JP2014011985A - Cell aggregation device, cell aggregation method, biological tissue forming method, laminated cell sheet manufacturing method, and biological tissue manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell aggregation device or the like capable of efficiently aggregating suitable cells in short time.SOLUTION: A holder 1 is formed by laminating a plate 3, a light absorber 5 and a transparent plate 9 in this order. The light absorber 5 is provided on a lower surface side of the transparent plate 9. The plate 3 is arranged on a lower surface side of the light absorber 5. The transparent plate 9 holds the light absorber 5 and the plate 3, and transmits laser beam entering from above. Quartz glass having the thickness of about 0.2 mm, for example, can be used as the transparent plate 9. The holder 1 has an arc-shaped cross-sectional shape which curves on one cross section. A large number of wells 7 are provided in the plate 3. Cells 13 are placed in the wells 7. In other words, the plate 3 functions as a cell holding section that holds cells.

Description

  The present invention relates to a cell assembling apparatus for assembling cells to create a cell sheet, a method for assembling cells, a method for forming a biological tissue, a method for producing a cell laminated sheet, a cell laminated sheet, and a holder used therefor It is.

  Conventionally, methods for forming cell sheets by stacking cells have been studied. In the cell sheet thus obtained, for example, a cell tissue structure is formed by stacking cells in a desired shape and thickness. In this case, it is necessary to collect and stack appropriate cells at high speed. Examples of the method of using such a cell sheet include a single-layer cell sheet, the same cell-layered sheet, and a multi-layered layered structure sheet, and these sheets are properly used depending on the tissue cells to be used.

  As a method of laminating such cells, a method of arranging cells at an arbitrary position on a substrate using an ink jet printing technique has been proposed (for example, Patent Document 1).

JP 2009-131240 A

  However, in the ink jet system described in Patent Document 1, since the size of cells is usually not constant, there is a problem of nozzle clogging when trying to eject cells of different sizes from the nozzle. Moreover, in order to avoid such nozzle clogging, the ink jet method has a problem that the ejection speed is limited and the cell extraction speed is slow. Furthermore, this method also has a problem that it is impossible to distinguish appropriate cells at the time of launch.

  For example, a cell aggregate having a cube size of 5 mm square is to be formed. In this case, when the cell size is 20 μm square and the porosity is 0, the cell aggregate contains 15 million or more cells. On the other hand, in the ink jet system, only about 500 cells / second can be taken out under the condition that the nozzle is not clogged. Therefore, when it is going to form the above-mentioned cell aggregate by an ink jet system, time for 4 hours or more will be needed. In addition, when such a long time is required, internal cells may be damaged during the formation of the cell aggregate. Furthermore, in the ink jet system, a sensor for discriminating appropriate cells cannot be attached in a nozzle having an inner diameter of 30 μm for ejecting cells.

  Further, for example, when a plurality of types of cells are assembled and assembled into a desired three-dimensional cell tissue, it is more difficult to control the apparatus for stacking appropriate cells at appropriate positions.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a cell collecting apparatus and the like that can efficiently collect appropriate cells in a short time.

  In order to achieve the above-mentioned object, the first invention is a cell assembling apparatus, comprising a holder for holding cells, a tissue table provided under the holder, and a laser source capable of irradiating the holder with a laser. The holder includes a transparent plate, a light absorber provided on a lower surface of the transparent plate, a plate provided on a lower surface of the light absorber and having a well for holding cells, and The microbubbles are generated in the well by the heat generated by irradiating the light absorber from the laser source with a laser formed on the inner surface side so as to be curved in at least one cross section. By the pressure of, the curved shape is formed on the tissue table or on the culture bed provided on the tissue table. It is a cell collection apparatus characterized by towards the center of curvature at each position of the lower surface of Ruda in the normal direction of each position it is possible to put forward the cells.

  Here, it is preferable that the cell launch position in the normal direction of the curved surface of the holder is a substantially focal position corresponding to the curved shape. Further, when the shape of the holder is a part of either a substantially cylindrical surface or a substantially spherical surface, and the shape of the holder is a part of a substantially cylindrical surface, the cell launch position is the curved shape. When a linear position corresponding to a substantially focal position is formed and the shape of the holder is a part of a substantially spherical shape, the cell launching position is one point that is a substantially focal position corresponding to the curved shape. This is a cell assembly device. The shape of the holder is a part of either a substantially cylindrical surface or a substantially spherical surface, and the tissue table is disposed at a position shifted from a substantially focal position corresponding to the curved shape, and the shape of the holder is When it is a part of a substantially cylindrical surface, the cell launch position is a band with a predetermined width, and when the shape of the holder is a part of a substantially spherical surface, the cell launch position is a circular shape with a predetermined range. It can also be.

  In addition, it further includes an imaging device that images the cells accommodated in the holder, selects a site where appropriate cells are held by the imaging device, and irradiates the selected site with the laser, Cells can be bombarded on the tissue table or on the culture bed.

