JP2011172533A - Method for three-dimensional high-density cell culture using microspace structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-density cell culture reactor enabling stable high-density culture of a large amount of cells over a long period and applicable e.g. to culture of artificial organ cells, and to provide a cell culture carrier substrate for the reactor. <P>SOLUTION: A cell culture carrier substrate having a porous structure part is used as the cell culture carrier substrate 6, wherein a through-hole 4 formed in the porous structure part has a polygonal form of from trigonal to hexagonal form having a side length or a diameter less than 50 to 500 μm. The cell culture reactor 0 contains two or more cell culture carrier substrates laminated together interposing a space between the substrates and placed in a cell culture tank having at least an inlet port and an outlet port for a fluid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

    The present invention relates to a cell culture carrier substrate useful for efficiently forming a high-density culture of cells and thereby forming a three-dimensional cell cluster (spheroid). The present invention also relates to a cell culture reactor using the cell culture support substrate and a cell culture system including the same. Furthermore, the present invention relates to a cell culture method using the cell culture support substrate, cell culture reactor or cell culture system.

    Living cells are associated with living tissue using various physical, chemical, and mechanical interactions. The mechanism is complex and has not been systematically interpreted. In order to culture viable cells by immobilizing them on a carrier such as a resin at a high density, it is necessary to control the interaction between the cells and the carrier. That is, it is necessary to control both the physical properties and morphology of the support surface. As the carrier, a resin having high affinity with cells and capable of fine processing is generally used, but there is no carrier that can stably hold cells over a long period of cell culture.

    Currently, proteins having physiological activity such as enzymes, monoclonal antibodies (including antibody drugs), vaccines, hormones, and the like are frequently used in various fields such as research and development, medicine, and general households. Many of these useful proteins are produced using animal cells and the like. However, in the conventional batch culture method, mass production and long-term continuous culture are impossible, and thus production efficiency is poor. Further, in the conventional batch culture method, the separation and purification process is complicated, and as a result, the product becomes expensive. Furthermore, the batch culture method is not suitable for multi-product small-volume production accompanying diversification of needs. In addition, it has been reported that cells cultured by such a two-dimensional and artificial method cannot exhibit the enzyme activity, biosynthetic activity and the like inherent in the cells (Non-patent Documents 1 and 2). reference).

    Therefore, in order to promote cell engraftment and proliferation, maintain and improve cell function, and maintain the cell morphology in a state that is closer to that in the living body, the cell is not a batch planar culture using a culture flask. Various three-dimensional high-density cultures using a carrier that serves as a scaffold for these have been proposed.

    Examples of the culture carrier used for such three-dimensional high-density culture include a porous cell culture carrier (Patent Document 1) formed of a foam material using polyurethane or the like, and a gap into which cells such as cellulose fibers and carbon fibers enter. Cell culture carrier (Patent Document 2) consisting of small pieces having a large number of spheroids, cell culture carrier having a solid body obtained by cutting the outside of a micro hollow body having an external shape of a sphere, ellipsoid, polyhedron, columnar body or cone (Patent Document 3) And the like can be mentioned.

    Patent Document 4 describes a cell culture chip provided with cell culture cells for aggregating and holding cells, which are regularly arranged in an array, honeycomb, or the like on a substrate surface. However, the cell culture cells regularly arranged in the honeycomb shape or the like are bottomed holes formed with dents on the substrate surface so that the cells can be held, or the openings of the bottomless holes are made water-permeable. It is formed by covering with a film or the like and does not have a through hole.

    As described above, even the conventionally proposed culture carriers have problems such that the cells hardly adhere to each other and that the cells hardly grow even if the cells adhere.

    In recent years, we have developed a high-density cell culture reactor that can be used for a long time with a small size, high quality, and continuous operation in order to realize high quality, wide variety, low volume production system and low cost to meet diversifying needs. It is requested to do.

JP-A-4-281784 JP 2004-135668 A JP 2009-247334 A JP 2005-27598 A

Nature, 424, 870 (2003) Science, 302, 46 (2003)

    The present invention relates to a cell culture carrier substrate having good cell adhesion and capable of culturing cells at high density, and a cell culture carrier useful for efficiently forming a three-dimensional cell conglomerate (spheroid). An object is to provide a substrate. Another object of the present invention is to provide a cell culture carrier substrate that is used by being installed inside a cell culture tank of a continuous ultra-high density cell culture reactor.

    The present invention also provides a cell culture reactor in which the cell culture support substrate is installed in a cell culture tank, a cell culture system including the cell culture reactor, and the cell culture support substrate, the cell culture reactor, and the cell culture system. It aims at providing the culture method of the used cell.

    Y. Utsumi et al., “Enhancement of the adhesive force of metal films on PTFE surface achieved bu fast-atom” By applying “-beam surface modification”, Microsystem Technology, 14, pp.1467-1473, 2008), it is possible to control the physical properties of the substrate surface layer having a nano-level thickness with high accuracy and stability. As a result of studies using this technique, the present inventors have already made it possible to make the substrate surface hydrophobic by providing a fine structure of several hundreds of nanometers on the surface of the cell culture carrier substrate. It has been found that an anchor effect for immobilizing cells can be obtained (unpublished).

    Based on these findings, we have intensively studied to develop a cell culture support substrate that enables high-density culture of cells and can be applied to continuous high-density cell culture reactors. By using a three-dimensional microstructure having a structure (hereinafter also referred to as “capillary structure”), it is possible to perform high-density culture inside the through-hole with cells attached to the inner wall, and cell proliferation efficiency And the inner wall of the through-hole has a curved surface rather than a curved surface, for example, a larger number of cells adhere, and the cell culture surface is roughened. Further, the inventors have found that the number of cell adhesion can be increased, and have completed the present invention.

That is, the present invention has the following configuration:
(I) Cell culture carrier substrate (I-1) A cell culture carrier substrate having a porous structure part, wherein the through-hole formed in the porous structure part has a length or diameter of one side of a triangle of less than 50 μm to less than 500 μm A cell culture carrier substrate which is a hexagonal polygonal through-hole.

  (I-2) The cell culture carrier according to (I-1), wherein the polygonal shape is a rhombus shape, a parallelogram shape or a honeycomb shape.

  (I-3) The cell culture carrier according to (I-1), wherein the polygonal shape is a rhombus shape or a parallelogram having an acute angle of 30 ° to 60 °.

  (I-4) The cell culture carrier substrate according to any one of (I-1) to (I-3), wherein at least the porous structure portion of the substrate has a thickness of 200 μm to 2 mm.

  (I-5) One or more selected from the group consisting of polymethyl methacrylate, polydimethylsiloxane, parylene, polyimide, polytetrafluoroethylene, epoxy photosensitive resin, polystyrene, polycarbonate, and cycloolefin polymer The cell culture carrier substrate according to any one of (I-1) to (I-4), which is formed from a resin.

  (I-6) The cell culture carrier substrate according to any one of (I-1) to (I-5), wherein at least a cell culture surface of the cell culture carrier substrate is processed.

  (I-7) The cell culture carrier substrate according to (I-6), wherein the surface of the cell culture carrier substrate is processed by chemical treatment or radiation irradiation treatment.

(II) Cell culture reactor and cell culture system (II-1) Cell culture according to any one of (I-1) to (I-7) in a cell culture tank having at least a fluid inlet and outlet A cell culture reactor in which two or more carrier substrates are arranged in a stacked state at intervals.

  (II-2) The cell according to (II-1), wherein the cell culture support substrate is formed by attaching live cells to be cultured or spheroids to the porous structure of the substrate. Culture reactor.

  (II-3) The cell culture carrier substrate is attached with live cells to be cultured in the through-holes of the porous structure portion of the substrate, and the liquid medium flows into the cell culture tank from the inflow port, A flow path of the liquid medium is formed so as to be discharged from the outlet through the through hole of the porous structure portion of the cell culture carrier substrate in the tank to the outside of the cell culture tank (II -1) The cell culture reactor described.

(II-4) In a cell culture tank having at least a fluid inlet and an outlet,
(A) Two or more cell culture carrier substrates formed by adhering live cells capable of forming spheroids in the through-holes are laminated at intervals,
(B) (a) at least one inflow path for allowing the liquid medium to flow into the cell culture tank from the inflow port;
(B) Three or more branch paths that branch from the inflow path and communicate with each other between two or more cell culture carrier substrates stacked at intervals, and (c) at least one of the three or more branch paths merges. Has an outflow channel,
(II-1) is characterized in that a liquid medium flow path is formed so that the liquid medium flowing in from the inlet is discharged from the outlet through the flow path to the outside of the cell culture tank. A cell culture reactor as described.

  (II-5) The inflow passage is formed so as to taper in the direction in which the fluid flows, and the outflow passage is formed so as to become wider in the direction in which the fluid flows. -4) The cell culture reactor described in the contract.

  (II-6) In addition to the cell culture reactor described in any one of (II-1) to (II-5), it is sent to the cell culture reactor in response to the dissolved oxygen amount, pH or electrolyte concentration of the liquid medium. A cell culture system comprising a device for adjusting a flow rate, pH or electrolyte concentration of a liquid medium to be liquefied.

(III) Cell culture method (III-1) Cell culture carrier substrate according to any one of (I-1) to (I-7), (II-1) to (II-5) A cell culture reactor, or a cell culture system described in (II-6).

  (III-2) The cell culture carrier substrate according to any one of (I-1) to (I-7), in which cells are attached in the through-holes of the porous structure, is placed in a cell culture tank containing a liquid medium. The method for culturing cells according to (III-1), comprising a step of arranging and culturing cells in the porous structure part of the cell culture support substrate.

  (III-3) Culturing the cell culture support substrate in a suspended state in a liquid medium, culturing the cell culture support substrate in a suspended state in a liquid medium, or stirring the cell culture support substrate The method for culturing cells according to (III-1) or (III-2), wherein the culture is performed while circulating the liquid medium in a suspended state in the liquid medium.

