of 300 nm or less and a length of 10 μm or more were arranged in a 10 mm square was obtained (FIG. 23). It has been confirmed that the produced nanoneedle array is efficiently inserted when an approaching operation is performed on cultured cells.

DESCRIPTION OF SYMBOLS 1 ... Columnar body, 2 ... Pillar array, 10 ... Glass substrate, 11 ... Coating, 12 ... Petri dish for cell culture, 13 ... Cell adhesion area | region, 14 ... Cell, 15 ... Cell array

Claims (13)

In a pillar array having a plurality of columnar bodies having substantially the same length on one main surface, a step of bringing a cell membrane modifier (Biocompatible Anchor for Membrane) solution into contact with the end surface of the columnar body;
Positioning the pillar array on one main surface of the substrate so that the end surface of the columnar body faces, and transferring the cell membrane modifier (Biocompatible Anchor for Membrane) solution adhering to the end surface to the one main surface of the substrate. A method for producing a cell array substrate.   The arrangement of the plurality of columnar bodies is a cell insertion member in which a plurality of needle-like objects are arranged on a support, and is arranged in proximity to a cell array formed by adhering cells to a cell array substrate. 2. The method for producing a cell array substrate according to claim 1, wherein the method corresponds to the arrangement of the plurality of needle-like objects in a cell insertion member capable of inserting the needle-like objects into cells.   2. The pillar array according to claim 1, wherein a plurality of substantially circular resist films are patterned on one main surface of a substrate made of an IV type semiconductor material, and the plurality of columnar bodies are formed by dry etching. The manufacturing method of the cell array substrate as described.   2. The method for producing a cell array substrate according to claim 1, wherein a bovine serum albumin film is formed in advance on one main surface of the substrate onto which the solution for cell membrane modifier (Biocompatible Anchor for Membrane) is transferred. .   2. The method for producing a cell array substrate according to claim 1, wherein the cell adhesive force is adjusted by the concentration of the cell membrane modifying agent contained in the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution.   2. The method for producing a cell array substrate according to claim 1, wherein the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution contains glycerin. In a pillar array having a plurality of columnar bodies having substantially the same length on one main surface, a step of bringing a cell membrane modifier (Biocompatible Anchor for Membrane) solution into contact with the end surface of the columnar body;
Positioning the pillar array on one main surface of the substrate so that the end surface of the columnar body faces, transferring the cell membrane modifier (Biocompatible Anchor for Membrane) solution attached to the end surface to the one main surface of the substrate;
And a step of bringing a cell suspension into contact with one main surface of the substrate to attach the cells to a region where the cell membrane modifier (Biocompatible Anchor for Membrane) solution has been transferred.   8. The process of adhering cells, wherein after the cell suspension is brought into contact with one main surface of the substrate, a process of removing cells not adhered on the substrate from the substrate is performed. A cell array production method.   The arrangement of the plurality of columnar bodies is a cell insertion member in which a plurality of needle-like objects are arranged on a support, and the needle-like objects are adhered to the substrate by being close to the cell array. The method for producing a cell array according to claim 7, wherein the method corresponds to the arrangement of the plurality of needle-like objects in the cell insertion member that can be inserted into the cell array.   8. The pillar array according to claim 7, wherein a plurality of substantially circular resist films are patterned on one main surface of a substrate made of an IV type semiconductor material, and the plurality of columnar bodies are formed by dry etching. The manufacturing method of the cell array of description.   8. The method for producing a cell array according to claim 7, wherein a bovine serum albumin film is formed in advance on one main surface of the substrate onto which the cell membrane modifier (Biocompatible Anchor for Membrane) solution is transferred.   8. The method for producing a cell array according to claim 7, wherein the cell adhesive force is adjusted by the concentration of the cell membrane modifying agent contained in the cell membrane modifying agent (Biocompatible Anchor for Membrane) solution.   8. The method for producing a cell array according to claim 7, wherein the cell membrane modifier (Biocompatible Anchor for Membrane) solution contains glycerin.

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