  A plurality of the holders are arranged on the upper part of the tissue table. By arranging a plurality of holders above the tissue table, cells can be efficiently assembled. Furthermore, at this time, a plurality of different cells can be assembled by disposing different cells in the plurality of holders and ejecting different cells from the respective holders.

  According to the first invention, the holder is curved, and the cells can be stacked in a desired form by causing the cells to be ejected to a focal position in the normal direction of the curved shape. For example, cells can be ejected in the normal direction of the curved surface by using a holder having a partial shape of a substantially cylindrical surface or a substantially spherical surface. Therefore, by arranging the tissue table so as to be at the focal position of the holder curved surface that forms a part of a substantially cylindrical surface, cells arranged on a plane (two-dimensional) are arranged on a straight line (one-dimensional). It can be launched as a stack.

  As described above, when the cells arranged two-dimensionally on the holder are ejected, for example, conversion into a one-dimensional array can facilitate the relative position control between the holder and the tissue table. For example, even when the relative movement between the tissue table and the holder is stopped, the cells can be stacked and assembled on the culture bed of the tissue table. Therefore, the movement of the holder can be reduced. As a result, it is possible to speed up the cell accumulation operation. In addition, when stacking cells on a straight line, the relative movement of the holder must be stopped and the holder moved relative only when the linear stacking position is changed. A cell sheet can be formed with minimal movement.

  Similarly, by placing the tissue table so as to be at the focal position of the curved curved holder curved surface that forms a part of a substantially spherical shape, one cell (0 dimension) is placed on the plane (two dimensions). ) It can also be punched out to be laminated on top. Usually, since tens of thousands of cells are contained in one plate, a necessary number of cells can be repeatedly accumulated at a predetermined position by collecting them at a focal position as necessary. In addition, by preparing a plurality of such substantially spherical tissues, a complex tissue including different cells can be formed. Further, as will be described later, by arranging (offset) slightly shifted from the focal position, a plurality of cells can be stacked in a predetermined range without moving the holder.

  Thus, in the present invention, cells can be ejected on a tissue table or a culture bed in a straight line or at one point without moving a holder or the like. Furthermore, even if the holder is not moved, it is possible to stack cells by expanding them into a band-like or circular region by launching with a shift from the focal position. In addition, as a laser, a high frequency pulse laser can be used and a laser can be irradiated to a desired site | part using a galvanometer mirror etc., for example. For this reason, by controlling the operation of the mirror and the irradiation pulse in the laser source in synchronism, desired cells can be launched at extremely high speed. As the high frequency laser, a laser of about 10 kHz to 100 kHz can be used, and even if a 10 kHz laser source is used, the above-described cell aggregate can be formed in several tens of minutes. Therefore, it is faster than the conventional ink jet method and there is no fear of nozzle clogging.

  In addition, by using an imaging device that images the cells accommodated in the holder, the size and operation of the cells can be detected. Therefore, only appropriate cells can be selectively used.

  In addition, by arranging a plurality of holders on the tissue table, a plurality of types of cells can be launched on the tissue table.

  A second invention uses the cell assembly device according to the first invention to repeatedly strike cells on the tissue table or the culture bed, thereby stacking the cells. Is the method. According to this cell assembling method, only appropriate cells can be reliably taken out in a short time and assembled in a straight line or at one point. In addition, by using this method, it is possible to directly stack cells on a tissue collected from a living body using the present technology, and it is also possible to directly stack cells on a living body. By using this cell assembly method, it is possible to obtain a method for forming a living tissue formed by accumulating one or more cells.

  For example, the cells can be stacked in an arbitrary shape on the tissue table or the culture bed in plan view. For example, cells can be stacked in a substantially honeycomb shape, and the vicinity of the center of the approximately hexagonal shape of the stacked cells can be used as a culture solution holding unit.

  In addition, when stacking cells, it is desirable not to stack directly on the tissue table but to stack on a culture bed. For example, the culture bed (culture dish) has a temperature-responsive culture bed that was developed by Professor Okano of Tokyo Women's Medical University and grafted onto a solid surface with a thickness of nanometer scale. A floor can be used. In addition, as long as it is a well-known culture bed as a culture bed, what kind of thing can be used according to the use purpose. Here, the culture bed in the present invention includes not only a simple culture bed but also an extracellular base material that serves as a base material when a cell sheet is prepared.

  3rd invention is the formation method of the biological tissue formed by accumulating 1 type, or 2 or more types of cells using the assembly method of the cell concerning 2nd invention. According to the third invention, a desired form of biological tissue can be obtained easily.

  A fourth invention uses the cell assembly device according to the first invention, repeats relative movement and stop of at least one of the holder or the tissue table, on the tissue table, or on the culture bed. A method for producing a cell laminate sheet, wherein cells are repeatedly ejected to form a cell laminate sheet by laminating the cells. According to 3rd invention, the cell lamination sheet of a desired form can be obtained easily.