  (III-4) having a step of culturing cells by placing the cell culture carrier substrate described in any of (I-1) to (I-7) on a polydimethylsiloxane-coated surface (III- The method for culturing cells described in 1) or (III-2).

  (III-5) The cell culture reactor described in any one of (II-1) to (II-5), in which the cell culture support substrate obtained by the cell culture method described in (III-4) is stacked The method for culturing cells described in (III-1), wherein

    According to the cell culture carrier substrate of the present invention, three-dimensional high-density cell culture can be performed using the microspace formed in the porous structure (in the plurality of through holes). Moreover, according to the cell culture support substrate of the present invention, the surface area required for cell adhesion can be increased. Furthermore, it is possible to further enhance cell adhesion by controlling the physical properties of the cell culture surface of the substrate, such as roughening the cell culture surface of the substrate, particularly the inner surface of the through hole. Can stably grow cells, and can also form three-dimensional cell clusters (spheroids). That is, according to the cell culture support substrate of the present invention, three-dimensional high-density cell culture is possible, so that it is possible to reduce the size and yield of the cell culture device (cell culture reactor).

    Further, by using the cell culture support substrate of the present invention or a laminate thereof, or a cell culture reactor or a cell culture system having these, the culture solution is continuously passed through the substrate or the laminate thereof for a long period of time. It is considered that cells having high activity and stable function can be cultured.

It is a schematic diagram which shows one aspect | mode of the cell culture reactor of this invention. It is a schematic diagram which shows another one aspect | mode of the cell culture reactor of this invention. FIG. 3 is a schematic diagram showing a preferred embodiment of the cell culture reactor shown in FIG. 2 (analysis of flow velocity distribution in the reactor by CFD analysis). As shown in the color diagram, the flow rate and flow rate of the liquid medium flowing in the cell culture carrier substrate (microcarrier) is maintained constant. The result of having observed the formation state of the cell aggregate with the fluorescence microscope is shown. (A) shows a cell aggregate with a diameter of about 200 μm, (B) shows a cell aggregate with a diameter of about 300 μm, and (C) shows an image of a cell aggregate with a diameter of about 500 μm. When the diameter is about 500 μm, it can be seen that the central part begins to be necrotic. An example (circular, quadrangular, pentagonal, star, rhombus) of the shape of the capillary structure prepared on the substrate made of polymethyl methacrylate (PMMA) in Experimental Example 1 is shown. The result of observing the state of cell adhesion after 3 days of culture on a cell culture carrier substrate (microcarrier) having a three-dimensional microstructure (capillary structure) of various shapes with a fluorescence microscope is shown. (a) (upper left) is a honeycomb shape, (b) (upper right) is a diamond, (c) (middle left) is a rectangle, (d) (middle right) is a pentagon, (e) (lower left) is a triangle , (F) (lower right) show the results in the case of a circle. Regarding the honeycomb shape and the rhombus shape, the state of cell adhesion after culturing for 3 days on a cell culture carrier substrate (microcarrier) having a three-dimensional microstructure of different sizes (length of one side) was observed with a fluorescence microscope. Results are shown. (a) (Left uppermost stage) honeycomb shape 200 μm, (b) (Right uppermost stage) honeycomb shape 150 μm, (c) (Left second stage) honeycomb shape 100 μm, (d) (Right second stage) honeycomb shape 50 μm, (E) (Left third stage) Honeycomb shape 30 μm, (f) (Right third stage) Diamond 200 μm, (g) (Left bottom) Diamond 100 μm, (h) (Right bottom) Diamond 50 μm Each is shown. The structure of a PMMA three-dimensional microstructure carrier (cell culture carrier substrate: microcarrier) used for qualitative evaluation of cell adhesion in Experimental Example 2 is shown (thickness: 1 mm, filter part diameter: 12 mm, pattern part diameter: 8 mm). ). The pattern portion has a porous structure portion having a honeycomb-shaped through hole having a side of 100 μm or a rhomboid shape having a side of 150 μm and an inner angle of 60 °. (A) Honeycomb-shaped cell culture substrate (microcarrier) having a side of 100 μm, (b) Diamond-shaped microcarrier having a side of 150 μm and an inner angle of 60 °, and (c) In a through hole (capillary) of a circular microcarrier The result of having observed the state of cell adhesion in a confocal laser microscope is shown (cell culture 4th day). A bottom view (left column of each figure) shows a microscope image of the bottom surface of the culture dish, and a side view (right column of each figure) shows a microscope image of the side surface of the culture dish. A state in which cells from the bottom side of the dish are developed is observed. The result of having observed the time-dependent state of the cell in the honeycomb-shaped through-hole (capillary) of a cell culture support substrate (micro support | carrier) is shown. (A) shows the result on the third day of culture, and (b) shows the result on the 11th day of culture. In each figure, the left column is a bottom view and the right column is a side view. Fig. 3 shows the structure of a PMMA three-dimensional microstructure carrier (cell culture carrier substrate: microcarrier) used for quantitative evaluation of cell adhesion in Experimental Example 3 (thickness: 1 mm, filter part diameter: 12 mm, pattern part diameter: 8 mm) . (A) is a three-dimensional microstructure carrier (microcarrier) having a porous structure consisting of honeycomb-shaped through-holes in the pattern portion, and a three-dimensional microstructure carrier (microcarrier) having a porous structure consisting of rhomboid-shaped through-holes. And (B) shows the shapes of (a) flat plate PMMA and (b) ring-shaped PMMA used as control PMMA. It is a figure which shows that there is a correlation between the number of HepG2-pEGFP cells (× 10 ⁵ cells) and the fluorescence intensity (GFP amount) of the cytoplasm fraction. (A) The results of comparing the number of cell adhesion over time against a PMMA carrier having a honeycomb structure (side: 50, 100, 150, 200, and 300 μm) and a control PMMA (flat PMMA, ring-shaped PMMA) are shown. . (B) Contrast of cell adhesion over time for PMMA carrier having a rhomboid shape (150μm on one side and acute angles: 30 °, 45 °, 60 °) and control PMMA (flat PMMA, ring-shaped PMMA) The results are shown. (C) The results of comparison of the number of cell adhesion over time to a PMMA carrier having a circular (diameter: 100, 200, 300 and 600 μm) capillary structure and a control PMMA (flat PMMA, ring-shaped PMMA) are shown. In addition, as the cell adhesion number, the amount of GFP (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction was used as an index (average value ± standard deviation, n = 3-8). (A) shows the number of cell adhesion over time per area of the inner wall of the capillary in a PMMA carrier having a honeycomb structure (one side: 50, 100, 150, 200 and 300 μm). (B) The number of cell adhesion over time per area of the inner wall of the capillary in a PMMA carrier having a rhombus-shaped (one side is 150 μm, acute angle: 30 °, 45 °, 60 °). The number of cell adhesions was evaluated using the amount of GFP (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction as an index (average value ± standard deviation, n = 3-8). The integrated value of the amount of intracellular GFP obtained up to the 14th day of culture was calculated using the shape pattern of through-holes (flat plate PMMA, circular (diameter 100 μm, 200 μm, 300 μm), honeycomb shape (side length 50 μm, 100 μm, 150 μm, 200 μm) , 300 μm) and rhombus (acute angle 30 °, 45 °, 60 °)). Cell adhesion when a PMMA carrier (microcarrier) having a honeycomb-shaped capillary structure with a side of 300 μm is cultured in a culture solution for 17 days, (a) stationary culture, (b) suspension culture, and (c) stirring culture The result of having observed a state with the fluorescence microscope is shown. A PMMA carrier (microcarrier) having a honeycomb-shaped capillary structure with a side of 300 μm is suspended in the culture solution, and the state of cell adhesion is observed with a confocal laser microscope when the culture solution is stirred for 17 days. The results are shown. (a) Oblique bottom view: It is recognized that cell aggregates are formed. (B) An image viewed from the side of the cell dish and (c) an image viewed from the bottom of the cell dish. 10 shows the results of quantitative evaluation of the number of adherent cells in stationary culture and suspension culture using a PMMA carrier (microcarrier) having a honeycomb shape (side length of 100 μm, 300 μm) in Experimental Example 4. The horizontal axis indicates the culture period (days), and the vertical axis indicates the GFP amount (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction as the number of cell adhesion. The result of Experimental example 4 is shown. From the left end, the number of adherent cells (GFP amount: μg) (white bar) when PMMA carrier (microcarrier) is left on the bottom of the cell culture dish for 17 days; PMMA on the 6th day after cell seeding Number of adherent cells (GFP amount: μg) (gray bar) when the carrier (microcarrier) is suspended in the culture for 11 days; PMMA carrier is suspended in the culture on the 6th day after cell seeding The number of adherent cells (GFP amount: μg) (black bar) when cultured for 11 days with stirring. Mean value ± standard deviation (n = 1-3). The result of having peeled the cell spheroid formed by the suspension culture from the microcarrier with a pipette and culturing on the cell culture dish is shown. (A) shows the result of culturing for 3 days after peeling, (b) shows the result of culturing for 10 days after peeling, and (c) shows the result of culturing for 14 days after peeling. (A) In Example 5, the number of cell adhesions between a roughened PMMA carrier (roughened PMMA) (-◆-) and an untreated PMMA carrier (flat PMMA) (-x-) The results of tracking changes over time of are shown. In addition, as the cell adhesion number, the amount of GFP (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction was used as an index (average value ± standard deviation, n = 4-5). (B) shows the result of estimating the integrated value of the amount of GFP up to the 14th day of culture by the trapezoidal method. The white bar shows the result of the untreated PMMA carrier (usually PMMA), and the black bar shows the result of the PMMA carrier (roughened PMMA) whose surface is roughened. The results of culturing cells on roughened PMMA and flat PMMA (untreated PMMA) and comparing cell adhesion and proliferation are shown ((1) 4th culture day, (2) 7th culture day, (3) ) Culture day 11). The state of cell adhesion to PMMA irradiated with synchrotron radiation ((a) dose 0, (b) dose 2000, (c) dose 5000, (d) dose 10000) is shown (Experimental Example 5 (2)). The quantitative evaluation result of the cell adhesion to the carrier made from PMMA irradiated with synchrotron radiation (dose 0, 2000, 5000, 10000) is shown (Experimental Example 5 (2)). Honeycomb 1 on the cell culture dish (PDMS-treated dish: “on PDMS” in the figure) and untreated cell culture dish (“on cell culture dish” in the figure)) with a thin coating of polydimethylsiloxane (PDMS) on the bottom A cell culture support substrate (microcarrier) having a capillary structure with a side of 100 μm is allowed to stand, seeded with HepG2-pEGFP cells, cultured, and the number of cells quantified from the amount of GFP (μg) is shown (experimental example) 6). 1 shows one embodiment of a cell culture reactor of the present invention. (A) Inner cell, (B) Outer cell, (C) State in which inner cell is incorporated in outer cell. There are a plurality of grooves inside the inner cell, and a plurality of cell culture carrier substrates (microcarriers) can be arranged in the groove (symbol 13) at regular intervals. In addition, a plurality of holes are formed in the side surfaces of the inner cell and the outer cell, respectively, and the holes communicate with each other at the time of assembly to form a connecting portion (symbol 14) with an external measuring instrument. It is a schematic diagram which shows the cross section of the cell culture reactor shown in FIG. Inside the outer cell (12) is stored the inner cell (11) in which 20 micro-carriers (6) are stacked at intervals (for convenience of drawing, 10 micro-carriers are stacked) Described). The inner cell has a step-like tapered groove for flowing the culture solution, and the culture solution flows through a space formed by the groove of the inner cell and the outer cell. Although the thickness of the created space changes in a staircase pattern, the width is set constant at both the top and bottom. The inner cell is formed with a hole through which the culture solution flows into the cell. In addition, the arrow which shows the flow of a fluid (culture solution) is only kept to description of half for simplification. 1 shows an embodiment of the cell culture system of the present invention. In Experimental example 7, the result of having investigated the influence which the difference in the length of one side of a honeycomb shape has on the number of cell adhesion is shown (result on the 12th day after culturing in a cell culture reactor). Delivery speed 10ml / hr. In addition, as the cell adhesion number, the GFP amount (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction was used as an index. In Experimental example 7, the influence which the difference in the liquid feeding amount (0.83, 3, 10 ml / h) to a cell culture reactor has on the number of cell adhesion is shown. In addition, as the cell adhesion number, the GFP amount (μg) calculated from the GFP fluorescence intensity of the cytoplasm fraction was used as an index. A schematic diagram of the cell culture reactor used in Experimental Example 7 is shown (analysis of flow velocity distribution in the reactor by CFD analysis). As shown in the color diagram, the flow rate and flow rate of the liquid medium flowing in the cell culture carrier substrate (microcarrier) is maintained constant. A schematic diagram of the cell culture reactor used in Experimental Example 7 is shown (analysis of flow velocity distribution in the reactor by CFD analysis). As shown in the color diagram, the flow rate and flow rate of the liquid medium flowing in the cell culture carrier substrate (microcarrier) is maintained constant.