  5th invention is the cell lamination sheet formed by the manufacturing method of the cell lamination sheet concerning 4th invention. According to 5th invention, the cell lamination sheet of a desired form can be obtained.

  In this manner, by placing cells on a culture bed provided on a tissue table, various cell sheets can be formed by laminating the same cells alone or in combination with a plurality of different types of cells. Here, the types of cell sheets to be formed by stacking are as follows: a single-layer cell sheet in which the same cells are formed in a single layer, a same cell stack sheet in which the same cells are stacked in a predetermined number of layers, and a heterogeneous cell in a predetermined number of layers Cell sheets with the desired dimensions required for regenerative medicine in a short period of time, such as patterned heterogeneous cell laminate sheets and patterned cell laminate sheets obtained by patterning and stacking different cells on specific cells as needed Can be formed. Further, for example, specific examples of the heterogeneous cell laminate sheet for laminating heterogeneous cells include epidermis cells, and a plurality of cells such as cardiomyocytes and endovascular skin cells within the same layer within the same laminate surface. A composite cell laminate sheet or the like that is composited and laminated can be obtained.

  6th invention is a holder used for a cell assembly apparatus, Comprising: The said holder forms the transparent plate, the light absorber provided on the said transparent plate, and the some well provided on the said light absorber And a plate that contains cells in the well and is curved to form a part of a substantially cylindrical surface having a curvature only in one direction with the well as the inner surface side. The holder for a cell assembly device is characterized in that the cells stored in the well are ejected by laser irradiation and the ejected cells are collected in a straight line corresponding to a substantially focal point of the well.

  A holder used in a cell assembly device, wherein the holder includes a transparent plate, a light absorber provided on the transparent plate, and a plate on which a plurality of wells provided on the light absorber are formed. The cell is housed in the well, and the well is curved so as to form a part of a spherical surface with the well as the inner surface side, and the cell housed in the well is ejected by laser irradiation. Thus, it is a holder for a cell assembly device, which collects the punched cells at one place which is substantially in focus.

  In this way, if the holder is formed so as to be curved in a substantially cylindrical surface so as to have a curvature only in one direction, the cells stored in the well are ejected by laser irradiation, and the well is substantially focused. It is possible to collect in a straight line corresponding to. In addition, this holder is formed so that the well is curved in a spherical shape with the inner surface side, and the cells stored in the well can be ejected by laser irradiation and collected in one place, which is a substantially spherical, substantially focal point. become.

  According to the fifth invention, it is possible to obtain a holder that can be used in a cell assembly device for collecting appropriate cells in a short time.

  According to the present invention, there is provided a cell collecting apparatus that can efficiently collect appropriate cells in a short time, and a cell collecting method that can efficiently collect appropriate cells in a short time using this apparatus. Can be provided. As a result, it is possible to obtain a living tissue or a cell laminate sheet necessary for regenerative medicine.

1 is an overall schematic diagram showing a cell assembly device 10. FIG. Schematic which shows the cell assembly apparatus 10. FIG. It is the schematic which shows the cell assembly apparatus 10, and is the sectional view on the AA line of FIG. The perspective view which shows the holder 1 Schematic which shows the state which strikes a cell from the holder 1. FIG. FIG. 4 is an enlarged view of a D part in FIG. 3, and shows a step of launching cells on the culture bed 16. The conceptual diagram which laminates | stacks the cell 13. FIG. Schematic which shows other embodiment. Schematic which shows other embodiment. It is a conceptual diagram which shows the aggregate | assembly form of the cell 13, (a) is a top view, (b) is the HH sectional view taken on the line of (a). (A) is a conceptual perspective view which shows the cell sheet 35, (b) is a conceptual perspective view which shows the cell sheet 35a.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is an overall schematic view showing the cell assembly device 10, and FIG. 2 is a schematic sectional view showing the cell assembly device 10. 3 is a cross-sectional view taken along line AA in FIG. 2, and is a schematic cross-sectional view in a direction perpendicular to FIG. FIG. 4 is a perspective view showing the holder 1. The cell assembly device 10 includes a holder 1, a tissue table 15, a culture bed 16, a laser source 17, a galvanometer mirror 19, an imaging device 21, and the like.

  As shown in FIG. 1, a cell table 29 that is moved by a drive mechanism 27 is disposed above the tissue table 15 and the like. After the properties of the cells are imaged in advance by the imaging device 21, the holder 1 is placed on the cell table 29 by the robot 2. A plurality of holders 1 can be placed on the cell table 29. Therefore, below the laser source 17, the holder 1 and the tissue table 15 (culture bed 16) can move relative to each other in the horizontal direction (in the direction of arrow G in the figure). The imaging device 21 is arranged on a precision table 30 having a window at the center, and the cells in the holder 1 are imaged by the imaging device 21 through the window from below the precision table 30. At this time, imaging may be performed with respect to the holder 1 placed on the cell table 29, but in this case, the imaging device 21 is driven, so that the drive mechanism 27 becomes larger than when the holder 1 is driven. . After the cells in the holder 1 are measured, they are transferred onto the cell table 29 by the robot 2.

  The imaging device 21 measures the shape of cells stored in the well 7 (in the direction of arrow B in FIG. 1). As the imaging device 21, for example, a cell stored in the well 7 (FIG. 2) of the holder 1 is magnified about 100 to 1000 times, the image is analyzed, and the shape of the cell is measured. A cell having an appropriate shape is determined from the measurement result, the position is stored, and the irradiation of the laser source 17 is cooperatively controlled to launch an appropriate cell. Furthermore, it is possible to measure the same cell a plurality of times over time, grasp the change of the cell at this time, and measure the activity level of whether the cell operation is active.

  As shown in FIG. 2, the holder 1 is formed by sequentially laminating a transparent plate 9, a plate 3, and a light absorber 5. The light absorber 5 is disposed on the lower surface side of the transparent plate 9. A plate 3 is provided on the lower surface side of the light absorber 5. The light absorber 5 has a light transmittance of about 30 to 50% and a film thickness of several tens to several hundreds of nanometers. When the laser is irradiated, a part of the laser is absorbed to generate heat. . As the light absorber 5, for example, a thin film or fine particles of titanium oxide, silicon oxide, gold, platinum, tantalum oxide or the like can be used, but a thin film of silicon oxide, gold or the like is preferably used.

  The transparent plate 9 holds the light absorber 5 and the plate 3 and transmits laser light from above. As the transparent plate 9, for example, quartz glass or transparent resin having a thickness of about 0.2 mm is used.

  As shown in FIGS. 3 and 4, the holder 1 has an arcuate cross-sectional shape (for example, a part of a cylindrical surface) curved in at least one cross-section. The plate 3 is provided with a large number of wells 7. Cells 13 are placed in the well 7. That is, the plate 3 functions as a cell holding unit that holds cells.

  The holder 1 may be formed by laminating the light absorber 5 on the transparent plate 9 and laminating the previously curved plate 3. However, the plate 3 is laminated on the flat transparent plate 9 or the like. After that, as shown in FIG. 3, the holder 1 may be formed by curving so that the well 7 side is the inner surface side.

  Such a plate 3 is formed, for example, by forming a light absorber 5 (thin film having a thickness of several tens of nm to 300 nm) on a quartz glass substrate having a thickness of about 0.1 to 0.3 mm, and further thereon. It can be formed by adhering a resin well 7 (a sheet having a 30 μm hole). Further, the substrate can be made of resin and the light absorber 5 can be bonded. In addition, the well 7 can be formed by etching on the glass substrate, and the light absorber 5 can be coated thereon (bottom surface of the well 7).

  The cell assembly device 10 shown in FIG. 2 shows an example in which one cell is held in each well 7. In this case, the size of the well 7 only needs to be slightly larger than the cell 13, and may be, for example, about 30 μmΦ. In addition, as a plate 3, things, such as resin and glass, can be used, for example. For example, the holder can form a predetermined number of regions in which 300 × 350 wells are arranged in a range of about 18 mm × 21 mm, with wells having a depth of 30 μm × 30 μmΦ arranged vertically and horizontally at a pitch of 60 μm. . If 12 such regions are arranged in a holder of a predetermined size (3 rows × 4 examples: 12), a well that can store about 1,200,000 or more cells can be formed in the holder.

  Each well 7 of the plate 3 is filled with cells 13 together with the culture medium. As the culture solution, for example, an organic acid salt having biocompatibility is used. The culture solution has a certain degree of viscosity (for example, about several hundred to several thousand cp), and the cells 13 are placed in the well 7 so that the cells 13 in the well 7 do not fall on the tissue table 15 or the culture bed 16 due to gravity. Can be held in.

  The culture solution is not particularly limited as long as it does not affect the cells 13, and preferably functions as nutrients for the cells 13. For example, sodium alginate can be used. Here, when Ca ion (calcium aqueous solution) is added to sodium alginate, it can be hardened easily. For example, when cells are put into a well, the viscosity of the sodium alginate aqueous solution is kept low without adding Ca, and Ca ions (calcium aqueous solution) are sprayed after the cells are injected into the well. Since the so-called ionic crosslinking occurs between ions, the viscosity can be increased by adding calcium to sodium alginate. In the present invention, the holder 1 in which the cells 13 are filled in the well 7 together with the gel-like member is referred to as a “cell holder”.

  A tissue table 15 is disposed below the holder 1 with a gap. A culture bed 16 is provided on the tissue table 15 as necessary. The tissue table 15 or the culture bed 16 is a site where a cell aggregate (cell sheet) is formed. The distance between the holder 1 and the tissue table 15 (culture bed 16) will be described in detail later.