(I) Cell Culture Carrier Substrate The cell culture carrier substrate of the present invention has a porous structure composed of a plurality of through-holes having a polygonal cross-sectional shape as a site for cells to adhere and proliferate. I have it.

    Examples of the polygonal shape include any one of a triangular shape to a hexagonal shape. Here, the triangle to hexagon means that the number of outside angles is 3 to 6, for example, a triangle (including a regular triangle and an isosceles triangle), a quadrangle (a square, a rectangle, a parallelogram, a trapezoid, and a rhombus) Etc.), pentagons (regular pentagons, and five star shapes each including outer and inner angles), hexagons (honeycomb shapes, parallelograms, and six stars each having outer and inner angles) ) And the like. A rhombus, a parallelogram, and a honeycomb shape are preferable. The honeycomb shape means a shape in which regular hexagons are arranged without gaps like a honeycomb.

    The polygonal shape preferably has a side length or diameter in the range of 50 μm to less than 500 μm. Here, “length of one side” means the length of one side of the polygon, and “diameter” means a line segment drawn from one vertex of the outer corner of the polygon to the opposite side through the center. It means length. More preferably, the length or diameter of one side is 50 μm to 300 μm, and particularly preferably 100 μm to 300 μm. Moreover, when aiming at formation of a three-dimensional cell conglomerate (spheroid), it is preferable that the length or diameter of one side of the polygonal shape of the cell culture carrier substrate is 50 μm to 150 μm.

    Of the polygonal shapes, the rhombus and the parallelogram are preferably those having an acute angle of 15 ° to 60 °. More preferably, it is a rhombus or a parallelogram having an acute angle of 30 ° to 60 °, particularly preferably 30 ° to 45 °.

    The through hole whose cross-sectional shape is the polygonal shape means a polygonal columnar hole (polygonal columnar capillary). Here, the length of the through hole (depth of the hole), that is, the thickness of at least the porous structure part of the cell culture support substrate is not limited, but is usually 100 μm to 10 mm, preferably 200 μm to 5 mm, more preferably 200 μm to 2 mm. Can be mentioned. The ratio (aspect ratio) of the hole depth (the thickness of the porous structure portion of the substrate) to the hole diameter is usually 0.1 or more, preferably 1 to 500, more preferably 1 to 200. That is, the cell culture support substrate of the present invention has a porous structure (high aspect ratio structure) having a large number of micropores with a high aspect ratio.

    The number of such high aspect ratio micropores is not limited, but may include at least 150 per cell culture support substrate. Preferably it is 1000 or more, More preferably, about 3000-20000 can be mentioned.

    The plurality of through-holes formed in one cell culture carrier substrate may all have the same shape, or may have two or more different shapes and sizes. It may be a thing.

    In the cell culture carrier substrate of the present invention having such a porous structure, cells adhere to the inner wall surface of the through-hole and grow and spontaneously aggregate to form a three-dimensional cell cluster (spheroid). As shown in Experimental Example 1, when the spheroid grows to a diameter of about 500 μm, it becomes difficult to supply nutrients to the center, and necrosis occurs from the cells in the center. According to the cell culture support substrate of the present invention, since the spheroids formed in the through hole reach the size of the through hole (the hole diameter, its shape, and the depth of the hole), the growth slows down. Accordingly, the size of the spheroid can be controlled to a desired size less than 500 μm in diameter. For this reason, spheroids can be formed efficiently without causing central necrosis.

    The cell culture carrier substrate may be formed from a resin having cell adhesion, or may be formed from a resin whose surface is coated (coated) with a substance having cell adhesion or cell affinity. .

    The resin having cell adhesiveness is preferably a resin having cell adhesiveness and capable of fine processing of the through hole. Examples of such resins include polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), parylene, polyimide, polytetrafluoroethylene (PTFE), epoxy photosensitive resin, polystyrene, polycarbonate, and cycloolefin polymer. . Here, examples of the epoxy photosensitive resin include SU-8 (trade name) represented by the following formula.

    PMMA, PDMS, parylene, polyimide, and PTFE are preferable, and PMMA and PDMS are more preferable.

    The substrate preferably has a plate shape, preferably a flat plate shape. As long as the shape is not limited, the shape can be appropriately set according to the size and shape of the cell culture tank in which the substrate is disposed, such as a triangle, a quadrangle, a polygon, and a circle.

    An example of a method for performing the micropore processing on the resin constituting the cell culture carrier substrate is a method using X-ray lithography (for example, Y. Ukita et al., “Fluid filter fabricated”). by deep X-ray lithography for micro fluidics ”, Microsystem Technology, 13, pp.435-439, 2007).

    In such a cell culture carrier substrate, the cell culture surface of the substrate, particularly the inner wall surface of the through hole, may be further subjected to processing such as roughening treatment in order to improve cell adhesion. Such roughening treatment is not particularly limited as long as it is a method capable of roughening the surface of the cell culture support substrate (formation of fine irregularities). For example, chemical treatment of treating a substrate with an organic solvent such as acetone, plasma on the surface of the substrate, nano-imprinted nano and sub-micron pattern molding can be exemplified. For example, when the above-mentioned resin such as PMMA or PDMS is used as the substrate material, chemical treatment using an organic solvent such as acetone is preferable, although it varies depending on the material of the substrate used.

    If necessary, the surface of the resin constituting the cell culture carrier substrate may be coated with collagen, phosphatidylcholine, phosphatidylserine, lecithin, hydrogenated of these unsaturated fatty acids, gelatin, fibronectin, or the like. About the cell culture carrier substrate by introducing a functional group having a positive charge or a negative charge, or modifying the resin surface with various cell adhesion factors (for example, RGD peptide (Arg-Gly-Asp)) Cell affinity and adhesion can also be increased.

    A hybridoma producing a monoclonal antibody has an intermediate property between adherent cells and blood cells. For this reason, although it couple | bonds with a cell culture dish with very weak force, the coupling | bonding will dissociate with slight stress. The above-mentioned various processing treatments including the roughening treatment of the cell culture carrier substrate can enhance the adhesion of cells having intermediate properties between such adherent cells such as hybridomas and blood cells to the substrate. Useful.

(II) Cell Culture Method Using Cell Culture Carrier Substrate Cell culture using the cell culture carrier substrate of the present invention described above is carried out so that the opening of the through-hole is preferably parallel to gravity. Placed in a cell culture vessel (for example, a cell culture vessel such as a cell culture dish) containing a liquid medium, and seeds the target cells on the upper surface (upper surface of the opening of the through hole) of the porous structure of the substrate. And can be carried out by culturing.

    By doing so, the cells enter the through-holes of the substrate and adhere and proliferate using the inner wall surface as a scaffold. In addition, cells spontaneously aggregate in the through-hole to form a three-dimensional cell cluster (spheroid).

    In the present invention, cell culture means maintaining at least the survival of cells, preferably adhesive cells, using a culture solution (liquid medium) containing components necessary for the survival of cells such as nutrients and oxygen. means. Preferably, cells (preferably adherent cells) are cultured and proliferated. When the cells are protein-producing cells, it means that the cells are cultured to produce the protein.

    As the cell culture tank, a general-purpose container or tank used for the above purpose can be used. For example, a petri dish, a plastic plate, a plastic tube, a glass tube, a column, a multiwell plate, a culture chamber for stirring culture, a cell holding portion of various cell assay devices, and the like can be mentioned.

    The type and composition of the liquid medium and the culture conditions (culture temperature and time) can be appropriately selected according to standard methods depending on the type of cells to be cultured.

    The target cells in the present invention are adhesive cells (animal cells, insect cells, plant cells, etc.), and the cells include the hybridomas described above having intermediate properties between adherent cells and blood cells. It is. Examples of such cells include tissues and cells derived from animals such as humans, pigs, monkeys, dogs, rats, mice, hamsters (eg, hepatocytes, kidney cells, nerve cells, skin keratinocytes, hair matrix cells, oral epithelial cells, Esophageal epithelial cells, gastric mucosal epithelial cells, small intestine absorbing epithelial cells, large intestine absorbing epithelial cells, bile duct epithelial cells, pancreatic insulin secreting cells, pancreatic glucagon secreting cells, bone cells, chondrocytes, smooth muscle cells, cardiomyocytes, muscle satellite cells, Primary cells obtained from hormone-secreting cells, white adipocytes, brown adipocytes, bone marrow, etc., or established cell lines; stem cells (embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, liver stem cells, pancreas) Stem cells, skin stem cells) and the like; iPS cells.

    Examples of adherent cell lines include CHO cells derived from Chinese hamster ovary cells, MDCK cells derived from canine kidney epithelial cells, NIH3T3 cells derived from mouse fetal skin, PC12 cells derived from rat adrenal medulla, and S2 derived from Drosophila. Examples include cells, sputum-derived Sf9 cells, African green monkey kidney-derived Vero cells, human uterine cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, human liver cancer-derived Huh7 cells, and HepG2 cells.

    The cells also include genetically modified cells such as cells configured to produce foreign proteins and cells deficient in specific genes. In addition, it is preferable that this protein producing cell is comprised so that the produced protein may be secreted out of a cell.

    Culturing can be carried out in a state where the cell culture carrier substrate of the present invention in which cells are seeded is left in a cell culture tank, but nutrients and oxygen are applied to cells that are engrafted on the inner wall surface of the through-hole. In order to supply without delay, the cell culture support substrate is not placed on the bottom surface of the cell culture tank, and is cultured in a suspended state in the tank, or while being stirred in the suspended state in the tank, Moreover, it is preferable to culture | cultivate, circulating a liquid culture medium in the state suspended in the tank.

    When culturing cells with the cell culture carrier substrate placed on the bottom of a cell culture vessel (cell culture dish or other cell culture vessel), apply polydimethylsiloxane (PDMS) to the bottom of the cell culture vessel. It is preferable to keep it. PDMS has the property of taking oxygen from a high concentration solution and releasing oxygen in a low concentration solution. For this reason, in cell culture on the PDMS-coated surface, oxygen is released from the PDMS when the number of cells increases and the oxygen concentration decreases, so that the cells can survive or grow for a long period of time.

    In the culturing, not only one cell culture carrier substrate of the present invention but also two or more substrates can be used in a laminated state, for example. Here, the term “lamination” includes not only the case where two or more substrates are bonded together, but also the case where each substrate is layered with an interval. When laminating in the former mode, it is desirable that the openings of the substrates are aligned in order to prevent the openings of the through holes from being blocked.

    Thus, the target cells can be proliferated three-dimensionally in the through hole of the substrate. In addition, spheroids can be efficiently formed without causing central necrosis. Furthermore, when the target cell is a protein-producing cell, the desired protein is produced and acquired by culturing the cell to accumulate the protein inside the cell or to secrete it outside the cell (in a liquid medium). Can do. In addition, since the spheroid grown in the through-hole of the cell culture carrier substrate can be easily removed from the through-hole, the extracted spheroid can be used as a tissue piece for transplantation or the like.

(III) Cell culture reactor or cell culture system, and cell culturing method using these cells The cell culture reactor of the present invention comprises the above-described cells of the present invention in a cell culture tank having at least a fluid inlet and an outlet. A state in which two or more culture carrier substrates are laminated at intervals (hereinafter, this state is referred to as “lamination”, and a laminated assembly is also referred to as “lamination”), that is, arranged as a laminate. It is characterized by being made. In addition, the living cell to culture | cultivate has adhered to the porous structure part of the said cell culture support substrate on the inner wall surface of the through-hole. Or the spheroid may be formed in the through-hole.

    As a first embodiment of the cell culture reactor (0) of the present invention, for example, as shown in FIG. 1, a cell culture reactor (1) having at least a fluid inlet (2) and an outlet (3) penetrates. Two or more cell culture carrier substrates (6) each having a living cell (5) attached to the inner wall surface of the hole (4) are laminated at intervals, and a liquid medium (7) corresponding to the fluid flows. It flows into the cell culture tank (1) from the inlet (2), passes through the through hole (4) of the porous structure part of the cell culture carrier substrate (6) arranged in the tank, and flows from the outlet (3) to the tank. Examples include a fluid (liquid medium) channel formed so as to be discharged to the outside. According to the cell culture reactor, it is possible to supply components necessary for survival and growth such as nutrients and oxygen to cells adhered in the through-holes of the cell culture support substrate without delay.

As a second embodiment of the cell culture reactor (0) of the present invention, for example, as shown in FIG. 2, in a cell culture tank (1) having at least a fluid inlet (2) and an outlet (3),
(A) Two or more cell culture carrier substrates (6) formed by adhering live cells (5) capable of forming spheroids (5 ') in the through-hole (4) are laminated at intervals,
(B) (a) at least one inflow channel (8) for allowing the liquid medium (7) to flow into the cell culture tank (1) from the inlet (2);
(B) Three or more branch paths (9) that branch from the inflow path (8) and communicate with each other between the layers of the cell culture support substrate (6) that are stacked at intervals of two or more, and (c) three or more Having at least one outflow channel (10) where
An example is one in which a flow path is formed so that the liquid medium (7) flowing in from the inflow port (2) is discharged out of the tank from the outflow port (3) through these flow paths. As shown in FIG. 2, the inflow path (8) and the outflow path (10) are provided between the side surface portion of the cell culture carrier substrate (laminate) and the inner peripheral surface of the cell culture tank facing it. And the branch path (9) is formed between the flat part of the cell culture carrier substrate and the flat part of the cell culture carrier substrate laminated thereon, or the flat part of the cell culture carrier substrate and the bottom part of the cell culture tank or It is formed between the upper surface part.

    According to the cell culture reactor, even when the through-hole of the porous structure part of the cell culture support substrate is blocked by the formation of spheroids, the spheroid in the through-hole stagnates components necessary for survival and proliferation such as nutrients and oxygen. Can be supplied without.

    As the second embodiment of the cell culture reactor (0) of the present invention, more preferably, as shown in the schematic view of FIG. 3, the inflow channel (8) is formed to taper in the fluid flow direction. In addition, the outflow path (10) is formed so as to become wider in the fluid flow direction. A mode in which the flow path is tapered or widened is not particularly limited, and may be gradually tapered or widened in a stepped manner, for example, or may be gradually tapered or widened with a smooth straight line. According to such a cell culture reactor, the flow rate of the liquid medium can be stably or uniformly maintained even when the through hole of the porous structure portion of the cell culture support substrate is blocked by the formation of spheroids.

    The cell culture reactor of the present invention preferably includes a liquid medium supply device or a liquid medium circulation device so that the liquid medium flows along the flow path in the cell culture tank described above. An example of such an apparatus is a pump for feeding a liquid medium into a cell culture tank (reference numeral 21 in FIG. 27). In addition, the cell culture reactor of the present invention may be equipped with, for example, a temperature control device for a culture tank or a thermostat having a hollow temperature control jacket in order to manage the temperature of the liquid medium in the cell culture tank. good.

    In addition, the cell culture reactor of the present invention relates to a device (such as a DOT probe) for measuring the dissolved oxygen content of a liquid medium, and the dissolved oxygen content in order to manage the state of oxygen supply to the cells. Accordingly, a device for adjusting the flow rate of the liquid medium and the amount of oxygen supplied to the medium may be included.

    Furthermore, in order to manage the pH of the liquid medium, the cell culture reactor of the present invention detects the pH of the liquid medium (such as a pH probe), and detects the pH, and adjusts the pH of the liquid medium accordingly. A device for adjustment (symbol 22 in FIG. 27) may be included.

    Furthermore, the cell culture reactor according to the present invention includes an apparatus for measuring the electrolyte concentration of the liquid medium in order to manage the electrolyte concentration of the liquid medium, and an apparatus for detecting the electrolyte concentration and adjusting the electrolyte of the liquid medium accordingly. (Symbol 23 in FIG. 27) may be included.

    By using a cell culture system equipped with such a cell culture reactor and various devices, cells can be efficiently and three-dimensionally cultured at the porous structure (in the through hole) of the cell culture carrier substrate of the present invention. The target cells are as described above. For example, when the cell is a protein-producing cell, the desired protein can be continuously produced in large quantities by such culture.