  As the tissue table 15, one made of glass or resin can be used. In addition, after assembling the cells 13 on the tissue table 15 or the culture bed 16, in order to peel the formed cell sheet or the like from the tissue table 15, the surface of the tissue table 15 is previously coated with, for example, fibrin glue or the like. You may keep it. Moreover, when using a culture bed, a well-known culture bed can be suitably selected and used according to the objective, For example, the above-mentioned temperature-responsive culture bed may be used.

  An Fθ lens 18, a laser source 17, and a galvanometer mirror 19 are provided on the upper portion of the holder 1. A galvanometer mirror 19 is disposed on the side of the laser source 17. By operating the galvanometer mirror 19, the laser oscillated from the laser source 17 can be irradiated in an arbitrary direction. The Fθ lens 18 irradiates the holder 1 with laser light whose direction is changed by the galvanometer mirror 19 substantially perpendicularly. That is, laser light can be irradiated toward an arbitrary well 7 by the Fθ lens 18 and the galvanometer mirror 19 (in the direction of arrow C in FIG. 3). As the laser source 17, for example, a pulse laser having a wavelength of 1000 to 1100 nm and a frequency of 10 kHz to 100 kHz may be used. The galvanometer mirror 19 is driven while the laser is not oscillating. In this case, it is possible to oscillate the laser in 10,000 to 100,000 target directions per second. By controlling the galvanometer mirror 19 to rotate in a desired direction during this pulse, it is possible to irradiate the well 7 at a desired position with high speed and reliability.

  The galvanometer mirror 19 is controlled similarly to the laser pulse of the laser source 17 by a control device (not shown). Moreover, the galvanometer mirror 19 should just be able to operate | move at high speed and accurately.

  FIG. 5 is a conceptual diagram showing the positional relationship between the holder 1 and the tissue table 15 (culture bed 16). As described above, the holder 1 is curved with the tissue table 15 side as the inner surface side. At this time, the shape of the holder 1 and the distance between the holder 1 and the tissue table 15 are set so that the focal position 25 in each part of the holder 1 is in the vicinity of the surface of the tissue table 15 (culture bed 16).

  Next, a method for collecting cells using the cell assembly device 10 will be described. FIG. 6A is an enlarged view of a portion D in FIG. 3 and is an enlarged view of the vicinity of the well 7 of the cell assembly device 10. First, the size and operation (activity) of each cell 13 are measured by the imaging device 21, and a cell 13 having a certain degree of appropriateness is selected based on the measurement result.

  Next, a laser is irradiated from the laser source 17 to the well 7 in which the selected cell 13 is placed (in the direction of arrow C in the figure). The laser irradiation direction is adjusted by the galvanometer mirror 19. When a laser is irradiated from above the transparent plate 9 using the laser source 17 (in the direction of arrow C in the figure), the laser passes through the transparent plate 9 and irradiates the light absorber 5. In the light absorber 5, laser light is absorbed and heat is generated. Therefore, the culture medium 23 in the well 7 is heated by this heat to generate microbubbles (E in the figure).

  When microbubbles are generated inside the well 7, as shown in FIG. 6B, the gel-like member (culture solution) filled in the well is ejected from the well 7 by the pressure. At this time, the cells 13 (and the culture solution 23) inside the well 7 are ejected toward the culture bed 16 on the tissue table 15 (in the direction of arrow F in the figure). Thus, cells 13 in any well can be taken out on the tissue table 15.

  Next, the process of laminating the cells 13 will be described. FIG. 7 is a detailed view of FIG. As described above, the irradiation direction of the laser irradiated from the laser source 17 can be controlled by the galvanometer mirror 19. Therefore, for example, each well 7 of the curved holder 1 can be irradiated with laser. Inside the well 7 irradiated with the laser, micro bubbles are generated as shown in FIG. Therefore, the cells 13 are ejected from the well 7 in which the microbubbles are generated toward the focal position in the normal direction of the curved surface of the holder 1 (in the direction of arrow F in the figure).

  In this way, the cells 13 in each well 7 of the holder 1 can be gathered at the focal position without moving the holder 1. For example, without moving the holder 1 or the like, the laser can be distributed by the galvanometer mirror 19 in the direction perpendicular to the paper surface of FIG. Therefore, the cells 13 can be stacked. Such irradiation can be performed while moving the holder 1.

  In addition, the position of the tissue table 15 can be deliberately shifted from the focal position with respect to the curved surface of the holder 1. In this way, the cells 13 can be launched into a predetermined range on the tissue table. Therefore, the cells 13 can be stacked in a band shape having a predetermined width (in which the cells 13 are arranged in parallel).

  Furthermore, by appropriately setting the shape of the holder 1 and the distance between the holder 1 and the tissue table 15 (culture bed 16), the cells 13 are three-dimensionally formed in an arbitrary form on the tissue table 15 (culture bed 16). Can be laminated. That is, cells 13 can be arranged in an arbitrary pattern on the culture bed 16 by a galvanomirror 19 or the like, and desired cells can be stacked three-dimensionally to form a cell sheet.

  If the culture bed 16 is not used, the cells 13 may be directly ejected onto the tissue table 15. Also in this case, the holder 1 has the same configuration. Further, the cells 13 can be launched while moving the tissue table 15.

  As described above, according to the present embodiment, only cells having appropriate properties can be selectively taken out on the tissue table 15 or the culture bed 16 in any form. In addition, since the holder 1 is curved, the cells 13 can be launched toward the focal direction. Therefore, cell stacking is easy.

  At this time, while the holder 1 is continuously moved, appropriate cells can be launched on the tissue table 15 or the culture bed 16 and the cells 13 can be stacked in a desired form. In the present invention, the holder is stopped. Even in the state, the cells 13 can be stacked.

  For example, first, a predetermined amount of cells 13 are stacked in a substantially straight line with the relative movement of the holder 1 and the tissue table 15 stopped. Next, after the holder is relatively moved left and right by the size of the cell 13, the movement of the holder 1 is stopped and the cell 13 is ejected again. As described above, the cells 13 can be stacked so that a plurality of cells 13 stacked in a straight line are provided.

  In this way, by repeatedly stopping and moving the holder 1, the cells 13 can be stacked in a desired form. At this time, since it is not necessary to always move the holder 1 continuously, the position control between the holder 1 and the tissue table 15 is easy, and the speed can be increased.

  Further, since the cells 13 are ejected onto the tissue table 15 or the culture bed 16 by the irradiation of the pulse laser, the cells 13 can be taken out by the number corresponding to the frequency of the laser. At this time, extremely high-speed pattern irradiation is possible by pulse laser irradiation frequency and rotation control of the galvanometer mirror 19. Therefore, a desired cell sheet can be formed in a short time. At this time, even if the speed is increased, there is no fear of nozzle clogging as in the ink jet method.

  Next, a second embodiment will be described. FIG. 8 is a diagram showing a cell assembly device 10a according to the second embodiment. In the following description, components having the same functions as those of the cell assembly device 10 are denoted by the same reference numerals as in FIGS. Although FIG. 8 shows an example in which the culture bed 16 is provided, the cells can be directly stacked on the tissue table 15 as described above. In the following drawings, the imaging device 21 is not shown.

  The cell aggregation device 10a has substantially the same configuration as the cell aggregation device 10, but differs in that a plurality of holders 1 are provided. That is, the laser source 17 and the imaging device 21 (not shown) are arranged for each of the plurality of holders 1. In this case, the focal positions 25 of the respective holders 1 may be arranged so as to substantially coincide with each other, or may be slightly shifted.

  In this way, different cells can be held in the respective holders 1. Therefore, different cells 13 can be stacked in any form. In the illustrated example, the holders 1 are arranged in two rows, but the present invention is not limited to this. For example, more holders 1 may be arranged. Further, the shapes of the holders 1 may not all be the same. Further, the holder 1 may be arranged in different directions without making the arrangement direction (curving direction) of the holder 1 the same direction.

  Next, a third embodiment will be described. FIG. 9 is a view showing a holder 1a which is still another embodiment. The holder 1a is a part of a substantially spherical shape. That is, the cells in the well are three-dimensionally arranged on a substantially spherical curved surface, but the cells to be ejected gather at the focal point (or one point), and thus become zero-dimensional. The holder 1 is linear without being curved in one of the cross-sectional directions, and has the largest curved shape with respect to the cross-section orthogonal to the holder 1a. It becomes a curved shape.

  A plurality of wells 7 are formed on the inner surface side of the holder 1a. Therefore, by irradiating the laser from above the holder 1a, the cells 13 can be ejected toward the focal direction of each well 7. Accordingly, since the cells are ejected to one point regardless of the cell position on the holder, cooperative control of the cell table and the tissue table is facilitated.

  Note that the position of the tissue table 15 can be deliberately shifted from the focal position with respect to the curved surface of the holder 1a. In this way, the cells 13 can be launched into a predetermined range on the tissue table. Therefore, the cells 13 can be stacked in a substantially circular shape extending over a predetermined range.

  Next, an example of the pattern of the cells 13 formed on the tissue table 15 or the culture bed 16 using the above-described cell assembly device will be described. FIG. 10A is a plan view showing an example of the pattern of the cells 13 on the tissue table 15, and FIG. 10B is a cross-sectional view taken along the line HH in FIG. Although FIG. 10 shows an example in which the cells 13 are stacked on the culture bed 16, the cells can be directly stacked on the tissue table 15 as described above.

  In the present invention, the cells 13 can be arranged in an arbitrary pattern in the planar direction by using the above-described cell assembly device. Therefore, as shown, the cells 13 can be arranged in a substantially hexagonal shape in a honeycomb shape and stacked. In addition, the arrangement | positioning of the cell 13 does not need to be arrange | positioned completely on each side of a substantially hexagon as shown in figure. Further, the number of cells 13 arranged on each side and the number of stacked layers are not limited to the illustrated example. The cells 13 are not necessarily arranged in a line, and a plurality of cells 13 may be provided side by side.