    Hereinafter, the configuration and effects of the present invention will be specifically described using experimental examples. However, these experimental examples are one aspect of the present invention and do not limit the invention.

Experimental Example 1 Qualitative evaluation of cell adhesion to three-dimensional microstructured carrier material (Part 1)
Usually, cell culture in vitro is performed in a cell culture dish, and in the case of adherent cells, only culture using a two-dimensional space on the bottom surface of the cell culture dish can be performed. However, in the method using a spheroid array chip made of polydimethylsiloxane (PDMS), cell aggregates are formed, and three-dimensional culture is possible. The cell aggregates of hepatocytes obtained by this method are attracting attention because they have advantages such as high albumin secretion activity and drug metabolism activity compared to two-dimensional cultured hepatocytes. However, when cell aggregates are formed, it becomes difficult to supply nutrients to the cells present in the central part of the aggregates, resulting in necrosis of the cells in the central part of the aggregates. As shown in FIG. 4, these cell aggregates are considered to require a solution because the cells in the central part thereof begin to be necrotic when they grow to a diameter of about 500 μm.

(1) Production of three-dimensional microstructured carrier In Experimental Example 1, circular and polygonal shapes (triangle, quadrangle, pentagon, star, rhombus, and honeycomb) having a side length or diameter of 50 μm to 300 μm. A substrate made of polymethylmethacrylate (PMMA) having a through-hole structure (hereinafter also referred to as “capillary structure” or “three-dimensional microstructure”) made of polymethyl methacrylate (PMMA) is prepared by X-ray lithography to determine whether cell adhesion is possible. It examined (refer FIG. 5).

(2) Cell culture in the through-hole of the three-dimensional microstructure carrier The substrate having the three-dimensional microstructure prepared above (hereinafter referred to as “three-dimensional microstructure carrier” or “microcarrier”) is made of 70% ethanol. After sterilization, place it on the bottom of a 100 mm cell culture dish with 10 ml of 10% fetal bovine serum and antibiotics (50 U / mL penicillin, 50 mg / mL streptomycin) added to Dulbecco's Modified Eagle's Medium. On top of this, HepG2-pEGFP cells (2 × 10 ⁶ cells) introduced with enhanced green fluorescent protein (EGFP) gene were seeded into HepG2 cells, which are human liver cancer-derived cells. Incubated with

    Since EGFP is a protein that emits fluorescence only with excitation light, it is possible to observe living cells over time under a fluorescence microscope and a confocal laser microscope. In addition, if the expression and production of EGFP can be confirmed in this culture system, it is possible to culture useful protein-producing cells in this cell culture system by introducing a foreign protein gene instead of the EGFP gene, that is, it is possible to produce useful protein. Conceivable.

    Cell adhesion and proliferation in the through-hole (hereinafter also referred to as “capillary”) of the microcarrier were observed with a fluorescence microscope (Olympus CKX41). The fluorescence microscope used a mercury lamp as an excitation light source, and measured under conditions of an excitation wavelength of 460-490 nm and an absorption wavelength of 520 nm.

(3) Results FIGS. 6 and 7 show the state of cell adhesion in the capillary of the microcarrier on the third day of culture observed under a fluorescence microscope. As shown in the figure, since HepG2-pEGFP cell luminescence was observed on the inner wall of the microcarrier capillary, it was confirmed that cell culture was possible in the microcarrier capillary. As shown in the figure, it was observed that the cell adhesion efficiency differs depending on the shape and size of the capillary structure (three-dimensional microstructure). Specifically, the cell inner surface of a capillary tends to be high, while the inner wall surface of the capillary tends to be high in a flat planar shape, in particular, a substrate having a honeycomb, rhombus, rectangle, pentagon, or triangle shape hole. Cell adhesion was hardly confirmed on the substrate having a curved wall surface, that is, a cylindrical hole (FIG. 6 (f)). Therefore, PMMA substrates having a capillary structure (three-dimensional microstructure) with polygonal, particularly rhomboid (parallelogram) and honeycomb-shaped through-holes are used as cell culture carriers and cell culture reactors. It was suggested to be useful as a carrier. In addition, when the same experiment was performed by changing the length of one side of the capillary shape (honeycomb shape, rhombus), the length of one side of the through hole was obtained even if the inner surface was a through hole having a linear planar shape. Was as short as 30 μm, cell adhesion was hardly confirmed (see FIG. 7 (e)). This is considered to be because the diameter of the HepG2-pEGFP cell is about 30-50 μm, which is larger than the length of one side of the through hole of the microcarrier.

Experimental Example 2 Qualitative evaluation of cell adhesion to three-dimensional microstructured carrier (Part 2)
Based on the result of Experimental Example 1, as shown in FIG. 8, a porous structure portion having a honeycomb-shaped through hole having a thickness of 1 mm, a filter diameter of 12 mm, and a pattern portion diameter of 8 mm and having a side of 100 μm on one side. A three-dimensional microstructure carrier (microcarrier) made of PMMA with a thickness of 10 mm was prepared. Further, instead of the honeycomb shape, a PMMA three-dimensional microstructure support (microcarrier) having a porous structure portion having a rhomboid through-hole with a side of 150 μm and an inner angle of 60 ° was prepared (see FIG. 8). With respect to these microcarriers, the possibility of cell adhesion was examined by the same method as in Experimental Example 1.

    Specifically, the microfabricated PMMA microcarriers subjected to the above microfabrication were placed on the bottom of a 100 mm cell culture dish to which the culture solution was added, and then HepG2-pEGFP cells were seeded and cultured on the 4th day of culture. The state of cell adhesion was observed with a confocal laser microscope (Carl Zeiss LSM510). The confocal laser microscope used an Ar laser as an excitation light source and measured under conditions of an excitation wavelength of 488 nm and an absorption wavelength of 505 nm.

    FIG. 9A shows the state of cell adhesion to a honeycomb-shaped microcarrier having a side of 100 μm, and FIG. 9B shows the state of cell adhesion to a rhombus-shaped microcarrier having a side of 150 μm and an inner angle of 60 °. As can be seen from this, as in the observation result under the fluorescence microscope (Experimental Example 1), if the inner surface of the through hole has a straight planar shape ((a) honeycomb, (b) rhombus) It was confirmed that cell adhesion was well performed on the wall surface. The cell growth distance from the bottom of the culture dish was about 100 μm for the honeycomb-shaped carrier and about 200 μm for the diamond-shaped carrier (see each side view). On the other hand, when the inner surface of the through hole has a curved circular shape, cell adhesion was hardly confirmed (see FIG. 6 (c)). From the above results, it became clear that the cell adhesion efficiency differs depending on the shape and size of the through hole.

    Furthermore, the state of cell change with time in the honeycomb-shaped microcarrier was observed (FIG. 10). The cell growth distance from the bottom of the culture dish was about 100 mm on the 3rd day of culture (FIG. 10 (a)), but on the 11th day of culture, cell growth was observed up to about 200-300 μm. A state in which cells were proliferating was observed (FIG. 10 (b)).

    As shown in the above results, cells adhere to the inner wall of the capillary of the PMMA substrate that has a capillary structure with honeycomb and rhombus-shaped through holes, and proliferate while progressing vertically from the bottom of the dish over time. It was confirmed that This suggests that a PMMA substrate having a capillary structure having honeycomb-shaped and diamond-shaped through-holes is a suitable material for a cell culture carrier used in a continuous cell culture reactor.

Experimental Example 3 Quantitative evaluation of cell adhesion to 3D microstructure carrier material (Part 3)
(1) As shown in FIG. 11 (A), it has a porous structure part having a honeycomb-shaped through-hole having a thickness of 1 mm, a filter diameter of 12 mm, and a pattern part diameter of 8 mm, and a side of the pattern part of 50 to 300 μm. PMMA three-dimensional microstructure support (microcarrier) and PMMA three-dimensional microstructure with a porous structure with rhomboid through-holes of 150 μm on each side and acute angles of 30, 45, 60 ° instead of honeycomb shape Each structure carrier (microcarrier) was prepared and the influence on cell adhesion was examined. As a control experiment, as shown in FIG. 11B, a disk-shaped PMMA substrate ((a) flat plate PMMA) having a diameter of 12 mm and a thickness of 1 mm that has not been finely processed, and a diameter of 1 mm and a thickness of 1 mm. The effect on cell adhesion of a ring-shaped PMMA substrate ((b) ring-shaped PMMA) (hereinafter collectively referred to as “control PMMA”) having a through-hole with a diameter of 8 mm in the center of the disk It was investigated. By using these two types of control PMMA, the result of the ring-shaped PMMA can be subtracted from the result of the flat plate PMMA, and can be regarded as a control of only the pattern portion (porous structure portion) of the carrier.

    Specifically, on the bottom of a 100 mm cell culture dish to which a culture solution has been added, a PMMA three-dimensional microstructure carrier (this is called “PMMA carrier”) and control PMMA (hereinafter referred to as this experiment) In the example, these were collectively referred to as “microcarriers”), and HepG2-pEGFP cells were seeded. Take out these microcarriers on the 4th, 7th, 10th, 12th, 14th and 18th days after sowing, wash them twice with phosphate buffered saline, and repeat freeze-thaw in 0.1% Tween-20 aqueous solution. Cells attached to each microcarrier were disrupted and then centrifuged to obtain a cytoplasmic fraction. The amount of GFP obtained by measuring the fluorescence intensity in this cytoplasmic fraction with a fluorometer (RF-5300PC manufactured by Shimadzu Corporation, excitation wavelength 490 nm, absorption wavelength 510 nm) and applying it to a calibration curve prepared with standard GFP Was used to evaluate the number of cell adhesion to each microcarrier. Prior to this study, it was confirmed that there was linearity between the number of HepG2-pEGFP cells and the fluorescence intensity in the cytoplasmic fraction (see FIG. 12).