  By disposing the cells 13 in a honeycomb shape, a culture solution holding portion 33 in which the cells 13 are not disposed is formed at the center of the substantially hexagonal shape. A culture solution for the cells 13 is placed in the culture solution holding unit 33. Therefore, when the cells 13 are stacked, the culture solution can be reliably supplied to the cells 13 located in the center. Therefore, for example, even in the case of a thick cell sheet in which several tens to several hundreds of cells are stacked, it is possible to prevent cells from being damaged without causing nutrients to reach the cells inside. it can.

  Fig.11 (a) is a figure which shows the cell sheet 35 formed using the above-mentioned cell assembly apparatus. The cell sheet 35 is formed by stacking cells 13. In addition, the lamination | stacking form of the cell 13 is not restricted to the illustrated example, It can also be set as the structure shown in FIG. According to the present invention, the cells 13 can be stacked on the culture bed 16 to obtain a cell sheet 35 having a desired size and thickness.

  Moreover, as shown in FIG.11 (b), the cell sheet 35a can also be formed by laminating | stacking several types of cells 13a and 13b. In addition, although the lamination | stacking method of the cells 13a and 13b may change for every layer as shown in figure, the cells 13a and 13b can also be arrange | positioned in a desired position also in the same layer.

  Thus, a cell sheet and a biological tissue using the same can be obtained by accumulating one type or two or more types of cells.

  As mentioned above, although embodiment of this invention was described referring an accompanying drawing, the technical scope of this invention is not influenced by embodiment mentioned above. It is obvious for those skilled in the art that various modifications or modifications can be conceived within the scope of the technical idea described in the claims, and these are naturally within the technical scope of the present invention. It is understood that it belongs.

DESCRIPTION OF SYMBOLS 1, 1a ......... Holder 2 ......... Robot 3 ......... Plate 5 ......... Light absorber 7 ......... Well 9 ...... Transparent plate 10, 10a ......... Cell assembly apparatus 13 ......... Cell 15 ……… Tissue table 16 ……… Culture bed 17 ……… Laser source 18 ……… Fθ lens 19 ……… Galvano mirror 21 ……… Imaging device 23 ……… Culture solution 25 ……… Focus position 27 …… ... Drive mechanism 29 ...... Cell table 30 ......... Precision table 33 ...... Medium fluid holding part 35, 35a ......... Cell sheet

In order to achieve the above-described object, the first invention is a cell assembly device, which is a holder for holding cells, a tissue table provided under the holder, a laser source capable of irradiating the holder with a laser, The holder includes a transparent plate, a light absorber provided on the lower surface of the transparent plate, and a plate provided on the lower surface of the light absorber and having a well for holding cells, The well is formed to be curved in a cross section in at least one direction with the inner surface side, and microbubbles are generated in the well by heat generated by irradiating the light absorber from the laser source, Curved shape on the tissue table or on the culture bed provided on the tissue table by the pressure of microbubbles It is a cell collection device characterized by to leave out the cells towards the center of curvature in the normal direction of each position in each position of the lower surface of a said holder.

A fourth invention uses the cell assembly device according to the first invention, uses a cell assembly device, repeats relative movement and stop of at least one of the holder or the tissue table, and on the tissue table, Or it is a manufacturing method of the cell lamination sheet | seat characterized by forming a cell lamination sheet | seat by selecting an appropriate cell on the said culture bed, repeatedly ejecting a cell, and laminating | stacking a cell. According to 4th invention, the cell lamination sheet of a desired form can be obtained easily.

A fifth invention is a cell laminate sheet formed by selecting and accumulating appropriate cells by the method for producing a cell laminate sheet according to the fourth invention. According to 5th invention, the cell lamination sheet of a desired form can be obtained.

6th invention is a holder used for a cell assembly apparatus, Comprising: The said holder forms the transparent plate, the light absorber provided on the said transparent plate, and the some well provided on the said light absorber A plate with a gel-like member in the well, and forming a part of a substantially cylindrical surface having a curvature only in one direction with the well as the inner surface side. A holder for a cell assembly device, wherein the holder is formed by bending, and the cells stored in the well are ejected by laser irradiation, and the ejected cells are collected in a straight line corresponding to a substantially focal point of the well. is there.

A holder used in a cell assembly device, wherein the holder includes a transparent plate, a light absorber provided on the transparent plate, and a plate on which a plurality of wells provided on the light absorber are formed. And the cells are housed in the well together with the gel-like member , and the well is formed to be curved so as to form a part of a substantially spherical shape with the well as the inner surface side. Is a holder for a cell assembly device, wherein the cells are ejected by laser irradiation, and the ejected cells are collected at a substantially focal point.