    FIG. 13A shows the result of comparing the number of cell adhesions between the PMMA carrier having a honeycomb-shaped capillary structure with a side of 50 to 300 μm and the control PMMA. As shown in FIG. 13 (A), the amount of GFP (adhesion) is higher in the PMMA carrier with the honeycomb-shaped microfabrication compared to the control PMMA (ring-shaped PMMA:-○-, flat plate PMMA:-●-). It was observed that the amount of GFP (the number of adherent cells) tended to increase as the length of one side was shorter in the honeycomb shape. It is considered that the smaller the one side of the honeycomb shape is, the larger the surface area of the inner wall of the through hole is, so that the number of cell adhesion increases.

    FIG. 13B shows the result of comparing the number of cell adhesions between the PMMA carrier having a rhomboid capillary structure with acute angles of 30, 45, and 60 ° and the control PMMA. As shown in FIG. 13 (B), the amount of GFP (adhesion) is higher in the PMMA carrier with diamond-shaped microfabrication compared to the control PMMA (ring-shaped PMMA:-○-, flat plate PMMA:-●-). The number of GFP (adherent cells) tended to increase as the interior angle became sharper, even among the diamond-shaped shapes. It is considered that the number of cell adhesion increases because the surface area of the inner wall of the through-hole is larger as the rhombus-shaped inner angle is sharper.

    On the other hand, in the circular pattern performed as a comparative experiment, the amount of GFP was almost equal to or less than that of flat plate PMMA (-●-) (see FIG. 13C). In addition, both the flat plate PMMA and the finely processed PMMA carrier showed a peak in the number of cells on the 10th to 12th days from the start of the culture, and a tendency to be maintained thereafter.

  (2) Furthermore, the amount of GFP per capillary inner wall surface area was calculated for a PMMA carrier having a honeycomb-shaped and rhombus-shaped capillary structure. The results are shown in FIG.

    As a result, in the honeycomb shape, as the length of one side is longer, the amount of GFP per surface area of the capillary inner wall tends to increase (FIG. 14A), and in the rhombus shape, the surface area of the capillary inner wall increases as the inner angle becomes obtuse. There was a tendency for the amount of per GFP to increase (FIG. 14B).

  (3) Moreover, the integral value of the amount of intracellular GFP obtained by the 14th day of culture | cultivation was analyzed for every shape pattern of the through-hole. From this analysis result, the number of cells present in the capillary of the microcarrier during the culture period was evaluated. Also, in order to evaluate the number of adherent cells in the 8 mm diameter pattern part (porous structure part) of the microcarrier, the value obtained by subtracting the integrated value of the GFP amount in the ring-shaped PMMA from the integrated value of the obtained GFP amount was obtained. The vertical axis is taken. The results are shown in FIG. The round shape and the honeycomb shape with one side length of 200 and 300 μm were almost equal to or less than the flat plate PMMA, but the honeycomb shape with one side of 50 to 150 μm and the acute angle was 30 ° ~ The 60 ° diamond shape is higher than the integrated value of the GFP amount of flat plate PMMA, especially the honeycomb shape with one side of 50μm, and the diamond shape with acute angles of 45 ° and 30 ° is about 3 times GFP compared to flat plate PMMA. The integral value of the quantity was obtained.

Further, when the number of cells is calculated from the amount of GFP based on the diagram of FIG. 12, the maximum number of adherent cells in the capillary of the microcarrier is 7.4 × 10 ^{5 with} respect to a pattern portion (capacity: 50.27 mm ² ) having a side of honeycomb of 50 μm. cells / scaffold (1.47 × 10 ⁷ cells / ml). Considering that cell culture can be performed at a density of about 5 × 10 ⁶ cells / ml on a cell culture petri dish, it is said that the culture is a high-density culture. It was shown that high-density culture is possible.

    From the results of Experimental Examples 1 to 3, PMMA is an appropriate material as a cell culture carrier material used in the cell culture reactor, and further, by applying a three-dimensional microstructure processing having a honeycomb-shaped or diamond-shaped through-hole to these materials. It was confirmed that cells can be cultured in a higher density state than a two-dimensional structure having the same area.

    In addition, as a result of comparing cell adhesion to a PMMA carrier with three-dimensional microfabrication of honeycomb structures or rhombus-shaped capillary structures of different sizes and angles, the number of cell adhesion per capillary inner wall surface area was 300 μm on a side. The shape of the capillary structure was the highest (FIG. 14A).

    As shown in Experimental Example 2, in the observation with a confocal laser microscope, the cell progress to the inner wall of the capillary remains at about 200 μm from the bottom surface. From this, among the three-dimensional structures used in this study, as a material for cell culture carriers or cell culture reactors, a honeycomb-shaped capillary structure with a side of 300 μm with the highest cell adhesion number per capillary inner wall surface area (three-dimensional A substrate made of PMMA having a fine structure) was considered most preferable.

Experimental Example 4 Cell Proliferation Effect by Suspension of Culture Carrier in Culture Medium and Stirring of Culture Solution As shown in the above experiment example, cell adhesion to the inner wall of the capillary in a PMMA carrier (microcarrier) with a three-dimensional microstructure However, for all the carriers, the number of adherent cells peaked on the 10th to 12th day from the start of the culture, and was maintained thereafter. The reason for this is that the supply of nutrients and oxygen to the cultured cells depends on molecular diffusion from the top of the cell culture dish on which the microcarriers are arranged, and the conventional culture methods use the carrier as the bottom surface of the cell culture dish. Because the carrier is 1 mm thick and the carrier is 1 mm thick, the supply of nutrients and oxygen necessary for further growth of cells that have grown to a certain amount over time is insufficient. Can be considered. In order to solve these problems, an attempt was made to float a microcarrier having cells attached to the inner wall of the capillary in the culture medium and to further culture the culture medium while stirring.

    Specifically, cells are seeded in a state where a PMMA carrier (microcarrier) having a honeycomb-shaped capillary structure with sides of 100 μm and 300 μm is placed on the bottom of a cell culture dish containing a culture solution, and the cells are microcarriers. Suspension culture was started by giving a scaffold to the microcarriers on the dish 6 days after sowing, which was considered to have adhered sufficiently in the capillaries. In the stirring culture, a sterilized rotator was sunk in a dish, and the culture was stirred with a magnetic stirrer. These were cultured in an incubator, and the state of the cells in each was observed with a fluorescence microscope and a confocal laser microscope. The culture conditions and microscope observation conditions were the same as in Experimental Examples 1 and 2.

    In each culture method ((a) static culture, (b) suspension culture, (c) stirring culture), the presence of cells in the capillary of the microcarrier on the 17th day of culture is shown in FIG. FIG. 17 shows a confocal laser microscope image of the presence state of the cells in the capillary of the microcarrier on the 17th day of culture in the stirring culture. As a result, there was a tendency for the number of cells to increase in suspension culture and stirring culture compared to static culture.

    As shown in FIG. 13 (a), it can be seen that in static culture, almost no cells are adhered to the inner wall surface of the capillary. On the other hand, as shown in FIGS. 16 (b) and 16 (c), it was confirmed that cells were in the form of spheroids (cell aggregates) in the capillary in suspension culture and stirring culture. The reason for this is that although the microcarriers floated in the culture medium and the supply of nutrients and oxygen were improved by the agitation of the culture liquid, the growth of cells attached to the inner wall of the capillary became active. It was inferred that cell growth did not easily occur toward the upper part of the capillary wall surface under the influence of the above, and as a result, it stayed at the lower part of the capillary wall surface and formed an aggregate.

    In addition, the cells obtained by suspension culture and stirring culture had strong fluorescence intensity compared to stationary culture, and the cell activity was very high. Further, as shown in FIG. 17, it was confirmed that a cell spheroid having a diameter of about 100 μm was formed in the confocal laser microscope image of the stirring culture.

    FIG. 18 shows the results of quantitative evaluation of the number of adherent cells in stationary culture and suspension culture. As shown in FIG. 18, the static culture reached a peak of the amount of GFP near the 14th and 10th days of culture, regardless of whether the side of the honeycomb shape was 100 μm or 300 μm. On the other hand, in suspension culture, the amount of GFP continued to increase until the 18th day of culture in both cases of honeycomb shapes of 100 μm and 300 μm on one side, and a higher GFP amount was obtained than in stationary culture. Moreover, the tendency for the amount of GFP to increase after the 18th day was also observed.

    FIG. 19 also shows the number of adherent cells (GFP amount: μg) (white bar) when cultured for 17 days in the state where the microcarriers are placed on the bottom of the cell culture dish in order from the left end. The number of adherent cells (GFP amount: μg) (gray bar) when the carrier is suspended in the culture medium and cultured for 11 days. The microcarrier is suspended in the culture medium on the sixth day after cell seeding and stirred. The number of adherent cells (GFP amount: μg) (black bar) when cultured for 1 day is shown. As shown in FIG. 19, the number of adherent cells was higher in suspension culture (gray bar) and stirring culture (black bar) than in stationary culture (white bar).

    In the static culture, since the microcarrier is left on the cell dish, there is almost no supply of nutrients and oxygen in the culture solution from the bottom of the microcarrier, and only the diffusion from the top of the microcarrier is performed. On the other hand, in suspension culture or stirring culture, the fresh culture solution also contacts the bottom of the microcarrier. Therefore, although there is an influence due to gravity, it seems that cells may gather at the bottom of the microcarrier having a good culture environment. From these results, it can be said that floating culture and stirring culture in which oxygen and nutrients are appropriately supplied into the capillaries of the porous structure of the microcarrier are useful for high-density cell culture. Moreover, since it can culture | cultivate in the state which adhere | attached the cell spheroid on the support | carrier, the application to a continuous cell culture reactor can also be performed. In addition, it is considered that higher density culture is possible by applying to continuous culture.