According to the sixth invention, it is possible to obtain a holder that can be used in a cell assembly device for collecting appropriate cells in a short time.
The seventh invention uses the cell assembly device according to the first invention, repeats relative movement and stop of at least one of the holder or the tissue table, and in a well plate containing different types of cells. A biological tissue manufacturing method characterized by collecting a plurality of types of cells at a focal position to repeatedly accumulate a number of appropriate cells at predetermined positions to form a biological tissue.
In addition, by using the cell assembly device according to the first invention, the relative movement and stop of at least one of the holder or the tissue table is repeated, and appropriate cells are stacked directly on the tissue collected from the living body or on the living tissue. A method for producing a biological tissue, characterized in that:

A fifth invention uses a cell assembly device according to the first invention, repeats relative movement and stop of at least one of the holder or the tissue table, and a plurality of types in a well plate containing different types of cells. In the method of manufacturing a biological tissue, the cells are collected at a focal position to form a biological tissue by repeatedly accumulating a number of appropriate cells at a predetermined position.
In addition, by using the cell assembly device according to the first invention, the relative movement and stop of at least one of the holder or the tissue table is repeated, and appropriate cells are stacked directly on the tissue collected from the living body or on the living tissue. A method for producing a biological tissue, characterized in that:

Claims (12)

A cell assembly device,
A holder for holding cells,
An organization table provided at the bottom of the holder;
A laser source capable of irradiating the holder with a laser;
Comprising
The holder includes a transparent plate, a light absorber provided on the lower surface of the transparent plate, and a plate provided on the lower surface of the light absorber and having a well for holding cells, and the well on the inner surface. As a side, it is formed to be curved in a cross section in at least one direction,
Microbubbles are generated in the wells by heat generated by irradiating the light absorber from the laser source, and provided on the tissue table or on the tissue table by the pressure of the bubbles. A cell assembly device characterized in that cells can be ejected onto a culture bed in the normal direction of each position toward the center of curvature at each position on the lower surface of the holder having a curved shape.   The cell assembly device according to claim 1, wherein a cell launch position in a normal direction of the curved surface of the holder is a substantially focal position corresponding to the curved shape.   When the shape of the holder is a part of either a substantially cylindrical surface or a substantially spherical surface, and the shape of the holder is a part of a substantially cylindrical surface, the cell launch position corresponds to the curved shape. When one linear position which is a substantially focal position is formed and the shape of the holder is a part of a substantially spherical shape, the cell launch position is one point which is a substantially focal position corresponding to the curved shape. The cell assembly device according to claim 1, wherein the device is a cell assembly device. The shape of the holder is a part of either a substantially cylindrical surface or a substantially spherical surface,
The tissue table is arranged at a position shifted from a substantially focal position corresponding to the curved shape,
When the shape of the holder is a part of a substantially cylindrical surface, the cell launch position is a band with a predetermined width, and when the shape of the holder is a part of a substantially spherical surface, the cell launch position is within a predetermined range. The cell assembly device according to claim 1, wherein the cell assembly device has a substantially circular shape extending in a circle. It further has an imaging device for imaging cells contained in the holder,
By selecting a part where appropriate cells are held by the imaging device and irradiating the selected part with the laser, the cells can be ejected onto the tissue table or the culture bed. The cell assembly device according to any one of claims 1 to 4, wherein the cell assembly device is possible.   The cell assembly device according to any one of claims 1 to 5, wherein a plurality of the holders are arranged on an upper portion of the tissue table.   A cell assembly device according to any one of claims 1 to 6, wherein the cells are repeatedly ejected on the tissue table or the culture bed to stack the cells. Aggregation method.   A method for forming a biological tissue formed by accumulating one or more types of cells using the cell assembly method according to claim 7. Using the cell assembly device according to any one of claims 1 to 6,
The relative movement and stop of at least one of the holder or the tissue table is repeated, the cells are repeatedly ejected on the tissue table or the culture bed, and the cells are stacked to form a cell laminated sheet. A method for producing a cell-laminated sheet characterized by the above.   A cell laminate sheet formed by the method for producing a cell laminate sheet according to claim 9. A holder used in a cell assembly device, wherein the holder is
A transparent plate,
A light absorber provided on the transparent plate;
A plate formed with a plurality of wells provided on the light absorber;
A cell is contained in the well, and the well is formed to be curved so as to form a part of a substantially cylindrical surface having a curvature only in one direction, with the well being an inner surface side. A holder for a cell assembly device, wherein the cells stored in the cell are ejected by laser irradiation, and the ejected cells are collected in a straight line corresponding to the approximate focus of the well. A holder used in a cell assembly device, wherein the holder is
A transparent plate,
A light absorber provided on the transparent plate;
A plate formed with a plurality of wells provided on the light absorber;
A cell is stored in the well, and the well is formed to be curved so as to form a part of a spherical surface with the well as the inner surface side, and the cell stored in the well is ejected by laser irradiation. A holder for a cell assembly device, wherein the punched cells are collected in one place which is substantially in focus.

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