    Furthermore, the cell spheroid formed by suspension culture was peeled off from the microcarrier by a pipette and cultured on a cell culture dish (culture day 21). The results are shown in FIG. On the third day of detachment, the state of the cell spheroid did not change (FIG. 20 (a)), but on the tenth day, it was confirmed that cells were proliferating in a monolayer from around the cell spheroid (FIG. 20 ( b)). Further, on the 14th day, it was observed that the cells were further proliferating into a monolayer from the periphery of the cell spheroid, and the central part of the cell spheroid was necrotized (FIG. 20 (c)). This is probably because if the cell spheroid grows too large, nutrients do not reach the central part of the spheroid and the central cell is necrotic. If necrosis occurs in the central part, it may affect surrounding cells. Therefore, it is necessary to devise a cell culture reactor using cell spheroids so that the cell spheroids do not become too large.

Experimental Example 5 Effect of surface roughening of PMMA carrier on cell adhesion Based on the results of Experimental Example 4, although cell adhesion to a PMMA carrier (microcarrier) subjected to three-dimensional microfabrication is possible, normal culture In the method, it was clarified that the cell growth to the upper part of the inner wall surface of the capillary did not progress so much, but only the adhesion at the lower part of the inner wall surface of the capillary. As a method for solving this problem, it was considered to roughen the surface of the microcarrier (particularly the inner surface of the capillary).

(1) Surface roughening with acetone First, a circular flat plate made of PMMA having a diameter of 12 mm and a thickness of 1 mm (flat plate PMMA) was immersed in acetone for 10 minutes (acetone treatment) to roughen the surface. The obtained roughened PMMA is seeded on the bottom surface of the cell culture dish containing the culture solution, seeded with cells, taken out on days 4, 7, 11, and 14 and washed with phosphate buffered saline. Thus, the cells attached to the carrier were collected. Next, the collected cells were repeatedly freeze-thawed in a 0.1% Tween-20 aqueous solution to obtain a cytoplasmic fraction. The fluorescence intensity in this cytoplasmic fraction was measured, and using the amount of GFP obtained by fitting to a calibration curve (FIG. 12) prepared with standard GFP, the change over time in the number of cells adhered to roughened PMMA was measured. evaluated.

    The results are shown in FIG. As a result, compared with flat plate PMMA (untreated PMMA) (-×-), roughened PMMA (-◆-) has a higher cell adhesion rate (cell proliferation rate) and a tendency to adhere to many cells. It was. Moreover, the result of estimating the integral value of the amount of GFP up to the 14th day of culture by the trapezoidal method is shown in FIG. As a result, the integrated value of roughened PMMA (FIG. 21 (b): black bar) was nearly twice as high as that of flat plate PMMA (untreated PMMA) (FIG. 21 (b): white bar). From this, it was found that roughening the surface of the PMMA carrier was effective for cell adhesion and cell proliferation.

    FIG. 22 shows the results of comparison of cell adhesion and growth on roughened PMMA and flat PMMA (untreated PMMA) ((1) 4th day of culture, (2) 7th day of culture, (3) Culture day 11). It can be seen that the cells adhere and proliferate in a single layer on the flat PMMA on the 4th and 7th day of culture. However, on the roughened PMMA, the cells seemed to be on top of each other, indicating that they were proliferating while forming spheroids. As described above, it was shown that the surface of the cell adhesion carrier is roughened to affect the cell adhesion and proliferation.

    Furthermore, it is considered that cell adhesion and cell proliferation can be further enhanced by chemical modification with a positive charge load or a biocompatible peptide on the surface of PMMA.

(2) Surface modification by radiation irradiation (2-1) Experimental method
PMMA was irradiated with synchrotron radiation at NewSUBARU synchrotron radiation facility BL-2 during 1.0 GeV operation, and four samples with X-ray exposure (dose) 0, 2000, 5000, and 10000 [mA * s] were prepared. .

    Cells were seeded with radiation-treated PMMA (radiation-treated PMMA) on the bottom of the cell culture dish containing the culture solution, and this was treated on days 2, 5, 7, and 9 after the start of culture. It was taken out from the culture dish. This was washed twice with PBS, and trypsin-EDTA was added to detach the cells. After detachment, the enzyme reaction was stopped by substitution with DMEM, and after centrifugation, the number of cells attached to the radiation-treated PMMA was measured with a hemocytometer.

(2-2) Experimental Results FIG. 23 shows the state of cell adhesion to PMMA irradiated with synchrotron radiation, and FIG. 24 shows the quantitative evaluation results. From FIG. 23, it can be seen that the number of cell adhesion increases as the exposure dose increases. Moreover, according to the quantitative evaluation result of FIG. 24, it can be seen that the number of cells is larger than the irradiated PMMA than the PMMA that was not irradiated with the emitted light until the seventh day of the culture. However, on day 9, the number of cells on irradiated PMMA generally decreased. This is thought to be because the cells on the exposed PMMA surface are saturated and the cells have an adverse effect. Since there is still room for cells to grow on the unexposed PMMA surface, it is thought to be growing. These results show that the structural change of PMMA caused by X-ray energy absorption worked well for cell culture.

    When PMMA is irradiated with X-rays, photoelectrons and Auger electrons are generated with the absorption of X-ray energy, and radicals are generated in PMMA and carry a positive charge. Since the cell membrane has a negative charge, it is considered that PMMA, which is a cell adhesion carrier, is charged positively by irradiating it with radiation, and electrostatic adhesion between PMMA and cells is improved electrostatically.

Experimental Example 6 Effect on cell adhesion in a culture vessel coated with polydimethylsiloxane (PDMS) From the result of Experimental Example 4, cell adhesion to a PMMA carrier (microcarrier) subjected to three-dimensional microfabrication is possible. In the normal culture method, it was found that the adhesion to the lower part of the inner wall of the capillary was limited, and the cell progress to the upper part of the inner wall of the capillary did not progress much. As a method for solving this problem, the inventors considered culturing cells on a cell culture dish in which PDMS, which is a cell non-adhesive material, was applied to the bottom surface.

    Specifically, on a cell culture dish (PDMS-treated dish) with a thin coating of polydimethylsiloxane (PDMS) on the bottom surface, a microcarrier having a capillary structure with a honeycomb side of 100 μm is allowed to stand and HepG2-pEGFP cells Sowing. After culturing for 3, 8, 10, 13, 15, 17, and 20 days after seeding, this was taken out and washed with phosphate buffered saline to recover the cells attached to the carrier. Next, the collected cells were repeatedly freeze-thawed in a 0.1% Tween-20 solution to obtain a cytoplasmic fraction. The fluorescence intensity in this cytoplasmic fraction was measured, and the change over time in the number of cells adhered to the microcarrier was evaluated using the amount of GFP obtained by fitting to a calibration curve (see FIG. 12) prepared with standard GFP. . For comparison, the same experiment was performed using a cell culture dish without PDMS (PDMS-untreated dish).

    The results are shown in FIG. The growth rate at the beginning of the culture was almost the same between the PDMS-untreated dish and the PDMS-treated dish. However, on the PDMS-untreated dish, there is a peak of GFP in about 10 days of culture, whereas on the PDMS-treated dish, the amount of GFP has increased even after the 20th day of culture. It was possible. As a factor of this result, good oxygen permeability of PDMS, a large amount of oxygen content in PDMS, etc. can be considered. That is, it is considered that PDMS has a property of taking in oxygen in a high concentration solution and releasing oxygen in a low concentration solution. In the in-capillary culture of the microcarrier on the cell culture dish, the number of cells in the capillary increases as the number of culture days increases, and it seems that oxygen supply cannot catch up. If this is on PDMS, oxygen supply is provided from PDMS, so cell growth is likely to continue for a longer time than when cultured on a cell culture dish.

    From this result, it is considered that cell culture in a microcarrier on PDMS is useful in high-density cell culture.

Further, from FIG. 25, the maximum number of adherent cells obtained in the microcarrier by culture on PDMS is 1.63 × in the pattern portion (porous structure) (capacity: 50.27 mm ² ) having a side of honeycomb of 100 mm. 10 ⁶ cells / scaffold (3.24 × 10 ⁷ cells / ml). Therefore, considering that cell culture can be performed at a density of about 5 × 10 ⁶ cells / ml on a cell culture dish, it is said that high-density culture is possible. It was shown that.

    From this result, it is considered that cell culture in a microcarrier on PDMS is effective for long-term high-density cell culture.

Experimental Example 7 Cell Culture by Cell Culture Reactor One embodiment of the cell culture reactor of the present invention is shown in FIG. The cell culture reactor has an inner groove portion (13) that can accommodate a plurality of substrates (microcarriers) (6) having a three-dimensional microstructure of the present invention (6) stacked in a predetermined interval. A cell (11) and an outer cell (12) for containing the inner cell and sealing the culture medium are constituted. The inner cell was provided with 20 groove portions for fixing the microcarriers, and 20 microcarriers were laminated. The microcarrier is rectangular and has a capillary area of 16 × 16 mm so as to obtain as many cell adhesions as possible (a size of 19 × 21 mm when an outer frame for supporting the capillary carrier is included). It was designed so that the outer frame of this capillary carrier fits into the groove of the inner cell. In addition, acrylic having excellent light transmittance was used as a reactor material so that the pH change of the culture solution in the cell culture reactor can be visually confirmed as a change in the color of the culture solution.

    FIG. 27 shows an outline of the internal cell of the cell culture reactor. Here, for convenience of explanation, a state is shown in which ten microcarriers (cell culture carrier substrates) are arranged in an inner cell of the cell culture reactor in a state of being stacked at intervals. Here, in the cell (1) having at least the fluid inflow port (2) and the outflow port (3), there are 10 microcarriers (6) formed by living cells attached to the inner wall surface of the capillary at intervals. So that the liquid medium flows into the cell from the inlet (2), passes through the stack of microcarriers arranged in the cell, and is discharged out of the cell from the outlet (3). A liquid medium flow path (inflow path (8), branch path (9), outflow path (10)) is formed.

More specifically, the cell culture reactor comprises at least a cell (1) having a fluid inlet (2) and an outlet (3),
(A) The microcarriers (6) in which the spheroids (5) are formed in the capillaries (4) are stacked with an interval of 20 sheets,
(B) (a) at least one inflow channel (8) for allowing the liquid medium (7) to flow into the cell (1) from the inlet (2);
(B) Three or more branch paths (9) and (c) 21 branch paths that branch from the inflow path (8) and communicate with each other between the layers of the microcarriers (6) stacked at intervals of two or more. (9) has at least one outflow channel (10) where it merges;
The flow path is formed so that the liquid medium (7) flowing in from the inflow port (2) is discharged out of the cell from the outflow port (3) through these flow paths. Further, as shown in the schematic diagram of FIG. 3, the cell culture reactor is formed so that the inflow path (8) is tapered toward the direction of fluid flow, and the outflow path (10) is in the direction of fluid flow. It is formed so as to become wider toward the front. By using these cell culture reactors, even when spheroids are formed by further growth of cultured cells and the capillaries of the microcarriers are blocked by spheroids, the flow path and flow rate of the liquid medium are ensured stably and uniformly. be able to.

    A cell culture system using the cell culture reactor is shown in FIG. In the cell culture area of the inner cell (11), 20 microcarriers (the length of one side of the honeycomb shape: 200 mm, 100 mm, 50 mm) (6) with cells seeded in the honeycomb-shaped through-hole (capillary) in advance (20) A stage was installed, and the culture solution was refluxed using a pump (21) at a flow rate of 0.83 ml / hour, 3 ml / hour, or 10 ml / hour. In addition, liquid feeding was performed with a syringe pump at a flow rate of 0.83 ml / hour and 3 ml / hour, and with a perista phone at a flow rate of 10 ml / hour. As a result, as shown in FIG. 29, it was confirmed that the cells proliferated well regardless of the capillary size (the length of one side of the honeycomb shape) and that the cells could be continuously cultured. In addition, as shown in FIG. 30, when the flow rate of the cell culture solution passing through the through-hole of the microcarrier was slow, the culture results tended to decrease because the dissolved oxygen concentration and nutrients were insufficient. For this reason, as shown in FIG. 28, the cell culture reactor, together with the “dissolved oxygen concentration measuring device” for measuring the dissolved oxygen concentration of the liquid medium, adjusts the flow rate of the liquid medium in response to the concentration. It is preferable to provide a “control device”. Examples of the “dissolved oxygen concentration measuring device” include an optical dissolved oxygen concentration measuring device (C) having a light source (24) such as an LED and a spectroscope (25). The apparatus is preferably linked with the “flow rate control unit” via the computer (D) so that the flow rate of the liquid medium can be adjusted.

    Further, as shown in FIG. 28, the cell culture reactor of the present invention further has a “pH measurement device” (22) or an “electrolyte concentration measurement device” (23), and is responsive to the pH or electrolyte concentration. It is preferable that a “pH control unit”, “electrolyte control unit” or “flow rate control unit” for adjusting the pH, electrolyte concentration or flow rate of the medium is provided. Examples of the “pH measurement device” (22) include an electrode for pH measurement, and examples of the “electrolyte concentration measurement device” (23) include an electrode for conductivity measurement. The measurement unit is linked with the “pH control unit”, “electrolyte control unit” or “flow rate control unit” via a computer, and the pH, electrolyte concentration or flow rate of the liquid medium can be adjusted. preferable.

    The flow velocity distribution in the cell culture reactor was analyzed by CFD analysis software FLUENT (manufactured by ANSYS JAPAN) by the method of Computational Fluid Dynamics (CFD). In accordance with Kirchhoff's law, the flow path was optimized by keeping the sum of the flow rates at the inlet and the branch path formed between the microcarrier layers branched from the inlet path. Each time the flow path branches off from the inflow path, the width of the flow path is set to 0.8, 0.6, 0.4, and 0.2 mm so that the inflow path tapers in the direction in which the liquid medium flows. The flow path widths were set to 0.2, 0.4, 0.6, and 0.8 mm each time they joined from the branch path so as to become wider in the flowing direction ((A) of FIG. 31). Here, the column (color gradation) shown on the left side of the figure indicates the flow velocity (m / s, meter / second) of the fluid (liquid medium) flowing in the flow path. As shown in FIG. 31 (color diagram), since the colors in the microcarriers are the same, it can be seen that there is little variation in flow rate between the microcarriers stacked in the cell.

    From this result, the inflow path is narrowed by the width of the flow path formed between the layers of the microcarrier each time the flow path is branched therefrom, and the outflow path is each time the microcarrier is joined from the branch path. It can be said that the reactor shape widened by the width of the channel formed between the layers is optimal.

In addition, FIG. 31B shows the result of CFD analysis performed in a shape in which the cell spheroid is not formed and the capillary part (through hole) is present. As a result, it was shown that since the flow also occurred in the through-holes of the microcarrier, the cell performance was possible without reducing the performance of the reactor even when the cell spheroids were not formed.
FIG. 32 shows the result of CFD analysis using a model having the same scale as that of an actual reactor. It can be seen that in the cell culture reactor in which 20 microcarriers having spheroids formed in the capillaries are stacked, a flow is almost uniformly generated in the microcarrier layer.

0: Cell culture reactor of the present invention 1: Cell culture tank 2: Fluid inlet in the cell culture carrier substrate 3: Fluid outlet in the cell culture carrier substrate 4: Through hole (capillary)
5: Live cells
5 ': Spheroid 6: Cell culture carrier substrate (micro carrier)
7: Liquid medium (fluid)
8: Inflow path 9: Branch path
10: Outflow channel
11: Inside cell culture layer
12: Outer cell of cell culture layer
13: Groove
14: Connection with measuring instrument
21: Pump
22: pH measuring device
23: Electrolyte concentration measuring device
24: Light source
25: Spectral intensity
26: Drain (A): Cell culture reactor section (B): pH, electrolyte concentration measurement system (C): Optical dissolved oxygen concentration measurement system (D) Computer

Claims (15)

  A cell culture support substrate having a porous structure, wherein the through-hole formed in the porous structure is a polygonal through-hole of any one of a triangle to a hexagon having a side length or diameter of 50 μm to less than 500 μm A cell culture carrier substrate characterized by the above.   The cell culture carrier substrate according to claim 1, wherein the polygonal shape is a rhombus shape, a parallelogram shape, or a honeycomb shape.   The cell culture support substrate according to claim 1 or 2, wherein at least the porous structure part of the substrate has a thickness of 200 µm to 10 mm.   Formed from one or more resins selected from the group consisting of polymethyl methacrylate, polydimethylsiloxane, parylene, polyimide, polytetrafluoroethylene, epoxy photosensitive resin, polystyrene, polycarbonate, and cycloolefin polymer. The cell culture carrier substrate according to any one of claims 1 to 3.   The cell culture carrier substrate according to any one of claims 1 to 4, wherein at least a cell culture surface of the cell culture carrier substrate is processed.   A cell culture in which at least two cell culture support substrates according to any one of claims 1 to 5 are disposed in a cell culture tank having at least a fluid inlet and an outlet and are stacked with an interval therebetween. reactor.   The cell culture carrier substrate has living cells attached to the through-holes of the porous structure portion, and the liquid medium flows into the cell culture tank from the inlet, and the cell culture carrier substrate in the tank The cell culture reactor according to claim 6, wherein a flow path of the liquid medium is formed so as to be discharged from the cell outlet through the through hole of the porous structure portion to the outside of the cell culture tank. In a cell culture vessel having at least a fluid inlet and an outlet,
(A) Two or more cell culture carrier substrates formed by adhering live cells capable of forming spheroids in the through-holes are laminated at intervals,
(B) (a) at least one inflow path for allowing the liquid medium to flow into the cell culture tank from the inflow port;
(B) Three or more branch paths that branch from the inflow path and communicate with each other between two or more cell culture carrier substrates stacked at intervals, and (c) at least one of the three or more branch paths merges. Has an outflow channel,
The liquid medium flow path is formed so that the liquid medium flowing in from the inflow port is discharged from the outflow port to the outside of the cell culture tank through these flow paths. Cell culture reactor. 9. The cell according to claim 8, wherein the inflow path is formed so as to taper in a direction in which the fluid flows, and the outflow path is formed so as to be wide in the direction in which the fluid flows. Culture reactor.   In addition to the cell culture reactor according to any one of claims 6 to 9, the flow rate, pH or electrolyte concentration of the liquid medium fed to the cell culture reactor in response to the dissolved oxygen amount, pH or electrolyte concentration of the liquid medium A cell culture system, comprising a device for regulating.   A cell culture carrier substrate according to any one of claims 1 to 5, a cell culture reactor according to any one of claims 6 to 9, or a cell culture system according to claim 10 is used. Culture method.   The cell culture carrier substrate according to any one of claims 1 to 5, wherein cells adhere to the through hole of the porous structure part, is placed in a cell culture tank containing a liquid medium, and the cell culture carrier substrate The cell culturing method according to claim 11, further comprising a step of culturing the cell.   Culturing the cell culture support substrate in a suspended state in a liquid medium, culturing the cell culture support substrate in a suspended state in a liquid culture medium, or suspending the cell culture support substrate in a liquid culture medium 13. The method for culturing cells according to claim 11 or 12, wherein the cells are cultured while circulating a liquid medium.   The method for culturing cells according to claim 11 or 12, comprising a step of culturing cells by disposing the cell culture carrier substrate according to any one of claims 1 to 5 on a polydimethylsiloxane-coated surface.   The cell culture reactor according to any one of claims 6 to 9, wherein a cell culture support substrate obtained by the cell culture method according to claim 14 is used. The cell culture method described.

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